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EditorJosef S Smolen (Austria)Associate EditorsFrancis Berenbaum (France)Dimitrios Boumpas (Greece)Gerd Burmester (Germany)Mary Crow (USA)Iain McInnes (UK)Thomas Pap (Germany)David Pisetsky (USA)Désirée van der Heijde (The Netherlands)Kazuhiko Yamamoto (Japan)Methodological and Statistical AdvisorStian Lydersen (Norway)Social Media AdvisorsAlessia Alunno (Italy)Mary Canavan (Ireland)Meghna Jani (UK) Elena Nikiphorou (UK)Caroline Ospelt (Switzerland)Christophe Richez (France)Paul Studenic (Austria)
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Annals of the Rheumatic Diseases publishes original work on all aspects of rheumatology and disorders of connective tissue. Laboratory and clinical studies are equally welcomeEditorial Board
Chairman of Advisory CommitteeJohannes Bijlsma (The Netherlands)
Advisory CommitteeFerry Breedveld (The Netherlands)Marco Matucci Cerinic (Italy)Michael Doherty (UK)Maxime Dougados (France)Paul Emery (UK)Daniel Furst (USA) Steffen Gay (Switzerland) Marc Hochberg (USA)Joachim Kalden (Germany)Edward Keystone (Canada)Lars Klareskog (Sweden)Tore Kvien (Norway)
Zhan-guo Li (China)Peter Lipsky (USA)Sir Ravinder Maini (UK)Emilio Martín-Mola (Spain)Haralampos Moutsopoulos(Greece)Karel Pavelka (Czech Republic)Yehuda Shoenfeld (Israel)Leo van de Putte (The Netherlands) Frank Wollheim (Sweden)Anthony Woolf (UK)
Daniel Aletaha (Austria) Johan Askling (Sweden) Sang-Cheol Bae (Korea) Xenofon Baraliakos (Germany) Anne Barton (UK) Maarten Boers (The Netherlands) Matthew Brown (Australia) Maya Buch (UK) Loreto Carmona (Spain) Carlo Chizzolini (Switzerland) Bernard Combe (France) Philip Conaghan (UK) Maurizio Cutolo (Italy) Nicola Dalbeth (Australia) Christian Dejaco (Austria) Oliver Distler (Switzerland) Thomas Dörner (Germany) Dirk Elewaut (Belgium) Axel Finckh (Switzerland) Roy Fleischmann (USA) Mary Goldring (USA) Laure Gossec (France) Walter Grassi (Italy) Ahmet Gül (Turkey) Frederic Houssiau (Belgium) Tom Huizinga (The Netherlands) Arthur Kavanaugh (USA) Robert Landewé (The Netherlands) Zhan-Gou Li (China)Rik Lories (Belgium)
Ingrid Lundberg (Sweden) Gary MacFarlane (UK) Xavier Mariette (France) Alberto Martini (Italy) Marco Mattuci Cerinic (Italy)Dennis McGonagle (UK) Fred Miller (USA) Peter Nash (Australia) Michael Nurmohamed (The Netherlands) Caroline Ospelt (Switzerland) Monika Østensen (Norway) Constatino Pitzalis (UK) Jane Salmon (USA) Georg Schett (Germany) José da Silva (Portugal)Hendrik Schulze-Koops (Germany) Nan Shen (China) Alexander So (Switzerland) Hiroshi Takayanagi (Japan) Tsutomu Takeuchi (Japan) Yoshiya Tanaka (Japan) Dimitrios Vassilopoulos (Greece) Douglas Veale (Ireland) Jiri Vencovsky (Czech Republic) Ronald van Vollenhoven (Sweden)Erwin Wagner (Spain) Michael Ward (USA) Kevin Winthrop (USA)Huji Xu (China)
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ARDThe EULAR Journal
Editorial867 Changing the wolf from outside: how
microbiota trigger systemic lupus erythematosusI Peene, D Elewaut
Heroes and pillars of rheumatology870 Henry G Kunkel and Rheumatoid Factor
E M Tan
Review872 Unmet need in rheumatology: reports from the
Targeted Therapies meeting 2018K L Winthrop, M E Weinblatt, M K Crow, G R Burmester, P J Mease, A K So, V Bykerk, R F Van Vollenhoven, M Dougados, J Kay, X Mariette, J Sieper, F Melchers, B N Cronstein, E Shevach, F C Breedfeld, J Kalden, J S Smolen, D E Furst
Recommendation879 Clinical relevance of HEp-2 indirect
immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspectiveJ Damoiseaux, L E C Andrade, O G Carballo, K Conrad, P L C Francescantonio, M J Fritzler, I Garcia de la Torre, M Herold, W Klotz, W d M Cruvinel, T Mimori, C von Muhlen, M Satoh, E K Chan
Rheumatoid arthritis890 Clinical outcomes in patients switched from
adalimumab to baricitinib due to non-response and/or study design: phase III data in patients with rheumatoid arthritisY Tanaka, B Fautrel, E C Keystone, R A Ortmann, L Xie, B Zhu, M Issa, H Patel, C L Gaich, S de Bono, T P Rooney, P C Taylor
899 Effects of the anti-RANKL antibody denosumab on joint structural damage in patients with rheumatoid arthritis treated with conventional synthetic disease-modifying antirheumatic drugs (DESIRABLE study): a randomised, double-blind, placebo-controlled phase 3 trialT Takeuchi, Y Tanaka, S Soen, H Yamanaka, T Yoneda, S Tanaka, T Nitta, N Okubo, H K Genant, D van der Heijde
908 Different classes of anti-modified protein antibodies are induced on exposure to antigens expressing only one type of modificationA S B Kampstra, J S Dekkers, M Volkov, A L Dorjée, L Hafkenscheid, A C Kempers, M van Delft, T Kissel, S Reijm, G M C Janssen, P A van Veelen, H Bang, T W J Huizinga, L A Trouw, D van der Woude, R E M Toes
917 Valine 11 and phenylalanine 13 have a greater impact on the T-cell response to citrullinated peptides than the 70–74 shared epitope of the DRB1 molecule in macaquesS Bitoun, P Roques, B Maillere, R Le Grand, X Mariette
Spondyloarthritis922 Aortic-vertebral interaction in ankylosing
spondylitis: syndesmophyte development at the juxta-aortic vertebral rimS Tan, A Dasgupta, J A Flynn, M M Ward
929 Identification of myeloid cells in the human enthesis as the main source of local IL-23 productionC Bridgewood, A Watad, T Russell, T M Palmer, H Marzo-Ortega, A Khan, P A Millner, R Dunsmuir, A Rao, P Loughenbury, M Wittmann, R J Cuthbert, D G McGonagle
Systemic lupus erythematosus934 Altered cognitive function in systemic
lupus erythematosus and associations with inflammation and functional and structural brain changesM Barraclough, S McKie, B Parker, A Jackson, P Pemberton, R Elliott, I N Bruce
941 Risk of severe herpes simplex virus infection in systemic lupus erythematosus: analysis of epidemiology and risk factors analysis in TaiwanT-H Li, C-C Lai, W-H Wang, W-S Chen, Y-P Tsao, C-Y Tsai, Y-S Chang
Contents Volume 78 Issue 7 | ARD July 2019ARDThe Eular Journal
EditorJosef S Smolen
Associate EditorsFrancis BerenbaumDimitrios BoumpasGerd BurmesterMary CrowIain McInnesThomas PapDavid PisetskyDésirée van der HeijdeKazuhiko Yamamoto
Editorial officeAnnals of the Rheumatic DiseasesBMJ Publishing Group LtdBMA HouseTavistock SquareLondon WCIH 9JR,UKT: +44 (0)20 3655 5889E: [email protected]: @ARD_BMJISSN: 0003-4967 (print)ISSN: 1468-2060 (online)Impact Factor: 12.350
Disclaimer: ARD is owned and published by BMJ Publishing Group Ltd (a wholly owned subsidiary of the British Medical Association) and the European League Against Rheumatism. The owners grant editorial freedom tothe Editor of ARD. ARD follows guidelines on editorial independence produced by the World Association of Medical Editors and the code on good publication practice of the Committee on Publication Ethics.ARD is intended for medical professionals and is provided without warranty, express or implied. Statements in the journal are the responsibility of their authors and advertisers and not authors’ institutions the BMJ Publishing Group, the European League Against Rheumatism or the BMA unless otherwise specified or determined by law. Acceptance of advertising does not imply endorsement.To the fullest extent permitted by law, the BMJ Publishing Group shall not be liable for any loss, injury or damage resulting from the use of ARD or any information in it whether based on contract, tort, or otherwise. Readers are advised to verify any information they choose to rely on.Copyright: © 2019 BMJ Publishing Group and European League Against Rheumatism. All rights reserved; no part of this publication may be reproduced stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying recording, or otherwise without prior permission
ARD is published by BMJ Publishing Group Ltd typeset by Exeter Premedia Services Private Ltd, Chennai, India and printed in the UK on acid-free paper.
Annals of the Rheumatic Diseases (ISSN No: 0003-4967) is published monthly by BMJ Publishing Group and distributed in the USA by Air Business Ltd. Periodicals postage paid at Jamaica NY 11431 POSTMASTER: send address changes to Annals of the Rheumatic Diseases, Air Business Ltd, c/o WN Shipping USA, 156-15, 146th Avenue, 2nd Floor, Jamaica, NY 11434, USA.
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This journal is a member of and subscribes to the principles of the Committee on Publication Ethicshttp://publicationethics.org/
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Member since 2008 JM00004
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Contents Volume 78 Issue 7 | ARD July 2019
947 Lupus nephritis is linked to disease-activity associated expansions and immunity to a gut commensalD Azzouz, A Omarbekova, A Heguy, D Schwudke, N Gisch, B H Rovin, R Caricchio, J P Buyon, A V Alekseyenko, G J Silverman
957 Low-density granulocytes activate T cells and demonstrate a non-suppressive role in systemic lupus erythematosusS Rahman, D Sagar, R N Hanna, Y L Lightfoot, P Mistry, C K Smith, Z Manna, S Hasni, R M Siegel, M A Sanjuan, R Kolbeck, M J Kaplan, K A Casey
Sjögren’s syndrome967 Video clip assessment of a salivary gland
ultrasound scoring system in Sjögren’s syndrome using consensual definitions: an OMERACT ultrasound working group reliability exerciseS Jousse-Joulin, M A D'Agostino, C Nicolas, E Naredo, S Ohrndorf, M Backhaus, G Tamborrini, I Chary-Valckenaere, L Terslev, A Iagnocco, P Collado, C Hernández-Díaz, F Gandjbakhch, W A Schmidt, G Filippou, C Dejaco, M H Stradner, M A Mortada, A Hočevar, S Chrysidis, G El Mardenly, J J de Agustín, R Thiele, D K MacCarter, S Finzel, P Hanova, A Zabotti, C Glaser, Z Alavi, D S Hammenfors, F Gatineau, G AW Bruyn
974 Blockade of CD40–CD154 pathway interactions suppresses ectopic lymphoid structures and inhibits pathology in the NOD/ShiLtJ mouse model of Sjögren’s syndromeG Wieczorek, M Bigaud, S Pfister, M Ceci, K McMichael, C Afatsawo, M Hamburger, C Texier, M Henry, C Cojean, M Erard, N Mamber, J S Rush
Systemic sclerosis979 Outcomes of patients with systemic sclerosis
treated with rituximab in contemporary practice: a prospective cohort studyM Elhai, M Boubaya, O Distler, V Smith, M Matucci-Cerinic, J J Alegre Sancho, M-E Truchetet, Y Braun-Moscovici, F Iannone, P I Novikov, A Lescoat, E Siegert, I Castellví, P Airó, S Vettori, E De Langhe, E Hachulla, A Erler, L Ananieva, M Krusche, F J López-Longo, J H W Distler, N Hunzelmann, A-M Hoffmann-Vold, V Riccieri, V M Hsu, M R Pozzi, C Ancuta, E Rosato, C Mihai, M Kuwana, L A Saketkoo, C Chizzolini, R Hesselstrand, S Ullman, S Yavuz, S Rednic, C Caimmi, C Bloch-Queyrat, Y Allanore, for EUSTAR network
Myositis988 Anti-Ro52 autoantibodies are associated
with interstitial lung disease and more severe disease in patients with juvenile myositisS Sabbagh, I Pinal-Fernandez, T Kishi, I N Targoff, F W Miller, L G Rider, A L Mammen
996 Focused HLA analysis in Caucasians with myositis identifies significant associations with autoantibody subgroupsS Rothwell, H Chinoy, J A Lamb, F W Miller, L G Rider, L R Wedderburn, N J McHugh, A L Mammen, Z E Betteridge, S L Tansley, J Bowes, J Vencovský, C T Deakin, K Dankó, L Vidya, A Selva-O'Callaghan, L M Pachman, A M Reed, Ø Molberg, O Benveniste, P R Mathiesen, T R D J Radstake, A Doria, J de Bleecker, A T Lee, M G Hanna, P M Machado, W E Ollier, P K Gregersen, L Padyukov, T P O'Hanlon, R G Cooper, I E Lundberg, on behalf of Myositis Genetics Consortium (MYOGEN)
Letters1003 Inflammatory back pain criteria perform
well in subset of patients with active axial psoriatic arthritis but not among patients with established axial diseaseM Haroon, P Gallagher, O FitzGerald
1004 Anticoagulation in patients with concomitant lupus nephritis and thrombotic microangiopathy: a multicentre cohort studyS Sciascia, J Yazdany, M Dall'Era, R Fenoglio, M Radin, I Aggarwal, M J Cuadrado, K Schreiber, A Barreca, M Papotti, D Roccatello
1006 Established organ damage reduces belimumab efficacy in systemic lupus erythematosusI Parodis, A Gomez, S Emamikia, K Chatzidionysiou
1007 Rituximab in moderate to severe non-renal systemic lupus erythematosus: a reanalysis of the EXPLORER studyM Scherlinger, C Carcaud, M-E Truchetet, T Barnetche, P Duffau, L Couzi, J Seneschal, P Blanco, E Lazaro, C Richez
1010 Low aspirin use and high prevalence of pre-eclampsia risk factors among pregnant women in a multinational SLE inception cohortA Mendel, S B Bernatsky, J G Hanly, M B Urowitz, A E Clarke, J Romero-Diaz, C Gordon, S-C Bae, D J Wallace, J T Merrill, J P Buyon, D A Isenberg, A Rahman, E M Ginzler, M Petri, M A Dooley, P R Fortin, D D Gladman, K Steinsson, R Ramsey-Goldman, M A Khamashta, C Aranow, M Mackay, G S Alarcón, S Manzi, O Nived, A Jönsen, A A Zoma, R F van Vollenhoven, M Ramos-Casals, G Ruiz-Irastorza, S Lim, K C Kalunian, M Inanc, D L Kamen, C A Peschken, S Jacobsen, A Askanase, J Sanchez-Guerrero, I N Bruce, N Costedoat-Chalumeau, É Vinet
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1012 Serial IL-6 measurements in patients with tocilizumab-treated large-vessel vasculitis detect infections and may predict early relapsesC T Berger, B Rebholz-Chaves, M Recher, T Manigold, T Daikeler
Electronic pagese61 To DAPSA or not to DAPSA? That is not the
questionM M Schoels, J S Smolen, D Aletaha
e62 Response to: ‘To DAPSA or not to DAPSA? That is not the question’ by Schoels et alL J J van Mens, M G H van de Sande, A W R van Kuijk, D L P Baeten, L C Coates
e63 Infrapatellar fat pad resection during total knee arthroplasty: yet another reason for?T J Ryan
e64 Response to: ‘Infrapatellar fat pad resection during total knee replacement: yet another reason?’ by RyanZ Zhu, W Han, G Ruan, S Zheng, C Ding
e65 Serum complement factor C5a in IgG4-related diseaseS Fukui, Y Fujita, T Origuchi, T Maeda, A Kawakami
e66 Response to: ‘Serum complement factor C5a in IgG4-related disease’ by Fukui et alH Umehara, M Kawano
e67 Drug-induced lupus erythematosus following immunotherapy with anti-programmed death-(ligand) 1J-M Michot, M Fusellier, S Champiat, C Velter, C Baldini, A-L Voisin, F-X Danlos, Y E Dakdouki, M Annereau, X Mariette, C Robert, K Cherif, A Marabelle, C Mateus, O Lambotte
e68 Checkpoint inhibitor-associated immune arthritisL Arnaud, B Lebrun-Vignes, J-E Salem
e69 Response to: ‘Drug-induced lupus erythematosus following immunotherapy with anti-programmed death-(ligand) 1’ by Michot et al and ‘Checkpoint inhibitor-associated immune arthritis’ by Arnaud et alM Kostine, C Richez, T Schaeverbeke, on behalf of the FHU-ACRONIM
e70 Checkpoint inhibitor-induced polymyalgia rheumatica controlled by cobimetinib, a MEK 1/2 inhibitorK K Chan, A R Bass
e71 Response to: ‘Checkpoint inhibitor-induced polymyalgia rheumatica controlled by cobimetinib, a MEK 1/2 inhibitor’ by Chan and BassM Kostine, L Dousset, T Schaeverbeke, on behalf of the FHU-ACRONIM
e72 Tofacitinib in steroid-dependent relapsing polychondritisA D Meshkov, P I Novikov, E V Zhilyaev, I D J Ilevsky, S V Moiseev
e73 Correction: ADA2 deficiency (DADA2) as an unrecognised cause of early onset polyarteritis nodosa and stroke: a multicentre national study
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Changing the wolf from outside: how microbiota trigger systemic lupus erythematosusIsabelle Peene,1,2 Dirk Elewaut 1,2
Systemic lupus erythematosus (SLE) is a prototypic autoimmune disorder affecting multiple organ systems including skin, joint and several internal organs such as kidney, heart or lung. A hallmark of this disease is the development of circulating antibodies against nuclear antigens and immune complex formation. The best-known antibodies are directed against native DNA and are prognostic for devel-opment of lupus nephritis. The processes resulting to the cascade of events culmi-nating in the clinical features of SLE are only partially understood and involve among others, defective clearance of apoptotic cells and enhanced DNA damage to ultraviolet irradiation, and netosis1 2 (figure 1). It has also been suggested for a long time that certain microbial triggers could play a role in the onset of SLE, supported by early reports that antibody responses to nucleic acids/nucleoproteins were found in association with certain bacterial infections. More recently, more definitive proof of concept studies using germ-free and reconstituted mice highlighted that intestinal microbiota are able to drive development of systemic autoantibody responses.3 4 However, whether similar processes underlie devel-opment of lupus nephritis and anti-DNA antibodies was unclear.
In Annals of Rheumatic Diseases, Azzouz et al5 describe the results of an in-depth analysis of the relationship between intes-tinal microbiota, development of anti-DNA antibodies and their link to development of lupus nephritis. 16S rRNA sequencing was conducted on faecal samples from a discovery SLE cohort of 61 patients. In this cohort, the investigators described a clear evidence of dysbiosis with the fivefold over-abundance of Ruminococcus gnavus (RG) versus controls being the most significant
finding. RG is an anaerobic gram-positive taxon in the Fimicutes phylum and Lachn-nospiraceae family and has been previously associated with ankylosing spondylitis (AS) and inflammatory bowel diseases (IBDs).6 Intriguingly, its relative abundance correlated with lupus disease activity. Outgrowth of Ruminococcus was associ-ated with reciprocal reductions of intes-tinal species such as Bacteroides uniformis. In addition to this, the investigators also revealed some evidence for impaired intestinal barrier integrity in a significant proportion of the patients with SLE. This could favour loss of systemic tolerance. To test the impact of overabundance of RG in patients with SLE, serum immunoglobulin (Ig)A and IgG antibody responses were measured. They were not only increased in SLE versus controls but the level of IgG anti-RG2 antibodies correlated with the faecal abundance of RG as well as the SLE Disease Activity Index score, a validated measure of disease activity. Based on these impressive correlations, the investigators evaluated the association between anti-RG and anti-native DNA antibodies, which are commonly found in patients with lupus nephritis. A number of observations listed below revealed a striking and selective asso-ciation between anti-RG IgG antibodies and occurrence of anti-DNA antibodies, including correlation with IgG reactivity against human glomerular extracts, and inverse association with C3 and C4 levels indicative of complement activation typi-cally observed in immune-complex medi-ated diseases such as lupus nephritis. The investigators then sought to unravel the molecular nature of this observed asso-ciation and found that RG2 but not RG1 antigens cross-react with lupus anti-dsDNA antibodies. Using a hydrophobic interac-tion chromatography-based strategy to characterise cell wall moeities, fractions of the RG2 strain were obtained and tested by nuclear magnetic resonance and mass spec-trometry analysis and tested for immune reactivity using SLE sera. The lipoglycan 3 cell wall expressed the immunodominant epitopes commonly recognised by SLE sera.
The association with RG2 antibodies and anti-native DNA autoantibodies could be replicated in two small cohorts of, respectively, 27 patients with SLE and 17 patients with active lupus nephritis but they were absent in patients with primary membranous glomuleronephritis and anti-neutrophil cytoplasmic antibody associated vasculitis suggesting their relative spec-ificity. In addition, the association with anti-RG2 antibodies was not found with other SLE autoantibodies such as anti-Ro. Collectively, the data shown by Azzouz et al reveal a novel and intriguing paradigm that SLE may be triggered or augmented through a molecular mimicry process between a lipoglycan expressed on the cell wall of intestinal RG2 and native DNA molecules (figure 1). However, whether these findings reflect causal associations is yet unclear. Some indirect evidence suggest that RG may foster barrier integrity loss as the Lachnnospiraceae family fill a special niche and degrade complex polysaccharides such as the intestinal mucus layer. On the other hand, expansion of RG strains has also been reported in both IBD and AS,6 yet whether they are identical as in SLE remains unclear. It is also unknown whether devel-opment of anti-RG antibodies is a SLE-spe-cific trait. In this regard, larger groups of controls including IBD, AS, spondyloar-thritis (SpA) need to be tested. In addition, patients with SLE and Sjogren’s syndrome (SS), two closely related autoimmune diseases, share a common restricted gut microbiota diversity, but their oral micro-biota are distinct.7 Knowledge of the gut microbiota diversity in relation to an auto-antibody status shared by different diseases (for instance, as for antiRo52 in SLE, SS, systemic sclerosis and idiopathic inflamma-tory myopathy) might further untangle this conundrum. An earlier study reported that sera from patients with SLE with antiRo60 antibodies immunoprecipitate orthologs Ro60 derived from commensal bacteria.8 In contrast to anti-DNA antibodies, this autoreactivity remains stable in the long term suggesting that it may be less influ-enced by microbiota alterations over time. Further research should elucidate whether the fluctuations of anti-DNA antibodies in patients run parallel to shifts in the diver-sity of gut microbiota. Furthermore, most definitive cause–effect studies such as gnotobiotic mouse models reconstituted with RG versus control microbiota on SLE features still need to be reported, but given the earlier reported association between intestinal microbiota, segmented filamen-tous bacteria (a strong inducer of Th17 responses) and development of antinuclear antibodies, it is tempting to assume that the
1Unit Molecular Immunology and Inflammation, Inflammation Research Center, VIB-Ghent University, Ghent, Belgium2Department of Rheumatology, University Hospital Ghent, Gent, Belgium
Correspondence to Dr Dirk Elewaut, Unit Molecular Immunology and Inflammation, Inflammation Research Center, VIB-Ghent University, Ghent 9052, Belgium; dirk. elewaut@ ugent. be
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current associations reflect a true causal association in genetically predisposed indi-viduals (figure 1). Alternatively, the asso-ciation could reflect a lowered threshold for systemic autoantibody development. Collectively, the data presented here reflect the relevance of an emerging theme that epithelial surfaces constitute a gateway to a myriad of rheumatic diseases including rheumatoid arthritis, SpA including AS and psoriatic arthritis, now joined by SLE.Correction notice This article has been corrected since it published Online First. The figure legend has been amended.
Handling editor Josef S Smolen
Contributors IP and DE wrote jointly this editorial. Figure concept was made by IP.
Funding This study was supported by Research Council of Ghent University and Fund for Scientific Research Flanders.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Commissioned; externally peer reviewed.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
To cite Peene I, Elewaut D. Ann Rheum Dis 2019;78:867–869.
Received 27 March 2019Revised 31 March 2019Accepted 1 April 2019
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214856
Ann Rheum Dis 2019;78:867–869.
Figure 1 Integrating intestinal microbiota into systemic lupus erythematosus (SLE) pathogenesis. (A) Exposure to various sterile and/or infectious stimuli can lead to apoptosis or NETosis contributing to release of modified self-antigen (DNA). Genetic susceptibility and/or defective clearance results in presentation of these modified self-antigens to T cells followed by B cell activation and eventually production of anti-DNA antibodies, well-known for their association with lupus nephritis. (B) Dysbiosis in patients with SLE with fivefold overabundance of Ruminococcus gnavus (RG) leads to barrier disruption and loss of systemic tolerance with subsequent development of anti-RG antibodies mainly directed towards the fragment RG2 (anti-RG2 antibodies). RG2-containing pools included moieties that were potent in vitro activators via toll-like receptor 2 (TLR2) (previously been implicated in lupus pathogenesis), illustrated by Azzouz et al5 by elevated serum interferon alpha 2. (C) SLE may be triggered or augmented through intestinal dysbiosis and a molecular mimicry process between a lipoglycan expressed on the cell wall of RG and native DNA molecules (1) Loss of systemic tolerance due to over abundance of RG and/or the formation of immune complexes may lead to augmented stimulation of innate immunity and subsequent enhanced presentation of self antigen followed by production of anti DNA antibodies. (2) Type1 interferon has a critical role in SLE pathogenesis. (3) Anti-DNA antibodies cross-react with RG2, more specifically the lipoglycan 3 cell wall expressing the immunodominant epitopes. Cross-reactivity of lupus autoreactive B cells to leaked stimulatory RG bacterial components, including TLR ligands(s), may contribute to the initiation and/or flares of SLE. (4) Kidney injury by direct antibody binding or by deposition of immune complexes.
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3 Van Praet JT, Donovan E, Vanassche I, et al. Commensal microbiota influence systemic autoimmune responses. Embo J 2015;34:466–74.
4 Van de Wiele T, Van Praet JT, Marzorati M, et al. How the microbiota shapes rheumatic diseases. Nat Rev Rheumatol 2016;12:398–411.
5 Azzouz D, Omarbekova A, Heguy A, et al. Lupus nephritis is linked to disease-activity associated expansions and immunity to a gut commensal. Ann Rheum Dis 2019;78:947–56.
6 Breban M, Tap J, Leboime A, et al. Faecal microbiota study reveals specific dysbiosis in spondyloarthritis. Ann Rheum Dis 2017;76:1614–22.
7 van der Meulen TA, Harmsen HJM, Vila AV, et al. Shared gut, but distinct oral microbiota composition in primary Sjögren’s syndrome and systemic lupus erythematosus. J Autoimmun 2019;97:77–87.
8 Greiling TM, Dehner C, Chen X, et al. Commensal orthologs of the human autoantigen Ro60 as triggers of autoimmunity in lupus. Sci Transl Med 2018;10:pii: eaan2306.
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870 Tan EM. Ann Rheum Dis 2019;78:870–871. doi:10.1136/annrheumdis-2018-214893
Heroes and pillars of rheumatology
Henry G Kunkel and Rheumatoid FactorEng M Tan
To cite: Tan EM. Ann Rheum Dis 2019;78:870–871.
Handling editor David S Pisetsky
Professor emeritus, The Scripps Research Institute, La Jolla, CA 92037, USA
Correspondence toEng M Tan, The Scripps Research Institute, La Jolla, CA 92037, USA; emtan@ scripps. edu
Received 23 January 2019Accepted 24 January 2019Published Online First 8 February 2019
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
Henry Kunkel was born in Brooklyn, New York in 1916. He died in 1983 at the early age of 67. He was an outstanding clinical scholar who had a deep passion for scientific facts. Almost all his scien-tific life was involved in biomedical research at the Rockefeller University in New York City. He had a laboratory on the third floor of Founder’s Hall (the first building at what was then Rockefeller Insti-tute for Medical Research). His office was a spare, unpretentious room with a view of the East River in Upper Manhattan. To this room came many scien-tists the world over as well as many of his former fellows who came back to see the old master. He was especially glad to talk about experimental data and would drop what he was doing to talk about the deeper significance of their observations. His parting words would often be, ‘Come again’
Kunkel has received numerous accolades which could be exemplified in the following story. His research grant application was being reviewed at an NIH Study section meeting and a reviewer pointed out the lack of details concerning techniques and procedures, but also said and I paraphrase, ‘but he (Kunkel) knows that we know that he knows how to do it.’ The application sailed smoothly through the meeting.
19S rHeumaToid faCTor iS anTibody To immune ComplexAmong all the outstanding work that Kunkel has done, I consider his studies on the characterisa-tion of rheumatoid factor (RF) to be a trailblazer in making us become aware of the intricacy of inter-acting cellular functions which unexpectedly enable production of outcomes which are pathogenic. In the 1950s, Kunkel and his associates used a Spinco Model E analytical ultracentrifuge to study proteins of different sedimentation constants in serum. These studies culminated in two papers,1 2 which described a population of proteins in rheumatoid arthritis (RA) sera with a high sedimentation constant of 22S. The 22S protein component was a complex of 19S and 7S proteins; the 19S protein was RF and the 7S compo-nent was identical to 7S gamma globulin. Further studies revealed that the 19S protein was like anti-body in that many other immune complexes could bind to the 19S protein dissociated from the 22S complex. The proposal that RF might be an antibody to antigen–antibody complexes was completely novel and surprising and aroused much scepticism; further evidence that 19S RF isolated from the 22S complex had other properties of 7S gamma globulin antibody convinced the sceptics. In quantitative immunopre-cipitation experiments, immunoprecipitates of 22S could be solubilised in zones of antigen excess. In an understated fashion, Kunkel and his associates said that, ‘a search for the hypothetical antigen–antibody
complex giving rise to the RF might yield consider-able further information.’2’
Synovial CaviTieS and oTHer SequeSTered SiTeS may be niduS for SuSTained anTibody and immune Complex-mediaTed inflammaTion in raThese seminal studies were suggestions that, to eluci-date the pathogenesis of RA, one should include studies in which antibody and immune complex-me-diated pathogenesis are involved. Many decades after the original observations, I believe we are beginning to unravel the complex scenario of RA pathogenesis. It is highly likely that the major candidate antigen in the ‘hypothetical antigen–antibody complex’ could be citrullinated filaggrin, a cytokeratin fila-ment aggregating protein. Initially called perinuclear factor, it is produced by deiminating enzymes in many cell types.3–5 As the Kunkel studies suggested, other antigen–antibody complexes may also be involved, but citrullinated proteins may be the major player. A renewable source of citrullinated proteins appears to be the synovial cavities of inflamed joints. Hollingsworth et al6 showed that the high percentage of neutrophils in synovial effusions was due to high turnover and not due to stagnation of neutrophils in synovial cavities. As studies showed, in RA synovial fluid, the major chemoattractant factor is complement component C5a and neutrophilia is augmented by C4a, a chemotaxis inhibitor for monocytes, allowing the preferential accumulation of neutrophils.7 The fact that complement-related fragments are plentiful in synovial fluids is a strong testament for the role of complement activation8–11 and the membrane attack complex (C5b–C9) in inducing hypercitrullination of neutrophil intracellular proteins.12 13 Basically, it can be said that the ‘RF is antibody to immune complex’ concept is the key to a reasonable construct on the chain of events in RA pathogenesis (also see detailed review in,14 and RA is probably an extreme form of immune complex illness.
Henry Kunkel received numerous honours during his lifetime, including the Gairdner Foundation Award (1962), Doctor of Medicine (Honoris Causa) University of Uppsala, Sweden (1964), Modern Medicine Distinguished Service Award (1973), Albert Lasker Award for Basic Medical Research (1975), Passano Foundation Award (1975), Avery Land-steiner Award (1975), American College of Physi-cians Award (1975), Louisa Gross Horwitz Award of Columbia University (1977), New York Academy of Medicine Award (1977), Kovalenko Medal of the National Academy of Science (1979), Lita Annen-berg Hazen Award (1980), Pasteur Medal (1980) and Doctor of Medicine (Honoris Causa) Harvard University (1982). The photo of Henry Kunkel (see figure 1) was taken in 1980 at a restaurant in Paris
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871Tan EM. Ann Rheum Dis 2019;78:870–871. doi:10.1136/annrheumdis-2018-214893
Heroes and pillars of rheumatology
figure 1 This picture of Henry Kunkel was taken in 1980 at a restaurant in Paris on the occasion of a meeting of the International Congress of Immunology. He had just been informed that he was the recipient of the Lita Annenberg Hazen Award for Excellence in Clinical Research (from the personal collection of the author).
when he was informed that he had received the Lita Annenberg Hazen Award for excellence in clinical research.acknowledgements The author is grateful to Mary Artley for her editorial assistance.
funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
patient consent for publication Not required.
provenance and peer review Commissioned; internally peer reviewed.
RefeRences 1 Franklin EC, Holman HR, Muller-Eberhard HJ, et al. An unusual protein component of
high molecular weight in the serum of certain patients with rheumatoid arthritis. J Exp Med 1957;105:425–38.
2 Edelman GM, Kunkel HG, Franklin EC. Interaction of the rheumatoid factor with antigen-antibody complexes and aggregated gamma globulin. J Exp Med 1958;108:105–20.
3 Nienhuis RL, Mandema E, Smids C. A new serum factor in patients with rheumatoid arthritis; the antiperinuclear factor. Ann Rheum Dis 1964;23:302–5.
4 Aho K, von Essen R, Kurki P, et al. Antikeratin antibody and antiperinuclear factor as markers for subclinical rheumatoid disease process. J Rheumatol 1993; 20:1278–81.
5 Youinou P, Serre G. The antiperinuclear factor and antikeratin antibody systems. Int Arch Allergy Immunol 1995;107:508–18.
6 Hollingsworth JW, Siegel ER, Creasey WA. Granulocyte survival in synovial exudate of patients with rheumatoid arthritis and other inflammatory joint diseases. Yale J Biol Med 1967;39:289–96.
7 Yamamoto T, Nishiura H, Nishida H. Molecular mechanisms to form leukocyte infiltration patterns distinct between synovial tissue and fluid of rheumatoid arthritis. Semin Thromb Hemost 1996;22:507–11.
8 Winchester RJ, Agnello V, Kunkel HG. Gamma globulin complexes in synovial fluids of patients with rheumatoid arthritis. Partial characterization and relationship to lowered complement levels. Clin Exp Immunol 1970;6:689–706.
9 Moxley G, Ruddy S. Elevated C3 anaphylatoxin levels in synovial fluids from patients with rheumatoid arthritis. Arthritis Rheum 1985;28:1089–95.
10 Sjöholm AG, Berglund K, Johnson U, et al. C1 activation, with C1q in excess of functional C1 in synovial fluid from patients with rheumatoid arthritis. Int Arch Allergy Immunol 1986;79:113–9.
11 Jose PJ, Moss IK, Maini RN, et al. Measurement of the chemotactic complement fragment C5a in rheumatoid synovial fluids by radioimmunoassay: role of C5a in the acute inflammatory phase. Ann Rheum Dis 1990;49:747–52.
12 Romero V, Fert-Bober J, Nigrovic PA, et al. Immune-mediated pore-forming pathways induce cellular hypercitrullination and generate citrullinated autoantigens in rheumatoid arthritis. Sci Transl Med 2013;5.
13 Zhou Y, Di Pucchio T, Sims GP, et al. Characterization of the Hypercitrullination reaction in human neutrophils and other leukocytes. Mediators Inflamm 2015;2015:1–9.
14 Tan EM, Smolen JS. Historical observations contributing insights on etiopathogenesis of rheumatoid arthritis and role of rheumatoid factor. J Exp Med 2016;213:1937–50.
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872 Winthrop KL, et al. Ann Rheum Dis 2019;78:872–878. doi:10.1136/annrheumdis-2018-214280
Review
Unmet need in rheumatology: reports from the Targeted Therapies meeting 2018Kevin L Winthrop,1 Michael E Weinblatt,2 Mary K Crow,3 Gerd R Burmester,4 Philip J Mease, 5 Alexander K So,6 Vivian Bykerk,3 Ronald F Van Vollenhoven,7 Maxime Dougados,8 Jonathan Kay, 9 Xavier Mariette,10 Joachim Sieper,11 Fritz Melchers,12,13 Bruce N Cronstein,14 Ethan Shevach,15 Ferdinand C Breedfeld,16 Joachim Kalden,17 Josef S Smolen, 18 Daniel E Furst5,19,20
To cite: Winthrop KL, Weinblatt ME, Crow MK, et al. Ann Rheum Dis 2019;78:872–878.
Handling editor Dimitrios T Boumpas
► Additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214280).
For numbered affiliations see end of article.
Correspondence toDr Kevin L Winthrop, Oregon Health and Science University, Portland, OR 97239, USA; winthrop@ ohsu. edu
Received 13 August 2018Revised 16 November 2018Accepted 19 January 2019Published Online First 2 February 2019
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
AbsTRACTTo develop a comprehensive listing of the greatest unmet scientific and clinical needs in rheumatology. The 20th annual international Targeted Therapies meeting brought more than 100 leading basic scientists and clinical researchers in rheumatology, immunology, epidemiology, molecular biology and other specialties. During the meeting, breakout sessions were convened, consisting of five disease-specific groups with 20–30 experts assigned to each group based on expertise. Specific groups included rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, systemic lupus erythematosus, connective tissue diseases and a basic science immunology group spanning all of these clinical domains. In each group, experts were asked to consider recent accomplishments within their clinical domain in the last year and update the unmet needs in three categorical areas: basic/translational science, clinical science and therapeutic development, and clinical care. While progress was noted among some of previously identified needs, both new needs were identified and themes from prior meetings were re-iterated: the need for better understanding the heterogeneity within each disease, and for identifying preclinical states of disease allowing treatment and prevention of disease in those at risk, and the elusive ability to cure disease. Within the clinical care realm, improved comorbidity management and patient-centred care continue to be unmet needs, and the need for new and affordable therapeutics was highlighted. Unmet needs for new and accessible targeted therapies, disease prevention and ultimately cure remain a priority in rheumatology.
bACkgRoundThe Targeted Therapies meeting has met annually for 20 years, with experts in the fields of clinical rheumatology, infectious diseases, epidemiology and basic scientific areas including immunology and molecular biology, presenting research devel-opments in their fields. The meeting focuses on medicine and translational research, and stimu-lates collaboration between basic scientists and clinicians. The meeting’s objective is to update participants regarding the latest insights regarding disease mechanism(s) and pathophysiology, and recent developments with both existing and novel targeted therapies in rheumatology. Previously, a consensus document describing the recommended use of targeted therapies within rheumatology was produced from this meeting.1 However, with the
expanse of biologic therapies and the recent clinical recommendations published from both American College of Rheumatology and the European Union League Against Rheumatism,2–4 a single consensus document covering all targeted therapies across all disease indications became too complex and volu-minous as a single manuscript. Accordingly, the annual meeting’s output was modified to debate and formulate a list of key unmet needs within the field, consistent with the meeting’s underlying objective of promoting innovation and collabora-tion within all areas of rheumatology.5 6 With the 2018 meeting, we conducted a similar process to review and update these unmet needs, with the goal of producing a roadmap for research in the future.
MeTHodsWe assigned conference participants to disease-spe-cific breakout groups which included Psori-atic Arthritis (PsA), Rheumatoid Arthritis (RA), Axial Spondyloarthritis (axSpA), Systemic Lupus Erythematous (SLE) and Connective Tissue Diseases (CTD). The CTD group was charged with specifically discussing Sjögren’s syndrome, myositis, systemic sclerosis, IgG4-related disease and vascu-litis including Behcet’s disease. Experts in each group were tasked with identifying unmet needs in three categorical areas: clinical care, clinical science and therapeutic development, and basic/trans-lational science. A ‘facilitator’ and ‘rapporteur’ lead each group’s discussion and summarised their results. The groups were asked to highlight notable progress made towards previously identified needs,5 identify new areas of need, and categorise all unmet needs as either primary or secondary. Subject-matter groups did not pursue formal measures of agreement or consensus, and needs were not ranked or further prioritised.
ResulTsThe RA group (table 1) reiterated the progress made to date with treatment of RA, including the strategy of ‘treat to target’ with rapid dose escala-tion of methotrexate followed by addition of either biologic disease-modifying antirheumatic drugs (DMARDs) or targeted small molecules. However, the committee also recognised that a significant proportion of patients continue to have moderate to high disease activity despite these therapeutic approaches, and the group highlighted the need
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Review
Tabl
e 1
Prim
ary
and
seco
ndar
y un
met
sci
entifi
c ne
eds
with
in rh
eum
atoi
d ar
thrit
is w
ith re
gard
to tr
ansl
atio
nal s
cien
ce, c
linic
al s
cien
ce a
nd th
erap
eutic
tria
ls, a
nd c
linic
al c
are∗
Prim
ary
unm
et n
eeds
seco
ndar
y un
met
nee
ds
Tran
slat
iona
l sc
ienc
eU
nder
stan
ding
the
role
of t
he m
icro
biom
e in
dis
ease
dev
elop
men
t, ca
usat
ion
and
mod
ulat
ion
Deve
lopm
ent o
f mol
ecul
ar d
efini
tions
of d
isea
se re
mis
sion
, flar
e, d
isea
se a
ctiv
ityId
entif
ying
bio
mar
kers
with
in ta
rget
tiss
ues
incl
udin
g im
agin
g th
at p
redi
ct o
r rap
idly
iden
tify
trea
tmen
t res
pons
eCo
ntin
ued
inve
stig
atio
n of
indi
vidu
al c
ell l
inea
ges
in R
A th
at in
clud
es b
ette
r int
egra
tive
anal
yses
of m
olec
ular
si
gnat
ures
, rel
atin
g si
gnat
ures
see
n in
focu
sed
stud
ies
on c
ell l
inea
ges
to th
eir p
atho
geni
c ro
les
to d
evel
op ta
rget
s
Iden
tifyi
ng s
ites
beyo
nd th
e jo
int (
eg, g
ut) t
hat m
ay b
e dr
ivin
g di
seas
eDe
velo
pmen
t of a
nim
al m
odel
s th
at b
ette
r refl
ect h
uman
dis
ease
Stud
y re
pair
and
rege
nera
tion
in th
e rh
eum
atoi
d jo
int
Und
erst
andi
ng th
e ro
le o
f the
vas
cula
r and
lym
phat
ic s
yste
ms
in th
e de
velo
pmen
t of R
A in
the
join
t
Clin
ical
sci
ence
an
d th
erap
euti
c tr
ials
Deve
lopm
ent o
f the
rape
utic
s th
at re
pair
dam
age,
incl
udin
g ou
tsid
e th
e jo
int (
eg, i
nter
stiti
al lu
ng d
isea
se, c
ardi
ac e
tc)
Eval
uatio
n of
exi
stin
g th
erap
ies
in c
ombi
natio
nCo
ntin
ued
need
for t
reat
men
t with
draw
al s
tudi
es c
ondu
cted
for l
onge
r tim
e pe
riods
Tria
ls th
at in
clud
e pa
tient
s w
ith c
omor
bidi
ties
and
elde
rly p
atie
nts
that
will
enh
ance
our
und
erst
andi
ng o
f the
saf
ety
of
exis
ting
ther
apie
s an
d w
ill a
sses
s th
e be
st tr
eatm
ents
for t
hese
com
orbi
ditie
sDe
velo
pmen
t of n
ew n
on-o
pioi
d an
alge
sia
Tria
ls e
valu
atin
g th
e be
nefit
s of
ear
ly tr
eatm
ent (
eg, c
hang
e th
e lo
ng-t
erm
pro
gnos
is o
f dis
ease
)Th
e de
velo
pmen
t of a
ppro
ache
s to
pre
vent
RA
(eg,
scr
eeni
ng, t
oler
isat
ion,
vac
cina
tion)
Defin
e tr
eatm
ent p
artia
l and
non
-res
pond
ers
desp
ite c
urre
nt tr
eatm
ent p
arad
igm
s an
d de
velo
p th
erap
eutic
tria
ls
targ
etin
g su
ch in
divi
dual
s w
ith m
oder
ate
and
high
dis
ease
act
ivity
Deve
lopm
ent o
f new
non
-imm
unos
uppr
essi
ve d
isea
se c
ontr
ol w
hich
can
be
used
as
mon
othe
rapy
or i
n co
mbi
natio
ns w
ith c
urre
nt tr
eatm
ents
Clin
ical
stu
dy o
f ext
rem
e ph
enot
ypes
: res
pond
ers
vs n
on-r
espo
nder
sBe
tter
und
erst
andi
ng o
f sec
onda
ry fa
ilure
(ant
i-dru
g an
tibod
y, m
etho
trex
ate
dose
or o
ther
mec
hani
sms)
Bett
er u
nder
stan
ding
and
cat
egor
isat
ion
of s
eron
egat
ive
patie
nts
with
incl
usio
n of
suc
h pa
tient
s w
ithin
cl
inic
al tr
ials
The
deve
lopm
ent o
f obs
erva
tion
stud
ies
incl
udin
g re
gist
ries
and
othe
r pop
ulat
ion-
base
d su
rvei
llanc
e sy
stem
s fo
r eva
luat
ing
the
safe
ty a
nd e
ffect
iven
ess
of d
rug
ther
apie
s in
clud
ing
bios
imila
rsDe
velo
pmen
t of i
nfra
stru
ctur
e fo
r usi
ng e
lect
roni
c he
alth
reco
rds
in c
linic
al re
sear
ch. T
his
incl
udes
m
echa
nism
s to
eas
ily a
cces
s in
dust
ry c
linic
al tr
ial d
ata
post
-dru
g ap
prov
al
Achi
evin
g cu
re a
nd d
efini
ng th
is a
s lo
ng-t
erm
rem
issi
on o
ff dr
ug th
erap
yId
entif
ying
pat
ient
s w
ho c
an ta
per t
heir
trea
tmen
t with
out a
flar
e of
art
icul
ar o
r ext
ra-a
rtic
ular
dis
ease
Mod
erat
ion
of d
rug
pric
ing
with
an
incr
ease
in a
cces
s to
exi
stin
g an
d ne
w th
erap
ies
incl
udin
g bi
osim
ilars
Achi
evin
g re
mis
sion
in g
reat
er p
ropo
rtio
ns o
f pat
ient
s (s
till n
ot m
ore
than
30%
)G
reat
er a
tten
tion
to m
anag
ing
dise
ase
outs
ide
the
join
t (ie
, RA
lung
, hea
rt d
isea
se)
*Seq
uenc
e of
nee
ds w
ithin
the
tabl
e is
rand
om a
nd is
not
mea
nt to
impl
y or
der o
f im
port
ance
.RA
, rhe
umat
oid
arth
ritis.
to further define ‘treatment-resistant disease’.7 These patients should be the priority for study with new molecules, as they remain the most difficult to treat. On the other hand, another unmet need highlighted the necessity of better identifying patients in low disease activity/remission in whom dose reduc-tion could be considered8 and, accordingly, highlighted a need for clinical trials to evaluate dose reduction strategies and their impact on both joint and extra-articular disease. There is also a need for registry or other population-based observational studies for postmarketing surveillance of biologics including originators and biosimilars, as well as newly approved small molecules.9 10 Identifying patients who should be able to dose reduce without a risk for flare remains challenging, and will require further development of biomarkers, particularly within target tissues, and imaging programmes.11 Future clinical trials should use such biomarkers to stratify patients.
Pain and fatigue were also recognised as important components of disease morbidity in a substantial portion of patients, despite there being numerous efficacious therapies for decreasing inflam-mation. Specific research regarding the multifactorial origin of fatigue and pain in RA should be developed using new methods of neuroimaging12 and ideally used to further the development of novel, non-opioid analgesics. Another domain of therapeutic unmet need was highlighted again this year: specifically the eval-uation and treatment of extra-articular disease including RA lung disease. The impact of DMARD therapy and low disease activity on cardiovascular disease was also highlighted as a continued important area of study. A major challenge remains access to care due to the high cost of biologic and new small-molecule DMARDs.13 This was a universal concern of group members independent of country of origin, as many patients fear the loss of access to treatment. The potential ability of biosimilars to allow for greater access to biologics was debated, although it remains unclear whether this will occur.
The group also identified the need for RA classification to extend beyond predefined clinical definitions; for example, using clinical phenotypes (eg, treatment response (ie, TNF vs B-cell responsive disease)) or developing a new mecha-nism-based taxonomy of disease. The role of the microbiome in RA development and natural history, the development of animal models more reflective of human disease, and the identification of extra-articular sites (eg, the gut) that could be driving joint inflammation were re-identified as areas of research needs.14
For PsA (table 2), advances have been escalating due to the convergence of greater understanding of the pathophysiology of PsA’s varied clinical domains and the coming to fruition of numerous therapeutic clinical trials.15 Whereas a core set of clinical domains to be assessed in PsA clinical trials and long-term observational studies was recently revised and updated,16 the identification of a core outcome measure set for clinical trials is in process.17 New therapies have been approved for PsA, confirming the importance of several pathophysiological path-ways including the cytokines TNF, IL-17 and IL-23, as well as various T-cell subset pathways.18–20 TNF inhibitors continue to represent standard of care, including one new approval—intra-venous golimumab. Other approvals include two additional oral agents, the JAK inhibitors tofacitinb and baricitinib, an additional IL-17 inhibitor ixekizumab, and the T-cell modulator abatacept. Numerous other agents which exploit these and other pathways are in phase II and III trials, including the IL-23 inhibitors gusel-kumab, risankizumab and tildrakizumab, and JAK inhibitors filgotinib and upadacitinib.21 Country-based or regional-based clinical registries around the world are maturing with substan-tial numbers of patients with clinically well-characterised PsA
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Review
Table 2 Primary and secondary unmet scientific needs within psoriatic arthritis with regard to translational science, clinical science and therapeutic trials, and clinical care∗
Primary unmet needs secondary unmet needs
Translational science
Understanding how to use molecular imaging modalities to interrogate tissue pathophysiology, especially the enthesiumBetter understanding the effect of genetic markers, skin and gut microbiome patterns, metabolic syndrome and central sensitisation (fibromyalgia) on disease subtypes and their outcomesFurther development of longitudinal, clinically well-characterised cohorts with appropriate imaging, tissue and fluid samples; improved data-sharing among investigators; these relate to inception cohorts as well as cohorts of patients with long-standing disease
Development of appropriate animal models for pathogenesisUnderstanding various mechanisms of pain, including central sensitisationUnderstanding the progression of skin psoriasis to that of associated arthritis.Understanding at the molecular level similarities or differences in inflammation between affected joints and skin
Clinical science and therapeutic trials
Standardisation of enthesitis and dactylitis measuresReliable and feasible imaging assessment of new bone formationCorrelation of physical examination with advanced imaging (US, MRI)Testing the benefits and risks of scores that use multiple disease domains vs scores that use single domains for approval of drugsUnderstanding differential therapeutic effects on different clinical domains in PsAFurther evaluation of combination therapies and strategic trials including the use of sequential therapies, controlled withdrawal, the treatment of early disease, and the treatment of monoarticular or oligoarticular diseaseHead-to-head comparison of therapies with different mechanisms of action
Development/validation of advanced imaging and other biomarkers including patient-reported outcomes to assess disease activity and clinical outcomes in the different clinical domains of PsAUse of NMR spectroscopy and other advanced imaging for metabolic syndromeSpecific interventions related to microbiome
Clinical care Standardisation in the characterisation and measurement of PsA clinical domainsThe development of a clinician-feasible measure(s) of PsA remission or low disease activity as a target of therapy.Developing better knowledge, communication and screening approaches (including the development of educational initiatives and cross-specialty clinics) for rheumatologists, dermatologists and primary care providers caring for patients with PsA to facilitate earlier diagnosisImproved clinical attention to PsA-related comorbidities, especially metabolic syndrome and central sensitisation
Use of serum and other types of biomarkers for diagnosis, disease severity categorisation and identifying structural damageThe development of guidance regarding therapeutic choice based on patient factors, clinical presentations and ‘to be developed’ biomarkers
*Sequence of needs within the table is random and is not meant to imply order of importance.PsA, psoriatic arthritis.
with, in some cases, paired biorepositories to pursue clinical and translational science in ‘real world’ settings distinct from clinical trial populations. Efforts to standardise clinical assess-ments have occurred in recent years, and publications regarding disease natural history, clinical features and treatment outcomes, and comorbidities are now emerging from these registries. The heterogeneous clinical domains include arthritis and skin mani-festations, and also enthesitis, dactylitis, spondylitis, enthesitis, uveitis, nail disease, and colitis are being studied translationally and clinically.22 Research on how to optimally assess these various clinical domains both individually and as part of composite measures is proceeding. This is important to holistically eval-uate PsA when targeting remission or low disease activity during treatment, but also to recognise how treatments may differen-tially affect different domains, raising the need for combination therapy and/or therapy intensification.20 21 23 At the moment, several composite measures, both categorical to measure multi-domain response, and continuous to measure disease activity as well as response, are being used in clinical trials and registries. There is not yet a consensus on which measure(s) will be the optimal one to use as a target of treatment.24 Numerous ques-tions remain at a basic science level. What mechanisms underlie the pathogenesis of different clinical domains; how similar and different? In situations where it is difficult to interrogate mech-anisms by tissue biopsy (eg, enthesitis), can advanced imaging techniques serve as a useful tool? There is a need to understand the transition from having just skin disease to the development of musculoskeletal features at the translational level: can arthritis development be preventable in those with skin psoriasis? Needed are serum biomarkers which predict that transition, as well as
assessment of disease activity and predicting progressive struc-tural damage in those with established PsA. Conversely, many patients with PsA have little or no skin involvement and do not even know they have skin psoriasis—this raises the question as to whether this group of patients is fundamentally different than those with significant skin disease. Biomarkers should be devel-oped using well-characterised clinical cohorts with appropriate imaging, tissue and fluid samples. An expert group has initi-ated a project to fund a collaborative consortium of centres to collect such samples.25 There is increasing understanding about genetic, gastrointestinal and skin microbiome profiles in PsA.26 It is associated with a number of comorbidities, including meta-bolic syndrome (obesity, hypertension, hyperlipidaemia), which increases the risk for early cardiovascular disease.27 A common but inadequately recognised comorbidity, the phenomenon of central sensitisation, which overlaps with fibromyalgia, charac-terised by augmented pain and fatigue, can have a significant impact on disease assessment and therapeutic response. There is a need for greater understanding of the impact of comorbidities, especially if modifiable, on disease course and outcomes through registry-based studies.28
Unlike RA, there have been a paucity of PsA studies evalu-ating combined or sequential therapies, controlled withdrawal and treatment of early or ‘pre-disease’. As more therapies with different mechanisms of action are proven efficacious in PsA, head-to-head comparisons are needed; several trials are underway, including comparison of TNF and IL-17 inhibition.29 It was highlighted in deliberations that ‘it takes a village’ to care for a patient with PsA. Optimal care teams include rheu-matologists and also dermatologists, ophthalmologists and
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Table 3 Primary and secondary unmet scientific needs within axial spondyloarthritis with regard to translational science, clinical science and therapeutic trials, and clinical care∗Translational/basic science
► What are the hallmarks of early SpA and pre-SpA? ► Better understanding of the links between intestinal disease and the
pathogenesis of SpA ► Improve international collaborations to set up registries and bio-banks on
SpA in order to share experience and resources for research ► Explore novel imaging modalities (PET, enhanced MRI etc) to enable early
diagnosis and define imaging parameters in interventional studies
Clinical science/therapeutic trials
► Improve our knowledge of peripheral SpA, particularly the disease course and the response to therapy. This can be addressed by large cohort studies
► Design strategy trials to study the optimal use and choice of biologics and small molecules that will deliver the best outcome in SpA
► Head-to-head trials that compare the efficacy of different biologics in SpA ► Can early treatment modify the disease course
► Understanding the biological basis of non-responders to current therapies, especially biologics
► Identify biomarkers and imaging and clinical markers that differentiate subsets of SpA that respond preferentially to one or another therapy
► Better understand the effects of treatment of SpA on inflammation of the eye and the gut
► International collaboration to conduct strategy trials ► Conduct T2T trials with clinical remission as the target
Clinical care ► Improved identification of early SpA and early referral to rheumatologists ► Improved patient education and patient involvement in clinical research and care
SpA, spondyloarthritis.
gastroenterologists with the ability to closely communicate their care of the disease’s key clinical features and associated condi-tions.30 31 Professional societies are nurturing the development of ‘combined’ clinics staffed by rheumatologists and derma-tologists, especially in academic centres for teaching as well as clinical consultation purposes. Similar efforts in the commu-nity among private practitioners are also occurring to improve communication and collaboration between specialists caring for these patients.32 Education of dermatologists, primary care providers and others who may see patients with PsA prior to the rheumatologist should be focused on screening patients with skin psoriasis for inflammatory musculoskeletal symptoms.
In axSpA (table 3), there is an urgent need to increase our knowledge of the intestinal microbiome and pathology and their influence on the development of SpA. Clinical and subclinical inflammation is a major feature of SpA, and there is increasing evidence of intestinal dysbiosis in SpA.33
Biomarkers for early disease stages (or ‘pre-disease’) are lacking, as is the clinical and pathophysiological understanding of the processes that characterise SpA at the time of onset or before.34 To accomplish these aims, international collaborations in terms of registries and bio-banks need to be established, inte-grated with the exploration of novel imaging modalities like enhanced MRI and PET scanning to detect early pathology.35 To improve clinical care, education should focus on early referral and diagnosis.36 This includes the appropriate use of imaging for both correctly interpreting disease classification criteria as well as making a correct diagnosis.34 Moreover, identifying patient subsets that can be best treated with a specific biologic agent could tremendously enhance treatment effectiveness.37 In the domain of clinical research and trials, peripheral SpA remains relatively unstudied, and there are little data to guide the treatment choice.38 For axial SpA, although current biologics have demonstrated efficacy, our knowledge of which biologics to choose is unsatisfactory, and head-to head trials as well as treatment strategy trials are needed to guide clinicians.37 Ideally, such studies should be complemented by detailed biomarker and genetic analyses in order to identify predictive factors asso-ciated with treatment response, as well as to understand the mechanisms of inadequate response to a biologic. Such studies will likely require international collaboration in order to be successful.
SLE (table 4), with its protean yet heterogeneous clinical manifestations as well as extensive autoimmunity and immune cell dysfunction, remains a disease that requires deeper insights
into its aetiology and mechanisms in addition to more effec-tive therapies. The breakout group discussing unmet needs and research priorities related to SLE noted considerable progress in those areas of scientific and clinical need that had been identi-fied in 2017.5 Technological advances continue to allow dissec-tion of the phenotypes and mechanisms of regulating T and B lymphocytes, including the important role of cell metabolism and effector cell populations.39 40 The significance of nucleic acid triggers of innate immune responses and type I interferon production continues to gain strong support.41 42 New insights regarding the contribution of brain microglial cells43 and tran-scriptome analysis of kidney tissue from patients with lupus nephritis,44 particularly data generated through the Accelerating Medicines Partnership programme, highlight the importance of tissue-specific mechanisms that contribute to the various disease phenotypes.45 The growing understanding of epigen-etic alterations46 and correlation of genome-wide association studies with clinical phenotypes have identified new genes and loci associated with risk of developing lupus, extending these analyses to include more samples from patients of African-Amer-ican and Asian ethnicities.47 Metabolic studies and studies of perturbations of signalling pathways, such as mTOR, have extended understanding of disease pathophysiology.47 48 Despite these advances addressing factors impacting susceptibility and immune and tissue-specific mechanisms, the group reiterated the view that the current classification criteria for SLE contribute to the observed heterogeneity of disease. Effective bioinformatics tools, including machine learning, will be required to effectively interpret in an unbiased manner the extensive and growing data-sets derived from patient cohorts followed longitudinally, which include genetic, epigenetic, transcriptomic, serological, immu-nological and microbiome data. These comprehensive analyses will likely yield actionable insights into the clinical heterogeneity of SLE. Despite continued progress in basic and clinical research, the group articulated the reality that the ‘cause’ of SLE remains elusive. Understanding the core aetiology of SLE, as well as the continued dissection of its disease mechanisms, were identified as the highest priority challenges for the research community.
Clinical trial design has been supported by the general accep-tance of SLE Responder Index 4 as an informative measure of response to therapy48; novel outcome measures also have been developed.49 Industry-sponsored clinical trials have generated valuable samples to be analysed for biologic factors associated with clinical response to therapy. Patients have identified ‘lupus fog’ and fatigue as the disease manifestations of greatest concern,
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Tabl
e 4
Prim
ary
and
seco
ndar
y un
met
sci
entifi
c ne
eds
with
in s
yste
mic
lupu
s er
ythe
mat
osus
with
rega
rd to
tran
slat
iona
l sci
ence
, clin
ical
sci
ence
and
ther
apeu
tic tr
ials,
and
clin
ical
car
e∗Pr
imar
y un
met
nee
dsse
cond
ary
unm
et n
eeds
Tran
slat
iona
l/ba
sic
scie
nce
Bett
er u
nder
stan
ding
of t
he ro
le o
f T a
nd B
lym
phoc
ytes
(and
sub
sets
), th
e ep
igen
etic
mod
ifica
tion
of v
ario
us c
ell t
ypes
(in
conn
exio
n w
ith e
nviro
nmen
tal f
acto
rs) a
nd m
etab
olic
per
turb
atio
ns in
the
path
ophy
siol
ogy
of d
isea
seId
entifi
catio
n of
fact
ors
that
lead
to ti
ssue
spe
cific
ity o
f SLE
dis
ease
man
ifest
atio
nsFu
rthe
r dev
elop
men
t of l
ongi
tudi
nal,
clin
ical
ly w
ell-c
hara
cter
ised
coh
orts
(im
mun
olog
ical
ly, g
enet
ical
ly a
nd m
etab
olic
ally
) with
ap
prop
riate
imag
ing,
tiss
ue a
nd fl
uid
sam
ples
; im
prov
ed d
ata-
shar
ing
amon
g in
vest
igat
ors
The
deve
lopm
ent o
f dia
gnos
tic to
ols
to id
entif
y pr
eclin
ical
dis
ease
sta
tes
Iden
tifica
tion
of c
ontr
ibut
ion
of g
enet
ic v
aria
nts
and
solu
ble
med
iato
rs to
risk
of
dise
ase
Bett
er u
nder
stan
ding
of t
he n
atur
al h
isto
ry o
f dis
ease
flar
es
Clin
ical
sci
ence
an
d th
erap
euti
c tr
ials
Furt
her r
efine
men
t of c
linic
al re
spon
se m
easu
res/
inde
xCr
itica
l app
rais
al a
nd p
oten
tial r
evis
ion
of th
e cu
rren
t ins
trum
ents
to a
sses
s di
seas
e ac
tivity
and
trea
tmen
t res
pons
eSt
anda
rdis
atio
n of
a d
efini
tion
of d
isea
se re
mis
sion
Clin
ical
tria
ls th
at in
corp
orat
e IF
N s
igna
ture
and
em
phas
ise
resp
onde
r ana
lyse
sLa
rge
prag
mat
ic tr
ials
of e
xist
ing
and
emer
ging
ther
apie
s. Th
ese
tria
ls s
houl
d fo
cus
also
on
prev
entio
n of
dis
ease
in th
ose
iden
tified
to b
e at
risk
Smal
l pro
of o
f mec
hani
sm tr
ials
for e
mer
ging
ther
apie
s
Impr
oved
iden
tifica
tion
and
targ
etin
g of
the
inna
te im
mun
e re
spon
seIm
prov
ed id
entifi
catio
n an
d us
e of
bio
mar
kers
with
in c
linic
al p
ract
ice
and
tria
lsBr
oade
n m
embe
rshi
p of
gro
ups
desi
gnin
g tr
ials
Clin
ical
car
eBe
tter
cha
ract
eris
e pa
tient
con
cern
s (v
s pr
ovid
er c
once
rns)
Opt
imis
atio
n of
ste
roid
-spa
ring
appr
oach
es to
trea
tmen
t inc
ludi
ng th
e de
velo
pmen
t of t
oxic
ity s
corin
g sy
stem
s, th
e de
velo
pmen
t of
sust
aine
d re
leas
e or
org
an-t
arge
ted
ster
oid
prep
arat
ions
, and
con
side
ratio
n of
diff
eren
t ‘ph
ases
’ of s
tero
id u
seIm
prov
ed u
nder
stan
ding
of t
arge
ting
spec
ific
ther
apie
s to
spe
cific
dis
ease
clin
ical
man
ifest
atio
ns
Iden
tifica
tion
of s
ocio
econ
omic
fact
ors
that
con
trib
ute
to lo
ng-s
tand
ing
dise
ase
Esta
blis
h pa
tient
sup
port
gro
ups
and
guid
es/a
dvoc
ates
to im
prov
e ad
here
nce
to
med
ical
regi
men
Bett
er u
nder
stan
d co
gniti
ve d
ysfu
nctio
n as
soci
ated
with
dis
ease
and
the
deve
lopm
ent
of a
usa
ble
inst
rum
ent t
o qu
antif
y in
clin
ical
pra
ctic
e
*Seq
uenc
e of
nee
ds w
ithin
the
tabl
e is
rand
om, a
nd is
not
mea
nt to
impl
y or
der o
f im
port
ance
.IF
N, in
tefe
ron;
SLE
, sys
tem
ic lu
pus
eryt
hem
atos
us.
challenging the research community to identify instruments that are sensitive and specific in their measure of these symptoms, and to design trials to test candidate therapeutic interventions.50 Clinical trials should be conducted in distinct clinical subpopu-lations to optimise the likelihood of detecting a significant clin-ical response. Development of practical interventions that might improve access to care, compliance with treatment and clinical outcomes represents yet another priority area to address the unmet needs of patients with lupus.
The CTD portion of this exercise included a number of diseases, including Sjogren’s syndrome, systemic sclerosis, inflammatory myopathies and vasculitides (table 5 and online supplementary table 5a–d). The unmet need in this group of diseases mirrored their inherent heterogeneity. The group agreed that compared with the prior year, little had changed with regard to identified unmet needs.
Emerging checkpoint inhibitor adverse events dominated the groups’ conversation early on, as these malignancy therapies can trigger severe rheumatic manifestations even among individuals who lack a history of CTD.51 52 Understanding most aspects of these events was highlighted again as an important unmet need including basic questions regarding incidence, risk factors, and optimal clinical management with glucocorticoids and other immunosuppressives. The ramifications of instituting immuno-suppression in these cases, in terms of the ability to continue using checkpoint inhibitors and control of the underlying malig-nancy, represent important and challenging clinical questions. This area is changing rapidly and is of increasing need for rheu-matological input.
In ANCA-associated vasculitis, there is a need to profile the genomic, proteomic ANCA subtypes MPO, PR3 and EGPA before therapy initiation as markers of prognosis. It is important to assess the use of these subsets to ascertain whether they can be used as therapeutic guides with current and novel treatments in patients with new onset and remission induction. Such ther-apies include rituximab, anti IL-6, JAK inhibitors, anti granu-locyte-macrophage colony-stimulating factor and synthetic chemotherapies (eg, cyclophosphamide, azathioprine, metho-trexate, mycophenolate mofetil) and anti-IL5/anti-IL5Rα (EGPA) as well as randomised control trials targeting the complement pathway. Secondary unmet needs include understanding the relationship between disease pathophysiology and autoantibody states, as well as the patterns of disease regarding organ involve-ment and treatments, and identification of strategies to predict when (if ever) ANCA vasculitis treatment can be discontinued without risk of recurrence.
Giant cell arteritis (GCA) and Takayasu arteritis (TAK) were the representative types of large vessel vasculitis (LVV) consid-ered. Recognising the important differences in the clinical features and response to various therapies used and in develop-ment, as well as effect on life expectancy and need for vascular surgery, the role, use and cost-effectiveness of approved IL-6 antagonist therapy in GCA, as well as its potential role in TAK are primary unmet needs.53 Other unmet secondary needs are efforts to understand the effects of anti-IL6 on the fundamental disease pathophysiology. The efficacy of abatacept in GCA requires ongoing study. Better outcome measures and disease activity measures and strategies for their use are needed for all forms of LVV, including improved biomarkers and imaging (PET, US, MRI, CT). For all forms of vasculitis, international prospec-tive observational studies of patient cohorts that include clinical imaging and bio-banking are needed.
For systemic sclerosis, similarly, the potential use of IL-6 inhibition remains important to further evaluate,54 as well as
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Table 5 Primary and secondary unmet scientific needs within other connective tissue disorders/vasculitis with regard to translational science, clinical science and therapeutic trials, and clinical care (these are overarching needs in common across individual diseases (including Sjogren’s syndrome, systemic sclerosis, inflammatory myopathies and vasculitides, including IgG4-related disease); individual diseases are presented in online supplementary appendices)∗
Primary unmet needs secondary unmet needs
Translational science
Need for better definition of the disease phenotype (heterogeneity of disease currently a problem in treatment, trial design etc)Multidisciplinary centres of excellence needed (led by experts to include a rheumatologist)Elucidating underlying pathophysiology of checkpoint inhibitor–induced connective tissue disease manifestations/reactions
Clinical science and therapeutic trials
Need for clinical trials with better defined outcome measures (clinician and patient)Determine the incidence of checkpoint inhibitor–induced connective tissue disease adverse events and to develop optimal treatment algorithms for such events
Need for new targets defined by elucidation of pathophysiological processes
Clinical care Early disease identification and treatment Identification and management (minimisation) of comorbidity
identifying those patients at high risk for lung cancer within the clinical care of patients with scleroderma.55 Unmet needs continue to include means for earlier diagnosis and consideration of haematopoietic stem cell transplantation for treatment.56 In Sjogren’s syndrome, lymphoma aetiology and pathophysiology remain poorly understood, although a recently developed algo-rithm is said to be able to predict this complication with high predictive value. This remains to be validated in a large multi-centre multinational cohort.57 58 For inflammatory myositis, identified unmet needs from the prior year were reiterated this year as unchanged. Questions remain regarding the associa-tion of anti-TIF1 and NXP2 antibodies with cancer-associated myositis, as well as the optimal management of these malignan-cies and associated myositis.59 60
suMMARyThe convening of the 20th annual Targeted Therapies meeting afforded the possibility to discuss and articulate major unmet needs in the field of rheumatology, and across domains there were several overarching perceived unmet needs. These included the need for the infrastructure necessary to study heterogeneity within each disease and develop predictive tools for thera-peutic response. This would best be facilitated by creation, or further development, of well-characterised, longitudinal patient cohorts (preferably inception cohorts) paired with bioreposi-tories of patient specimens. Within clinical care, a commonly identified unmet need was improved management of comor-bidities as well as patient-centred care. Within clinical science and therapeutic development, the abilities to identify ‘pre-dis-ease’ with subsequent prevention in those at risk, and to cure disease were identified. Other primary unmet needs included accessible and affordable new therapeutics including the devel-opment of non-opiate therapies for pain control. Issues of improved management of comorbidities and cross-specialty training/education and co-management continued to be a theme, although progress has been made in some disease areas. Finally, despite the relatively rapid evolution of new therapies for some rheumatic diseases, continued development of new therapeu-tics across all disease states, better access and better targeting of existing therapies continues to represent a major unmet need for patients and rheumatologists.
Author affiliations1Oregon Health and Science University, Portland, Oregon, USA2Brigham and Women’s Hospital, Boston, Massachusetts, USA3Hospital for Special Surgery, New York City, New York, USA4Charité-Universitätsmedizin Berlin, Berlin, Germany5Swedish Medical Center, University of Washington, Seattle, Washington, USA
6University of Lausanne, Lausanne, Switzerland7Amsterdam Rheumatology and Immunology Center, Amsterdam, Netherlands8Hôpital Cochin, Paris, France9University of Massachusetts Medical School, Worcester, Massachusetts, USA10Paris-Sud University, Orsay, France11Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Charité, Berlin, Germany12Max Planck Institute for Infection Biology, Berlin, Germany13Deutsches Rheumaforschungszentrum, Berlin, Germany14NYU School of Medicine, New York City, New York, USA15National Institutes of Health, Bethesda, Maryland, USA16Leiden University Medical Center, Leiden, Netherlands17University of Erlangen, Erlangen, Germany18Division of Rheumatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria19Los Angeles Medical Center, University of California, Los Angeles, California, USA20University of Florence, Florence, Italy
Contributors All coauthors contributed to this manuscript’s creation in all aspects including data gathering, analysis, writing and critical revision of the manuscript.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
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22 Haroon M, FitzGerald O. Psoriatic arthritis: complexities, comorbidities and implications for the clinic. Expert Rev Clin Immunol 2016;12:405–16.
23 Mease PJ GM, Weinblatt M, Peloso PM, et al. Safety and efficacy of ABT-122, a TNF and IL-17–Targeted Dual Variable Domain (DVD)–Ig™, in psoriatic arthritis patients with inadequate response to methotrexate: results from a phase 2 trial. Arthritis Rheumatol 2016;68(suppl 10).
24 Coates LC, FitzGerald O, Merola JF, et al. Group for research and assessment of psoriasis and psoriatic arthritis/outcome measures in rheumatology consensus-based recommendations and research agenda for use of composite measures and treatment targets in psoriatic arthritis. Arthritis Rheumatol 2018;70:345–55.
25 Jadon DR, Chandran V, Stober C, et al. Proceedings of The 2017 GRAPPA Collaborative Research network meeting. J Rheumatol Suppl 2018;94:54–61.
26 FitzGerald O, Haroon M, Giles JT, et al. Concepts of pathogenesis in psoriatic arthritis: genotype determines clinical phenotype. Arthritis Res Ther 2015;17.
27 Ogdie A, Schwartzman S, Husni ME. Recognizing and managing comorbidities in psoriatic arthritis. Curr Opin Rheumatol 2015;27:118–26.
28 Mease PJ, Fibromyalgia MPJ. Fibromyalgia, a missed comorbidity in spondyloarthritis: prevalence and impact on assessment and treatment. Curr Opin Rheumatol 2017;29:304–10.
29 Genovese MC, Weinblatt ME, Aelion JA, et al. ABT-122, a bispecific DVD-Immunoglobulin targeting TNF- and IL-17A, in RA with inadequate response to methotrexate: a randomized, double-blind study. Arthritis Rheumatol 2018;70:1710–20.
30 Cobo-Ibáñez T, Villaverde V, Seoane-Mato D, et al. Multidisciplinary dermatology–rheumatology management for patients with moderate-to-severe psoriasis and psoriatic arthritis: a systematic review. Rheumatol Int 2016;36:221–9.
31 Velez NF, Wei-Passanese EX, Husni ME, et al. Management of psoriasis and psoriatic arthritis in a combined dermatology and rheumatology clinic. Archives of Dermatological Research 2012;304:7–13.
32 Okhovat JP, Ogdie A, Reddy SM, et al. Psoriasis and Psoriatic Arthritis Clinics Multicenter Advancement Network Consortium (PPACMAN) survey: benefits and challenges of combined rheumatology-dermatology clinics. J Rheumatol 2017;44:693–4.
33 Van de Wiele T, Van Praet JT, Marzorati M, et al. How the microbiota shapes rheumatic diseases. Nature Reviews Rheumatology 2016;12:398–411.
34 Sieper J, Poddubnyy D. Axial spondyloarthritis. Lancet 2017;390:73–84. 35 Hall-Craggs MA, Bray TPJ, Bainbridge AP. Quantitative imaging of inflammatory
disease: are we missing a trick? Ann Rheum Dis 2018;77:1689–91. 36 Poddubnyy D, van Tubergen A, Landewé R, et al. Development of an ASAS-
endorsed recommendation for the early referral of patients with a suspicion of axial spondyloarthritis. Ann Rheum Dis 2015;74:1483–7.
37 Poddubnyy D, Sieper J. What is the best treatment target in axial spondyloarthritis: tumour necrosis factor alpha, interleukin 17, or both? Rheumatology 2017.
38 Mease P, Sieper J, Van den Bosch F, et al. Randomized controlled trial of adalimumab in patients with nonpsoriatic peripheral spondyloarthritis. Arthritis Rheumatol 2015;67:914–23.
39 Li W, Sivakumar R, Titov AA, et al. Metabolic factors that contribute to Lupus pathogenesis. Critical Reviews in Immunology 2016;36:75–98.
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51 Cappelli LC, Gutierrez AK, Baer AN, et al. Inflammatory arthritis and sicca syndrome induced by nivolumab and ipilimumab. Ann Rheum Dis 2017;76:43–50.
52 Johnson DB, Sullivan RJ, Ott PA, et al. Ipilimumab therapy in patients with advanced melanoma and preexisting autoimmune disorders. JAMA Oncol 2016;2:234–40.
53 Nakaoka Y, Isobe M, Takei S, et al. Efficacy and safety of tocilizumab in patients with refractory Takayasu arteritis: results from a randomised, double-blind, placebo-controlled, phase 3 trial in Japan (the TAKT study). Ann Rheum Dis 2018;77:348–54.
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Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspectiveJan Damoiseaux, 1 Luis Eduardo Coelho Andrade,2 Orlando Gabriel Carballo,3,4 Karsten Conrad,5 Paulo Luiz Carvalho Francescantonio,6 Marvin J Fritzler,7 Ignacio Garcia de la Torre,8 Manfred Herold,9 Werner Klotz,10 Wilson de Melo Cruvinel,11 Tsuneyo Mimori,12 Carlos von Muhlen,13 Minoru Satoh,14 Edward K Chan15
To cite: Damoiseaux J, Andrade LEC, Carballo OG, et al. Ann Rheum Dis 2019;78:879–889.
Handling editor Josef S Smolen
► Additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214436).
For numbered affiliations see end of article.
Correspondence toDr Jan Damoiseaux, Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht 6229 HX,The Netherlands; jan. damoiseaux@ mumc. nl
Received 13 September 2018Accepted 23 January 2019Published Online First 12 March 2019
© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
AbsTRACTThe indirect immunofluorescence assay (IIFA) on HEp-2 cells is widely used for detection of antinuclear antibodies (ANA). The dichotomous outcome, negative or positive, is integrated in diagnostic and classification criteria for several systemic autoimmune diseases. However, the HEp-2 IIFA test has much more to offer: besides the titre or fluorescence intensity, it also provides fluorescence pattern(s). The latter include the nucleus and the cytoplasm of interphase cells as well as patterns associated with mitotic cells. The International Consensus on ANA Patterns (ICAP) initiative has previously reached consensus on the nomenclature and definitions of HEp-2 IIFA patterns. In the current paper, the ICAP consensus is presented on the clinical relevance of the 29 distinct HEp-2 IIFA patterns. This clinical relevance is primarily defined within the context of the suspected disease and includes recommendations for follow-up testing. The discussion includes how this information may benefit the clinicians in daily practice and how the knowledge can be used to further improve diagnostic and classification criteria.
InTRoduCTIonAutoantibodies, as detected by the indirect immu-nofluorescence assay (IIFA) on HEp-2 cells (IIFA HEp-2), are recognised as important diagnostic markers in a plethora of autoimmune diseases, in particular the systemic autoimmune rheumatic diseases (SARD).1 Although somewhat dated by today’s standards, members of the American College of Rheumatology (ACR) prepared an evidence-based guideline for the usefulness of the HEp-2 IIFA results for diagnostic and prognostic purposes and also for meeting diagnostic criteria.2 That guideline was based on reactivity with nuclear antigens as detected by IIFA on rodent tissue or HEp-2 cells. More recently, the IIFA on HEp-2 cells was reinforced as the gold standard for autoanti-body screening in SARD.3
Interestingly, the HEp-2 IIFA test reveals much more information than the mere absence or pres-ence of autoantibodies, that is, the level of antibody as well as the HEp-2 IIFA pattern. Based on titra-tion or appropriate evaluation of the fluorescence intensity, the antibody level can be determined and this information has general concordance with the
clinical relevance of the test result. Indeed, higher antibody levels are better associated with SARD and have an increased likelihood to identify the auto-antigen in follow-up testing.4–6 The importance of the level of autoantibodies is also recognised in the ACR guideline as well as by the recommendations issued by the European Autoimmunity Standardiza-tion Initiative (EASI) and the International Union of Immunologic Societies (IUIS) Autoantibody Stan-dardization Subcommittee.2 7
The HEp-2 IIFA pattern may also reveal clini-cally relevant information. This information is not restricted to giving direction to follow-up testing for antigen-specificity, but, for instance, the centromere pattern is included in the classification criteria for systemic sclerosis,8 while the nuclear dense fine speckled pattern is reported to be more prevalent in apparently healthy individuals as compared with patients with SARD.9 To harmonise the names and descriptions of the distinct HEp-2 IIFA patterns, an ordered classification taxonomy was proposed.10 This proposal was subsequently elaborated on by the International Consensus on ANA Patterns (ICAP), initiated in parallel to the 12th Interna-tional Workshop on Autoantibodies and Autoim-munity (2014) held in Sao Paulo, Brazil. During this workshop, a consensus was reached on the nomen-clature and definitions of 28 HEp-2 IIFA patterns. Each HEp-2 IIFA pattern was ascribed an alphanu-meric code from AC-1 to AC-28.11 The consensus nomenclature for each pattern and representative images were also made available online at the ICAP website (http://www. ANApatterns. org).
In addition to the nuclear patterns, important cytoplasmic and mitotic patterns may also be observed in HEp-2 IIFA analysis. Although reporting non-nuclear patterns is considered clin-ically relevant,7 for various jurisdictional reasons there is no clear-cut consensus viewpoint on reporting non-nuclear patterns as a negative or positive test.12 With the understanding that the term ‘Antinuclear antibody (ANA) test’ may be inappropriate to designate a test that also addresses autoantibodies to antigens in the cytoplasm and mitotic apparatus, an alternative name, anticellular antibodies, was suggested in the EASI/IUIS recom-mendations.7 Recent publications from ICAP have preferred the term HEp-2 IIFA as it covers the
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whole spectrum of patterns that can be observed when using the HEp-2 cells as substrate.13 14
Originally, the HEp-2 IIFA patterns were associated with diseases, but it was anticipated that many of these associa-tions are only valid if the antigen-specificity was confirmed by follow-up testing. In subsequent ICAP workshops, it was agreed that the disease associations should be replaced by clinical rele-vance. In this current paper, we present the consensus on the clinical relevance of the distinct HEp-2 IIFA patterns as achieved by consecutive workshops and discussions among the executive ICAP members.
MATeRIAls And MeTHodsFor discussion about the structure of clinical relevance templates were prepared for AC-2 (LECA), AC-3 (JD) and AC-5 (MS). This formed the basis of a guideline for description of each AC pattern (EC). Of highest importance, it was agreed that the information should be objective and helpful for the clinician, the pattern–antigen associations should be put in the right clinical context and information should be evidence-based.
In preparation for the third ICAP workshop in Kyoto (2016), composition of the clinical relevance documents was started for the nuclear patterns (JD, LECA, MS), cytoplasmic patterns (CAvM, EKLC) and mitotic patterns (MH, TM). As far as already available, the documents were commented on by the ICAP exec-utive board and, after appropriate adjustment, discussed with the workshop participants. The feedback from participants mainly focused on the structure of the information provided, on the required level of detail and the format of recommended follow-up testing.
In anticipation of the fourth ICAP workshop in Dresden (2017), the set of clinical relevance documents was completed for all patterns. Further comments from the ICAP executive board were included. The resulting documents were individ-ually discussed with the workshop participants for nuclear (JD), cytoplasmic (CAvM) and mitotic (MH) patterns. Besides several substantive comments, there was general agreement that the information should be provided in tabular format at two distinct levels. The first level should contain information on relevant follow-up testing in the respective clinical context, the recommended follow-up tests should be commercially available and detailed test characteristics should not be given because of potential geographic and jurisdictional differences. Information based on case reports or small patient cohorts, as well as infor-mation on possible follow-up testing that is only available in specialised research laboratories, should only be provided in the second level information.
Tables for nuclear, cytoplasmic and mitotic patterns were prepared for first and second level information (JD). These tables were commented by the ICAP executive board and final-ised by JD. Of note, since the starting point of the tables on clinical relevance is the HEp-2 IIFA pattern and not the clinically suspected disease, the tables do not list all autoantibodies related to the respective disease.
ResulTsnuclear Hep-2 IIFA patternsTo date, a total of 15 nuclear HEp-2 IIFA patterns have been described, that is, AC-1–AC-14 and AC-29. Table 1 summarises the clinical relevance of these patterns.8 9 14–79 Since AC-29 was only recently described,14 the advice for follow-up testing for autoantibodies to topoisomerase I (Scl-70) in case of clinical suspicion of systemic sclerosis is also added as a note to the
clinical relevance of AC-1. In particular, disease-specific immu-noassays, like autoimmune liver disease profile, inflammatory myopathy profile, systemic sclerosis profile, are often only avail-able in specialty clinical laboratories.
For six nuclear HEp-2 IIFA patterns (AC-3, 5, 7, 8, 12 and 13), additional information about clinical relevance is summarised in online supplementary table S1. Although some assays for anti-CENP-A antibodies are commercially available, these anti-bodies are included in online supplementary table S1 because the majority of sera revealing the AC-3 pattern are also reactive with CENP-B. In contrast to CENP-A, CENP-B is included in many routine extractable nuclear antigens profiles.
Cytoplasmic Hep-2 IIFA patternsTable 2 summarises the clinical relevance of the nine cyto-plasmic HEp-2 IIFA patterns, that is, AC-15–AC-23.26 33 80–101 It is recognised that the distinction between AC-19 (dense fine speckled) and AC-20 (fine speckled) can be challenging. More-over, within the spectrum of anti-tRNA synthetase antibodies, not all produce an HEp-2 IIFA pattern and only some anti-Jo-1 antibodies are considered to give the AC-20 pattern, while the other anti-tRNA synthetase antibodies (EJ, KS, OJ, PL-7 and PL-12) are more likely to reveal the AC-19 pattern. Solid infor-mation on the pattern of two additional anti-tRNA synthetase antibodies (Ha and Zo) is lacking. Overall, the relation between these two cytoplasmic HEp-2 IIFA patterns and the distinct anti-tRNA synthetase antibodies is subject to further discussion. In clinical practice, the complete spectrum of the anti-tRNA synthe-tase antibodies should be determined irrespective of the subtype of cytoplasmic speckled pattern, that is, AC-19 or AC-20.
For seven cytoplasmic HEp-2 IIFA patterns (AC-15–AC-19, AC-22 and AC-23), more detailed information is provided in online supplementary table S2. In particular, for AC-16–AC-18, the clinical associations are quite diverse, depending on the antigen recognised. Overall, the clinical associations provided are primarily based on antigen-specific immunoassays and not on the HEp-2 IIFA pattern as such.
Mitotic Hep-2 IIFA patternsThe clinical relevance of the five mitotic patterns is summarised in table 3,102–122 with more detailed information in online supplementary table S3. As for the cytoplasmic patterns, clinical associations for the mitotic patterns are primarily based on anti-gen-specific immunoassays and not on the HEp-2 IIFA pattern as such.
dIsCussIonIn the current paper, we present the ICAP consensus on the clin-ical relevance of 29 HEp-2 IIFA patterns defined by ICAP.11 14 The consensus on clinical relevance is defined in the clinical context of the patient, that is, suspected disease, and includes recommended follow-up testing within the spectrum of anti-gen-specificities that are commercially available. Obviously, if follow-up testing identifies the antigen, the clinical relevance can be further refined.123
Defining the clinical relevance of HEp-2 IIFA patterns in the context of disease manifestations is meant to be an important tool for the clinician in the diagnostic work-up of patients suspected of SARD. Unfortunately, good data on the associa-tion between HEp-2 IIFA patterns and the distinct diseases are lacking, probably due to reasons summarised below. There are several reasons for not finding a perfect association between HEp-2 IIFA patterns and diseases. First, pattern assignment in
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Table 1 Nuclear HEp-2 IIFA patterns
Code AC pattern—clinical relevance Refs
AC-1 HOMOGENEOUS
► Found in patients with SLE, chronic autoimmune hepatitis or juvenile idiopathic arthritis
► If SLE is clinically suspected, it is recommended to perform a follow-up test for anti-dsDNA antibodies, alone or in combination with dsDNA/histone complexes (nucleosomes/chromatin); anti-dsDNA antibodies are included in the classification criteria for SLE
15, 16
► If chronic autoimmune hepatitis or juvenile idiopathic arthritis is suspected, follow-up testing is not recommended because the respective autoantigens revealing the AC-1 pattern are not completely defined
17
Notes: Although autoantibodies to Topoisomerase I (formerly Scl-70) may be reported as nuclear homogeneous, they typically reveal a composite AC-29 HEp-2 IIFA pattern; as such, clinical suspicion of SSc may warrant follow-up testing for reactivity to this antigen.
14, 18
Although AC-1 is the most prevalent pattern in chronic autoimmune hepatitis, other HEp-2 IIFA patterns may occur, but also for these patterns the autoantigens are not completely defined.
19
AC-2 DENSE FINE SPECKLED
► Commonly found as high titer HEp-2 IIFA-positive in apparently healthy individuals or in patients who do not have a systemic autoimmune rheumatic disease (SARD)
9
► The negative association with SARD is only valid if the autoreactivity is confirmed as being directed to DFS70 (also known as LEDGF/p75) and if no other common ENA is recognized
20, 21
► Both in apparently healthy individuals as well as patients who do not have a SARD the AC-2 pattern may be caused by autoantibodies to other antigens than DFS70
22
Note: Confirmatory assays for anti-DFS70 antibodies may be available only in specialty clinical laboratories.
AC-3 CENTROMERE (see online supplementary table S1 for further details
► Commonly found in patients with limited cutaneous SSc, and as such included in the classification criteria for SSc
8, 15, 23
► In combination with Raynaud phenomenon, the AC-3 pattern is prognostic for onset of limited cutaneous SSc
15, 23
► Strongly associated with antibodies to CENP-B; especially in case of low titers, confirmation by an antigen-specific immunoassay is recommended to support the association with limited cutaneous SSc; the CENP-B antigen is included in many routine ENA profiles
15
► The AC-3 pattern is also apparent in a subset of patients with PBC; these patients often have both SSc as well as PBC
15
AC-4 FINE SPECKLED
► Present to a varying degree in distinct SARD, in particular SjS, SLE, subacute cutaneous lupus erythematosus, neonatal lupus erythematosus, congenital heart block, DM, SSc, and SSc-AIM overlap syndrome
15
► If SjS, SLE, subacute cutaneous lupus erythematosus, neonatal lupus erythymatosus, or congenital heart block is clinically suspected, it is recommended to perform follow-up tests for anti-SS-A/Ro (Ro60) and anti-SS-B/La antibodies; in most laboratories these antigens are included in the routine ENA profile
15
► Autoantibodies to SS-A/Ro are part of the classification criteria for SjS (the criteria do not distinguish between Ro60 and Ro52/TRIM21)
25
► If SSc, AIM, or to a lesser extend SLE, is clinically suspected, it is recommended to perform follow-up tests for detecting autoantibodies to Mi-2, TIF1γ, and Ku; these antigens are typically included in disease specific immunoassays (i.e., inflammatory myopathy profile*)
26
► Autoantibodies to Mi-2 and TIF1γ are associated with DM; autoantibodies to TIF1γ in patients with DM, although rare in the overall AC-4 pattern, is strongly associated with malignancy in old patients
26, 27
► Autoantibodies to Ku are associated with SSc-AIM and SLE-SSc-AIM overlap syndromes 26
Notes: Anti-SS-A/Ro (Ro60) and AIM-specific autoantibodies may be undetected in HEp-2 IIFA-screening. 28
AC-5 LARGE/COARSE SPECKLED (see online supplementary table S1 for further details)
► Present to a varying degree in distinct SARD, in particular SLE, SSc, MCTD, SSc-AIM overlap syndrome, and UCTD (i.e, patients with rheumatic symptoms without a definite SARD diagnosis)
29
► If SLE is clinically suspected, it is recommended to perform follow-up tests for anti-Sm and anti-U1RNP antibodies; these antigens are commonly included in the routine ENA profile; anti-Sm antibodies are included in the classification criteria for SLE
16, 30, 31
► If SSc is clinically suspected, it is recommended to perform a follow-up test for anti-RNApol III antibodies (e.g., SSc profile*); the anti-RNApol III antibodies are included in the classification criteria for SSc
8
► If MCTD is clinically suspected, it is recommended to perform a follow-up test for anti-U1RNP antibodies; the antigen is commonly included in the routine ENA profile; anti-U1RNP antibodies are included in the diagnostic criteria for MCTD
32
► If the SSc-AIM overlap syndrome is clinically suspected, it is recommended to perform follow-up tests for anti-U1RNP and anti-Ku antibodies; these antigens are included in the routine ENA profile (U1RNP), or in disease specific immunoassays (Ku, i.e., inflammatory myopathy profile* and SSc profile*)
26, 33
► In non-SARD individuals in the general population, the presence of the AC-5 pattern is not associated with the autoantigens mentioned above and most often concerns low antibody titers
Continued
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Code AC pattern—clinical relevance Refs
AC-6 MULTIPLE NUCLEAR DOTS
► Found in a broad spectrum of autoimmune diseases, including PBC, AIM (DM), as well as other inflammatory conditions
34
► If PBC is clinically suspected, it is recommended to perform follow-up tests for anti-Sp100 (and PML/Sp140) antibodies; in particular anti-Sp100 antibodies have the best clinical association with PBC and have added value, especially when associated with AMA; the Sp100 (and PML-Sp140) antigen is included in disease specific immunoassays (ie, liver profile*)
17, 35, 36
► If DM is clinically suspected, it is recommended to perform a follow-up test for anti-MJ/NXP-2 antibodies; these anti-MJ/NXP-2 antibodies are highly specific for AIM, are found in up to one third of patients with juvenile DM, and have been reported to be associated with malignancies in adult AIM patients; the antigen is included in disease specific immunoassays (i.e., inflammatory myopathy profile*)
37–39
AC-7 FEW NUCLEAR DOTS (see online supplementary table S1 for further details)
► The AC-7 pattern has low positive predictive value for any disease 40, 41
► Antigens primarily localized in the dots include p80-coilin and SMN complex; specific immunoassays for these autoantibodies are currently not commercially available
42, 43
AC-8 HOMOGENEOUS NUCLEOLAR (see online supplementary table S1 for further details)
► Found in patients with SSc, SSc-AIM overlap syndrome, and patients with clinical manifestations of other SARD
44–46
► If limited cutaneous SSc is clinically suspected, it is recommended to perform a follow-up test for anti-Th/To antibodies; the antigen is included in disease specific immunoassays (ie, SSc profile*)
44, 45
► If SSc-AIM overlap syndrome is clinically suspected, it is recommended to perform a follow-up test for anti-PM/Scl antibody reactivity; the antigen may be included in the routine ENA profile and is included in disease specific immunoassays (i.e., inflammatory myopathy profile* and the SSc profile*); in general, anti-PM/Scl antibodies yield a diffuse nuclear fine speckled staining in addition to the AC-8 pattern
46
► Other antigens recognized include B23/nucleophosmin, No55/SC65, and C23/nucleolin, but the clinical significance of these autoantibodies is not well established; specific immunoassays for these autoantibodies are currently not commercially available
Notes: Although some anti-Th/To antibody immunoassays are commercially available, technical issues relating to the limited sensitivity of these immunoassays should be taken in to consideration.
44, 47
AC-9 CLUMPY NUCLEOLAR
► Found in patients with SSc 48
► If SSc is clinically suspected, it is recommended to perform a follow-up test for anti-U3RNP/fibrillarin antibodies; the antigen is included in disease specific immunoassays (i.e, SSc profile*)
48
► If confirmed as anti-U3RNP/fibrillarin reactivity by immunoassay, the clinical association is with diffuse SSc, increased incidence of pulmonary arterial hypertension, skeletal muscle disease, severe cardiac involvement, and gastrointestinal dysmotility
23, 48–50
► Among SSc patients, anti-U3RNP/fibrillarin antibodies are most commonly found in African American and Latin American patients
48, 49, 51
Notes: Although some anti-U3RNP/fibrillarin immunoassays are commercially available, technical issues relating to the limited sensitivity of these immunoassays should be taken into consideration.
24
AC-10 PUNCTATE NUCLEOLAR
► The AC-10 pattern can be seen in various conditions, including SSc, Raynaud’s phenomenon, SjS, and cancer
52–56
► If the AC-10 pattern is observed in the serum of patients with conditions mentioned above, follow-up testing for anti-NOR90(hUBF) antibodies is to be considered; the antigen is included in disease specific immunoassays (i.e. SSc profile*)
54, 55
► While AC-10 is associated with anti-RNApol I antibodies, these antibodies almost always coexist with anti-RNApol III antibodies which reveal the AC-5 pattern; therefore, if SSc is clinically suspected, it is recommended to perform a follow-up test for anti-RNApol III antibodies (See also AC-5); specific immunoassays for anti-RNApol I antibodies are currently not commercially available
52, 53, 57
AC-11 SMOOTH NUCLEAR ENVELOPE
► The AC-11 pattern is infrequently found in routine autoantibody testing and has been described in autoimmune-cytopenias, autoimmune liver diseases, linear scleroderma, APS, and SARD; current information on clinical associations is based mainly on case reports and small cohorts
58–60
► Antigens recognized include lamins (A, B, C) and LAP-2; specific immunoassays for these autoantibodies are currently not commercially available
58–60
AC-12 PUNCTATE NUCLEAR ENVELOPE (see online supplementary table S1 for further details)
► Found in patients with PBC, as well as patients with other autoimmune liver diseases and SARD 61
► If PBC is clinically suspected, it is recommended to perform a follow-up test for anti-gp210 antibodies; the antigen is included in disease specific immunoassays (ie, extended liver profile*)
62–64
► Other antigens recognized include p62 nucleoporin, LBR, and Tpr; specific immunoassays for these autoantibodies are currently not commercially available
65–68
Table 1 Continued
Continued
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Code AC pattern—clinical relevance Refs
AC-13 PCNA-like (see online supplementary table S1 for further details)
► The AC-13 pattern has formerly been considered highly specific for SLE, but this specificity is debated 69, 70
► If SLE is clinically suspected, it is recommended to perform a follow-up test for anti-PCNA antibodies; the antigen is included in several routine ENA profiles
69
► Recent studies with antigen-specific immunoassays show clinical associations also with SSc, AIM, RA, HCV, and other conditions
70–73
AC-14 CENP-F-like
► The majority of sera exhibiting the AC-14 pattern are from patients with a diversity of neoplastic conditions (breast, lung, colon, lymphoma, ovary, brain); paradoxically, the frequency of the AC-14 pattern in patient cohorts with these malignancies is low
► The AC-14 pattern is also seen in inflammatory conditions (Crohn’s disease, autoimmune liver disease, SjS, graft-versus-host disease); current information on clinical associations is based mainly on case reports and series of cases
► Possible associations only hold if the reactivity to CENP-F is confirmed in an antigen-specific immunoassay; current information on clinical associations is based mainly on case reports and series of cases; specific immunoassays for this autoantibody are currently not commercially available
74–78
AC-29 TOPOI-like
► The AC-29 pattern is highly specific for SSc, in particular with diffuse cutaneous SSc and more aggressive forms of SSc
14, 18, 23
► If SSc is clinically suspected, it is recommended to perform a follow-up test for anti-Topoisomerase I (formerly Scl-70) antibodies; the anti-Topoisomerase I antibodies are included in the classification criteria for SSc and the antigen is included in routine ENA profiles
8, 23, 79
*Availability of the inflammatory myopathy profile, the SSc profile and the (extended) liver profile may be limited to specialty clinical laboratories.AIM, autoimmune myopathy; AMA, antimitochondrial antibodies; APS, antiphospholipid syndrome; CENP, centromere-associated protein; DFS, dense fine speckled; DM, dermatomyositis; ENA, extractable nuclear antigens; HCV, hepatitis C virus; IIFA, indirect immunofluorescence assay; LAP, lamin-associated polypeptide; LBR, lamin B receptor; LEDGF, lens epithelial derived growth factor; NOR, nucleolus organiser region; NXP, nuclear matrix protein; PBC, primary biliary cholangitis; PCNA, proliferating cell nuclear antigen; PML, promyelocytic leukaemia; PM/Scl, polymyositis-scleroderma; RA, rheumatoid arthritis; RNApol, RNA polymerase; RNP, ribonucleoprotein; SARD, systemic autoimmune rheumatic diseases; SLE, systemic lupus erythematosus; SMN, survival of motor neuron; SSc, systemic sclerosis; SjS, Sjögren’s syndrome; TIF, transcription intermediary factor; TRIM, tripartite motif; Tpr, translocated promoter region; UCTD, undifferentiated connective tissue disease; dsDNA, double stranded DNA; hUBF, human upstream binding factor.
Table 1 Continued
clinical laboratories is rather inconsistent as shown by external quality assessments.14 124 125 This is exactly the reason why ICAP was initiated: the consensus on nomenclature and definitions of HEp-2 IIFA patterns allows to align pattern description across laboratories. Also, the integration of computer-aided immu-nofluorescence microscopy (CAIFM) may further improve the consistency in pattern assignments.126–131 As such, it is promising that several companies involved in CAIFM have declared their intention to accommodate to the ICAP classification. Second, even apparently healthy individuals may have autoantibodies as detected by the HEp-2 IIFA. Such autoantibodies, being either innocent bystander antibodies or predictive antibodies, may still be present on development of SARD and interfere with the SARD-related pattern. Interestingly, the pattern best asso-ciated with apparently healthy individuals is the nuclear dense fine speckled pattern (AC-2), but this association only holds if the specificity is confirmed as monospecific for DFS70.20 21 132 Third, the HEp-2 IIFA patterns may slightly differ depending on the cellular substrate used. For this reason, the ICAP website contains for each pattern multiple pictures taken from different brands of HEp-2 slides. Fourth, diseases like systemic lupus erythematosus and autoimmune inflammatory myopathies may be associated with distinct autoantibodies, each associated with a distinct HEp-2 IIFA pattern. If the autoantigens are ill defined, as is the case, for instance, in autoimmune hepatitis, only the most prevalent patterns are included. Altogether, it is evident that, with the exception of the centromere pattern (AC-3), all patterns are to be confirmed by antigen-specific immunoassay for a solid association with the respective autoimmune diseases.
While consensus statements have been generated for all 29 HEp-2 IIFA patterns, and it is highly recommended to report
patterns,7 11 it is anticipated that laboratories may restrict their reports to the so-called ‘competent level’ patterns (http://www. ANApatterns. org).133 Although, for instance, the nucleolar patterns may not be reported as distinct entities (AC-8, AC-9 and AC-10), all three subtypes represent autoantibodies reactive with antigens associated with systemic sclerosis, either alone or in combination with autoimmune inflammatory myopathies. Follow-up testing, therefore, anyhow involves the systemic sclerosis multiparameter assay including all the relevant auto-antibodies. Traditionally, only nuclear HEp-2 IIFA patterns have been considered as a true positive HEp-2 IIFA test, and this is most likely related to the time-honoured terminology ‘Antinuclear Antibody Test’,12 but it is evident from this report that even for nuclear HEp-2 IIFA patterns, the clinical associ-ations are quite diverse. In particular, the nuclear dense fine speckled pattern (AC-2) seems to have an inverse association with SARD.9 134 On the other hand, the cytoplasmic HEp-2 IIFA patterns, and to a lesser extent the mitotic patterns, are also clinically relevant and may demand dedicated follow-up testing in daily clinical practice. Therefore, the ICAP executive board advocates that information on HEp-2 IIFA patterns should be reported to the clinician and should also be incorporated in diag-nostic and classification criteria instead of the simple assignment ‘ANA-positive’.135
Although the HEp-2 IIFA has been considered the gold stan-dard for autoantibody detection in SARD,3 the limitations of this assay are understood.136–138 Indeed, up to 35% of healthy controls may be positive if a screening dilution of 1/40 is used.139 Therefore, in the EASI/IUIS recommendations, it is advocated that each laboratory verifies that the screening dilution is defined by a cut-off set at the 95th percentile.7 However, by
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Table 2 Cytoplasmic HEp-2 IIFA patterns
Code AC pattern–—clinical relevance Refs
AC-15 FIBRILLAR LINEAR (see online supplementary table S2 for further details)
► Found in patients with AIH type 1, chronic HCV infection, and celiac disease (IgA isotype); rare in SARD
17
► If AIH type 1 is clinically suspected, it is recommended to confirm reactivity with smooth muscle antibodies (IgG isotype), typically detected by IIFA on rodent tissue (liver, stomach, kidney); anti-smooth muscle antibodies are included in the international criteria for AIH type 1
17,80
► F-actin is the main target antigen of anti-smooth muscle antibodies in AIH type 1; autoantibodies to F-actin are of more clinical importance than antibodies to G-actin
81–83
Notes: Although anti-F-actin immunoassays are commercially available, technical issues relating to the sensitivity of these immunoassays should be taken into consideration.
AC-16 FIBRILLAR FILAMENTOUS (see online supplementary table S2 for further details)
► Found in various diseases, but AC-16 is not typically found in SARD
► Antigens recognized include cytokeratins 8, 18, & 19, tubulin, and vimentin; specific immunoassays for these autoantibodies are currently not commercially available
AC-17 FIBRILLAR SEGMENTAL (see online supplementary table S2 for further details)
► Found very infrequently in a routine serology diagnostic setting
► Antigens recognized include α-Actinin and Vinculin; specific immunoassays for these autoantibodies are currently not commercially available
AC-18 DISCRETE DOTS (see online supplementary table S2 for further details)
► Autoantibodies revealing the AC-18 pattern have been reported in distinct SARD and in a variety of other diseases; their prevalence in unselected or specified disease cohorts has not been thoroughly studied
84
► Antigens recognized include GW-body (Processing or P body) antigens (Ge-1/Hedls, GW182, and Su/Ago2) and endosomal antigens (EEA1, CLIP-170, GRASP-1, and LBPA); specific immunoassays for these autoantibodies are currently not commercially available
Notes: Autoantibodies to GW-bodies and endosomes may yield slightly different HEp-2 IIFA patterns.
84, 85
AC-19 DENSE FINE SPECKLED (see online supplementary table S2 for further details)
► Found in patients with SLE and the anti-synthetase syndrome (a subset of AIM), interstitial lung disease, polyarthritis, Raynaud’s phenomenon, and mechanic’s hands; these features may occur in various combinations or as an isolated manifestation, especially interstitial lung disease
33, 86, 87
► If SLE is clinically suspected, follow-up tests for antibodies to ribosomal P phosphoproteins (P0, P1, P2, C22 RibP peptide) are recommended; these antigens may be included in the routine ENA profile
► Anti-RibP antibodies have been associated in some studies with neuropsychiatric lupus, and in childhood-onset SLE with autoimmune hemolytic anemia
86, 88, 89
► If AIM, in particular the anti-synthetase syndrome, is suspected, it is recommended to perform follow-up tests for antibodies to tRNA synthetases; antigens are included in disease specific immunoassays (ie, inflammatory myopathy profile*)
26, 33
► If AIM, in particular necrotizing myopathy, is suspected, it is recommended to perform follow-up tests for anti-SRP antibodies; the antigen is included in disease specific immunoassays (ie, inflammatory myopathy profile*)
26
Notes: The fine distinction between AC-19 and -20 may depend on HEp-2 substrates and/or antibody concentration; antibodies to both RibP as well as tRNA synthetases may be undetected in HEp-2 IIFA-screening.
AC-20 FINE SPECKLED
► Found in patients with the anti-synthetase syndrome (a subset of AIM), interstitial lung disease, polyarthritis, Raynaud’s phenomenon, and mechanic’s hands; these features may occur in various combinations or as an isolated manifestation, especially interstitial lung disease
33, 90
► Autoantibodies associated with the AC-20 pattern are primarily reported for the anti-Jo-1 antibody, which recognizes histidyl-tRNA synthetase; since AC-20 is not specific for Jo-1, it is recommended to perform a follow-up test for anti-Jo-1 antibodies; the antigen is included in the routine ENA profile, as well as in disease specific immunoassays (i.e., inflammatory myopathy profile*); the anti-Jo-1 antibodies are included in the classification criteria for AIM
91, 92
Notes: The fine distinction between AC-19 and -20 may depend on HEp-2 substrates and/or antibody concentration; antibodies to Jo-1 may be undetected in HEp-2 IIFA-screening.
Continued
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Code AC pattern–—clinical relevance Refs
AC-21 RETICULAR/AMA
► Commonly found in PBC, but also detected in SSc, including PBC-SSc overlap syndrome and PBC-SjS overlap syndrome
93–97
► If PBC is clinically suspected it is recommended to perform a follow-up test for AMA, historically detected by IIFA on rodent tissue (liver, stomach, kidney); these autoantibodies are primarily directed to the PDH complex, and in particular the E2-subunit (PDH-E2); the antigen is included in disease specific immunoassays (i.e., liver profile*) as well as in some routine ENA profiles
93, 94
► Additional antigens recognized include the E1α and E1β subunits of PDH, the E3-binding protein of PDH, and the 2-OGDC; these antigens are only included in extended disease specific immunoassays (i.e., extended liver profile*)
93, 94
AC-22 POLAR/GOLGI-like (see online supplementary table S2 for further details)
► Found in small numbers of patients with a variety of conditions
► Antigens recognized include giantin/macrogolgin and distinct golgin molecules; specific immunoassays to detect autoantibodies directed to specific Golgi antigens are currently not commercially available
85
AC-23 RODS and RINGS (see (online supplementary table S2 for further details)
► Most commonly found in HCV patients who have been treated with pegylated interferon-α/ribavirin combination therapy, but autoantibodies revealing the AC-23 patterns were undetected prior to treatment; as the use of interferon-α/ribavirin in HCV treatment is decreasing, the frequency and clinical associations of the AC-23 pattern may change
98–101
► Specific immunoassays to detect autoantibodies directed to specific Rods and Rings antigens, for instance IMPDH2, are not commercially available
Note: Presence of the AC-23 pattern depends on the HEp-2 cell substrate.
*Availability of the inflammatory myopathy profile, the SSc profile and the (extended) liver profile may be limited to specialty clinical laboratories.AIH, autoimmune hepatitis; AIM, autoimmune myopathy; AMA, anti-mitochondrial antibodies; APS, antiphospholipid syndrome; Ago, argonaute protein; CENP, centromere-associated protein; CLIP, class II-associated invariant chain peptide; DFS, dense fine speckled; DM, dermatomyositis; EEA, early endosome antigen; ENA, extractable nuclear antigens; HCV, hepatitis C virus; IFA, indirect immunofluorescence assay; LAP, lamin-associated polypeptide; LBR, lamin B receptor; LEDGF, lens epithelial derived growth factor; NOR, nucleolus organizer region; NXP, nuclear matrix protein; PBC, primary biliary cholangitis; PCNA, proliferating cell nuclear antigen; PML, promyelocytic leukaemia; PM/Scl, polymyositis-scleroderma; RA, rheumatoid arthritis; RNApol, ribonucleic acid polymerase; RNP, ribonucleoprotein; SARD, systemic autoimmune rheumatic diseases; SLE, systemic lupus erythematosus; SMN, survival of motor neuron; SRP, signal recognition protein; SSc, systemic sclerosis; SjS, Sjögren’s syndrome; TIF, transcription intermediary factor; TRIM, tripartite motif; Tpr, translocated promoter region; dsDNA, double stranded deoxyribonucleic acid; hUBF, human upstream binding factor; tRNA, transfer ribonucleic acid.
Table 2 Continued
taking into account that the HEp-2 IIFA nowadays is ordered by a wide spectrum of clinical disciplines,1 the number of clin-ically unexpected positive results, that is, positive test results with no clinical evidence of an associated autoimmune disease, is ever increasing and may even equal the likelihood of a clini-cally true-positive result.140 141 A study performed in a commu-nity setting concluded that many patients with a positive ANA test are incorrectly given a diagnosis of systemic lupus eryther-matosus and sometimes even treated with toxic medications.142 These arguments are used to introduce a gating strategy in order to restrict test-ordering to those cases that have a sufficiently high pretest probability for having a SARD. However, it can also be argued that patients with a low pretest probability should be tested using the HEp-2 IIFA in order to prevent true cases, espe-cially those with very early disease manifestations, from being missed. This is a paradigm shift to disease prediction and preven-tion.143 In this strategy, the HEp-2 IIFA could be integrated in multianalyte ‘omic’ profiles for case finding and establishing an early diagnosis and preventing severe complications.143 144 Obvi-ously, it is anticipated that the added value of the HEp-2 IIFA in this approach can be increased by incorporating information on both patterns as well as titres in combination with well-directed advices on follow-up testing.
Although the current consensus on the clinical relevance of HEp-2 IIFA patterns has come across after extensive discussion and debate within the ICAP executive board as well as with the workshop participants, the information provided is not based on a systematic review or meta-analysis of the existing litera-ture. Because of the short history of ICAP, being founded in 2014, inclusion of older literature might have been hampered
by potential differences in pattern nomenclature and defi-nitions. For instance, the nuclear dense fine speckled (AC-2) and topo I-like (AC-29) patterns were previously often consid-ered homogeneous, speckled or even mixed patterns. The centromere pattern (AC-3) or the cytoplasmic reticular/AMA (AC-21) patterns, on the other hand, are examples that prob-ably have been less prone to change in pattern definition over time. The universal use of the ICAP nomenclature and pattern definitions, both in daily clinical practice as well as in the scien-tific literature, may enable systematic reviews in the future, and may well fine-tune current consensus based on expert opinions only.
In conclusion, the consensus statements on clinical relevance should be readily available to clinicians and this will enable further harmonisation of test-result interpretation with respect to HEp-2 IIFA patterns. Obviously, clinicians should be aware of the clinical suspicion for the respective patient, and therefore should order specific tests accordingly, also taking into account the anticipation of prevalence of HEp-2 IIFA negative (AC-0)13 results in SARD. The information on clinical relevance of HEp-2 IIFA patterns is intended to support the decision strategy of the clinician. Information presented in the online supplementary tables 1–3 is primarily intended to be used for complex cases in the consultation of the laboratory specialist by the clinician. Depending on various jurisdictional regulations, follow-up testing can be automated in predefined algorithms which even-tually will shorten the diagnostic delay. Eventually, appropriate integration of HEp-2 IIFA pattern information may help to better define disease criteria and even enable a paradigm shift in the pretest probability paradox.
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Table 3 Mitotic HEp-2 IIFA patterns
Code AC pattern—clinical relevance Refs
AC-24 CENTROSOME (see online supplementary table 3 for further details)
► The AC-24 pattern has low positive predictive value for any disease
► Within the spectrum of the SARD, the AC-24 pattern is found in patients with Raynaud’s phenomenon, localized scleroderma, SSc, SLE and RA, either alone or in combination with other SSc-associated antibodies;
102–105
► Antigens recognized include α-enolase, γ-enolase, ninein, Cep-250, Mob1, PCM-1/2, pericentrin; specific immunoassays for these autoantibodies are currently not commercially available
104, 106–108
AC-25 SPINDLE FIBERS (see online supplementary table 3 for further details)
► The AC-25 pattern has low positive predictive value for any disease
109
► Found very infrequently in a routine serology diagnostic setting
109
► Antigen recognized includes HsEg5; specific immunoassays for this autoantibody, or other spindle fiber targets, are currently not commercially available
110, 111
AC-26 NuMA-like
► Approximately one-half of the patients with the AC-26 pattern have clinical features of a SARD (SjS, SLE, UCTD, limited SSc, or RA); the AC-26 pattern is also observed in patients with organ-specific autoimmune diseases and less frequently in non-autoimmune conditions, especially when in low titer
109, 111–114
► Found very infrequently in a routine serology diagnostic setting
109
► Antigens recognized include NuMA, centrophilin, SP-H antigen and NMP-22; specific immunoassays for these autoantibodies are currently not commercially available
115
AC-27 INTERCELLULAR BRIDGE (see online supplementary table 3 for further details)
► The AC-27 pattern has low positive predictive value for any disease
116
► Found very infrequently in a routine serology diagnostic setting
117
► Antigens recognized include, among other, CENP-E, CENP-F, TD60, MSA36, KIF-14, MKLP-1, MPP1/KIF20B, and INCENP; specific immunoassays for these autoantibodies are currently not commercially available
116, 118, 119
AC-28 MITOTIC CHROMOSOMAL (see online supplementary table 3 for further details)
► The AC-28 pattern has low positive predictive value for any disease
► Found very infrequently in a routine serology diagnostic setting
120
► Antigens recognized include DCA, MCA1, and MCA5; specific immunoassays for these autoantibodies are currently not commercially available
120–122
CENP, centromere-associated protein; Cep, centrosomal protein; DCA, dividing cell antigen; IIFA, indirect immunofluorescence assay; INCENP, inner centromere protein; KIF, kinesin family; MCA, mitotic chromosomal antigen; MKLP, mitotic kinesin-like protein; MPP, M-phase phosphoprotein; MSA, mitotic spindle apparatus; NMP, nuclear matrix protein; NuMA, nuclear mitotic apparatus; PCM, pericentriolar material; RA, rheumatoid arthritis; SARD, systemic autoimmune rheumatic diseases; SLE, systemic lupus erythematosus; SSc, systemic sclerosis; SjS, Sjögren’s syndrome; UCTD, undifferentiated connective tissue disease.
Author affiliations1Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands2Rheumatology Division, Universidade Federal de Sao Paulo, Sao Paulo, Brazil3Department of Immunology, Instituto Universitario del Hospital Italiano de Buenos Aires, Buenos Aires, Argentina4Laboratory of Immunology, Hospital General de Agudos Carlos G Durand, Buenos Aires, Argentina5Immunology, Medical Faculty TU Dresden, Dresden, Germany6Pontificio Universidade Catolica de Goias, Goias, Brazil
7Medicine, Health Sciences Centre, Calgary, Alberta, Canada8Department of Immunology and Rheumatology, Hospital General de Occidente, Guadalajara, Mexico9Rheumatology Unit, Clinical Department of General Internal Medicine, Innsbruck Medical University, Innsbruck, Austria10Department of Internal Medicine II, Medical University of Innsbruck, Innsbruck, Austria11Pontificia Universidade Catolica de Goias, Goiania, Brazil12Department of Rheumatology and Clinical Immunology, Kyoto University Graduate school of Medicine, Kyoto, Japan13Brazilian Society of Autoimmunity, Porto Alegre, Brazil14Department of Clinical Nursing, University of Occupational and Environmental Health, Kitakyushu, Japan15Department of Oral Biology, University of Florida, Gainesville, Florida, USA
Acknowledgements We thank all the workshop participants for their constructive comments and fruitful discussions.
Contributors All authors actively participated in the respective workshops in Kyoto and Dresden. They also participated in the discussions of the executive ICAP committee. The draft of the manuscript was made by JD and was commented on by all authors. Final discussions have taken place at the international autoimmunity meeting in Lisbon. Required amendments were made by JD and approved by all authors.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests The ICAP committee is funded by unrestricted educational grants by several in vitro diagnostics companies (for details see www. anapatterns. org/ sponsors. php). JD has received lecture fees from Euroimmun and Thermo Fisher. MJF is a consultant to Inova Diagnostics and Werfen International; none of the other authors declare any competing interest.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
data sharing statement No additional data are available.
open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.
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Rheumatoid arthritis
CliniCal sCienCe
Clinical outcomes in patients switched from adalimumab to baricitinib due to non-response and/or study design: phase III data in patients with rheumatoid arthritisYoshiya Tanaka,1 Bruno Fautrel,2 edward C Keystone,3,4 Robert a Ortmann,5 li Xie,5 Baojin Zhu,5 Maher issa,5 Himanshu Patel,5 Carol l Gaich,5 stephanie de Bono,5 Terence P Rooney,5 Peter C Taylor 6
To cite: Tanaka Y, Fautrel B, Keystone eC, et al. Ann Rheum Dis 2019;78:890–898.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214529).
For numbered affiliations see end of article.
Correspondence toDr Yoshiya Tanaka, First Department of internal Medicine, University of Occupational and environmental Health, Japan, Kitakyushu 807-8555, Japan; tanaka@ med. uoeh- u. ac. jp
Received 4 October 2018Revised 8 March 2019accepted 27 March 2019Published Online First 30 april 2019
© author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-nC. no commercial re-use. see rights and permissions. Published by BMJ.
Key messages
What is already known about this subject? ► In patients who do not respond adequately to first-line therapies or who discontinue initial treatment due to intolerance, alternative treatment strategies are used, including adding or switching to other disease-modifying antirheumatic drugs.
► Because switching therapies is common in clinical practice, the safety and efficacy of new therapies should be assessed when used as replacement treatment following initial regimens.
What does this study add? ► In the phase III RA-BEAM study, patients taking adalimumab or baricitinib could be rescued to baricitinib at week 16 or later due to inadequate response; at the end of RA-BEAM, and on entry to a long-term extension (LTE) study, patients were switched/continued to baricitinib with no adalimumab washout period.
► In both baricitinib-rescued and adalimumab-rescued patients, there were significant improvements in all efficacy and patient-reported outcome measures up to 12 weeks after rescue compared with the time of rescue. Patients who switched from adalimumab to baricitinib showed improvements in disease control through 12 weeks in the LTE.
► Incidence rates for adverse events and infections, including serious events, were similar for patients who switched from adalimumab to baricitinib and for those who continued baricitinib.
ABSTRACTObjective To evaluate clinical outcomes in patients who changed treatment from adalimumab to baricitinib, an oral Janus kinase (JaK)1/JaK2 inhibitor, during a phase iii programme.Methods in phase iii Ra-BeaM, patients were randomised 3:3:2 to placebo, baricitinib 4 mg once daily, or adalimumab 40 mg biweekly. at week 16 or subsequent visits, non-responders were rescued to open-label baricitinib 4 mg. at week 52, patients could enter a long-term extension (lTe) and continue on baricitinib or switch from adalimumab to baricitinib 4 mg with no adalimumab washout period. Percentage of patients achieving low disease activity and remission were assessed, along with physical function, patient’s assessment of pain, and safety.Results Thirty-five (7%) baricitinib-treated and 40 (12%) adalimumab-treated patients were rescued to baricitinib in Ra-BeaM; 78% (381/487) of baricitinib-treated and 72% (238/330) of adalimumab-treated patients who were not rescued in Ra-BeaM, entered the lTe and continued/were switched to baricitinib. in both baricitinib-rescued and adalimumab-rescued patients, there were significant improvements in all measures up to 12 weeks after rescue compared with the time of rescue. Patients who switched from adalimumab to baricitinib showed improvements in disease control through 12 weeks in the lTe. exposure-adjusted incidence rates for treatment-emergent adverse events (Teaes) and infections, including serious events, were similar for patients who switched from adalimumab to baricitinib and those who continued on baricitinib.Conclusions switching from adalimumab to baricitinib (without adalimumab washout) was associated with improvements in disease control, physical function and pain during the initial 12 weeks postswitch, without an increase in Teaes, serious adverse events or infections.Trial registration numbers nCT01710358, nCT01885078.
InTROduCTIOnThe combined use of conventional synthetic disease-modifying antirheumatic drugs, targeted synthetic disease-modifying antirheumatic drugs (tsDMARDs) or biological disease-modifying antirheumatic drugs (bDMARDs), as well as the
application of the treat-to-target strategy, has revo-lutionised the treatment of rheumatoid arthritis (RA), and clinical remission or low disease activity (LDA) is now a realistic target.1–3 Not all patients respond adequately to first-line therapies or may discontinue initial treatment due to intolerance. In these cases, for optimal management of disease, alternative treatment strategies are used, including adding or switching to other disease-modifying
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Rheumatoid arthritis
Key messages
How might this impact on clinical practice or future developments?
► Transition from adalimumab to baricitinib does not require lengthy washout from the prior treatment in patients with rheumatoid arthritis and is associated with improvements in clinical disease control, with acceptable safety.
antirheumatic drugs (DMARDs). Because switching therapies is common in clinical practice, the safety and efficacy of new ther-apies should be assessed when used as replacement treatment following initial regimens.
Baricitinib is a recently developed tsDMARD and an oral selec-tive inhibitor of Janus kinase (JAK)1 and JAK2, which belong to a family of protein tyrosine kinases that mediate signal transduc-tion for a variety of cytokines involved in inflammatory condi-tions, including RA.4 5 Baricitinib is approved for the treatment of moderately to severely active RA in adults in over 50 coun-tries, including European countries, the USA and Japan. The 52-week RA-BEAM study (NCT01710358) of methotrexate-in-adequate responder (MTX-IR) patients with active RA showed that baricitinib 4 mg demonstrated superior efficacy compared with placebo and adalimumab, a tumour necrosis factor (TNF) inhibitor, based on 20% response according to the criteria of the American College of Rheumatology at 12 weeks, as well as secondary measures, including mean change in disease activity score for 28 joints with the use of high-sensitivity C reactive protein and percent of patients achieving LDA based on Simpli-fied Disease Activity Score (SDAI) and Clinical Disease Activity Score (CDAI).6
During RA-BEAM, non-responders were rescued to baric-itinib, and at the completion of RA-BEAM, patients had the option to enrol in a long-term extension (LTE) study, RA-BE-YOND (NCT01885078), in which all patients were switched to baricitinib. The objectives of this analysis were to evaluate the efficacy, physical function, pain and safety in two groups of patients from RA-BEAM: patients taking adalimumab or baric-itinib who were rescued to baricitinib during RA-BEAM and those who were switched to baricitinib on entering the LTE study RA-BEYOND.
MeTHOdSThe eligibility criteria for the originating study, RA-BEAM, have been previously published.6 In brief, RA-BEAM included patients aged ≥18 years with a confirmed diagnosis of active RA, defined as ≥6/68 tender and ≥6/66 swollen joints, serum high-sensitivity C reactive protein (hsCRP) ≥6 mg/L and ≥3 joint erosions (patients with one to two erosions could enrol if they were rheumatoid factor or anti-citrullinated protein anti-bodies positive). Patients with prior bDMARD therapy were excluded from RA-BEAM. All patients completing the 52 weeks of RA-BEAM, regardless of rescue status, were eligible to enrol in RA-BEYOND. Safety-related exclusion criteria for RA-BE-YOND included having significant medical issues that devel-oped during RA-BEAM that, in the opinion of the investigator, posed an unacceptable risk to the patient if baricitinib/study drug continued to be administered.
Study design and treatmentsRA-BEAM was a 52 week, double-blind, placebo-controlled and active-controlled phase III study in adult MTX-IR patients
with active RA (online supplementary figure S1). Patients were randomised 3:3:2 to receive oral placebo once daily, oral baric-itinib 4 mg once daily or subcutaneous adalimumab 40 mg biweekly. Patients continued receiving background MTX (stable dose of 7.5–25 mg/week). At week 16 in RA-BEAM, non-re-sponders (lack of improvement of ≥20% in tender joint and swollen joint count at both weeks 14 and 16 compared with baseline) received rescue treatment (baricitinib 4 mg). After week 16, patients received rescue treatment at the investiga-tor’s discretion on the basis of tender and swollen joint counts. Rescued patients received open-label baricitinib once daily, but no biweekly subcutaneous injection, for the remainder of the study. From the time of rescue (but not before), background corticosteroids (maximum dose of prednisone 10 mg daily or equivalent), non-steroidal anti-inflammatories and analgesics could be added or increased in dose. Rescuing all groups to baricitinib (including those already receiving baricitinib with inadequate response) therefore increased baricitinib exposures for safety evaluation by regulators, while still offering patients some escalation of (background) treatment. At week 24, patients receiving placebo were switched to baricitinib, unaware of the change in treatment. Rescued/switched patients and their inves-tigators remained blinded to original treatment assignment.
RA-BEYOND is a phase III LTE study of the efficacy and safety of baricitinib in patients with RA (study duration up to 7 years). On entering RA-BEYOND, all patients from RA-BEAM continued on baricitinib 4 mg once daily or switched from adalimumab therapy to baricitinib 4 mg QD. All patients entering RA-BEYOND remained blinded to original treatment. No washout period of adalimumab occurred in patients who were rescued during RA-BEAM or switched at the entry to RA-BEYOND.
The current analysis includes two separate patient popu-lations: (1) rescued patients in RA-BEAM: patients who were rescued from blinded baricitinib or adalimumab to open-label baricitinib between weeks 16 and 24 due to non-response (data after rescue from placebo are presented in the online supple-mentary material); and (2) switched patients in RA-BEYOND: patients who were not rescued in RA-BEAM, who were origi-nally randomised to baricitinib and continued baricitinib or who switched from adalimumab to baricitinib at the entry of RA-BE-YOND, and entered RA-BEYOND at least 24 weeks before the data cut-off date (1 September 2016). In RA-BEAM, efficacy data collection (visits) for rescued patients occurred at weeks 16 (first rescue opportunity), 20, 24, 28, 32, 40 and 52. Efficacy data are not presented in patients who were rescued after week 24 because data acquisition (visits) occurred less frequently than in patients rescued earlier.
RA-BEAM and RA-BEYOND were conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice Guidelines and approved by each centre’s institutional review board or ethics committee. All patients provided written informed consent. The studies were designed by the sponsors, Eli Lilly and Company and Incyte Corporation, with input from an academic advisory board in which non-Lilly authors of this article participated. All authors participated in the preparation and review of this manuscript and approved the final version.
Patient involvementThis research was done without patient involvement. Patients were not invited to comment on the study design and were not consulted to develop patient relevant outcomes or interpret the
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Table 1 Characteristics and disease activity at baseline of RA-BEAM for the overall baricitinib-treated and adalimumab-treated patients and for patients rescued to baricitinib between weeks 16 and 24
Overall patients in RA-BeAM Patients rescued to baricitinib
Baricitinib4 mg (n=487)
Adalimumab(n=330)
Baricitinib4 mg (n=35)
Adalimumab(n=40)
Age, years 53.5 (12) 52.9 (12) 52.3 (12) 51.9 (14)
Female, n (%) 375 (77) 251 (76) 30 (86) 31 (78)
Duration of RA,* years 8.7 (8.6) 8.3 (7.9) 8.0 (7.8) 7.0 (6.2)
ACPA positive, n (%) 427 (88) 295 (89) 28 (80) 37 (93)
RF positive, n (%) 439 (90) 301 (91) 30 (86) 38 (95)
Swollen joint count, of 66 15 (8) 15 (9) 19 (10) 19 (12)
Tender joint count, of 68 23 (13) 23 (14) 25 (13) 23 (11)
hsCRP, mg/L 22 (23) 22 (21) 20 (20) 31 (29)
ESR, mm/hour 49 (26) 48 (26) 46 (25) 56 (30)
DAS28-hsCRP 5.8 (0.9) 5.8 (0.9) 6.0 (0.9) 6.1 (0.9)
DAS28-ESR 6.5 (0.9) 6.4 (1.0) 6.7 (1.0) 6.8 (1.0)
CDAI 38 (12.0) 38 (13) 43 (13.6) 42 (13.0)
SDAI 40 (12.7) 40 (13.4) 45 (14.2) 45 (14.0)
MTX average weekly dose, mg 14.9 (4.6) 14.6 (4.4) 15.5 (5.3) 14.4 (4.2)
HAQ-DI 1.57 (0.68) 1.59 (0.70) 1.69 (0.64) 1.75 (0.64)
Patient’s assessment of pain 61.8 (21.8) 61.0 (22.7) 67.6 (23.2) 67.1 (20.9)
Data are mean (SD) unless otherwise stated.*Time from RA diagnosis.ACPA, anti-citrullinated protein antibodies; CDAI, Clinical Disease Activity Index; DAS28-ESR, disease activity score using 28-joint count with erythrocyte sedimentation rate; DAS28-hsCRP, disease activity score for 28 joints with the use of high-sensitivity C reactive protein; ESR, erythrocyte sedimentation rate; HAQ-DI, Health Assessment Questionnaire Disability Index; MTX, methotrexate-inadequate responder; RA, rheumatoid arthritis; RF, rheumatoid factor; SDAI, Simplified Disease Activity Index; hsCRP, high-sensitivity C reactive protein.
results. Patients were not invited to contribute to the writing or editing of this document for readability or accuracy.
efficacy and patient-reported outcomesEfficacy assessments included the proportion of patients achieving LDA and remission based on CDAI (LDA ≤10, remis-sion ≤2.8), SDAI (LDA ≤11, remission ≤3.3) and disease activity score using 28-joint counts with erythrocyte sedimen-tation rate (DAS28-ESR; LDA ≤3.2, remission <2.6), as well as change from baseline of these measures. Patient-reported outcomes (PROs) included assessment of physical function using the Health Assessment Questionnaire Disability Index (HAQ-DI) and patient’s assessment of pain (0–100 mm, visual analogue scale). Continuous data for the individual acute phase markers (erythrocyte sedimentation rate and hsCRP) are also presented. In patients who were rescued during RA-BEAM, efficacy and PROs are reported up to 12 weeks after rescue. In patients who switched to baricitinib on entry to RA-BEYOND, outcomes were reported at the baseline of the LTE and up to 24 weeks after entry into the LTE.
SafetyTreatment-emergent adverse events (TEAEs), including infec-tions, specifically herpes zoster and gastrointestinal disorders; discontinuations; and serious adverse events (SAEs), including serious infections, were assessed. Safety was assessed through 24 weeks after the transition to baricitinib 4 mg.
Statistical analysesStatistical analyses were conducted for patients’ clinical outcomes and PROs before and after treatment change due to rescue or switch. In rescued patients, comparisons were between
the time of rescue and 4, 8 and 12 weeks after rescue. In patients who switched treatment on entry to the LTE, comparisons were between the time of switch and 4, 12 and 24 weeks after switch. For continuous measures, a mixed model for repeated measures approach was used with treatment, visit and treatment-by-visit interaction included. For categorical measures, the proportion of patients who achieved LDA and remission was summarised; non-responder imputation was applied for missing data and after permanent discontinuation of the study drug. Safety data are expressed as percentages and exposure-adjusted incidence rates (EAIRs) up to 24 weeks for the rescued and switched patients separately. Due to the non-randomised nature of this analysis, no between-group statistical comparisons were made.
ReSulTSPatientsA total of 1307 patients were randomised and 1305 (488 placebo, 487 baricitinib 4 mg and 330 adalimumab) patients received treatment. Between weeks 16 and 24, 35 (7%) patients treated with baricitinib and 40 (12%) treated with adalim-umab were rescued to open-label baricitinib in RA-BEAM. The majority of rescued patients completed RA-BEAM through week 52: 25 patients (74%) treated with baricitinib-to-barici-tinib and 35 patients (90%) treated with adalimumab-to-baric-itinib. A total of 78% (381/487) of baricitinib-treated patients and 72% (238/330) of adalimumab-treated patients were not rescued in RA-BEAM and entered the LTE ≥24 weeks before the data cut-off date. The disposition of patients is shown in online supplementary figure S2. Baseline demographics and clin-ical characteristics of all patients and patients rescued during RA-BEAM are listed in table 1. Overall, patients had similar demographics across the treatment groups; 80% of rescued
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Table 2 Change in efficacy and patient-reported outcomes 4, 8 and 12 weeks after rescue in rescued patients from RA-BEAM
Baricitinib to baricitinib (n=35) Adalimumab to baricitinib (n=40)
Score at time of rescue
Change from rescue visitScore at time of rescue
Change from rescue Visit
Week 4 (n=34) Week 8 (n=33) Week 12 (n=30) Week 4 (n=40) Week 8 (n=39) Week 12 (n=33)
DAS28-ESR 6.1 (1.3) −0.93 (0.18)*** −1.51 (0.20)*** −1.73 (0.22)*** 6.2 (1.3) −1.38 (0.17)*** −1.69 (0.19)*** −1.92 (0.21)***
CDAI 39.3 (15.2) −14.1 (2.1)*** −20.5 (2.2)*** −22.3 (2.3)*** 36.4 (14.5) −14.8 (2.0)*** −17.8 (2.0)*** −20.2 (2.2)***
SDAI 40.8 (16.0) −14.1 (2.3)*** −21.0 (2.3)*** −23.1 (2.5)*** 38.9 (14.5) −16.2 (2.1)*** −19.2 (2.2)*** −21.8 (2.3)***
Patient’s assessment of pain
53.8 (21.0) −13.1 (3.8)*** −13.8 (3.9)*** −18.3 (4.0)*** 56.4 (25.4) −14.5 (3.5) *** −21.5 (3.6)*** −24.1 (3.7)***
HAQ-DI 1.42 (0.72) −0.19 (0.08)* −0.28 (0.08)*** −0.34 (0.09)*** 1.46 (0.66) −0.37 (0.07) *** −0.47 (0.08)*** −0.49 (0.09)***
CRP, mg/L 14.9 (23.5) −0.45 (4.35) −4.79 (4.49) −8.50 (5.04) 25.3 (29.8) −13.50 (4.00)*** −14.10 (4.13)*** −16.00 (4.69)***
ESR, mm/h 36.5 (27.7) 0.71 (3.40) −4.49 (4.16) −5.05 (4.76) 51.9 (35.4) −16.60 (3.16)*** −21.80 (3.85)*** −19.06 (4.48)***
Score at the time of rescue data are mean (SD); change from baseline data is LSM (SE).*p≤0.05; **p≤0.01; ***p≤0.001 from within-group mean change from the last visit prior to rescue using MMRM.CDAI, Clinical Disease Activity Index;CRP, C reactive protein; DAS28-ESR, disease activity score using 28-joint count with erythrocyte sedimentation rate;ESR, erythrocyte sedimentation rate; HAQ-DI, Health Assessment Questionnaire Disability Index; LSM, least squares mean; MMRM, mixed-effects model repeated measures; SDAI, Simplified Disease Activity Index.
patients were female, the mean age was 51.8 years, and the mean RA duration was 7.9 years (table 1).
efficacy and PROs of rescued patientsIn patients rescued in RA-BEAM, both patients treated with baricitinib and patients treated with adalimumab showed signif-icant improvements in all measures at weeks 4, 8 and 12 after rescue compared with values at the time of rescue (table 2). Online supplementary table 1 shows results of placebo patients who were rescued. At the time of rescue, no patients were in LDA or remission based on CDAI or SDAI; 1 (3%) baricitinib patient was in LDA and 1 (3%) adalimumab patient was in LDA and remission based on DAS28-ESR (figure 1). By week 12 after rescue, 21%, 33% and 33% of baricitinib and 17%, 37% and 43% of adalimumab patients had reached LDA based on DAS28-ESR, CDAI, and SDAI, respectively (figure 1). Acute phase markers decreased significantly in patients rescued from adalimumab to baricitinib, but not in those remaining on baric-itinib (table 2). Rescued patients showed significant improve-ments in pain and physical function 4 weeks after rescue, which were sustained through 12 weeks (table 2). After week 24, an additional eight baricitinib patients and 11 adalimumab patients were rescued. These patients were not included in this study, but a sensitivity analysis using all patients rescued between weeks 16 and 52 showed consistent findings to the patients rescued from weeks 16 to 24 (data not shown).
efficacy and PROs of switched patients in the lTePatients who switched from adalimumab to baricitinib main-tained disease response in the LTE, with improvement in the first 12 weeks post-switch. Patients who continued treatment with baricitinib maintained disease response (figure 2). In patients initially treated with adalimumab in RA-BEAM, mean CDAI, SDAI and DAS28-ESR showed statistically significant improve-ment through 24 weeks of the LTE after switching to baricitinib 4 mg (figure 2). Overall, the percentage of switched patients who achieved LDA based on CDAI and SDAI increased from the time of switch (entry to RA-BEYOND) to week 24 for both patients treated with baricitinib-to-baricitinib and patients treated with adalimumab-to-baricitinib (figure 3).
During the LTE improvements in physical function and pain were maintained in patients continuing baricitinib and patients who switched from adalimumab to baricitinib; by week 24 of
the LTE, both groups had similar outcomes related to pain and physical function (figure 2).
SafetyIn rescued patients, EAIRs for infections, SAEs and AEs leading to discontinuation were numerically higher in patients who continued baricitinib. The most common TEAEs were infections and gastrointestinal disorders (table 3). The most common infec-tions were nasopharyngitis (n=2 for baricitinib-to-baricitinib and n=2 for adalimumab-to-baricitinib) and urinary tract infec-tion (n=2 for baricitinib-to-baricitinib). In switched patients in the LTE, EAIRs for TEAEs, infections and serious infections were similar in patients who switched from adalimumab to baricitinib and patients who continued baricitinib (table 3).
dISCuSSIOnOn treatment failure with adalimumab, patients rescued with baricitinib 4 mg showed sustained and clinically relevant improvements in efficacy and PROs, representing measures that are important to patients. After switching from adalimumab to baricitinib on entering the LTE without adalimumab washout, disease control and maintenance in PROs were sustained through 24 weeks postswitch. There was no increase in overall TEAEs, SAEs or infections after transitioning to baricitinib, when patients could have still been exposed to therapeutic levels of adalimumab during the first 12 weeks after the treatment change. Safety findings were consistent with the known safety profile of baricitinib.
Within the RA patient population, the magnitude of efficacy responses to therapeutic intervention is heterogeneous. The contemporary treatment goal is to achieve LDA or remission.3 In the absence of a satisfactory clinical response, in patients initiated on MTX, options exist to titrate the dose upwards or change to parenterally administered drugs. In contrast, for patients on targeted bDMARD and tsDMARD therapies, there are limited options for dose titration; therefore, current practice is most often to switch between different therapies, either within class, in the case of biologic TNF inhibitors, or from one mech-anism of action to another. The data emerging from the present analyses are therefore of contemporary relevance with respect to two sets of patients: first, patients requiring rescue to a new drug due to lack of clinical response, and second, patients who
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Figure 1 Low disease activity and remission at the time of rescue and 4, 8 and 12 weeks after rescue in rescued patients from RA-BEAM. NRI was used for missing data imputation. CDAI, clinical disease activity index; DAS28-ESR, disease activity score using 28-joint count with erythrocyte sedimentation rate; NRI, non-responder imputation; SDAI, simplified disease activity index.
have a clinical response but choose to switch therapies for other reasons.
The proportion of patients with RA with inadequate response or intolerance to TNF inhibitors is increasing with wider use of these agents.7 The phase III RA-BEACON study showed that baricitinib is an effective and well-tolerated once daily oral treat-ment option for such patients.8 Phase III trials of novel targeted DMARDs in TNF-IR RA patients typically require discontin-uation of TNF inhibitors prior to enrolment. Commonly, the
required period of discontinuation for the previous TNF inhib-itor is based on a multiple of half-lives for the specific agent. This results in lengthy washout periods prior to commencing the novel investigational treatment. In an effort to more closely reflect routine clinical practice, RA-BEACON (a phase III randomised controlled trial of baricitinib in patients with inad-equate response to TNF inhibitors) adopted a more pragmatic minimum of 4 weeks’ discontinuation for prior TNF inhibitors before patients could enter the study screening period. The
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Figure 2 Mean observed values for CDAI (A), SDAI (B), DAS28-ESR (C), physical function (HAQ-DI) (D) and patient’s assessment of pain (E) through 24 weeks in the switched patients from RA-BEYOND. *p≤0.05; **p≤0.01; ***p≤0.001 for within-group change based on mixed model for repeated measures. Week 0=time of switch (or entry to RA-BEYOND). CDAI, clinical disease activity index; DAS28-ESR, disease activity score using 28-joint count with erythrocyte sedimentation rate; HAQ-DI, Health Assessment Questionnaire Disability index; SDAI, simplified disease activity index.
present dataset is of relevance as it may better approximate real-world practice in that patients with inadequate response to adalimumab in RA-BEAM who were rescued to baricitinib at a scheduled visit did so only 2 weeks following the last dose of adalimumab. Unsurprisingly, given the prior findings of RA-BEACON, these patients showed clinical improvement with baricitinib (although as noted below, present observations are limited by the open-label nature of rescue). Perhaps the more practically important novel finding from the present study is that adverse event data from the period immediately following the treatment change suggest that a prompt transition from adalim-umab to baricitinib can be executed with acceptable safety and tolerability.
Switching between therapeutics may occur even in patients who exhibit clinical responses and tolerate a given initial treat-ment. Patients randomised to adalimumab who completed the 52 week RA-BEAM without requiring rescue were switched to baricitinib in RA-BEYOND, with maintained efficacy and acceptable safety. These findings are of practical clinical rele-vance where patients receiving a biologic TNF inhibitor who have not necessarily exhibited intolerance or poor response to that agent are contemplating transition to baricitinib. This scenario could arise, for instance, due to patient preference for oral over injectable treatment options.6 As patients communicate with their healthcare providers regarding preferred treatments, they can be assured that important quality-of-life outcomes will
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Figure 3 Low disease activity and remission at weeks 0, 12 and 24 after switch in switched patients from RA-BEYOND. Week 0=time of switch (or entry to RA-BEYOND). NRI was used for missing data imputation. CDAI, clinical disease activity index; DAS28-ESR, disease activity score using 28-joint count with erythrocyte sedimentation rate; NRI, non-responder imputation; SDAI, simplified disease activity index.
be maintained after switching to baricitinib as physical func-tion and pain improvements that occurred in RA-BEAM with adalimumab were maintained into the LTE after switching to baricitinib.
The switch from adalimumab to baricitinib occurred without a washout period, and adalimumab has a mean circulating half-life of approximately 14 days.9 Patients would therefore have received several weeks of dual TNF and JAK1/JAK2 inhibition
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Table 3 Safety in rescued patients from RA-BEAM and patients switched in RA-BEYOND
Rescued patients in RA-BeAM(24 weeks after rescue)
Switched patients in RA-BeYOnd(weeks 0–24 after switch)
Baricitinib to baricitinib(n=35, PYe=15.8)
Adalimumab to baricitinib(n=40, PYe=19.6)
Baricitinib to baricitinib(n=381, PYe=189.4)
Adalimumab to baricitinib(n=238, PYe=118.2)
Patients with ≥1 TEAE 21 (60.0) (132.8) 26 (65.0) (132.4) 206 (54.1) (108.8) 124 (52.1) (104.9)
Infections 11 (31.4) (69.6) 7 (17.5) (35.6) 87 (22.8) (45.9) 49 (20.6) (41.5)
Herpes zoster 1 (2.9) (6.3) 1 (2.5) (5.1) 5 (1.3) (2.6) 5 (2.1) (4.2)
Gastrointestinal disorders 6 (17.1) (37.9)* 8 (20.0) (40.7)† 36 (9.4) (19.0) 18 (7.6) (15.2)
AEs that led to permanent study drug discontinuation
4 (11.4) (25.3) 1 (2.5) (5.1) 8 (2.1) (4.2) 3 (1.3) (2.5)
Patients with ≥1 SAE 6 (17.1) (37.9) 2 (5.0) (10.2) 26 (6.8) (13.7) 11 (4.6) (9.3)
Serious infections 0 0 8 (2.1) (4.2) 4 (1.7) (3.4)
Data are n (%) (EAIR).*Gastrointestinal disorders included nausea, dental caries, constipation, gastritis, vomiting, diarrhoea, inguinal hernia, mouth ulceration and upper gastrointestinal haemorrhage.†Gastrointestinal disorders included dyspepsia, abdominal pain, constipation, stomatitis, upper abdominal pain, diarrhoea, enterocolitis and hyperchlorhydria.AE, adverse event; EAIR, exposure-adjusted incidence rate; PYE, patient-years of exposure; SAE, serious adverse event; TEAE, treatment-emergent adverse event.
after treatment change. Hence, although the observed improve-ments in CDAI and SDAI were sustained over 24 weeks (figure 2) and may simply reflect the improved efficacy seen with baricitinib over adalimumab in the randomised period of RA-BEAM, some of the initial benefit evident after switch might in fact be accounted for by the combination. From a biological perspective, there are reasons to consider that a combination of TNF and JAK inhibition could have merit. TNF inhibition using bDMARDs provides near-complete, sustained inhibition of the TNF pathway and its downstream effects. Baricitinib, on the other hand, provides partial and transient inhibition of the broader variety of JAK1/JAK2-dependent pathways. Accordingly, whole blood microarray evaluation in RA-BEAM has shown that baricitinib and adalimumab exert pharmacodynamic effects that impact substantially non-overlapping sets of mediators.10 The observation of added efficacy, without apparent acute safety signal during the weeks when patients were exposed to both adalimumab and baricitinib, is of interest given the findings from earlier, dedicated combination targeted DMARD studies, which showed that compared with single targeted therapy, selected combinations (TNF inhibition with either interleukin-1 or T-cell costimulation blockade) increased safety risks without adding benefit.11 12
The current analysis has a number of relevant limitations. First, although initial randomised treatment assignment remained blinded at all times, baricitinib was administered in an open-label manner after rescue or switch. This likely influenced subjective responses thereafter, in particular for RA-BEAM rescue, where patients knew they were moving to baricitinib from a treatment that was ineffective for them, and which could have been placebo. Given these dynamics, the psychological effects of ‘rescue’ may have masked detectable differences between groups based on prior treatment. This could account for the counterintuitive finding that, in contrast to acute phase markers, which signifi-cantly improved only in those patients rescued from adalimumab to baricitinib, improvements in composite disease activity scores (which include subjective components) after rescue to open-label baricitinib did not appear different for patients rescued from blinded baricitinib (ie, no treatment change, apart from background therapy) compared with those rescued from blinded adalimumab (ie, a change of treatment and a setting (TNF-IR) where baricitinib has been proven to be efficacious).8 This bias was likely less of a factor for the switch to baricitinib on entering the LTE, as at the time of switch, all patients were knowingly
receiving active treatment to which they had exhibited a reason-able response; the psychological placebo effect of rescue from an ineffective treatment was not in play. In addition, rescue criteria in RA-BEAM differed during the period of interest; at week 16, patients were automatically rescued without at least a minimal improvement in swollen/tender joint counts, whereas thereafter, investigators decided whether to rescue. Finally, the transitions to baricitinib (rescue and switch) were not randomised.
In conclusion, these data support and extend the findings of prior baricitinib studies,6 8 that in patients with RA, transi-tion from adalimumab to baricitinib does not require lengthy washout from the prior treatment and is associated with main-tained clinical disease control, with acceptable safety.
Author affiliations1The First Department of internal Medicine, school of Medicine, University of Occupational and environmental Health, Japan, Kitakyushu, Japan2Dept of Rheumatology, sorbonne Universite,Pierre louis institute of epidemiology and Public Health, Pépites team; aPHP, GH Pitie salpetriere, Paris, France3Rebecca MacDonald Centre for arthritis and autoimmune Diseases, Mount sinai Hospital, Toronto, Ontario, Canada4University of Toronto, Toronto, Ontario, Canada5eli lilly and Company, indianapolis, indiana, Usa6Botnar Research Centre, nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal sciences, University of Oxford, Oxford, United Kingdom
Acknowledgements The authors thank Kathy Oneacre, Ma, of syneos Health for medical writing support and assistance with preparation and submission of this article. PCT would like to acknowledge support by the national institute for Health Research Oxford Biomedical Research Centre and by aR UK. This article is based on work presented at the 2016 aCR/aRHP annual Meeting: Taylor PC, Keystone e, Ortmann R, issa M, Xie l, Muram D, Bradley JD, de Bono s, Rooney T, Tanaka Y. efficacy and safety of switching from adalimumab to Baricitinib: Phase 3 Data in Patients with Rheumatoid arthritis (abstract). arthritis Rheumatol. 2016; 68 (suppl 10) and at the 2018 aCR/aRHP annual Meeting: Weinblatt Me, Taylor PC, Keystone eC, Ortmann Ra, issa M, Xie l, de Bono s, Tanaka Y. efficacy and safety of switching from adalimumab to Baricitinib: long-Term Data from Phase 3 extension study in Patients with Rheumatoid arthritis (abstract).10arthritis Rheumatol. 2018; 70 (suppl 10).
Contributors YT participated in the conception of the work, the interpretation of data for the work and the critical revision of the manuscript for important intellectual content; BF participated in the interpretation of data for the work and the critical revision of the manuscript for important intellectual content; eK, RO, lX and BZ participated in in analysis and interpretation of data for the work and critical revision of the manuscript for important intellectual content; Mi participated in the conception and design of the work, acquisition, analysis and interpretation of data for the work, drafting of the manuscript, and critical revision of the manuscript for important intellectual content. HP participated in analysis and interpretation of data for the work and drafting of the manuscript; CG participated in the acquisition and interpretation of data for the work and in the drafting of the manuscript
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for important intellectual content; sDB participated in the design, analysis and interpretation of data for the work and in the critical revision of the manuscript for important intellectual content; TR participated in the acquisition, analysis and interpretation of data for the work and in the critical revision of the manuscript for important intellectual content; PCT participated in the conception and design of the work, interpretation of data for the work, and drafting and critical revision of the manuscript for important intellectual content.
Funding The studies were sponsored by eli lilly and Company and incyte Corporation.
Competing interests YT has received speaking fees and/or honoraria from Daiichi-sankyo, astellas, eli lilly, Chugai, sanofi, abbvie, Pfizer, Yl Biologics, Bristol-Myers, Glaxo-smithkline, UCB, Mitsubishi-Tanabe, novartis, eisai, Takeda, Janssen and asahi-kasei, and has received research grants from Mitsubishi-Tanabe, Bristol-Myers, eisai, Chugai, Takeda, abbvie, astellas, Daiichi-sankyo, Ono, MsD and Taisho-Toyama; BF reports grants and personal fees from abbVie, eli lilly, Pfizer and MsD, and personal fees from Biogen, BMs, Celgene, Janssen, Medac, nordic, novartis, Roche, sOBi, sanofi-Genzyme and UCB outside the submitted work; eK reports funding for research from abbVie, amgen, Bristol-Myers squibb, F. Hoffmann-la Roche inc, Gilead, Janssen, lilly Pharmaceuticals, Pfizer Pharmaceuticals, sanofi-aventis, consulting agreements with abbVie, amgen, astraZeneca Pharma, Biotest, Bristol-Myers squibb Company, Celltrion, Crescendo Bioscience, F. Hoffmann-la Roche inc, Genentech inc, Gilead, Janssen inc, lilly Pharmaceuticals, Merck, Pfizer Pharmaceuticals, sandoz and UCB, and has received speaker honoraria from amgen, abbVie, Bristol-Myers squibb Canada, F. Hoffmann-la Roche inc., Janssen, Merck, Pfizer Pharmaceuticals, sanofi Genzyme and UCB; RO, lX, BZ, Mi, HP, CG, sDB and TR are employees and stockholders of eli lilly and Company; PCT reports grants from eli lilly and Company, Celgene, UCB and Galapagos, and personal fees from eli lilly and Company, abbVie, Gilead, and Pfizer during the conduct of the study.
Patient consent for publication not required.
Provenance and peer review not commissioned; externally peer reviewed.
Open access This is an open access article distributed in accordance with the Creative Commons attribution non Commercial (CC BY-nC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially,
and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.
RefeRences 1 smolen Js, aletaha D, Mcinnes iB. Rheumatoid arthritis. The Lancet
2016;388:2023–38. 2 smolen Js, Breedveld FC, Burmester GR, et al. Treating rheumatoid arthritis to target:
2014 update of the recommendations of an international Task Force. Ann Rheum Dis 2016;75:3–15.
3 smolen Js, landewé R, Bijlsma J, et al. eUlaR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2016 update. Ann Rheum Dis 2017;76:960–77.
4. Fridman Js, scherle Pa, Collins R, et al. selective inhibition of JaK1 and Jak2 is efficacious in rodent models of arthritis: preclinical characterization of inCB028050. J Immunol 2010;184:5298–307.
5 O’shea JJ, Holland sM, staudt lM. JaKs and sTaTs in immunity, immunodeficiency, and cancer. N Engl J Med 2013;368:161–70.
6 Taylor PC, Keystone eC, van der Heijde D, et al. Baricitinib versus placebo or adalimumab in rheumatoid arthritis. N Engl J Med Overseas Ed 2017;376:652–62.
7 emery P. Optimizing outcomes in patients with rheumatoid arthritis and an inadequate response to anti-TnF treatment. Rheumatology 2012;51(suppl 5):v22–30.
8. Genovese MC, Kremer J, Zamani O, et al. Baricitinib in patients with refractory rheumatoid arthritis. N Engl J Med 2016;374:1243–52.
9 anon. Humira (Adalimumab) [package insert. north Chicago, il: abbVie inc, 2017. 10 emery P, Taylor PC, Weinblatt M, et al. Microarray Pathway analysis Comparing
Baricitinib and adalimumab in Moderate to severe Rheumatoid arthritis Patients, from a Phase 3 study [abstract]. Arthritis Rheumatol 2017;69(suppl 10).
11 Genovese MC, Cohen s, Moreland l, et al. Combination therapy with etanercept and anakinra in the treatment of patients with rheumatoid arthritis who have been treated unsuccessfully with methotrexate. Arthritis Rheum 2004;50:1412–9.
12 Weinblatt M, schiff M, Goldman a, et al. selective costimulation modulation using abatacept in patients with active rheumatoid arthritis while receiving etanercept: a randomised clinical trial. Ann Rheum Dis 2007;66:228–34.
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CliniCal sCienCe
Effects of the anti-RANKL antibody denosumab on joint structural damage in patients with rheumatoid arthritis treated with conventional synthetic disease-modifying antirheumatic drugs (DESIRABLE study): a randomised, double-blind, placebo-controlled phase 3 trialTsutomu Takeuchi, 1 Yoshiya Tanaka,2 satoshi soen,3 Hisashi Yamanaka,4 Toshiyuki Yoneda,5 sakae Tanaka,6 Takaya nitta,7 naoki Okubo,8 Harry K Genant,9 Désirée van der Heijde 10
To cite: Takeuchi T, Tanaka Y, soen s, et al. Ann Rheum Dis 2019;78:899–907.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214827).
For numbered affiliations see end of article.
Correspondence toDr Tsutomu Takeuchi, Division of Rheumatology, Department of internal Medicine, Keio University school of Medicine, Tokyo 160-8582, Japan; tsutake@ z5. keio. jp
TT and YT contributed equally.
Received 27 november 2018Revised 19 March 2019accepted 20 March 2019Published Online First 29 april 2019
© author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-nC. no commercial re-use. see rights and permissions. Published by BMJ.
AbsTRACTObjective To evaluate the efficacy of denosumab in suppressing joint destruction when added to conventional synthetic disease-modifying antirheumatic drug (csDMaRD) therapy in patients with rheumatoid arthritis (Ra).Methods This was a multi-centre, randomised, double-blind, parallel-group, placebo-controlled phase 3 study in Japan. Patients with Ra aged ≥20 years receiving csDMaRDs were randomly assigned (1:1:1) to denosumab 60 mg every 3 months (Q3M), denosumab 60 mg every 6 months (Q6M) or placebo. The change in the modified total sharp score (mTss) and effect on bone mineral density (BMD) at 12 months was evaluated.Results in total, 654 patients received the trial drugs. Denosumab groups showed significantly less progression of joint destruction. The mean changes in the mTss at 12 months were 1.49 (95% Ci 0.99 to 1.99) in the placebo group, 0.99 (95% Ci 0.49 to 1.49) in the Q6M group (p=0.0235) and 0.72 (95% Ci 0.41 to 1.03) in the Q3M group (p=0.0055). The mean changes in bone erosion score were 0.98 (95% Ci 0.65 to 1.31) in the placebo group, 0.51 (95% Ci 0.22 to 0.80) in the Q6M group (p=0.0104) and 0.22 (95% Ci 0.09 to 0.34) in the Q3M group (p=0.0001). no significant between-group difference was observed in the joint space narrowing score. The per cent change in lumbar spine (l1–l4) BMD in the placebo, Q6M and Q3M groups were −1.03%, 3.99% (p<0.0001) and 4.88% (p<0.0001). no major differences were observed among safety profiles.Conclusions Denosumab inhibits the progression of joint destruction, increases BMD and is well tolerated in patients with Ra taking csDMaRD.
InTROduCTIOnRheumatoid arthritis (RA) is characterised by inflammatory synovitis that causes joint cartilage and bone destruction1 2 and increases fracture risk through bone erosion and osteoporosis.3 4 Bone damage is localised to the periarticular cortical
areas of inflamed joints in early RA, but oste-oporosis extends to the diaphyses becoming generalised in advanced stages. On infiltration into the periarticular region, activated T and B cells express two essential osteoclast mediators,
Key messages
What is already known about this subject? ► Denosumab, an antibody targeting receptor activator of nuclear factor κB ligand, can successfully inhibit the progression of bone erosion and increase bone mineral density (BMD) in patients with rheumatoid arthritis (RA) receiving methotrexate.
What does this study add? ► The DESIRABLE study is the largest study performed to date investigating the efficacy of denosumab in patients with RA.
► Denosumab significantly inhibits the progression of joint destruction: denosumab inhibited the progression of the modified total Sharp score and erosion scores and led to an increase in BMD but did not affect the joint space narrowing or disease activity scores in patients with RA receiving conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) (including methotrexate).
► Denosumab was generally well tolerated in patients receiving background treatment with csDMARDs.
How might this impact on clinical practice or future developments?
► Denosumab has potential as a novel therapeutic option for suppression of bone erosion and bone loss in patients with RA with or without concomitant osteoporosis, particularly in patients who are contraindicated for biological disease-modifying antirheumatic drugs (bDMARDS).
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one being the receptor activator of nuclear factor κB ligand (RANKL).1 2 5 The proliferated synovium erodes from the osteochondral junction into the bone tissue, with osteoclasts destroying the local joint site.
Disease-modifying antirheumatic drugs (DMARDs) modu-late the inflammatory immune responses slowing radiographic damage.6 Conventional synthetic DMARDs (csDMARDs) are commonly prescribed for patients with RA, although meth-otrexate remains the gold standard. Biological DMARDs (bDMARDs) are also used to target specific proteins and can potently suppress RA disease; however, they may cause serious infection. Some patients do not respond fully to bDMARDs,1 possibly because their joint destruction is unconnected to the clin-ical scores of inflammation.7 These issues may be compounded by the worsening of RA pathology by steroid-induced and disuse osteoporosis.8
RANKL is essential for osteoclast development, activation and survival.9 Denosumab, a fully human monoclonal IgG2 antibody, binds and neutralises the activity of human RANKL suppressing bone resorption10 and may also prevent progression of bone erosion. Two phase 2 studies of denosumab have been conducted in patients with RA receiving methotrexate: in a study in the USA and Canada, denosumab 60 or 180 mg was administered every 6 months,11 while in a Japanese study, denosumab 60 mg was administered every 2, 3 or 6 months.12 Both demonstrated significant inhibition of bone erosion progression compared with placebo. Therefore, we conducted this phase 3 study to evaluate the effect of denosumab on the progression of joint damage in patients with RA being treated with csDMARDs.
MeTHOdsstudy design and patientsThis was a randomised, double-blind, parallel-group, place-bo-controlled phase 3 study conducted at 104 hospitals in Japan. We enrolled patients aged ≥20 years who fulfilled the 1987 American College of Rheumatology (ACR) criteria13 or 2010 ACR/European League Against Rheumatism criteria14 for RA, who had RA for 6 months to <5 years and were receiving treat-ment with one or more csDMARDs. Based on a treat-to-target strategy, investigators adjusted csDMARD dosages for RA disease activity and/or added other treatments as appropriate (including nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids and csDMARDs such as salazosulfapyridine, bucillamine and tacrolimus in addition to methotrexate). Eligible patients had bone erosions (≥1) on radiographs or met the following criteria: C-reactive protein (CRP) levels of ≥1.0 mg/dL or an erythrocyte sedimentation rate (ESR) of ≥28 mm/hour, and positive anticy-clic citrullinated peptide antibodies or rheumatoid factor (>20 IU/mL). The main exclusion criteria were RA functional class IV15 and previous or current treatment of RA with any biolog-ical agents. Use of bisphosphonates, parathyroid hormones, oral glucocorticoids (prednisolone equivalent doses of >10 mg/day), biologics and tofacitinib6 were prohibited. However, hormone replacement therapy was allowed. Participants continued csDMARDs treatment and received ≥600 mg/day calcium with vitamin D.
Treatment and randomisationThe study drugs were denosumab (one prefilled syringe containing 1 mL of a sterile colourless solution of denosumab 60 mg) and denosumab-matching placebo. Patients received either denosumab or placebo at 0, 3, 6 and 9 months.
Patients were stratified by baseline glucocorticoid use and randomly assigned in a 1:1:1 ratio by the Interactive Web Response System to receive one of three treatments for 12 months: subcutaneous injection of denosumab 60 mg every 6 months (Q6M), every 3 months (Q3M) or placebo. Assignment was done using the RANUNI function of SAS software (release V.9.1.3). Treatment was masked to patients, investigators, spon-sors and trial personnel involved in measuring outcomes.
Collected patient data and assessmentsRadiographs were taken at baseline, 6 months and 12 months and reviewed at a central imaging core lab. Independent readers evaluated hand and foot radiographs by the modified Sharp/van der Heijde method.16 Dual energy X-ray absorptiometry scans for bone mineral density (BMD) were performed to evaluate the lumbar spine (L1–L4) at baseline and 12 months, using Hologic Inc (USA) or GE Healthcare Ltd (UK) devices, and analyses were performed by BioClinica Inc (USA).
Clinical assessments at baseline, 6 months and 12 months included: physician’s and patient’s global assessment of disease activity by visual analogue scale (VAS), patient’s assessment of pain by VAS, Health Assessment Questionnaire-Disability Index (HAQ-DI),17 the 66-joint count for swollen joints and the 68-joint count for tender joints. Blood and urine samples were obtained at fixed times and analysed at a central laboratory (LSI Medience Corporation, Tokyo, Japan) for haematology, blood biochemistry, serum C-telopeptide of type I collagen (CTX-I), urine C-telopeptide of type II collagen (CTX-II) and serum carti-lage oligomeric matrix protein (COMP). CTX-II was adjusted for creatinine (CTX-II/Cre). The ACR response18 and disease activity score 28 (DAS28) CRP and ESR19 were calculated using these outcomes. Antidenosumab antibodies were assessed at baseline and 12 months by PPD Development, LLC (USA).
Outcome measuresThe primary endpoint was the change in the modified total Sharp score (mTSS) from baseline to 12 months. Secondary endpoints were the changes from baseline to 6 months in the mTSS, changes in the erosion score at 6 months and 12 months, changes in the joint space narrowing (JSN) score at 12 months, per cent change in lumbar spine (L1–L4) BMD and the per cent change in markers of bone and cartilage metabolism. Explor-atory variables of efficacy were assessed at 12 months: the proportion of patients without radiographic progression (change in the radiographic score of ≤0.5)20 according to the mTSS, the erosion score or the JSN score; the per cent change in BMD in the subgroup using glucocorticoids or the subgroup with base-line osteoporosis; and the proportions of patients achieving an ACR20/50/70 response, a change in HAQ-DI, DAS28-CRP and DAS28-ESR from baseline. Safety was assessed by adverse events, laboratory tests and antidenosumab antibody levels and summarised according to the Medical Dictionary for Regulatory Activities (V.19.0).
statistical analysesA sample size of 214 patients per group was estimated to be sufficient to obtain a power of 0.9 and a two-sided significance level of 0.05, assuming a probability of 0.593 that an individual change in mTSS score randomly drawn from the denosumab group is less than that from the placebo group (based on the phase 2 study12) and allowing 5% missing radiographic data. The full analysis set (FAS) for the efficacy analyses included all patients who received the assigned study drug and had an
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Figure 1 Trial profile. *As one patient assigned to denosumab Q6M was administered placebo by mistake, the patient was included in the placebo group for safety analysis. †No mTSS measurements available at baseline or after the first administration of treatment. §207 (per-protocol set). ¶202 (per-protocol set). ǂ200 (per-protocol set). mTSS, modified total Sharp score; PRT, protocol; Q3M, every 3 months; Q6M, every 6 months.
available mTSS score at baseline and at least one postbaseline assessment. Missing values for the radiographic score were imputed by linear extrapolation/interpolation. For other vari-ables, no imputation was implemented.
The primary analysis compared the change in mTSS from baseline to 12 months between each denosumab group and the placebo group using the van Elteren stratified rank test adjusted for randomised strata (baseline glucocorticoid use). A hierarchical testing procedure was used to control the overall type I error at 0.05 for multiple comparisons of the primary endpoint; if there was a significant difference between the Q3M and placebo groups, comparison between the Q6M and placebo groups was formally tested. The same analysis method was used without multiple adjustments for the other radiographic score endpoints.
The proportions of patients without radiographic progression, with rapid radiographic progression (yearly progression of ≥5 in mTSS), and of patients achieving ACR20/50/70 were analysed using the Cochran-Mantel-Haenszel test adjusted for randomised strata. In a post hoc analysis, the proportion of patients with minimal clinically important progression of HAQ-DI (change from baseline ≥0.22)21 was analysed by the same method. For changes from baseline in the DAS28-CRP, DAS28-ESR and HAQ-DI, comparisons of each denosumab group with the placebo group were performed using repeated measures analysis adjusted for treatment, visit, baseline value, randomised strata and treatment-by-visit interaction. The per cent changes from baseline in lumbar spine (L1–L4) BMD, including for subgroups,
were assessed using the analysis of covariance model. Safety was analysed with the data set from patients who received at least one dose of the study drug. All analyses were conducted using SAS V.9.2.
ResulTsPatient disposition and baseline characteristicsBetween 29 October 2013 and 1 December 2015, 679 patients were randomised (figure 1). The FAS included 654 patients. One patient in the Q6M group received placebo by mistake, and therefore, that patient’s safety data were included in the placebo group. Baseline characteristics were similar across treatment groups (table 1). Dose adjustments of csDMARD and glucocor-ticoid were made similarly among treatment groups.
efficacyRegarding the primary endpoint, the mTSS increased in all groups despite csDMARD treatment (figure 2A). The Q3M and Q6M groups showed significantly smaller changes in the mTSS from baseline to 12 months versus the placebo group: mean changes were 1.49 (95% CI 0.99 to 1.99) in the placebo group; 0.99 (95% CI 0.49 to 1.49), Q6M group (p=0.0235); and 0.72 (95% CI 0.41 to 1.03), Q3M group (p=0.0055). Compared with placebo, changes in the mTSS from baseline to 6 months were significantly smaller in the Q6M and Q3M groups (p=0.0360 and 0.0028, respectively) (figure 2A), as were the changes in the erosion score from baseline to 12 months (p=0.0104 and
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Table 1 Baseline demographics and characteristics
denosumab
Placebo(n=218)
60 mg Q6M(n=217)
60 mg Q3M(n=219)
Total(n=654)
Female, n (%) 167 (76.6) 168 (77.4) 154 (70.3) 489 (74.8)
Age (years) 55.8 (11.70) 58.1 (12.30) 58.2 (12.04) 57.4 (12.04)
≥65 years, n (%) 55 (25.2) 77 (35.5) 69 (31.5) 201 (30.7)
BMI (kg/m2) 22.65 (3.49) 22.38 (3.68) 22.64 (3.62) 22.56 (3.59)
Osteoporosis, n (%) 44 (20.2) 42 (19.4) 46 (21.0) 132 (20.2)
Duration of RA (years) 2.07 (1.30) 2.20 (1.33) 2.20 (1.30) 2.16 (1.31)
RF status positive, n (%) 137 (62.8) 140 (64.5) 128 (58.4) 405 (61.9)
ACPA positive, n (%) 145 (66.5) 158 (72.8) 149 (68.0) 452 (69.1)
Modified total Sharp score (0–448) 13.14 (21.44) 15.92 (22.21) 15.17 (18.97) 14.75 (20.91)
Modified Sharp erosion score (0–280)
6.55 (10.58) 7.53 (10.11) 7.16 (9.41) 7.08 (10.04)
Modified Sharp JSN score (0–168) 6.59 (11.94) 8.39 (13.82) 8.01 (10.86) 7.66 (12.27)
Swollen joint count (0–66) 9.35 (4.43) 9.46 (4.88) 8.96 (4.30) 9.25 (4.54)
Tender joint count (0–68) 6.62 (6.39) 7.48 (8.20) 7.32 (8.00) 7.14 (7.57)
DAS28-CRP 3.43 (1.02) 3.62 (1.09) 3.52 (1.04) 3.52 (1.05)
DAS28 >3.2, n (%) 119 (54.6) 141 (65.0) 138 (63.0) 398 (60.9)
CRP (mg/dL) 0.36 (0.57) 0.65 (1.25) 0.50 (1.03) 0.51 (1.00)
HAQ-DI (0–3) 0.31 (0.39) 0.40 (0.51) 0.38 (0.45) 0.36 (0.45)
MTX use, n (%) 190 (87.2) 176 (81.1) 189 (86.3) 555 (84.9)
MTX weekly dose (mg) 9.80 (3.33) 9.33 (3.05) 9.80 (2.97) 9.65 (3.13)
Other major DMARDs
Salazosulfapyridine, n (%) 44 (20.2) 59 (27.2) 49 (22.4) 152 (23.2)
Bucillamine, n (%) 35 (16.1) 28 (12.9) 23 (10.5) 86 (13.1)
Iguratimod, n (%) 14 (6.4) 12 (5.5) 11 (5.0) 37 (5.7)
Tacrolimus, n (%) 2 (0.9) 10 (4.6) 5 (2.3) 17 (2.6)
Gold sodium thiosulfate, n (%) 3 (1.4) 4 (1.8) 7 (3.2) 14 (2.1)
Glucocorticoid use, n (%) 69 (31.7) 73 (33.6) 68 (31.1) 210 (32.1)
Glucocorticoid dose (mg/day) 3.73±1.89 3.96±2.16 4.10±2.27 3.93±2.11
NSAID use, n (%) 145 (66.5) 152 (70.0) 151 (68.9) 448 (68.5)
Hormone replacement therapy for osteoporosis treatment
0 (0.0) 2 (0.9) 1 (0.5) 3 (0.5)
Lumbar spine (L1–L4) BMD by machine type (g/cm2)
Hologic* 0.89 (0.16) 0.89 (0.16) 0.90 (0.16) 0.89 (0.16)
Lunar* 1.11 (0.19) 1.01 (0.19) 1.02 (0.15) 1.05 (0.18)
CTX-I (ng/mL)† 0.46 (0.29, 0.59) 0.45 (0.28, 0.56) 0.48 (0.26, 0.64) 0.47 (0.28, 0.59)
COMP (U/L)† 9.43 (7.00, 11.40) 9.61 (7.80, 11.00) 9.58 (7.70, 11.50) 9.54 (7.45, 11.30)
CTX-II (ng/mmol Cre)† 397.09 (184.00, 480.00) 442.90 (189.00, 563.00) 389.27 (184.00, 457.00) 409.73 (184.00, 492.00)
n=number of patients who received ≥1 dose of study drug and had a baseline and at least 1 postbaseline measurement of the radiograph score.Values are mean (SD) unless otherwise indicated.*Hologic machine use: 142, 151 and 145 patients; Lunar machine use: 76, 66 and 74 patients; placebo, Q6M and Q3M, respectively.†Values are medians (quartile 1, quartile 3).ACPA, anticyclic citrullinated peptide antibody; BMD, bone mineral density; BMI, body mass index; COMP, cartilage oligomeric matrix protein; CRP, C reactive protein; CTX-I, C-telopeptide of type I collagen; CTX-II, C-telopeptide of type II collagen; Cre, creatinine; DAS, disease activity score; DMARDs, disease-modifying antirheumatic drugs; HAQ-DI, Health Assessment Questionnaire-Disability Index; JSN, joint space narrowing; MTX, methotrexate; NSAID, non-steroidal anti-inflammatory drug; Q3M, every 3 months; Q6M, every 6 months; RA, rheumatoid arthritis; RF, rheumatoid factor.
0.0001, respectively). At 6 months, only the Q3M group showed a significantly lower result in the erosion score versus placebo (p=0.0002) (figure 2B). Overall, the changes in the JSN score from baseline to 12 months were not significantly different among groups (figure 2C).
Cumulative probability plots for changes in the mTSS, erosion score and JSN score at 12 months are shown in figure 3. The proportions of patients without radiographic progression at 12 months were greater in the denosumab groups versus the placebo group: 64.2% in the placebo group (140/218), 75.6% in the Q6M group (164/217; p=0.0097) and 78.1% in the Q3M
group (171/219; p=0.0014). Likewise, significant increases in the proportions of patients without progression of radiographic erosion (change in erosion score ≤0.5) were observed in both denosumab groups. The proportions of patients with progres-sion of JSN were similar among the groups. The proportions of patients with rapid radiographic progression at 12 months in the placebo, Q6M and Q3M groups were 10.6% (23/218), 6.9% (15/217; p=0.1785 vs placebo) and 5.0% (11/219; p=0.0310 vs placebo), respectively.
The per cent changes in lumbar spine (L1–L4) BMD from baseline to 12 months were −1.03%, 3.99% and 4.88% in the
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Figure 2 Mean changes from the baseline in the radiographic scores by the van der Heijde-modified Sharp method. (A) Modified total Sharp score, (B) modified Sharp erosion score and (C) modified Sharp joint space narrowing score. Missing values were imputed using linear extrapolation/interpolation. Mean and 95% CIs are presented. P values were calculated by two-sided van Elteren stratified rank test adjusting for baseline use of glucocorticoid. BL, baseline; n, number of patients who received ≥1 dose of investigational product and had a baseline and at least one postbaseline measurement of the radiograph score; Q3M, every 3 months; Q6M, every 6 months.
placebo, Q6M and Q3M groups, respectively (p<0.0001 for both denosumab groups vs placebo) (figure 4A). Lumbar spine (L1–L4) BMD was significantly increased in the denosumab groups versus the placebo group, regardless of glucocorticoid use or osteoporosis (p<0.0001 for both subgroups vs placebo) (figure 4B and C).
Denosumab treatment significantly decreased the bone metab-olism marker CTX-I level at 1 month, and the decrease was maintained over the study period (online supplementary file 1A). Urine CTX-II/Cre was decreased by both doses of denosumab for the first 3 months; thereafter, a decrease was observed only in the Q3M group (online supplementary file 1B). However, denosumab showed no effect on the cartilage turnover serum marker COMP (online supplementary file 1C).
No significant differences were observed between groups in the ACR20/50/70 at 12 months (online supplementary file 2). No significant changes in the DAS28-CRP or DAS28-ESR at 12 months were observed. Changes in the HAQ-DI at 12 months were only significant in the Q6M group (−0.09, p=0.0028). Post hoc analysis revealed that the proportions of patients with minimal clinically important progression of HAQ-DI (≥0.22)21 were lower in both denosumab groups versus the placebo group.
safetyThe incidence of all adverse events was similar among the groups, except for stomatitis, which was observed more frequently in the denosumab groups (table 2). One death was reported due to interstitial lung disease in the Q3M group. This was judged to be drug related; however, the patient was known to have rheumatic interstitial pneumonia concomitantly.
dIsCussIOnRA is a systemic autoimmune inflammatory disease that causes joint destruction and osteoporosis, primarily through osteoclast activation.3 4 We conducted this phase 3 study to evaluate the efficacy and safety of denosumab in patients with RA, in whom disease activity can be controlled by 1 year treatment with csDMARDs irrespective of whether they were receiving gluco-corticoids. In this study, denosumab significantly inhibited the progression of joint destruction and increased lumbar spine BMD in patients receiving concomitant csDMARD treatment. These results are consistent with those of previous research in patients with RA.11 12
Overall efficacy assessmentAlthough the effects of Q3M versus Q6M regimens were not compared in this study, the data for the Q3M regimen were
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Figure 3 Cumulative probability plots of changes from the baseline in the radiographic score at 12 months. (A) Modified total Sharp score, (B) modified Sharp erosion score and (C) modified Sharp joint space narrowing score. n, number of patients who received ≥1 dose of investigational product and had a baseline and at least one postbaseline measurement of the radiograph score; Q3M, every 3 months; Q6M, every 6 months.
better than those for the Q6M regimen in all the mean changes at 12 months, the percentage of progression-free patients (ΔmTSS≤0.5), the proportion of rapid radiographic progression and the per cent change in BMD. Notably, progression of mTSS was lower with the Q3M regimen versus the Q6M regimen, suggesting that the Q3M regimen is numerically more effective.
Progression of joint damage is irreversible, and the small amounts accrued over 1 year can gradually accumulate, causing extensive damage. Moreover, radiographic damage is related to clinical outcomes important to patients such as work and physical function. Therefore, preventing joint destruction is important. Denosumab suppressed the mean change in mTSS (0.72 in the Q3M group) and increased the proportion of patients without mTSS progression (78.1% in the Q3M group). However, true clinical relevance cannot be assessed over the short duration of a trial.
Comparison with studies of bDMARDS is difficult as demo-graphic factors and populations are different. However, the Certolizumab–Optimal Prevention of joint damage for Early RA study of certolizumab reported a mean change in mTSS in week 52 of 1.58 and 0.36, and a proportion of subjects with mTSS ≤0.5 of 70.7% and 82.9% in the placebo and certolizumab groups, respectively.22
Denosumab suppressed joint margin erosion but did not block JSN or affect the proportion of patients without JSN change, compared with placebo. Furthermore, we found no significant
differences in key indicators of disease activity. Denosumab did not affect the cartilage turnover marker serum COMP, although a clear inhibition was observed for bone metabolism markers. These results were consistent with past studies.11 12 Accordingly, distinct from DMARDs, we can conclude that denosumab did not exert an anti-inflammatory effect in patients with RA. These observations were expected, given that denosumab has only been shown to inhibit RANKL–receptor activator of nuclear factor κB (RANK) signalling during osteoclastogenesis, without affecting other inflammatory pathways.10
Functional assessmentThe HAQ-DI assesses physical functional status in adults with arthritis and correlates with the disease activity score.23 In this study, no clinically meaningful improvement in the HAQ-DI was obtained, consistent with the finding that denosumab has no effect on disease activity. As joint destruction assessed by the mTSS correlates with functional disability over time,24 denos-umab seems likely to have a preferential effect on physical func-tion. Therefore, evaluating the long-term effects of denosumab in this area may be worthwhile.
effects on lumbar spine bMdIndividuals with RA have an increased risk of bone loss and fracture.3 4 In this study, denosumab prevented a decrease in
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Figure 4 Per cent change in lumbar spinal bone mineral density (BMD) at 12 months from baseline in all patients (A), patients stratified by baseline use of glucocorticoid (B) and patients stratified by osteoporosis status (C). Data are for full analysis set (observed data). Coloured bars show least square mean values. P values are calculated using the analysis of covariance model after adjusting for treatment, baseline value, machine type, baseline value-by-machine type interaction and baseline use of glucocorticoid. Q3M, every 3 months; Q6M, every 6 months.
lumbar spine BMD and significantly increased the BMD. This increase was observed regardless of glucocorticoid use or base-line osteoporosis, as has been reported previously.25 Patients at risk of osteoporosis tend to be treated with bisphosphonates, which inhibit osteoclastic bone resorption,26 but non-adherence reduces its efficacy.27 Annually administered parenteral zoledro-nate could be superior to oral bisphosphonates due to improved compliance; notably, only zoledronate has been reported to suppress progression of bone erosions in patients with RA.28 One study showed that denosumab prevents bone erosion, but alendronate had no effect.29 Another reported that denosumab was more effective than zoledronate for menopause-related osteoporosis.30 The differences in effects between bisphospho-nates and denosumab may be explained by their distributions and mechanisms of action.31 Bisphosphonates are preferentially distributed in the gaps of bone resorption present in trabecular bone, taken up by osteoclasts and inhibit bone resorption with greater effect on cancellous bone. In contrast, denosumab is distributed throughout the extravascular space, without binding to the bone surface, inhibiting bone remodelling in both cortical and cancellous bone by blocking the RANKL–RANK interaction, a common pathway for osteoclast differentiation in osteopo-rosis and inflammation. Denosumab is the first drug shown to have potent suppressive effects on both osteoporosis and joint destruction in patients with RA who are concomitantly receiving csDMARDs. As osteoporosis and joint destruction have been
observed in some patients with RA treated with bDMARDS,32 and because bDMARDs are contraindicated in some patients due to infection risk or costs, a combination of csDMARDs and denosumab could be a new treatment option for patients in whom csDMARDs are used to control disease activity.
safety profileThe incidence of adverse events was similar across treatment groups. Specifically, hypocalcemia, an identified risk of deno-sumab,33 and fractures were comparable between groups. Overall, the safety profile of each denosumab group was broadly comparable to the placebo group, consistent with previous studies.11 12 33
study limitationsThis study has some limitations, including the prohibited concomitant use of biologics and tofacitinib and the short study duration. This short duration was chosen for ethical reasons: to treat patients optimally in the placebo group. We conducted an open-label extension study following the phase 3 study to evaluate long-term effects and are planning a follow-up study of patients who completed the phase 3 extension study to assess the rebound effects and safety of denosumab on bone erosion and BMD in patients with RA.
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Table 2 Summary of adverse events
Aes, n (%)Placebo(n=224)
denosumab
60 mg Q6M(n=221)
60 mg Q3M(n=222)
Patients with adverse events 186 (83.0) 187 (84.6) 185 (83.3)
Patients with serious adverse events 13 (5.8) 19 (8.6) 19 (8.6)
Patients with related adverse events* 38 (17.0) 38 (17.2) 36 (16.2)
Patients with related serious adverse events* 3 (1.3) 4 (1.8) 4 (1.8)
Death 0 (0.0) 0 (0.0) 1 (0.5)
Any adverse events in ≥5% of patients in any treatment group
Nasopharyngitis 73 (32.6) 79 (35.7) 69 (31.1)
Pharyngitis 16 (7.1) 13 (5.9) 16 (7.2)
Influenza 12 (5.4) 8 (3.6) 10 (4.5)
Stomatitis 13 (5.8) 22 (10.0) 27 (12.2)
Dental caries 9 (4.0) 12 (5.4) 7 (3.2)
Back pain 7 (3.1) 5 (2.3) 12 (5.4)
Upper respiratory tract inflammation 9 (4.0) 18 (8.1) 8 (3.6)
Hepatic function abnormal 20 (8.9) 14 (6.3) 13 (5.9)
Drug-related serious adverse events*
Lymphoproliferative disorder 0 (0.0) 0 (0.0) 1 (0.5)
Rectal cancer 0 (0.0) 0 (0.0) 1 (0.5)
Squamous cell carcinoma of lung 0 (0.0) 1 (0.5) 0 (0.0)
Lung neoplasm malignant 1 (0.4) 0 (0.0) 0 (0.0)
Interstitial lung disease 0 (0.0) 1 (0.5) 1 (0.5)
Abscess jaw 0 (0.0) 1 (0.5) 0 (0.0)
Diverticulitis 1 (0.4) 0 (0.0) 0 (0.0)
Ventricular tachycardia 0 (0.0) 0 (0.0) 1 (0.5)
Platelet count decreased 0 (0.0) 0 (0.0) 1 (0.5)
Brain stem infarction 0 (0.0) 1 (0.5) 0 (0.0)
Sudden hearing loss 1 (0.4) 0 (0.0) 0 (0.0)
n=Number of patients who received ≥1 dose of investigational product. Classifications of adverse events are based on the Medical Dictionary for Regulatory Activities. Only includes treatment-emergent adverse events.*This includes events for which the investigator indicated there was a reasonable possibility they may have been caused by the investigational product.MACE, major adverse cardiovascular events; Q3M, every 3 months; Q6M, every 6 months.
Clinical impact and future potentialCurrently, not all patients with RA receive treatment to suffi-ciently suppress joint destruction for many reasons. Due to safety concerns, csDMARDs have a dose limit, and some csDMARDs cannot be combined. Furthermore, biologics are not used in all patients who insufficiently respond to csDMARDs for safety or economic reasons, even if indicated.34 In clinical trials using denosumab, no additive anti-inflammatory effect was observed in patients with RA, but its inhibitory effect on joint destruction was confirmed by use in combination with csDMARDs. Hence, we believe that this drug is a novel treatment option for patients with RA who do not respond well to csDMARDs, are unable to adjust or start other DMARDs due to safety concerns or costs or require treatment for osteoporosis.
Author affiliations1Division of Rheumatology, Department of internal Medicine, Keio University school of Medicine, Tokyo, Japan2First Department of internal Medicine, University of Occupational and environmental Health, Kitakyushu, Japan3Department of Orthopedics and Rheumatology, Kindai University nara Hospital, ikoma, Japan4institute of Rheumatology, Tokyo Women’s Medical University, Tokyo, Japan5Department of Biochemistry, Osaka University Graduate school of Dentistry, Osaka, Japan6Department of Orthopedic surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan7Clinical Development Department, Daiichi sankyo Co., ltd, shinagawa-ku, Tokyo, Japan
8Biostatistics & Data Management Department, Daiichi sankyo Co., ltd, shinagawa-ku, Tokyo, Japan9Department of Radiology, Medicine and Orthopedic surgery, University of California, san Francisco, California, Usa10Department of Rheumatology, leiden University Medical Center, leiden, The netherlands
Contributors TT and YT contributed equally as first authors. TT, YT, ss and HY provided substantial contributions to the study conception and design. TY diagnosed the oral adverse events. sT diagnosed the atypical femoral fracture events. Tn was involved in the design and conduct of the study and in collecting data. nO was involved in the design of the study and data analysis. DvdH supervised scoring of the radiographs. all authors interpreted the data. all authors discussed and agreed on the content of the manuscript before submission.
Competing interests TT has received research grants from abbVie, asahi Kasei, astellas, aYUMi, Chugai, Daiichi sankyo, eisai, Mitsubishi Tanabe, nippon Kayaku, novartis, Pfizer and Takeda and has received personal fees from abbVie, astellas, astra Zeneca, Bristol-Myers squibb, Chugai, Daiichi sankyo, eisai, eli lilly, GlaxosmithKline, Janssen, Mitsubishi Tanabe, nippon Kayaku, novartis, Pfizer, sanofi, Taiho, Taisho Toyama, Takeda, Teijin and UCB. YT has received research grants from abbVie, astellas, Chugai, Bristol-Myers squibb, Daiichi sankyo, eisai, Kyowa Hakko Kirin, Mitsubishi Tanabe, MsD, Ono, Pfizer and Takeda and has received personal fees from astellas, Bristol-Myers squibb, Chugai, Daiichi sankyo, eli lilly, Janssen, Mitsubishi Tanabe, Pfizer, sanofi, UCB and Yl Biologics. ss has received grant/research support from Chugai and Daiichi sankyo and has received personal fees from asahi-Kasei Pharma, astellas, MsD, Chugai, Daiichi sankyo, eli lilly, Mitsubishi-Tanabe, Pfizer, Takeda and Teijin. HY has received research grants from abbVie, astellas, aYUMi, BMs, Chugai, Daiichi sankyo, eisai, Kaken, Mitsubishi Tanabe, MsD, nippon shinyaku, Ono, Pfizer, Takeda, Teijin, Torii and UCB and has received consulting fees from astellas, BMs, Chugai, Daiichi sankyo, Mitsubishi Tanabe, nippon Kayaku, Pfizer, Takeda, Teijin and Yl Biologics. TY has received Grants-in-aid for scientific Research from the Ministry of education, Culture, sports, science and
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Technology of Japan (MeXT #17H04377) and has received consulting fees from Daiichi sankyo. sT has acted as a consultant for abbVie, asahi Kasei Pharma, amgen, astellas, Daiichi sankyo, eli lilly, MsD, Ono and Teijin Pharma. Tn is an employee of Daiichi sankyo. nO is a shareholder and employee of Daiichi sankyo. HKG has received consulting fees from amgen, agnovos, Bioclinica, Biomarin, Clementia, Daiichi sanyo, eli lilly, Janssen, Medimmune, Merck, novartis, Pfizer, Regeneron, servier and Takeda. DvdH has received consulting fees from abbVie, amgen, astellas, astraZeneca, BMs, Boehringer ingelheim, Celgene, Daiichi sankyo, eli lilly, Galapagos, Gilead, GlaxosmithKline, Janssen, Merck, novartis, Pfizer, Regeneron, Roche, sanofi, Takeda and UCB and is the director of imaging Rheumatology BV.
Patient consent for publication not required.
Provenance and peer review not commissioned; externally peer reviewed.
data availability statement Deidentified individual patient data and supporting documents pertaining to this study, such as the study protocol, statistical analysis plan and clinical study report, are provided upon request via the data sharing portal (https:// vivli. org/ ourmember/ daiichi- sankyo/) in accordance with the data sharing policy of Daiichi sankyo Co., ltd.
Open access This is an open access article distributed in accordance with the Creative Commons attribution non Commercial (CC BY-nC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.
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9 Yasuda H, shima n, nakagawa n, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRanCe/RanKl. Proc Natl Acad Sci U S A 1998;95:3597–602.
10 Kostenuik PJ, nguyen HQ, McCabe J, et al. Denosumab, a fully human monoclonal antibody to RanKl, inhibits bone resorption and increases BMD in knock-in mice that express chimeric (murine/human) RanKl. J Bone Miner Res 2009;24:182–95.
11 Cohen sB, Dore RK, lane ne, et al. Denosumab treatment effects on structural damage, bone mineral density, and bone turnover in rheumatoid arthritis: a twelve-month, multicenter, randomized, double-blind, placebo-controlled, phase ii clinical trial. Arthritis Rheum 2008;58:1299–309.
12 Takeuchi T, Tanaka Y, ishiguro n, et al. effect of denosumab on Japanese patients with rheumatoid arthritis: a dose-response study of aMG 162 (Denosumab) in patients with Rheumatoid arthritis on methotrexate to Validate inhibitory effect on bone erosion (DRiVe)-a 12-month, multicentre, randomised, double-blind, placebo-controlled, phase ii clinical trial. Ann Rheum Dis 2016;75:983–90.
13 arnett FC, edworthy sM, Bloch Da, et al. The american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.
14 aletaha D, neogi T, silman aJ, et al. Rheumatoid arthritis classification criteria: an american College of Rheumatology/european league against rheumatism collaborative initiative. Arthritis Rheum 2010;2010:2569–81.
15 Hochberg MC, Chang RW, Dwosh i, et al. The american College of rheumatology 1991 revised criteria for the classification of global functional status in rheumatoid arthritis. Arthritis Rheum 1992;35:498–502.
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18 Felson DT, anderson JJ, Boers M, et al. american College of rheumatology. Preliminary definition of improvement in rheumatoid arthritis. Arthritis Rheum 1995;38:727–35.
19 Prevoo Ml, van Gestel aM, van T Hof Ma, et al. Remission in a prospective study of patients with rheumatoid arthritis. american rheumatism association preliminary remission criteria in relation to the disease activity score. Br J Rheumatol 1996;35:1101–5.
20 van der Heijde D, simon l, smolen J, et al. How to report radiographic data in randomized clinical trials in rheumatoid arthritis: guidelines from a roundtable discussion. Arthritis Rheum 2002;47:215–8.
21 Wells Ga, Tugwell P, Kraag GR, et al. Minimum important difference between patients with rheumatoid arthritis: the patient’s perspective. J Rheumatol 1993;20:557–60.
22 atsumi T, Yamamoto K, Takeuchi T, et al. The first double-blind, randomised, parallel-group certolizumab pegol study in methotrexate-naive early rheumatoid arthritis patients with poor prognostic factors, C-OPeRa, shows inhibition of radiographic progression. Ann Rheum Dis 2016;75:75–83.
23 van der Kooi e, Klarenbeek nB, Güler-Yüksel M, et al. a decrease in disease activity score (Das) level is associated with a decrease in health assessment Questionnaire (HaQ) score, independent of follow-up duration, during 5 years of tightly controlled treatment: results from the best study. Ann Rheum Dis 2011;70:168–71.
24 Drossaers-Bakker KW, de Buck M, van Zeben D, et al. long-term course and outcome of functional capacity in rheumatoid arthritis: the effect of disease activity and radiologic damage over time. Arthritis Rheum 1999;42:1854–60.
25 Dore RK, Cohen sB, lane ne, et al. effects of denosumab on bone mineral density and bone turnover in patients with rheumatoid arthritis receiving concurrent glucocorticoids or bisphosphonates. Ann Rheum Dis 2010;69:872–5.
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TranslaTional science
Different classes of anti-modified protein antibodies are induced on exposure to antigens expressing only one type of modificationarieke suzanna Berendina Kampstra, 1 Jacqueline stephanie Dekkers,1 Mikhail Volkov,1 annemarie l Dorjée,1 lise Hafkenscheid,1 ayla c Kempers,1 Myrthe van Delft,1 Theresa Kissel,1 sanne reijm,1 George M c Janssen,2 Peter a van Veelen,2 Holger Bang,3 Tom W J Huizinga,1 leendert a Trouw,4 Diane van der Woude,1 rené e M Toes 1
To cite: Kampstra asB, Dekkers Js, Volkov M, et al. Ann Rheum Dis 2019;78:908–916.
Handling editor Prof Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214950).
1Department of rheumatology, leids Universitair Medisch centrum, leiden, The netherlands2center of Proteomics and Metabolomics, leids Universitair Medisch centrum, leiden, The netherlands3research and development, orgentec Diagnostika, Mainz, Germany4Department of immunohematology and Bloodtransfusion, leids Universitair Medisch centrum, leiden, The netherlands
Correspondence toarieke suzanna Berendina Kampstra, rheumatology, leids Universitair Medisch centrum, leiden 2333 Za, netherlands; a. s. b. kampstra@ lumc. nl
asBK and JsD contributed equally.
received 19 December 2018revised 5 March 2019accepted 30 March 2019Published online First 31 May 2019
© author(s) (or their employer(s)) 2019. no commercial re-use. see rights and permissions. Published by BMJ.
Key messages
What is already known about this subject? ► Antibodies targeting different post-translational modified proteins have been described for patients with rheumatoid arthritis (RA). Different classes of these antibodies can be present simultaneously. Nevertheless, the mechanisms behind the concurrent presence of different anti-modified protein antibody (AMPA) classes in RA are unclear.
What does this study add? ► Our data show that, in mice, a protein expressing one particular post-translational modification can induce cross-reactive AMPA against other post-translational modifications as well.
► Different AMPA from patients with RA show similar cross-reactivity.
How might this impact on clinical practice or future developments?
► Our results indicate a ‘common’ B-cell response from which different AMPA responses originate, thereby providing a conceptual framework for the mutual relationship and simultaneous presence of different AMPA ‘classes’ in RA.
AbsTRACTObjectives autoantibodies against post-translationally modified proteins (anti-modified protein antibodies or aMPas) are a hallmark of rheumatoid arthritis (ra). a variety of classes of aMPas against different modifications on proteins, such as citrullination, carbamylation and acetylation, have now been described in ra. at present, there is no conceptual framework explaining the concurrent presence or mutual relationship of different aMPa responses in ra. Here, we aimed to gain understanding of the co-occurrence of aMPa by postulating that the aMPa response shares a common ’background’ that can evolve into different classes of aMPas.Methods Mice were immunised with modified antigens and analysed for aMPa responses. in addition, reactivity of aMPa purified from patients with ra towards differently modified antigens was determined.Results immunisation with carbamylated proteins induced aMPas recognising carbamylated proteins and also acetylated proteins. similarly, acetylated proteins generated (autoreactive) aMPas against other modifications as well. analysis of anti-citrullinated protein antibodies from patients with ra revealed that these also display reactivity to acetylated and carbamylated antigens. similarly, anti-carbamylated protein antibodies showed cross-reactivity against all three post-translational modifications.Conclusions Different aMPa responses can emerge from exposure to only a single type of modified protein. These findings indicate that different aMPa responses can originate from a common B-cell response that diversifies into multiple distinct aMPa responses and explain the presence of multiple aMPas in ra, one of the hallmarks of the disease.
InTROduCTIOnThe presence of anti-citrullinated protein anti-bodies (ACPAs) is one of the hallmarks of rheuma-toid arthritis (RA). ACPAs recognise citrullinated proteins and display an extensive citrulline-de-pendent cross-reactivity towards multiple citrul-linated antigens.1 2 Interestingly, the citrullinated epitope-recognition profile expands before clinical onset of disease, possibly as a consequence of the activation of new ACPA-expressing B cells and/or
progressive somatic hypermutation of individual B-cell clones.3–7 Also, other post-translationally modified (PTM) proteins, in particular carbamy-lated and acetylated proteins, have been found to be recognised by RA autoantibodies.8 Carbamyla-tion and acetylation do not modify arginine, the target of citrullination, but lysine into respectively homocitrulline and acetyl-lysine. Homocitrulline is an amino acid resembling citrulline, but containing an additional methylene group. Anti-Carbamy-lated protein (anti-CarP) antibodies are present in approximately 45% of patients with RA.9 These antibodies can be cross-reactive to citrullinated anti-gens, but can also display a more restricted recogni-tion profile directed against carbamylated proteins only. Indeed, 10%–20% of ACPA-negative patients with RA are positive for anti-CarP antibodies,
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indicating that these antibodies represent a different class of anti-modified protein antibodies.9 10 Acetylation, on the other hand, is mediated by intracellular acetyltransferases. Anti-acetyl-ated protein antibodies (AAPAs) are present in approximately 40% of patients with RA11 and are mainly found in ACPA-pos-itive RA, although also ACPA-negative patients with RA can be AAPA positive. Inhibition experiments showed limited cross-re-activity between anti-acetylated, anti-carbamylated and ACPAs, indicating that also AAPAs represent another class of anti-modi-fied protein antibody (AMPA).11
These previous observations are interesting as they indicate that AMPAs, due to their concurrent presence in RA, have a commonality that is currently not understood. Here, we studied the possibility that the AMPA response originates from a common ‘event’ by analysing whether exposure to one partic-ular class of modified proteins can generate different AMPA responses.
MATeRIAls And MeTHOdsProteins, modifications and immunisationsAll procedures for protein modification, mass spectrometry and immunisations are previously described and further detailed in the online supplementary materials.9 12 13 Animal experiments were approved by the Ethical Committee for Animal Experimen-tation. All immunised mice were healthy and showed no signs of arthritis throughout the experiment.
Mass spectrometryProcedure for the mass spectrometry analysis is described in detail in online supplementary materials and methods.
detection of AMPAsFor the detection of AMPAs in mice, the following ELISA was performed: Modified proteins and their non-modified coun-terparts were coated at a concentration of 10 µg/mL in 0.1 M carbonate–bicarbonate buffer (pH 9.6) overnight on Nunc Maxi-sorp plates (Thermo Scientific). The plates were blocked with phosphate buffered saline (PBS)+1% bovine serum albumin. The mouse sera were diluted 1:100 in RIA buffer (10 mM Tris (pH 7.6), 350 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycho-late, 0.1% SDS) and incubated overnight. Binding of mouse IgG was detected with horseradish peroxidase–conjugated goat-an-ti-mouse IgG1 (cat. no. 1070-05; Southern Biotech) and subse-quently visualised with ABTS. Washing steps were performed between each incubation with PBS+0.05% Tween 20. All incu-bations, aside from the incubations with goat-anti-mouse IgG1 and ABTS, were performed at 4°C, and the final two steps were performed at room temperature. Arbitrary units were calculated using a reference serum in serial dilution. The reference serum was acquired from CaOVA-immunised or Ac-OVA-immunised mice for the carbamylated or acetylated protein ELISA, respec-tively. For the inhibition experiments, the sera were pre-incu-bated with 0–0.2 mg/mL protein for 1 hour before transferring them to the ELISA plate.
Reactivity of purified ACPA and anti-CarP antibodies, obtained from sera and synovial fluid (SF) of patients with RA, was measured using modified vimentin peptides (plates and reagents were kindly provided by Orgentec), as previously described.11 In addition, purified ACPA and anti-CarP antibodies were tested on CCP2 and Ca-FCS, respectively, according to protocols previ-ously described.9 14 15
Patients with RAThe material of the ACPA-positive patients with RA was selected for ACPA purification based on the ACPA status and levels. The patients with RA fulfilled the European League Against Rheu-matism/American College of Rheumatology 2010 classification criteria. Similar to the material from ACPA-positive patients, the material from anti-CarP-positive patients used for anti-CarP-an-tibody isolation was derived from patients screened for anti-CarP status and levels.
IgG-AMPA purificationSpecific AMPAs are isolated as has been previously described for ACPA in Scherer et al.16 In short, plasma or serum samples and SF were acquired from patients. The plasma, serum and SF samples were subsequently filtered (0.2 µM filters; Millipore) before purifying AMPA with affinity chromatography (ÄKTA; GE Healthcare). Purification was performed using HiTrap streptavidin HP 1 mL columns (GE Healthcare) coupled with biotinylated CCP2 peptides (obtained from J.W. Drijfhout, IHB LUMC) for the isolation of ACPA17 18 or in-house prepared biotinylated (Ca-)FCS for the isolation of anti-CarP antibodies. PTM specificity was controlled by attaching a control column coated with the native version (CCP2 arginine or FCS) before the column coated with the modified version (CCP2 citrul-line or Ca-FCS). Antibodies were eluted using 0.1 M glycine hydrogen chloride (HCl) pH 2.5 and neutralised with 2 M Tris. ACPA-IgG1,2,4 was subsequently purified from ACPA with Prot A and Prot G HiTrap columns.
statisticsStatistical tests were performed with Prism V.7 (GraphPad). Significance of AMPA reactivity on proteins was tested with paired t-test. Differences in titre were tested with Mann-Whitney U tests. Correlations were assessed with Spearman. A p value of <0.05 was considered significant.
ResulTsCross-reactive AMPAs are induced upon vaccination with one defined modified antigenTo analyse whether AMPA recognising different classes of PTMs can be induced with an antigen expressing one defined modifi-cation, we immunised mice with either non-modified, carbamy-lated, citrullinated or acetylated ovalbumin (OVA). The presence of either homocitrulline as a result of carbamylation or acetyl-ated lysine as a consequence of acetylation was confirmed by mass spectrometry and commercially available antibodies against either carbamylated or acetylated lysines in ELISA (online supplementary figure 1). Non-modified OVA was found to be acetylated, but not carbamylated, at the N terminus by mass spectrometry and therefore the latter antigen was included in all immunisation experiments as additional specificity control.
To discriminate between reactivity against the PTM and protein backbone used for immunisation, we employed modi-fied fibrinogen (Fib) as read-out. In doing so, antibodies recog-nising OVA were not interfering with the detection of AMPA.13 To control for possible baseline reactivity towards modified proteins, sera from non-immunised mice were taken along with the ELISA experiments. Indeed, no reactivity was observed to non-modified fibrinogen or its modified counterparts in naïve animals, indicating that without immunisations, AMPA responses are not present towards either modified fibrin-ogen (figure 1A) or mouse albumin (figure 2A).13 19 Likewise, although a strong reaction against OVA was noted (data not
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Figure 1 Immunisation with Ca-OVA or Ac-OVA induces antibody responses towards modified fibrinogen. Antibody reactivity towards modified fibrinogen in sera derived from non-immunised (A), OVA-immunised (B), Cit-OVA-immunised (C), Ca-OVA-immunised (D) or Ac-OVA-immunised (E) mice was measured by ELISA. Reactivity is depicted with OD values measured at 415 nm. For all groups, n=6. Representative data from two experiments is shown. Ac, acetylated; Ca, carbamylated; Cit, citrullinated; Fib, fibrinogen; OD, optical density; OVA, ovalbumin.
shown), indicating proper immunisation, mice immunised with non-modified OVA did not react to modified Fib (figure 1B) nor modified mouse albumin (figure 2B).13 These results indi-cate that neither non-modified OVA nor the adjuvant used is driving AMPA production. We were unable to detect reactivity towards citrullinated Fib (Cit-Fib) in mice immunised with citrullinated OVA (Cit-Ova) (figure 1C). As ACPAs have been reported in some murine models,20–23 we additionally tested the sera on modified myelin basic protein (MBP), but again we were unable to detect citrulline reactivity (online supple-mentary figure 2). Mice immunised with carbamylated OVA
(Ca-OVA), however, displayed a strong reactivity towards Ca-Fib, but not non-modified-Fib (figure 1D). Remarkably, sera of mice immunised with Ca-OVA also reacted to Ac-Fib and to some extent to Cit-Fib. This reactivity was further vali-dated using modified MBP (online supplementary figure 2). Moreover, these sera also reacted to both Ac-mouse albumin (Ac-mAlb) and Ca-mAlb (figure 2C), indicating that exposure to modified foreign proteins is capable of inducing a breach of tolerance towards self-antigens carrying different classes of modifications. These data are intriguing as they indicate that antibody responses induced by carbamylated antigens are able
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Figure 2 Break of tolerance towards modified self-proteins in Ca-OVA-immunised and Ac-OVA-immunised mice. Reactivity towards carbamylated and acetylated mouse albumin was tested by ELISA (A) with sera derived from non-immunised (A), OVA-immunised (B), Ca-OVA-immunised (C) and Ac-OVA-immunised (D) mice. Results show representative data from two immunisation experiments. p<0.05 depicts significance. Ac, acetylated; Ca, carbamylated; OD, optical density; OVA, ovalbumin
to recognise multiple modifications, pointing to the generation of cross-reactive (autoreactive) AMPAs induced by exposure to only one class of modified antigen.
Next, we wished to determine whether cross-reactive anti-bodies could also be induced by immunisation with acetylated OVA. We observed reactivity to Ac-Fib as expected and also towards Ca-Fib (figure 1E). Reactivity towards Cit-Fib was only moderately apparent. This could not be validated using Cit-MBP (online supplementary figure 2). Similar reactivity patterns were observed when modified mouse albumin was used as model autoantigen (figure 2D). Together, these results indicate that immunisation with Ac-OVA induces (auto-)antibodies cross-re-active to acetyl-lysine and homocitrulline.
Cross-reactive antibody responses harbour different PTM recognition profilesTo further investigate the cross-reactive nature of these AMPA responses in more detail, we next analysed the autoantibody titre through dilution of sera from immunised animals. Similar antibody titres were observed towards Ac-Fib and Ca-Fib in Ca-immunised mice (figure 3A). In contrast, the titre of anti-bodies recognising Ac-Fib was considerable higher than the antibody titre against Ca-Fib in Ac-OVA-immunised mice (figure 3B). These data indicate that in contrast to anti-CarP antibodies in Ca-OVA-immunised mice, the AAPA response in Ac-OVA-immunised mice is only partly cross-reactive to both modifications.
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Figure 3 Antibody titres and avidity in sera of Ca-OVA-immunised and Ac-OVA-immunised mice. Antibody titres as measured by ELISA on Ca-Fib and Ac-Fib for Ca-OVA-immunised (A) and Ac-OVA-immunised (B) mice. EC50 depicts the dilution at which half of the maximum effective reactivity is present. Representative data from two immunisation experiments are shown. Ac, acetylated; Ca, carbamylated; Fib, fibrinogen; EC50, effective concentration at 50%; OD, optical density; OVA, ovalbumin.
The data presented on antibody titre also predict that the AMPA response present in Ca-OVA-immunised mice (highly cross-reactive) can be readily inhibited by both acetylated and carbamylated proteins, whereas the AMPA reaction in Ac-OVA-immunised mice can only be fully inhibited by acetylated proteins. To confirm this notion, the binding capacity towards Ca-Fib and Ac-Fib was analysed by inhibition experiments with modified fibrinogen. Indeed, for Ca-OVA-immunised mice, anti-body reactivity towards either modified antigen could be inhib-ited by Ac-Fib (figure 4A, B), whereas for Ac-OVA-immunised mice, Ac-Fib reactivity could not be inhibited by competing with Ca-Fib (figure 4C, D). These data confirm that the AMPA response generated by Ca-OVA-immunisation is highly cross-re-active, whereas part of the antibodies induced by Ac-OVA immu-nisation are cross-reactive towards both modifications.
Cross-reactive antibodies towards different modifications are present in patients with RAThe data presented above show that exposure of mice to a protein carrying one defined PTM can induce cross-reactive AMPAs. To address whether also in humans AMPA are cross-reactive towards different classes of modified antigens, we next isolated ACPA-IgG from SF or plasma of seven patients with RA as previ-ously described.17 18 We focused on ACPA as the ACPA response is the most prominent AMPA response in RA. As depicted in figure 5A, B, ACPA-IgG were strongly enriched following isola-tion, whereas the flow-through contained low to no levels of ACPA-IgG (online supplementary figure 3). Next, the purified ACPA-IgG were analysed for their reactivity towards a citrulli-nated, carbamylated or acetylated peptide from vimentin. In all cases, purified ACPA also showed a highly enriched reactivity towards these differently modified peptides. These data indicate that ACPA-IgG from patients with RA are cross-reactive towards carbamylated antigens as observed previously9 and that they can also recognise acetylated antigens. To analyse whether also anti-CarP antibodies display cross-reactivity towards different classes of PTMs, we next isolated anti-CarP antibodies from sera of two anti-CarP-positive patients. As shown in figure 5C, the isolated antibodies were highly enriched for anti-CarP reactivity. Likewise, as observed for isolated ACPA, also purified anti-CarP
antibodies showed strongly enriched reactivity towards the three different classes of modified antigen. Together, these data indi-cate that different families of human AMPA are cross-reactive towards different classes of modified antigens, including acetyl-ated antigens.
dIsCussIOnRA is characterised by the presence of autoantibodies against different PTMs, including citrullinated, carbamylated and acetyl-ated proteins. As different AMPAs target different PTMs and are generally seen as distinct autoantibody families, it is intriguing that their presence often goes together in RA. At present, there is no conceptual framework explaining the concurrent presence of different AMPA responses in RA. Here, we show that exposure to a protein carrying one defined PTM can lead to cross-reactive (auto)antibody responses towards different PTMs. Interestingly, we have shown that AMPA from patients with RA purified with antigens carrying one particular PTM can recognise different classes of PTMs too, indicating a cross-reactive nature of these autoantibodies as well. These findings are important as they indicate that the different AMPA responses observed in RA can potentially be generated by antigen(s) carrying only one partic-ular modification. Similarly, they provide a rationale for the simultaneous manifestation of multiple AMPA reactivities in RA.
Given the observations that different AMPAs target different antigens and are generally seen as distinct autoantibody fami-lies, it has been intriguing to note that their presence often go together in RA. In contrast, AMPAs are less frequently present in other rheumatic diseases and their co-occurrence is rarely observed outside RA. The co-occurrence of different AMPAs represent an interesting conundrum as it is unclear why, after activation of a B cell with a receptor for a particular modified protein, another B cell expressing a receptor recognising a differently modified protein would also be activated in the same subject. In general, the activation of a particular B cell will not directly influence the activation of other B cells directed against other antigens, although it has been shown in a transgenic mouse model for SLE that epitope spreading to other antigens can occur once tolerance is broken for one self-antigen.24 Our data indicate that exposure to a defined antigen displaying a particular class of
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Figure 4 Inhibition of antibody binding by pre-incubation of mouse sera with modified fibrinogen. Cross-reactivity of antibodies is studied by assessment of the inhibitory capacity of pre-incubating sera with modified fibrinogen. Sera from Ca-OVA-immunised mice were pre-incubated with varying concentrations of modified fibrinogen before testing the antibody reactivity on Ca-Fib (A) or Ac-Fib (B). Sera from Ac-OVA-immunised mice were pre-incubated with varying concentrations of modified fibrinogen before testing the antibody reactivity on Ca-Fib (C) or Ac-Fib (D). Results show representative data of two experiments. Ac, acetylated; Ca, carbamylated; Fib, fibrinogen; OD, optical density; OVA, ovalbumin.
PTM can lead to a cross-reactive antibody response recognising several classes of modified antigens, conceivably explaining the co-occurrence of multiple AMPA reactivities in RA.
It has been shown that ACPA and anti-CarP antibodies can be cross-reactive towards citrullinated and carbamylated antigens.9 Citrulline and homocitrulline are highly similar in structure as they differ in only one methyl group, even though they are conversions from different amino acids. We now show that also acetylated antigens can be recognised by these antibodies. This was unexpected as acetyl-lysine shares less structural homology to citrulline/homocitrulline (online supplementary figure 1). The cross-reactivity towards acetylated antigens was even more prominent in mice because AMPA induced by Ca-OVA immuni-sation did not recognise citrullinated proteins, even though they are able to recognise acetylated lysines.
The finding that exposure to, for example, an acetylated protein leads to the formation of autoantibodies against proteins carrying other classes of PTM as well is also relevant for consid-erations on the breach of tolerance and induction of AMPA responses. From our findings, it can be postulated that the inciting antigen responsible for the induction of, for example, ACPA or anti-CarP antibodies does not have to be citrullinated or carbamylated, but could be represented by, for example, an acetylated protein. Clearly, at present, we cannot conclude from our data whether a particular PTM antigen initiates AMPA induction in RA. Nonetheless, it will be relevant to study in pre-disease samples whether a breach of tolerance towards, for example, acetylated or carbamylated proteins precedes ACPA production or vice versa and whether this is similar in all patients or can vary from patient to patient.
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Figure 5 Cross-reactivity of purified human ACPA or anti-CarP antibodies towards modified vimentin peptides. ACPA and anti-CarP antibodies were isolated from patients with rheumatoid arthritis (RA). ACPA from synovial fluid (A, n=4) and serum (B, n=3) from patients were tested on CCP2 and modified vimentin peptides. Anti-CarP antibodies from serum of patients with RA (c, n=2) were tested on Ca-FCS and modified vimentin peptides. Reactivity is depicted as arbitrary units per milligram IgG and calculated based on standards. AcLys, acetylated lysine; Arg, arginine control; AU/mg IgG, arbitrary units per milligram immunoglobulin G; Ca, carbamylated; CArgP2, cyclic arginine control peptide; CCP2, cyclic citrullinated peptide; Cit, citrullinated; FCS, fetal calf serum; hCit, homocitrulline (carbamylated); Lys, lysine control; Vim, vimentin peptide.
An increasing number of studies suggest that mucosal surfaces, specifically the periodontium, the gut and the lungs, could be sites of disease initiation of RA and indicate the microbiome as an important driver of the initiation of autoimmunity. In this respect, especially protein acetylation by bacteria might now also be incriminated in the induction of autoantibody responses against PTM proteins. Recent evidence shows that many bacte-rial species are able to acetylate proteins,25 including bacteria proposed as link between periodontal infection and RA.26 Given our observation that AMPAs recognising citrullinated
and carbamylated proteins can be cross-reactive to acetylated proteins, these findings together provide a novel and stimu-lating angle to the notion that the microbiome contributes to the induction of autoimmunity in RA. Therefore, a logical next step is to test faecal extracts from patients with RA also for the presence of acetylated bacterial proteins to obtain more insight on the possible link between the microbiome, the presence of acetylated proteins and RA. Through the formation of acetylated proteins, disturbances of the microbiome (eg, through infection) could lead to the formation of acetylated proteins detected
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by the immune system and thereby to the induction of AMPA responses. In doing so, the origin of the T-cell help required for the B cell to undergo isotype switching and somatic hyper-mutation could come from different sources. In this scenario, it is conceivable that microbe-specific T cells help the B cell initially recognising the microbe-derived modified protein. On further somatic hypermutation, the B-cell response could be selected/start recognising other modified proteins explaining the cross-reactive nature of AMPAs and the observation that different AMPAs often appear together in patients. Likewise, the diversification of an initial AMPA response towards other PTMs could, potentially, also explain the observation that the HLA-shared-epitope (SE) alleles are associated with ACPA-posi-tive RA, whereas the first appearance of ACPA in healthy subjects is HLA-SE-allele independent.27 28 Possibly, by diversification towards citrulline recognition, an initially HLA-SE-indepen-dent AMPA reaction against, for example, acetylated proteins could recruit new HLA-SE-restricted T cells required for further broadening of the AMPA/ACPA response associated with disease precipitation. Thus, in this scenario, the link to the microbiome, the cross-reactive nature of AMPAs, the breach of tolerance to modified self-proteins, the HLA-SE association with the ‘second hit’, as well as the concurrent presence of AMPAs in disease can be explained.
Our study has several limitations as we did not show that also in humans the inciting antigen carrying a particular PTM will lead to the induction of a cross-reactive AMPA response. Obviously, studies immunising a host with a defined modified antigen, as was performed in mice, is not feasible in humans and therefore the concepts obtained from such animal studies will be difficult to demonstrate in the human system. Nonetheless, the observation that also human AMPAs are cross-reactive to several different PTM does support such views. Furthermore, we would like to emphasise that, despite the advantages of using a controlled setting for the immunisation of mice, a major pitfall of studying RA-associated antibodies in mice is the inability to induce detectable production of ACPAs with our standard immunisation protocol, that is, two subsequent immunisations in aluminium hydroxide. Consequently, the analysis of antibody cross-reactivity towards citrullinated antigens is limited and restricted to the human setting. In addition, our antibody exper-iments are focused on polyclonal antibody responses. Neverthe-less, our inhibition studies do suggest that individual antibodies are capable of cross-recognising multiple PTM, though isola-tion of monoclonal antibodies will be necessary to validate this notion. Interestingly, recent studies have shown two monoclonal ACPAs able to interact with an acetylated histone peptide29 as well as one able to recognise a carbamylated vimentin peptide.30
In conclusion, our data show that induction of cross-reactive AMPA can be achieved by the encounter with a protein carrying one specific PTM and indicate that the different AMPAs present in RA could have a common ‘background’, thereby providing novel insight into the concurrent presence of these antibodies in RA, an important hallmark of the disease.Acknowledgements We thank Dr Jan Wouter Drijfhout (lUMc, leiden) for providing the ccP2 peptide.
Contributors asBK, JsD and rT have designed the experiments. asBK and JsD have done the animal experiments. asBK, JsD, MV and alD have performed the elisas (murine and human). lH, acK and MvD have performed the aMPa purification from patients with ra. GMcJ and PavV have done the mass spectrometry analysis of the modified antigens. asBK, JsD, MV, HB, TWJH, laT, DvdW and rT were involved in critically revising the manuscript for intellectual improvement. asBK, JsD, MV, alD, TK, sr, laT, DvdW and rT have been extensively involved in the interpretation and analysis of the results. all authors have contributed to the writing and editing of the manuscript.
Funding This work has been financially supported by the eU/eFPia innovative Medicines initiative 2 Joint Undertaking rTcure grant no. 777357, by reumanederland (13-3-401) and by Target to B! (grant no. lsHM18055-sGF). in addition, this work is part of the research programme investment Grant nWo Medium, project no. 91116004, which is (partly) financed by ZonMw. l.a.T. has received funding from the european research council (erc) under the european Union’s Horizon 2020 research and innovationprogramme (grant agreement no. 724517).
Competing interests none declared.
Patient consent for publication obtained.
ethics approval all animal experiments were approved by the ethical committee for animal experimentation of the lUMc, leiden. The study with human material was conducted with the approval of the regional ethics committee at leiden University Medical center.
Provenance and peer review not commissioned; externally peer reviewed.
data sharing statement all data relevant to the study are included in the article or uploaded as supplementary information.
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8 Trouw la, rispens T, Toes reM. Beyond citrullination: other post-translational protein modifications in rheumatoid arthritis. Nat Rev Rheumatol 2017;13:331–9.
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10 chemin K, Pollastro s, James e, et al. a novel Hla-DrB1*10:01-restricted T cell epitope from citrullinated type ii collagen relevant to rheumatoid arthritis. Arthritis Rheumatol 2016;68:1124–35.
11 Juarez M, Bang H, Hammar F, et al. identification of novel antiacetylated vimentin antibodies in patients with early inflammatory arthritis. Ann Rheum Dis 2016;75:1099–107.
12 Guan K-l, Yu W, lin Y, et al. Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides. Nat Protoc 2010;5:1583–95.
13 Dekkers Js, Verheul MK, stoop Jn, et al. Breach of autoreactive B cell tolerance by post-translationally modified proteins. Ann Rheum Dis 2017;76:1449–57.
14 Kerkman PF, rombouts Y, van der Voort eiH, et al. circulating plasmablasts/plasmacells as a source of anticitrullinated protein antibodies in patients with rheumatoid arthritis. Ann Rheum Dis 2013;72:1259–63.
15 van Delft MaM, van Beest s, Kloppenburg M, et al. Presence of autoantibodies in erosive hand osteoarthritis and association with clinical presentation. J Rheumatol 2019;46:101–5.
16 scherer HU, Wang J, Toes reM, et al. immunoglobulin 1 (igG1) Fc-glycosylation profiling of anti-citrullinated peptide antibodies from human serum. Proteomics Clin Appl 2009;3:106–15.
17 rombouts Y, Willemze a, van Beers JJBc, et al. extensive glycosylation of acPa-igG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis. Ann Rheum Dis 2016;75:578–85.
18 Hafkenscheid l, Bondt a, scherer HU, et al. structural analysis of variable domain glycosylation of anti-citrullinated protein antibodies in rheumatoid arthritis reveals the presence of highly sialylated glycans. Mol Cell Proteomics 2017;16:278–87.
19 stoop Jn, Fischer a, Hayer s, et al. anticarbamylated protein antibodies can be detected in animal models of arthritis that require active involvement of the adaptive immune system. Ann Rheum Dis 2015;74:949–50.
20 cantaert T, Teitsma c, Tak PP, et al. Presence and role of anti-citrullinated protein antibodies in experimental arthritis models. Arthritis Rheum 2013;65:939–48.
21 Hill Ja, Bell Da, Brintnell W, et al. arthritis induced by posttranslationally modified (citrullinated) fibrinogen in Dr4-ie transgenic mice. J Exp Med 2008;205:967–79.
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22 Kidd Ba, Ho PP, sharpe o, et al. epitope spreading to citrullinated antigens in mouse models of autoimmune arthritis and demyelination. Arthritis Res Ther 2008;10.
23 Mohamed BM, Boyle nT, schinwald a, et al. induction of protein citrullination and auto-antibodies production in murine exposed to nickel nanomaterials. Sci Rep 2018;8.
24 Degn se, van der Poel ce, Firl DJ, et al. clonal evolution of autoreactive germinal centers. Cell 2017;170:913–26.
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26 Butler ca, Veith PD, nieto MF, et al. lysine acetylation is a common post-translational modification of key metabolic pathway enzymes of the anaerobe Porphyromonas gingivalis. J Proteomics 2015;128:352–64.
27 Hensvold aH, Magnusson PKe, Joshua V, et al. environmental and genetic factors in the development of anticitrullinated protein antibodies (acPas) and acPa-positive
rheumatoid arthritis: an epidemiological investigation in twins. Ann Rheum Dis 2015;74:375–80.
28 Terao c, ohmura K, ikari K, et al. effects of smoking and shared epitope on the production of anti-citrullinated peptide antibody in a Japanese adult population. Arthritis Care Res 2014;66:1818–27.
29 lloyd Ka, Wigerblad G, sahlström P, et al. Differential acpa binding to nuclear antigens reveals a PaD-independent pathway and a distinct subset of acetylation cross-reactive autoantibodies in rheumatoid arthritis. Front Immunol 2018;9.
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TranslaTional science
Valine 11 and phenylalanine 13 have a greater impact on the T-cell response to citrullinated peptides than the 70–74 shared epitope of the DRB1 molecule in macaquessamuel Bitoun, 1 Pierre roques,2 Bernard Maillere,3 roger le Grand,2 Xavier Mariette1
To cite: Bitoun s, roques P, Maillere B, et al. Ann Rheum Dis 2019;78:917–921.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2019- 215114).
1rheumatology Department, Hôpitaux Universitaires Paris-sud-assistance Publique-Hôpitaux de Paris (aP-HP), Université Paris-sud- cea-inserM U1184 ’immunology of viral infections and autoimmune diseases’, le Kremlin-Bicêtre, France2iDMiT infrastructure, cea - Université Paris sud 11 - inserM U1184, immunology of viral infections and autoimmune diseases, Fontenay-aux-roses, France3iBiTecs, service d’ingenierie Moleculaire des Proteines (siMoPro), labex lerMiT, labex Vri, cea, Gif sur Yvette, France
Correspondence toProfessor Xavier Mariette, service de rhumatologie, Hôpitaux Universitaires Paris-sud 78, rue du Général leclerc - 94270, le Kremlin-Bicêtre, France; xavier. mariette@ aphp. fr
received 22 January 2019revised 4 april 2019accepted 7 april 2019Published online First 25 april 2019
© author(s) (or their employer(s)) 2019. no commercial re-use. see rights and permissions. Published by BMJ.
Key messages
What is already known about this subject? ► Due to a strong linkage disequilibrium (LD) in humans between the 70–74 shared epitope (SE), Val11 and Phe13 on the HLA-DRB1 molecule, it is impossible to determine what position on HLA-DRB1 is the most important for predicting anti-citrullinated protein antibody-positive rheumatoid arthritis.
What does this study add? ► The absence of LD between Val11 and SE in macaques allowed us to demonstrate that the most important HLA positions to induce a T-cell response against citrullinated peptides were Val11 and Phe13 and not the 70–74 SE.
► A dose effect was observed between the number of copies of Val11 and Phe13 and the T-cell response against citrullinated peptides, further confirming the impact of these two positions.
How might this impact on clinical practice or future developments?
► Val11 and Phe13, rather than the 70–74 SE, have to be considered in the in vitro models of presentation of citrullinated peptides by HLA and in the human studies exploring the possibility to induce a specific tolerance to citrullinated peptides.
AbsTRACTObjectives Various rheumatoid arthritis (ra) Hla-DrB-1 risk haplotypes have been regrouped under the shared epitope (se) in position 70–74. The presence of Valine in position 11 (Val11) and phenylalanine in position 13 (Phe13) are also associated with ra, but it is impossible to differentiate their role compared with the se since they are in strong linkage disequilibrium (lD) in humans. similar to humans, certain macaques express the se (H6). We analysed the effect of various DrB1 haplotypes on T-cell response to citrullinated peptides (cit-P) in macaques.Methods six H6 and six non-H6 macaques were immunized with four cit-P. T-cell response was assessed using interferon γ enzyme-linked immunospot.Results animals developed a specific anti-cit-P T-cell response. surprisingly, H6 animals had a significantly lower T-cell response than non-H6. in macaques, the 70–74 se and the Val11 are on separate haplotypes. Presence of Val11 was strongly associated with the anti-cit-P T-cell response, whatever the 70–74 sequence was. This response was amplified in case of presence of Phe13.Conclusion The absence of lD between Val11 and se in macaques allowed us to demonstrate that the most important Hla positions to induce a T-cell response against cit-P were Val11 and Phe13 and not the 70–74 se.
InTROduCTIOnAmong genetic risk factors of rheumatoid arthritis (RA), the HLA-DRB1 locus is considered to have the biggest effect with odds ratios up to 4.4.1 A common amino-acid motif in positions 70–74 among RA-risk alleles of the DRB-1 molecule (QKRAA or QRRAA) is called the shared epitope (SE).2 Its association with RA has recently been more precisely defined to anti-citrullinated protein antibody (ACPA)-positive RA.3 Recent work challenged the SE hypothesis and showed that positions 11, 13, 71 and 74 explained most of the association with RA.1 4 5 There have then been attempts to find the missing link between anti-citrullinated peptides (Cit-P) production and carriage of certain alleles of the HLA-DRB1 mole-cule that favours RA. In RA patients, few studies have identified that SE carriage increased T-cell response to certain Cit-P compared with healthy controls.6–8 Binding studies showed that SE would
have a higher affinity to Cit-P.9 Mouse models require to be transgenic to express human SE. Even though T-cell response and arthritis were obtained in mice immunised with human Cit-P, these results remain controversial.10 Macaque models offer the advantage of closer genetic proximity to human DRB1 molecules. Indeed, some animals display exactly the same SE QRRAA motif as the human HLA-DRB1*01:01. This motif is present in the macaque H6 haplotype.
Based on this observation, we previously set-up a macaque model of RA by immunising these animals (H6 or not H6) with Cit-P. Using six animals we showed a trend to a specific T-cell response against Cit-P, but it was independent of the H6 haplotype.11
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Table 1 Amino-acid positions of Mauritius macaque haplotypes of the DRB-1 molecule compared with the HLA-DRB1*01:01, one of the human-shared epitope alleles
Allele nameAmino-acid sequence11/13/70–74
Haplotype name
Human allele
HLA-DRB1*01:01 L/F/QRRAA Shared epitope
Macaques allele
MAFA-DRB1*W21:01 V/S/QKRGQ H1
MAFA-DRB1*W04:02 V/S/QKRGQ H2
MAFA-DRB1*10:02 V/F/DRRAS H3
MAFA-DRB5*W03:01 D/F/RRRAE H4
MAFA-DRB5*W03:01 D/F/RRRAE H5
MAFA-DRB1*W04:01 F/P/QRRAA H6
We could not get polyarthritis but we got monarthritis after a boost of Cit-P in the joint.11
The objective of this study was to set up an experimental model by immunising macaques with Cit-P to study the HLA restriction of the T-cell response against Cit-P.
MeTHOdsMacaques and genotypingAdult captive-bred 3−5-year-old female cynomolgus macaques (Macaca fascicularis) from Mauritius island were subject to HLA genotyping by microsatellite analysis as described in Blancher et al12 and in supplementary methods.
ImmunisationAnimals were immunised eight times with Cit-P. The peptides were derived from human proteins vimentine (59–71 and 66–78), fibrinogen alpha (79-91) and aggrecan (89-103), as previously described in Bitoun et al.11 Two are strictly identical between macaques and humans, and human fibrinogen alpha and Vimentine 66 have one amino-acid difference (online supple-mentary table 1). One hundred micrograms of each peptide was emulsified with CpG and Montanide adjuvant (kindly provided by Seppic, La Garenne-Colombes, France) and injected intrader-mally in the shaved back of animals.
Immune response assessmentAnti-Cit-P and anti-arginine-peptides T-cell response were assessed using precoated interferon gamma detecting enzyme-linked immunospot plates specifically developed for non-human primates (Mabtech, Nacka Strand, Sweden) as described in supplementary methods.
statisticsTo compare continuous variables, non-parametric Mann-Whitney and Kruskal-Wallis tests were used. P value inferior to 0.05 were considered significant and graphs display *<0.05; **<0.01; ***<0.0001; ****<0.0001. Analyses were performed using GraphPad Prism V.8 software, USA.
ResulTsGenotypingSix animals carried at least one H6 allele consisting of the QRRAA motif on positions 70–74 (H6 group) (table 1) known to favour RA in humans. Six animals did not carry any H6 allele, four of them carrying the H3 haplotype (supplementary methods). Concerning position 11 of the DRB1 molecule, eight
animals carried an at-risk valine while nine animals were posi-tive for the RA risk amino-acid phenylalanine in position 13. Sequence alignment of macaque haplotypes showed 45 variable amino-acid positions in the DRB1 gene (online supplementary table 2).
Anti-Cit-P T-cell response is genotype dependentFrom week 13, the animals showed a specific anti-Cit-P response compared with the response to arginine-containing peptides (figure 1A). Despite repeated immunisation and close clinical and ultrasound follow-up, no sign of arthritis was observed. One animal died of peritoneal inflammation unrelated to the protocol during follow-up. Dividing the animals in H6 carriers and non-H6 carriers led to surprising unexpected results: the T-cell response was significantly lower in the H6 group (figure 1B). This was further confirmed by the fact that the only H6 homozy-gous animal did not show any response above the threshold for the whole length of the study (data not shown).
In order to understand these surprising results, we tried to divide the animals depending on the presence or absence of valine in position 11 (Val11) in the DRB1 molecule. In humans, Val11 is in linkage disequilibrium (LD) with the SE. But in macaques, Val11 is present on the H1-3 haplotypes and not associated with the SE on the H6 haplotype (table 1). Interest-ingly, the eight Val11-positive animals showed a significantly higher T-cell response against Cit-P than the four Val11-negative animals (figure 2A). To further comfort the role of Val11, we showed that there was a significant difference in the area under the curve of the T-cell response between carriage of zero, one or two Val11 (figure 2B p=0.0179).
Using our experimental approach, we also studied the effect of the amino-acid with the second strongest association to the risk of RA in humans, which was available in our macaques model: phenylalanine in position 13 (Phe13). In macaques, conversely to humans, Val11 and Phe13 are not in strong LD (table 1). There was an increased anti-Cit-P T-cell response in animals carrying Phe13 at two time points (figure 2C). Dividing animals according to the copy number of Phe13 and Val11 led to non-significant results regarding T-cell response to Cit-P (figure 2D). But combining copy numbers of the two risk alleles Val11 and Phe13 significantly defined different T-cell response groups (figure 2E). Thus, Phe13 had a significant impact on T-cell response to Cit-P only in combination with Val11.
Finally, animals carrying Phe11 and Pro13 (in complete LD with the H6 SE) are protected against the T-cell response to Cit-P (Figure 1B and supplementary Figure 1A), whereas Asp11 and Ser13 are neutral regarding this specific T-cell response (online supplementary figure 1B and C).
dIsCussIOnIn this study, we confirmed that the T-cell response against Cit-P obtained after immunisation with these peptides was specifically directed against Cit-P and not against arginine peptides. We then showed that carriage of the QRRAA SE led to a paradoxically significantly diminished T-cell response to Cit-P. This is due to absence of LD between the SE and Val11 (the contrary of what exists in humans). Indeed, carriage of Val11 was associated with a significantly higher T-cell response to Cit-P with a dose effect with higher response in homozy-gotes. This was amplified by the presence of Phe13 also asso-ciated with RA in humans.
The SE hypothesis defines position 70–74 motifs QKRAA and QRRAA as the main genetic risk factors for RA. In recent
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Figure 1 T-cell response to citrullinated peptides is citrulline specific and is reduced in shared epitope carrying animals. Twelve macaques were immunised eight times (arrows) with citrullinated peptides. (A) T-cell response directed against either citrullinated peptides or their arginine counterpart was regularly assessed using enzyme-linked immunospot and is displayed here. (B) T-cell response to citrullinated peptides was compared between six animals carrying at least one H6 haplotype and six animals carrying none. Mean and SEM are plotted. Individual time points are compared with Mann-Whitney test. PBMC, peripheral blood mononuclear cell.
years, individual amino-acid positions have challenged this dogma. Raychaudhuri and colleagues identified three positions on the DRB1 molecule that were associated with ACPA-pos-itive RA. Besides positions 71 and 74 that are part of the SE, position 11 was mentioned. Strikingly, this study also showed that Val11 had the strongest association to ACPA-positive RA compared with positions 71 and 74.1 The importance of Val11 was further confirmed in several cohorts of RA patients. It was associated with mortality,13 radiographic progression13 14 and disease activity.15 However, in humans, the respective roles of the SE and Val11 are very difficult to decipher since there are in strong LD.14 Similar to our results, in the studies that tried to specifically differentiate the effect of Val11 and the SE, Val11 seemed to have a stronger effect than the SE.1 13 Compared with humans, the LD of Val11 and the SE does not occur in macaques. The H6 haplotype-carrying macaques had the advantage of displaying the SE in position 70–74 and no valine in position 11. This allowed to independently assess the effect of both elements in this experimental model. Likewise, positions 11 and 13 are in even higher LD in humans. We showed that Phe13 moderately influenced the anti-Cit-P T-cell response on its own, but when combined with Val11, had a striking effect on the T-cell response. The number of copies of the SE is known to influence the risk of RA.3 This was also shown in patients for the Val11 association where homo-zygotes showed the highest association with radiographic progression.14 A similar dose effect of the four allele risks (two Val11 and two Phe13) was shown in our study in macaques.
One limitation to this study is the restricted number of animals imposed by ethical considerations, which prevents
exploring the effect of other amino-acid sites. We, thus, focused on amino-acids that had previously been shown to have the strongest effect in humans, SE and positions 11 and 13, without adjusting for the confounding effect of all the 45 variable amino-acid genetic positions (online supplementary table 2). We would need larger numbers of animal for drawing robust conclusions. Another limitation is the fact that only six haplotypes exist in regard to eight theoretical combinations between positions 11, 13 and the SE motif. It is due to the fact that there is complete LD between the H6 haplotype and Phe11 and Pro13 that are always present together. Interest-ingly, since in our study the H6 haplotype is protective, this protection could be linked to the Phe11 and Pro13 combina-tion, which reinforces the putative role of these two positions.
While the SEs are entirely embedded into the pocket 4 (P4) of the HLA-DRB1 class II molecules16 positions 11 and 13 are both outside this pocket and lie in pocket 6 (P6). Because of the preferential role of Val11 and Phe13 when compared with the SE on the level of T-cell response to Cit-P, our data strongly suggest that the amino-acid composition of P6 exerts a more important role in this response than that of the P4 in macaques. Of note, positions 11 and 13 have also been found to be associated with susceptibility to SLE but with different amino-acids: Pro 11 and Arg 13.17 Thus, it is a further indi-cation that these two positions are important probably by binding different peptides and stimulating different T-cells involved in several autoimmune diseases.
While a possible pathogenic role of ACPA in RA is recognised by most authors, the presence of a T-cell response against Cit-P is still debated. Recently, Auger et al suggested that the B-cell
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Figure 2 Valine in position 11 and to a lesser extent phenylalanine in 13 lead to greater T-cell response to citrullinated peptides. T-cell response to citrullinated peptide was assessed regularly using interferon gamma enzyme-linked immunospot and is plotted in this figure. Mean and SEM are displayed. (A) Immunised animals were divided on carriage or not of at least one valine in position 11 of the DRB1 molecule. Each time point is compared with Mann-Whitney test. (B) Individual animal AUC was calculated, and animals were then divided into three groups according to the number of Val11 copies they displayed. These three groups were compared using a one-way ANOVA namely the Kruskal-Wallis test, p=0.0179. (C) Animals were divided according to carriage of at least one Phe 13 or absence of carriage. Each time point is compared with Mann-Whitney test. (D) Individual animal AUC was calculated, and animals were then divided into groups according to the number of Phe 13 copies they displayed. The three groups were tested using a one-way ANOVA, namely, the Kruskal-Wallis test, p=0.20. (E) Individual animal AUC was calculated, and animals were then divided into groups according to the number of Phe 13 and Val11 copies they displayed. The four groups were tested using a one-way ANOVA namely the Kruskal-Wallis test, p=0.006. AUC, area under the curve; PBMC, peripheral blood mononuclear cell; Phe13, phenylalanine13.
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immunisation against Cit-P may occur without any anti-Cit-P T-cell response, but via an hapten-carrier model, PAD4 being the target of the T-cell response.18 On the contrary, numbers of studies confirmed the existence of an anti-Cit-P T-cell response in RA in humans,6 9 19 in mice transgenic with human SE, and we confirm its existence in the macaques.
While we observed an effect of genotype on the T-cell response to immunisation against Cit-P, it was insufficient to induce arthritis even in Val11 homozygotes animals. Anti-Cit-P response has been shown to be insufficient to trigger disease10 even in macaques.11 This is confirmed by the knowl-edge that patients often exhibit ACPA years before the onset of symptoms,20 and that maturation of these ACPA for a long time and second hits are probably necessary to induce clinical symptoms.
In conclusion, thanks to the absence of LD between the SE and Val11 in macaques, this study shows the predominant role of Val11 and Phe13 of the DRB-1 molecule in the T-cell response to immunisation to Cit-P. This effect is largely inde-pendent of the 70–74 SE. Further work is needed to study the specific binding of Cit-P rather than arginine peptides to HLA motifs carrying Val11 and Phe13.Acknowledgements We thank the iDMiT infrastructure staff (christophe Joubert, Benoit Delache, sebastien langlois, Jean-Marie robert, raphael Ho Tsong Fang and Julie Morin) for excellent technical assistance at the macaque facility.
Contributors sB, Pr, rlG, BM and XM designed the study, supervised the experimental design and the data analysis. sB and Pr made the experiments.sB performed statistical analysis. sB and XM wrote the original draft of the manuscript, and all authors reviewed and edited the manuscript, and provided final approval of the version to be published.
Funding The research leading to these results was supported by the labex in research on Medication and Therapeutic innovation (lerMiT) (anr10). The iDMiT infrastructure is supported by the French government ’Programme d’investissements d’avenir’ (Pia), under grant anr-11-inBs- 0008. sB was supported by two PhD grants from société Française de rhumatologie and inserM.
Competing interests none declared.
Patient consent for publication not required.
ethics approval The macaque study was approved by the regional animal care and ethics committee (comité régional d’ethique sur l’expérimentation animale Île de France sud, Fontenay-aux-roses, France; decision #a15_016). The cea institute was approved as compliant with eTs123 recommendations for animal breeding (european Union Directive 2010/63/eU, september 22, 2010) and with standards for Human care and Use of laboratory animals (animal Welfare assurance, olaW no. #a5826-01). The study was also approved by the French Department of education and research (Menesr; study no. 2015070114504151v3) as defined in French law ’décret 2013-118 from 2013 Feb 1st’.
Provenance and peer review not commissioned; externally peer reviewed.
data availability statement all data relevant to the study are included in the article or uploaded as supplementary information.
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Spondyloarthritis
CliniCal sCienCe
Aortic-vertebral interaction in ankylosing spondylitis: syndesmophyte development at the juxta-aortic vertebral rimsovira Tan,1 abhijit Dasgupta,1 John a Flynn,2 Michael M Ward 1
To cite: Tan s, Dasgupta a, Flynn Ja, et al. Ann Rheum Dis 2019;78:922–928.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214675).
1intramural Research Program, national institute of arthritis and Musculoskeletal and skin Diseases, national institutes of Health, Bethesda, Maryland, Usa2University of Chicago Medical Center, Chicago, illinois, Usa
Correspondence toDr Michael M Ward, national institutes of Health, Bethesda, MD 20892, Usa; wardm1@ mail. nih. gov
Received 31 October 2018Revised 26 February 2019accepted 23 March 2019Published Online First 6 april 2019
© author(s) (or their employer(s)) 2019. no commercial re-use. see rights and permissions. Published by BMJ.
Key messages
What is already known about this subject? ► Paravertebral ossification in DISH occurs less commonly on the left side of the spine.
► Whether the aorta is associated with the local presence or absence of syndesmophytes in ankylosing spondylitis has not been investigated.
What does this study add? ► Using CT scans, we show that syndesmophytes are less frequent and smaller in the area of the vertebral rim adjacent to the aorta than in neighbouring regions in the lower thoracic and upper lumbar spine.
How might this impact on clinical practice or future developments?
► Local inhibition of syndesmophyte growth may be due to mechanical effects of aortic pulsations.
► Factors other than vertebral inflammation can influence syndesmophyte formation in ankylosing spondylitis.
AbSTrACTObjectives The aorta inhibits paravertebral ossification in diffuse idiopathic skeletal hyperostosis. We investigated if syndesmophytes in ankylosing spondylitis (as) occurred less often at the vertebral rim near the aorta.Methods We performed thoracolumbar CT scans in 60 subjects in this cross-sectional study. The mid-thoracic spine was also scanned in 22 subjects. We divided the rim of each intervertebral disc space (iDs) into 72 angular sectors, each of 5°. We computed syndesmophyte height in each sector, and the distance from the sector to the aorta. We evaluated if syndesmophyte size or frequency in a sector was associated with its distance from the aorta.results in the 180° region of the vertebral rim centered on the sector closest to the aorta, syndesmophyte height and/or frequency varied with the distance of the sector to the aorta, with the lowest frequency and smallest mean syndesmophyte height at the sector along the rim nearest the aorta. additionally, syndesmophytes were less common in subjects and at iDss where the aorta was anatomically closer to the vertebra. no syndesmophytes were present in the sector closest to the aorta in subjects whose aorta-vertebral distance was less than 2 mm, but syndesmophytes were progressively more common among subjects whose aortas lay further from the rim.Conclusions syndesmophytes occurred less commonly and were smaller at the thoracolumbar vertebral rim near the aorta. These findings suggest that mechanical factors extrinsic to the spine and not solely vertebral inflammation, influence syndesmophyte development in as.
In ankylosing spondylitis (AS), spinal fusion can result from the development of syndesmophytes that join adjacent vertebral bodies, and from fusion of the zygapophyseal joints.1–3 Syndesmophytes are thought to develop in response to local inflam-mation in the enthesis at the annulus-bone junc-tion or in the subcortical vertebral body, but the processes that govern syndesmophyte development are incompletely understood.4–6 Vertebral osteitis and fat metaplasia, detected by MRI, is associated with future development of syndesmophytes at the adjacent vertebral rim, but this association has only modest sensitivity and specificity.7 8 Clinical factors other than inflammation that may influence the development of syndesmophytes have not been widely considered.
Emerging evidence suggests that mechanical stress may modify syndesmophyte development and their distribution.9 Patients with certain
physically demanding occupations, particularly those with exposure to whole body vibration, have more extensive syndesmophytes than those with less active occupations.10 In the lower thoracic and upper lumbar vertebrae, syndesmophytes commonly develop at the posterolateral vertebral rim, which is the portion of the vertebral body typi-cally subjected to the most mechanical stress.11 In contrast, syndesmophytes are less likely to develop at the anterior vertebral rim. The distribution of syndesmophytes within a disc space may provide clues to their pathogenesis.
Considering the anatomic proximity of the aorta to the vertebral rim, we hypothesised that the aorta might influence syndesmophyte development at the neighbouring region of the vertebrae. In diffuse idiopathic skeletal hyperostosis (DISH), paraverte-bral ossification in the thoracic spine preferentially develops contralateral to the aorta, as demonstrated by cases with situs inversus.12–14 This inhibition of paravertebral ossification has been attributed to the effects of constant aortic pulsation on the adja-cent soft tissue.12 15 We hypothesised that similar inhibition due to extrinsic mechanical forces from the aorta may occur in AS. We investigated the
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Table 1 Subject characteristics (n=54)
Value range
Age, years 46.4±10.9 24–69
Men 47 (87)
Duration of AS, years 19.0±11.4 2–53
HLA-B27 present 40 (85)*
Bath AS Functional Index (0–100) 27.5±23.4 0–89.0
Bath AS Disease Activity Index (0–10) 2.9±2.0 0–7.3
Peripheral arthritis 7 (13)
Hip or shoulder involvement 6 (11)
Median Lumbar modified Stoke AS Spine score (0–36) 4 0–26
Radiographic lumbar syndesmophytes 41 (76)
Plus-minus values are mean±SD; other values are number (per cent).*Of 47 subjects tested.AS, ankylosing spondylitis.
Figure 1 Examples of intervertebral disc spaces (from T10-T11 to L3-L4) where the portion of the rim closest to the aorta is free of syndesmophyte but is surrounded by syndesmophytes on both sides. The first row shows an axial slice of the CT scan, with yellow arrows pointing to syndesmophytes and the red arrow pointing to the aorta. The second row shows the corresponding three-dimensional surface reconstructions, with yellow surfaces denoting syndesmophytes and semitransparent red showing the aorta. Each of the six examples is from a different subject.
association between the aorta and the local presence of syndes-mophytes in the thoracolumbar spine in patients with AS, using the precise localisation provided by CT. Our research question was whether, in an affected intervertebral disc space (IDS), syndesmophytes were more likely or less likely to be present at
the vertebral rim closest to the aorta compared with other areas of the rim.
MeTHOdSPatients and study protocolWe invited adults age 18 or older with AS who were attending our clinics to participate in a research study that used spinal CT to quantitate syndesmophyte growth.11 All subjects fulfilled the modified New York criteria for the classification of AS and had a Bath AS Radiology Index (BASRI) lumbar spine score of 0 to 3 (ie, not completely fused) at study entry.16 17 Subjects with radiographic syndesmophytes were preferentially enrolled, as these were most informative. All subjects completed a phys-ical examination, health questionnaires, laboratory tests, spine radiographs and spinal CT at study entry. Some subjects were followed prospectively, while others participated in substudies that required only a single evaluation. In this cross-sectional analysis, we included only data from the baseline visit. The study protocol was approved by the institutional review boards, and all subjects gave written informed consent.
CT scanning and image analysisSixty subjects had CT scans of the lower thoracic and lumbar spine, which provided data on six IDS: T10-T11, T11-T12, T12-L1, L1-L2, L2-L3 and L3-L4. Four subjects were scanned too low and did not contribute data on T10-T11. The 22 most-recently enrolled subjects also had CT scans of the mid-tho-racic spine.18 These subjects contributed data on five additional IDSs: T5-T6, T6-T7, T7-T8, T8-T9 and T9-T10. Therefore, 22 subjects had scans of 11 IDSs, 34 subjects had scans of only 6 IDSs and 4 subjects had scans of only 5 IDSs. Earlier scans were done using either a Philips Brilliance 64 (slice thickness 1.5 mm) or GE Lightspeed Ultra (slice thickness 1.25 mm) scanner, while more recent scans were done using Siemens Somatom Flash or Somatom Force (slice thickness 1.0 mm) scanners. The estimated absorbed radiation dose was 8.01 mSv.
We computed syndesmophyte height around the vertebral rim using a validated semiautomated method.19 At each IDS, the 360° of the entire vertebral rim was divided into 72 angular sectors of 5° each. In each angular sector, height was computed for both ascending and descending syndesmophytes, which were summed and normalised to the local IDS height so that bridging had a value of 1.0, values between 0 and 1.0 represented the proportion of the IDS spanned by syndesmophytes and 0 indi-cated the absence of syndesmophytes. This measure is both reli-able and valid.19 20
We next segmented the aorta, and computed the distance of each vertebral angular sector at a given IDS to the aorta at that level. Distance was calculated to the portion of the aortic wall closest to the rim, taken as the average distance from the supe-rior and inferior rims. For each IDS, we identified the angular sector closest to the aorta, which we termed the abutting sector, and defined a 180° region around this sector (ie, 90° on either side) as the region for analysis (online supplementary figure 1). We used an IDS-specific region rather than a fixed anatomic region because the aorta migrates medially as it descends.21 We computed syndesmophyte heights in this semicircle of angular sectors and included those subjects who had a syndesmophyte in at least one of these 37 angular sectors in the analysis of a given IDS. These subjects had syndesmophytes in the region near the aorta and therefore would provide an adequate test of associations with the aorta. Subjects who did not have syndes-mophytes at a given IDS or who only had syndesmophytes far
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Figure 2 (A) Relationship between syndesmophytes and the distance of angular sectors to the aorta among all intervertebral disc spaces. The X axis represents the location of the angular sectors on the vertebral rim in relationship to the aorta. Plots are centered around the abutting sector. Angular sectors 90° on either side of the abutting sector were analysed. The blue bars represent the mean distance of each angular sector to the closest part of the aortic wall, in millimetres (left Y axis). Syndesmophyte frequency (green line) and mean syndesmophyte height (red line) are plotted for each angular sector (right Y axis). (B) Relationship stratified by distance of the aorta to the abutting sector (minimum aortovertebral (AV) distance <2.0 mm, 2.0–2.99 mm or ≥3.0 mm). IDS, intervertebral disc space.
from the aorta (ie, further than 90° away in either direction) would not be informative. Different subjects could therefore contribute data for different IDSs, depending on the location of their syndesmophytes.
Aortic wall calcifications and vertebral flattening were eval-uated to examine whether local inflammatory or mechanical mechanisms might underlie the aortic-vertebral association. We noted the presence and location of aortic wall calcifications by visual inspection of the CT scans to determine if these were associated with syndesmophytes at the adjacent vertebral rim. We also visually examined the scans for evidence of flattening of the contour of the vertebral body by the aorta.22 Flattening of the vertebral centrum is an anatomic feature attributed to pressure on the bone from the adjacent aorta and was used as another indicator of mechanical aortic-vertebral interactions.23 Flattening was assessed qualitatively by one reader (MMW) as a left-right asymmetry in the contour of the anterior vertebral rim, with either a depression or straightening of the rim on the left side.
Statistical analysisWe analysed the aortic-syndesmophyte association in relation to three factors: the angular sectors (ie, position in the arc along the rim), the IDS level and the distance of the aorta to the abut-ting sector (hereafter, minAV distance). To investigate if the angular sectors close the aorta were less likely to have syndes-mophytes than other nearby sectors, we first plotted syndesmo-phyte frequency (presence or absence of syndesmophyte at any angular sector) and mean syndesmophyte height in each angular sector in relation to the distance of the sector to the aorta. We then stratified the plots by IDS, and by minAV distance (in three groups:<2.0 mm, 2.0–2.99 mm or ≥3.0 mm). We also exam-ined the frequency of syndesmophytes in the abutting sector as a function of the minAV distance at each IDS level.
We tested whether the presence of syndesmophytes in a given angular sector was associated with its location on the vertebral rim (ie, from −90° to +90° from the abutting sector), the minAV distance or IDS level, using a multivariable logistic regression model. We also tested the interaction between the position of the angular sector on the rim and the minAV distance because the plots suggested a dependence on the distance from aorta to rim. The model was implemented using generalised estimating equations (GEE) with an exchangeable correlation structure to account for multiple IDSs per patient. We computed robust SEs using sandwich estimators. To provide global estimates of the associations, we first represented the angular sector as a contin-uous variable. To provide detailed estimates of the interaction effects, we next modelled angular sector as a categorical vari-able. Results represent the relative odds of a syndesmophyte in a given angular sector. We also examined if local aortic wall calci-fication was associated with the presence of syndesmophytes by comparing model results in IDSs with or without adjacent aortic calcification. R programs were used for analysis.
reSulTSOf the 60 subjects enrolled, 54 subjects had syndesmophytes on CT in the 180° region of the vertebral rim neighbouring the aorta in at least one IDS and were included in the analysis. Most subjects were middle-aged men with long-standing AS (table 1). The number of subjects that contributed data to the analysis of a given IDS ranged from 9 subjects at T9-T10 to 42 subjects at T11-T12. The angular sector closest to the aorta at T5-T6 was at the left lateral aspect of the vertebral body, while at L3-L4 it was near the midline (online supplementary figure 2). Characteristics of subjects excluded from analysis are provided in online supple-mentary table 1.
Inspection of axial CT images revealed a relative absence of syndesmophytes at the vertebral rim adjacent to the aorta. In
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Figure 3 Association between syndesmophyte frequency in the abutting sector and the absolute distance (in millimetres) from the vertebral rim to the aorta by intervertebral disc space. The number (N) of subjects that contributed data varied among the different intervertebral disc spaces.
several cases, syndesmophytes bracketed the region of the rim closest to the aorta (figure 1).
In plots of the association between the presence of syndes-mophytes in a particular angular sector and the distance of the sector from the aorta, syndesmophyte frequency was lowest at the abutting sector and progressively increased at sectors along the rim further from the aorta (figure 2A). Mean syndesmophyte height had a similar pattern. Individual subject data showing syndesmophytes and their location relative to the aorta as heat-maps are presented in online supplementary figure 3.
When individual IDSs were examined, the association was strongest at T10-T11, T11-T12, T12-L1 and L1-L2, and some-what less pronounced at T7-T8, T8-T9 and L2-L3 (online supplementary figure 4). The minAV distance varied among IDSs, with means<3 mm at the thoracolumbar junction, but >4 mm for other IDSs (online supplementary figure 5). This pattern suggested that the differences across IDSs were mediated by how far the aorta was from the vertebra at different levels. There was a marked difference in the association by minAV distance. Syndesmophytes were not present near the abutting sector when minAV distance was <2.0 mm, while there was no association when minAV distance was ≥3.0 mm (figure 2B). The minAV distance also varied considerably among subjects. For example, at T11-T12, this distance ranged from 0.89 mm to 6.11 mm among different subjects. There was a graded associ-ation between the frequency of syndesmophytes and the minAV distance (figure 3). Syndesmophytes occurred less often near the aorta in those subjects whose aorta was closer to the vertebral rim. No syndesmophytes were present in the abutting sector in patients whose aorta was <2 mm from the vertebral rim.
In the GEE model, both the location of the angular sector on the rim in relation to the aorta (p<0.0001) and minAV distance (p<0.0001) were associated with the likelihood of a syndesmo-phyte at a given angular sector (online supplementary table 2).
There was also a significant interaction between these measures (p<0.0001), such that the association of minAV distance was strongest at the abutting sector and nearby sectors. At the abut-ting sector, the odds of a syndesmophyte decreased by one-half with each millimetre decrease in minAV distance. However, at sectors more than 45° from the abutting sector, there was no association between syndesmophytes and minAV distance (figure 4 and online supplementary table 3). IDS level was not associated with the presence of syndesmophytes (online supple-mentary tables 2 and 3).
Aortic wall calcification was present in 25 patients, mostly below L2, and only 11 patients had calcifications in the posterior aortic wall near an IDS. Associations between minAV distance and angular sector were similar in IDSs with and without adja-cent aortic calcification (online supplementary figure 6).
Flattening of the left anterior vertebral rim was assessed for all vertebrae regardless of the presence of syndesmophytes. Flat-tening was observed primarily at the thoracolumbar junction, affecting 32% of T10 vertebrae, 37% of T11 vertebrae, 50% of T12 vertebrae and 20% of L1 vertebrae, with rare or no involve-ment at other levels (figure 5). Flattening was present in 17% of vertebrae with minAV distance <3.0 mm, compared with 5.8% with minAV distance ≥3.0 mm.
diSCuSSiOnUnderstanding the processes that regulate the development of syndesmophytes could lead to the discovery of treatments to slow or stop spinal fusion. Studies to date have focused on spinal inflammation as the precursor of syndesmophytes.6–8 Our results indicate that factors extrinsic to the spine also influence the presence of syndesmophytes in AS. Syndesmophytes occurred less commonly and were smaller at the vertebral rim near the aorta, suggesting an inhibition of syndesmophyte growth. This
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Figure 4 (A) Adjusted ORs of the association between minAV distance and presence of a syndesmophyte in an angular sector. The OR presents the likelihood for every 1 mm closer that the aorta is to the vertebral rim. At the abutting sector (labelled 0), for each 1 mm closer the aorta is to the rim, the odds that a syndesmophyte was present decreased by one-half. For angular sectors more distant from the abutting sector, the association of syndesmophytes with the distance of the aorta from the vertebral rim was less strong and was not significant at sectors more than 45° away from the abutting sector. Error bars represent 95% CIs. This is a graphic representation of the model results provided in online supplementary table 3. (B) Corresponding p values for each OR.
Figure 5 Examples of vertebral rims flattened by the aorta compared with not flattened rims at the same vertebral level. Each example was from a different subject.
association was stronger in those subjects whose aorta was closer to the spine, supporting a ‘dose-response’ association.
It is well known that paravertebral ossification in DISH mainly develops contralateral to the aorta.12–14 Ossification in DISH is easily visible on plain radiographs, which undoubtedly facilitated detection of its predominance on the right side of the spine. It is less well known that vertebral osteophytes also display later-ality. In both imaging studies and studies of anatomic specimens, osteophytes in the thoracic spine mostly develop on the right side of the vertebral bodies.24–28 This laterality is not present in the lumbar spine inferior to the aortic bifurcation, suggesting that the aorta acts to inhibit osteophyte development on left side of the thoracic spine.25 27 Our findings related to syndes-mophytes were quite similar, notwithstanding the inflammatory nature of AS. These similarities suggest that common processes may influence the development of both syndesmophytes and vertebral osteophytes.29 The small size of syndesmophytes rela-tive to the ossification in DISH, and the very local effects of the aorta, likely helped conceal this association.
The inhibition of paravertebral bone growth in DISH and of vertebral osteophytosis has been attributed to the mechanical effects of aortic pulsations on the vertebrae and surrounding tissue.12 15 24 While this is also the most likely explanation for our observations in AS, direct testing of this hypothesis is not possible. It is not completely understood how mechanical effects inhibiting bone growth are transduced at the molecular level. Mechanisms other than mechanical effects, such as para-crine effects of substances released by vascular or perivascular
tissue, may also be hypothesised. Periaortic fat secretes a variety of cytokines, chemokines, adipokines and growth factors that could potentially influence adjacent vertebrae.30 31 Most substances characterised from periaortic fat have been proin-flammatory, and therefore might be thought to promote syndes-mophyte development if diffusion to the area of the enthesis occurred. We did not find any association between aortic
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wall calcification and syndesmophyte distribution, suggesting that atheromatous inflammation in the adjacent aorta did not mediate this association, although proinflammatory media-tors may be present in the absence of aortic calcification, and processes other than atherosclerosis may have paracrine effects on bone. However, to our knowledge, studies demonstrating that abnormalities in periaortic tissue can promote osteopro-liferation have not been reported. Aortitis likely does not have a role, given its rarity and that it affects the aortic root and ascending aorta.
In contrast, the presence of vertebral flattening supports a mechanical hypothesis by demonstrating that the aorta is both close enough and powerful enough to shape vertebral morphology.22 In our study, flattening was most often seen at the thoracolumbar junction and when the aorta was close to the vertebrae. In the extreme, pressure from aortic aneurysms can cause extensive erosion of vertebral bodies.32 33 We hypothe-sise that aortic pulsation causes local movement of soft tissue to inhibit syndesmophyte formation in what may be the tissue-level analogue of exercise.
The strengths of this study include the use of a well-validated and sensitive measure of syndesmophytes, testing multiple IDSs, and examining associations with the distance to the aorta. The study also has some limitations. We studied a modest number of subjects; in particular, few were included in analyses of the mid-thoracic IDSs. However, the number was sufficient to detect strong associations with aortic distance. No subject had a right-side aorta to confirm an association with the aorta specifically, but the syndesmophyte deficit tracked with the position of the aorta down the spine and was not limited to the same angular sectors across IDSs or patients. Also, we did not directly inves-tigate potential mechanisms that might mediate this association. Our goal was to investigate the interaction between syndesmo-phytes and the aorta and not to test a global model of syndesmo-phyte development.
Our results indicate that the aorta is associated with less frequent development of syndesmophytes in AS, possibly through mechanical effects. These findings suggest a complicated relationship between mechanical forces and syndesmophyte development, where forces with differing characteristics (loca-tion, directionality, intensity or constancy) may either promote or inhibit syndesmophyte growth. Skeletal tissues are mech-anoresponsive and require loading to maintain normal struc-ture.34 35 Mechanical stress that is supraphysiological or that affects inflamed tissues may initiate ectopic bone forma-tion.9 36 37 These findings, together with our results, suggest that the responses of bone to forces generated within the skeletal system (from muscle contraction or gravity) may differ from responses to extrinsic forces, as from the aorta. It is important to recognise that this influence is relative rather than absolute, as bridging syndesmophytes can encase the IDS, including the vertebral rim near the aorta. This indicates that the association between the aorta and syndesmophyte growth may be best char-acterised as a local slowing of growth. This local slowing may contribute to the low concordance between vertebral inflam-mation on MRI and future syndesmophytes, where syndesmo-phytes are not seen after 2 years in 78%–90% of anterior lumbar vertebral corners with inflammation.7 8 38 39 Most importantly, our results indicate that factors other than vertebral inflamma-tion may affect syndesmophyte development, including factors extrinsic to the spine.Acknowledgements We thank lori Guthrie, Rn and amanda Bertram for assistance.
Contributors MMW conceived the study. sT, JaF and MMW designed the study and sT and aD did the analysis. MMW drafted the manuscript and all authors provided critical review and approval of the final version.
Funding This work was supported by the intramural Research Program, national institute of arthritis and Musculoskeletal and skin Diseases, national institutes of Health and by the Clinical Center, national institutes of Health and the Johns Hopkins University school of Medicine General Clinical Research Center (grant number M01-RR00052 from the national Center for Research Resources/niH).
Competing interests none declared.
Patient consent for publication not required.
Provenance and peer review not commissioned; externally peer reviewed.
data availability statement Data will be available on request at the conclusion of the study.
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25 nathan H. Osteophytes of the vertebral column. an anatomic study of their development according to age, race, and sex with considerations as to their etiology and significance. J Bone Joint Surg 1962;44-a:243–68.
26 nathan H. Osteophytes of the spine compressing the sympathetic trunk and splanchnic nerves in the thorax. Spine 1987;12:527–32.
27 Watanabe s, Terazawa K. age estimation from the degree of osteophyte formation of vertebral columns in Japanese. Leg Med 2006;8:156–60.
28 Goldberg RP, Carter Bl. absence of thoracic osteophytosis in the area adjacent to the aorta: computed tomography demonstration. J Comput Assist Tomogr 1978;2:173–5.
29 appel H, Maier R, loddenkemper C, et al. immunohistochemical analysis of osteoblasts in zygapophyseal joints of patients with ankylosing spondylitis reveal repair mechanisms similar to osteoarthritis. J Rheumatol 2010;37:823–8.
30 Thalmann s, Meier C. local adipose tissue depots as cardiovascular risk factors. Cardiovasc Res 2007;75:690–701.
31 G. spiroglou s, G. Kostopoulos C, n. Varakis J, et al. adipokines in periaortic and epicardial adipose tissue: differential expression and relation to atherosclerosis. JAT 2010;17:115–30.
32 Hertzer nR. aneurysms with backbone—an inside view of vertebral erosion. Ann Vasc Surg 2014;28:1813–5.
33 Wansink J, van der Kleij FGH. Vertebral-Body erosion in thoracic aortic aneurysm. N Engl J Med 2016;374:e10.
34 Rosa n, simoes R, Magalhães FD, et al. From mechanical stimulus to bone formation: a review. Med Eng Phys 2015;37:719–28.
35 sun HB, schaniel C, leong DJ, et al. Biology and mechano-response of tendon cells: progress overview and perspectives. J Orthop Res 2015;33:785–92.
36 Tsukamoto n, Maeda T, Miura H, et al. Repetitive tensile stress to rat caudal vertebrae inducing cartilage formation in the spinal ligaments: a possible role of mechanical stress in the development of ossification of the spinal ligaments. J Neurosurg Spine 2006;5:234–42.
37 sherlock JP, Joyce-shaikh B, Turner sP, et al. il-23 induces spondyloarthropathy by acting on ROR-γt+ CD3+CD4−CD8− entheseal resident T cells. Nat Med 2012;18:1069–76.
38 Maksymowych WP, Chiowchanwisawakit P, Clare T, et al. inflammatory lesions of the spine on magnetic resonance imaging predict the development of new syndesmophytes in ankylosing spondylitis: evidence of a relationship between inflammation and new bone formation. Arthritis Rheum 2009;60:93–102.
39 van der Heijde D, Machado P, Braun J, et al. MRi inflammation at the vertebral unit only marginally predicts new syndesmophyte formation: a multilevel analysis in patients with ankylosing spondylitis. Ann Rheum Dis 2012;71:369–73.
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TranslaTional science
Identification of myeloid cells in the human enthesis as the main source of local IL-23 productioncharlie Bridgewood, 1 abdulla Watad,1,2,3 Tobias russell,1 Timothy M Palmer,4 Helena Marzo-ortega,1,5 almas Khan,6 Peter a Millner,6 robert Dunsmuir,6 abhay rao,6 Peter loughenbury,6 Miriam Wittmann,1,5 richard J cuthbert,1 Dennis G McGonagle1,5
To cite: Bridgewood c, Watad a, russell T, et al. Ann Rheum Dis 2019;78:929–933.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214944).
1leeds institute of rheumatic and Musculoskeletal Medicine (lirMM), University of leeds, leeds, UK2Department of Medicine ’B’ and Zabludowicz center for autoimmune Diseases, sheba Medical center, Tel aviv, israel3sackler Faculty of Medicine, Tel aviv University, Tel aviv, israel4centre for atherothrombosis and Metabolic Disease, Hull York Medical school, University of Hull, Hull, UK5national institute for Healthresearch (niHr) leeds Biomedical research centre (Brc), leeds Teaching Hospitals, leeds, UK6leeds Teaching Hospitals nHs Trust, leeds, UK
Correspondence toProf Dennis G McGonagle, leeds institute of rheumatic and Musculoskeletal Medicine, University of leeds, leeds, United Kingdom; d. g. mcgonagle@ leeds. ac. uk
received 18 December 2018revised 19 March 2019accepted 23 March 2019Published online First 24 april 2019
© author(s) (or their employer(s)) 2019. re-use permitted under cc BY-nc. no commercial re-use. see rights and permissions. Published by BMJ.
Key messages
What is already known about this subject? ► Interleukin (IL)-23-producing myeloid cells are thought to be key to the pathogenesis of closely related disease such as psoriasis and inflammatory bowel disease.
► The enthesis is considered the cardinal lesion in psoriatic arthritis (PsA) and spondyloarthropathy (SpA) but it was previously unknown if this tissue contained a population of myeloid cells capable of IL-23 production.
► It has been considered that intestinal or systemically derived IL-23 may have been responsible for triggering enthesitis.
What does this study add? ► We demonstrate that normal spinal enthesis soft tissue and bone has resident myeloid cells.
► It was possible to induce IL-23 protein which was almost exclusive to a population of CD14+ myeloid cells.
How might this impact on clinical practice or future developments?
► This study increases the understanding of innate immunity at the normal healthy human enthesis that needs further study to understand how this might contribute to blocking IL-23 efficacy, or not in some settings, in PsA and SpA.
AbSTrACTObjective We investigated whether the normal human spinal enthesis contained resident myeloid cell populations, capable of producing pivotal proinflammatory cytokines including tumour necrosis factor (TnF) and interleukin (il)-23 and determined whether these could be modified by PDe4 inhibition.Methods normal human enthesis soft tissue (sT) and adjacent perientheseal bone (PeB) (n=15) were evaluated using immunohistochemistry (iHc), digested for myeloid cell phenotyping, sorted and stimulated with different adjuvants (lipopolysaccharide and mannan). stimulated enthesis fractions were analysed for inducible production of spondyloarthropathy disease-relevant mediators (il-23 full protein, TnF, il-1β and ccl20). Myeloid populations were also compared with matched blood populations for further mrna analysis and the effect of PDe4 inhibition was assessed.results a myeloid cell population (cD45+ HlaDr+ cD14+ cD11c+) phenotype was isolated from both the sT and adjacent PeB and termed ’cD14+ myeloid cells’ with tissue localisation confirmed by cD14+ iHc. The cD14− fraction contained a cD123+ HlaDr+ cD11c− cell population (plasmacytoid dendritic cells). The cD14+ population was the dominant entheseal producer of il-23, il-1β, TnF and ccl20. il-23 and TnF from the cD14+ population could be downregulated by a PDe4i and other agents (histamine and 8-Bromo-caMP) which elevate caMP. entheseal cD14+ cells had a broadly similar gene expression profile to the corresponding cD14+ population from matched blood but showed significantly lower ccr2 gene expression.Conclusions The human enthesis contains a cD14+ myeloid population that produces most of the inducible il-23, il-1β, TnF and ccl20. This population has similar gene expression profile to the matched blood cD14+ population.
InTrOduCTIOnSynovitis is the primary lesion in rheumatoid arthritis (RA), whereas in spondyloarthropathy (SpA)-related diseases, enthesitis is the cardinal lesion.1 Psoriatic arthritis (PsA) and the associ-ated SpA disorders are linked to multiple genetic polymorphisms in the interleukin (IL)-23/17 axis. Manipulation of this pathway results in an enthesi-tis-dependent murine arthritis and several popula-tions of IL-23R positive cells reside at the normal
murine and human enthesis.1–4 The observed difference in efficacy of IL-23/17 axis blockers and phosphodiesterase 4 inhibitor (PDE4I) therapy in RA and SpA may be a result of immunopathogenic differences linked to the location of the primary site of inflammation.1 5 6
The prevailing theories have proposed that the IL-23 driving enthesitis originates at remote sites including the gut.3 In both human psoriasis and mouse models of psoriasis, the major producers of IL-23 are skin resident myeloid cells.7 Studies also implicate myeloid cells in human enthesitis with macrophages being present in early SpA-related enthesitis.8 A recent mouse model of mechanical strain also confirmed enthesitis was dependent on myeloid infiltration.9
Accordingly, we hypothesised that the human enthesis harbours a population of myeloid cells
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Figure 1 Immunohistochemistry of CD14+ cells in (A) perientheseal bone (PEB) and (B) entheseal soft tissue (ST). Enthesis samples were separated into PEB and ST and CD14+ and CD14− cells sorted. (C–J) CD14+ and CD14− fractions were stimulated with LPS/interferon gamma (IFNү) or mannan for 48 hours. TNF, IL-23, CCL20 and IL-1β levels were measured by ELISA. n=5. Two-way analysis of variance (ANOVA) with Bonferroni multiple comparisons test was performed. *Significance from relevant unstimulated control, for example, CD14+ or CD14− unstimulated. (K) PEB was stimulated with LPS/IFNү in the presence of Golgi plug for 16 hours. Cells were surface stained for CD14+ and CD14− cells and intracellular IL-23p19 were measured. Blue histograms represent unstimulated and red stimulated. (L) Change in IL-23p19 median fluorescence intensity (MFI), following LPS/IFNү stimulation in both CD14+ and CD14− gates. n=3. Paired t-test. *p<0.05; **p<0.01; ***p<0.001. IL, interleukin; LPS, lipopolysaccharide; PEB, perientheseal bone; ST, soft tissue; TNF-α, tumour necrosis factor alpha.
capable of proinflammatory cytokine production. We investi-gated whether potential IL-23 secretion could be modulated by PDE4 inhibition, a target pathway known to show efficacy in PsA but not RA.
MATerIAlS And MeTHOdSComprehensive materials and methods can be found in online supplementary materials. Briefly, for tissue collection, normal spinous process enthesis was obtained from 15 patients (6 men and 9 women; median age 46 years) who were undergoing spinal decompression or surgery for scoliosis correction of thoracic or lumbar vertebrae using previously described methods4 (see online supplementary figure 1). The study protocol of the current investigation was approved by North West-Greater Manchester West Research Ethics Committee.
Statistical analysisStatistical significance was calculated using a one or two-way anal-ysis of variance with Bonferroni's multiple comparisons test, unless stated. Analysis was performed using Prism software (GraphPad software V.8.0, La Jolla, CA, USA). Error bars represent the SEM.
reSulTSMyeloid cells are present in entheseal soft tissue and bone and can produce Il-23Following the digestion of both the perientheseal bone (PEB) and soft tissue (ST) enthesis and flow cytometry analysis, a myeloid CD45+ CD14+ HLADR+ CD11c+ (hereafter referred to as the CD14+ population) and a CD45+ CD14− (hereafter
referred to as the CD14− population) of cells were evident (see online supplementary figures 2–4). Within the CD14− fraction, a CD123+HLADR+ CD11c− (plasmacytoid dendritic cell) popu-lation was identified, accounting for 1% of CD45+ cells (online supplementary figure 2). Due to demands of using multiple colours for flow cytometry, we decided to exclude the analysis of ILC3 cells, which we have previously identified at the human enthesis.4 Histo-logical staining of both the PEB and ST showed CD14+ enthesis immune cell populations (figure 1A, B). For control staining and isotype control, see online supplementary figure 5.
Following the isolation of CD14+ and CD14− fractions by fluorescence-activated cell sorting (FACS), they were stimulated with either mannan or lipopolysaccharide (LPS) and interferon gamma (IFNγ). Disease-relevant cytokines and chemokines, tumour necrosis factor (TNF), IL-23 (full protein p40+p19), CCL20 and IL-1β were measured by ELISA (figure 1C–J). IL-23 and CCL20 were only inducible from CD14+ cells (red box plots) following stimulation (figure 1E–H). While both frac-tions CD14+ and CD14− showed inducible TNF and IL-1β following stimulation, this was only statistically significant in the CD14+ fraction (figure 1C, D, I, J).
In terms of weight, the PEB contained more CD14+ per gram than the ST (mean=7.9×104 vs 6.8×103). Because of the scarcity of available cells in the ST, CD14+ and CD14− cells were seeded at a lower density than those from the bone (see the Materials and methods section). The individual CD14+ myeloid cell production of IL-23 and TNF expressed as pg/cell was not significantly different between PEB and ST (online supplementary figure 6). Intracel-lular staining for IL-23 of both CD14+ and CD14− populations
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Figure 2 (A–D) CD14+ fraction cells were isolated from the PEB and ST as before (n=5), and stimulated with LPS/IFNү and mannan as before, with and without PDE4I rolipram, IL-23 and TNF were measured by ELISA. (E, F) Healthy blood CD14+ cells were stimulated with LPS/IFNү (E) or mannan (F) with and without cAMP-elevating agents; rolipram, 8-Br-cAMP and histamine for 48 hours (n=7), IL-23 secretion was measured by ELISA. One-way analysis of variance (ANOVA) with Bonferroni multiple comparisons test was performed. (E, F) Significance from stimulated (LPS or mannan). (G) CD14+ cells were isolated from the PEB or matched blood and transcript analysis was performed (n=4). Black bars represent those higher expressed in blood, white bars those with higher expression in PEB. Genes highlighted in blue were undetectable in both blood and PEB. A paired t-test was performed on ΔCt values for PEB versus blood. *p<0.05; **p<0.01; ***p<0.001. IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; PEB, perientheseal bone; ST, soft tissue; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor.
following LPS/IFNγ stimulation also confirmed CD14+ as the exclusive IL-23 producer in the PEB (figure 1K, L).
Pde4I and other cAMP-elevating agents inhibit Il-23 from Cd14+ entheseal and blood myeloid cellsFrom figure 1, it was evident that CD14+ cells were the exclu-sive IL-23 producers. We next tested the ability of a PDE4I (rolipram) to modulate IL-23 secretion. Rolipram is widely used in vitro to study the effects of PDE4 inhibition and has a similar profile to apremilast.10 Blockade of the PDE4 pathway is effi-cacious in PsA but not RA. Rolipram effectively inhibited both mannan/IFNγ and LPS/IFNγ-induced IL-23 and TNF from the CD14+ fraction from both ST and PEB (figure 2A–D).
PDE4Is are thought to modulate inflammatory pathways via elevating cAMP. Due to the low number of cells yielded from the enthesis, cAMP pathway exploratory research on IL-23 secretion was conducted on the corresponding CD14+ population in blood from healthy donors. CD14+ cells were isolated by FACS and stimulated with LPS/IFNγ (figure 2E) or mannan/IFNγ (figure 2F) as before. To access the importance of the cAMP pathway in PDE4I attenuation of IL-23 production, other molecules known to activate the cAMP pathway (histamine and 8-Bromo-cAMP) were tested for their ability to inhibit IL-23 secretion and confirm the importance of the PDE4 pathway on these cells (figure 2E, F).
entheseal myeloid cells share a similar gene profile to matched blood cellsIn order to provide further functional characterisation of CD14+ myeloid cells in the enthesis, entheseal cells were compared with those isolated from matched blood (figure 2G).
Both populations had a broadly similar gene expression profile. The majority of genes analysed did not have a greater than twofold difference and only CCR2 expression was statistically significant (figure 2G). However, the majority of inflammatory cytokines and inflammatory signalling pathways trended higher in the blood population.
dISCuSSIOnIn this study, we report that both normal entheseal ST and adjacent bone have a population of CD45+ CD14+ HLDR+ CD11c+ myeloid cells. Entheseal CD14+ cells are capable of producing IL-23, TNF, IL-1β and CCL20. Moreover, IL-23 and TNF induction can be reduced by PDE4 inhibition. These obser-vations may also be of relevance to disease, given that both PDE4 inhibitors and IL-23 blockers show efficacy in PsA but not RA.
In a landmark IL-23-dependent enthesitis mouse model of SpA, it was shown that in vivo hepatic expression of IL-23 induced enthesitis via an effect on resident enthesis T cells and it was considered that the IL-23 effect was via systemically released cyto-kine.3 Presently, there are no reports of normal enthesis myeloid cells with the ability to secrete IL-23 locally. Herein we show that cells capable of producing IL-23 are present at the enthesis and that virtually all of the IL-23 was from the CD14+ fraction as opposed to the CD14− fraction. This highlights the potential importance of this myeloid cell population in disease initiation. Both bacterial and fungal adjuvants were able to induce IL-23 production from the CD14+ fraction. In agreement with this, in the SKG mouse model of arthritis where pathology originates as enthesitis, both curdlan and chlamydia promote disease.11 12 Moreover, previous
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research has shown myeloid cells such as macrophages in the early inflamed SpA enthesis.8
It was previously unknown if IL-23-producing myeloid cells at the enthesis were resident or tissue infiltrating. Classification of myeloid cell subsets within tissues is notoriously difficult and remains controversial. A new single-cell RNA-seq approach may be the only way to truly define the myeloid subsets.13 During inflammation, classical monocytes (defined as CD14high CD16low) enter tissues in a CCR2-CCL2 dependent manner. In a recent mouse model of biomechanical loading, arthritis was dependent on CCL2 production and subsequent monocyte recruitment.9 The myeloid cells we described at the enthesis may be non-classical monocytes. It is known that non-classical mono-cytes migrate and ‘patrol’ peripheral tissues, so are not defined as tissue resident macrophages in the sense of microglial or Kupffer cells.14 Non-classical monocytes (defined as CD14low CD16high) are known to express less CCR2 and inflammatory mediators when compared with classical monocytes, which would align with our findings.14 Entheseal myeloid cells in our study indeed showed statistically significantly less expression of CCR2, while basal production of inflammatory mediators such as IL-1β, IL-6 and TNF trended downwards.
We also report that fungal and bacterial adjuvants are able to induce CCL20 from enthesis CD14+ cells, but not CD14– cells. CCL20 is a chemokine that attracts IL-17-expressing cells via its ligand CCR6. In agreement with this, PsA synovial fluid levels of CCL20 correlate with disease severity.15 IL-17-expressing ILC3 and T cells are also CCR6+ from the synovial fluid of patients with PsA.16 17 These findings suggest a link between enthesis myeloid cells and the cells that preferentially accumulate in the adjacent joint cavity in PsA.
While therapeutic blockage of IL-23 has demonstrated effi-cacy in PsA, recent results from a clinical trial in patients with ankylosing spondylitis have proved disappointing.18 It has been hypothesised that variation in efficacy may be due to a differing immunopathogenesis including different immune cell popula-tions between the spine and peripheral joint.19 A recent paper using an HLA-B27 rat model showed that SpA pathology is dependent on IL-23 for initiation but not disease persistence.20 This may indeed point towards the idea of IL-17 independent of IL-23 for disease progression, while IL-23 is required for disease initiation. Related research has shown that torn supraspinatus tendon contains IL-17+ cells including T cells; however, it is not known if these cells can produce IL-17 independently of IL-23.21
PDE4Is are thought to mediate anti-inflammatory effects by elevating cAMP. Other cAMP pathway activators such as hista-mine and 8-Bromo-cAMP are known to attenuate inflammatory cytokine production from peripheral blood mononuclear cells; however, this is the first report showing this on tissue myeloid cells.22 Previous research has suggested cAMP-elevating agents downregulate IL-12p40, the common chain between IL-12 and IL-23, which in theory may also account for the downregulation of full IL-23 protein (p19+p40) observed (figure 2A–D).22 Further work is required to decipher the exact mechanisms in which cAMP elevation regulates individual IL-12 family member chains.
In conclusion, these data demonstrate that the normal enthesis contains a population of myeloid cells capable of local innate cytokine production including TNF and IL-23. While CD14+ HLADR+ CD11c+ confirm the presence of a myeloid pheno-type, further work is required to fully characterise this popu-lation. The in vivo triggers of myeloid-derived TNF and IL-23 remain to be fully elucidated but our findings show that the normal enthesis contains myeloid cells capable of locally contrib-uting to disease.
Acknowledgements The authors thank abed el rahman Wattad for providing the online supplementary figure 1. This article/paper/report presentsindependent research funded/supported by the national institute for Healthresearch (niHr) leeds Biomedical research centre (Brc). The views expressed arethose of the author(s) and not necessarily those of the niHr or the Departmentof Health and social care.
Contributors cB, aW, TP, Tr, MW, rJc and DGM contributed to the acquisition, analysis or interpretation of data and critical revision of the manuscript. aK, HMo PaM, rD, aB and Pl contributed to the acquisition of data and drafting of the manuscript.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests none declared.
Patient consent for publication not required.
ethics approval The current study has been approved by the University of leeds ethical approval committee.
Provenance and peer review not commissioned; externally peer reviewed.
data sharing statement all data relevant to the study are included in the article or uploaded as supplementary information.
Open access This is an open access article distributed in accordance with the creative commons attribution non commercial (cc BY-nc 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.
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Systemic lupus erythematosus
CliniCal sCienCe
Altered cognitive function in systemic lupus erythematosus and associations with inflammation and functional and structural brain changesMichelle Barraclough, 1,2 shane McKie,3 Ben Parker,2,4 alan Jackson,5 Philip Pemberton,6 Rebecca elliott,7 ian n Bruce1,2
To cite: Barraclough M, McKie s, Parker B, et al. Ann Rheum Dis 2019;78:934–940.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214677).
For numbered affiliations see end of article.
Correspondence toProfessor ian n Bruce, arthritis Research UK Centre for epidemiology, Division of Musculoskeletal and Dermatological sciences, school of Biological sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9Pl, UK; ian. bruce@ manchester. ac. uk
Barraclough M, elliott R, Parker B, McKie s, Jackson a, Pemberton P, Bruce in. altered cognitive function in systemic lupus erythematosus and associations with inflammation and functional brain changes [abstract]. arthritis Rheumatol. 2018; 70 (suppl 10). https:// acrabstracts. org/ abstract/ altered- cognitive- function- in- systemic- lupus- erythematosus- and- associations- with- inflammation- and- functional- brain- changes/.
Received 31 October 2018Revised 15 February 2019accepted 2 March 2019Published Online First 12 april 2019
© author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.
AbSTrACTObjectives Cognitive dysfunction (CD) is common in systemic lupus erythematosus (sle) but the cause remains unclear and treatment options are limited. We aimed to compare cognitive function in sle and healthy controls (HCs) using both behavioural and neuroimaging techniques.Methods Patients with sle with stable disease and HCs were recruited. Clinical and psychological data were collected along with a blood sample for relevant biomarkers. neurocognitive function was assessed using tests from the Cambridge neuropsychological Test automated Battery (CanTaB) and functional magnetic resonance imaging (fMRi) was used to examine brain responses to working memory (WM) and emotional processing (facial emotional recognition task, FeRT) tasks.results Compared with HCs (n=30), patients with sle (n=36) scored higher on measures of depression, fatigue and had higher hsCRP (p=0.013), il-6 (p=0.003) and B lymphocyte stimulator (p<0.001). Patients with sle had poorer performance on a task of sustained attention (p=0.002) and had altered brain responses, particularly in default mode network (DMn) regions and the caudate, during the WM task. Higher organ damage and higher VCaM-1 were associated with less attenuation of the DMn (p=0.005 and p=0.01, respectively) and lower BOlD signal in the caudate areas (p=0.005 and p=0.001, respectively). increased il-6 was also associated with lower BOlD signal in caudate areas (p=0.032).Conclusions sustained attention was impaired in patients with sle. Poor attenuation of the DMn may contribute to cognitive impairments in sle and our data suggest that in addition to mood and fatigue inflammatory mechanisms and organ damage impact cognitive functioning in sle. The multifaceted nature of CD in sle means any therapeutic interventions should be individually tailored.
InTrOduCTIOnCognitive dysfunction (CD) is one of the most commonly reported neuropsychiatric symptoms in patients with systemic lupus erythematosus (SLE) and significantly affects quality of life. While it has been reported in up to 90% of patients,1 treatment options remain limited in large part due to uncer-tainty around the cause(s), the lack of a consistent measure and the observation that patients with SLE
may perform similarly to healthy controls (HCs) on objective testing.2
CD is common in other chronic conditions, such as inflammatory bowel disease and multiple scle-rosis3 and factors associated with chronic disease such as mood disorders, medications and fatigue can all affect cognition. Specific SLE-factors are also hypothesised to play a role,4 with reported associa-tions between autoantibodies and CD, particularly anti-N-methyl-D-asparate, anti-dsDNA and anti-phospholipid (aPL) antibodies.5 Structural brain alterations in SLE such as cerebrovascular events, and the increased number of white matter hyper-intensities6 also may contribute, although others have suggested that such structural changes are not directly associated with CD. As such, clinical and imaging biomarkers of CD in SLE remain elusive.
Key messages
What is already known about this subject? ► Cognitive dysfunction (CD) is a significant problem in systemic lupus erythematosus (SLE), reported to affect up to 90% of patients.
► The cause is unclear and as such treatment is limited.
► No consistent measure of CD in SLE. ► Limited correlations found between structural brain abnormalities in SLE and cognitive function.
► Increasing interest in the use of functional magnetic resonance imaging (fMRI) in the assessment of cognitive function in SLE.
What does this study add? ► One of the largest fMRI studies in SLE. ► Assesses behavioural, functional and structural abnormalities in relation to cognitive function in SLE.
► Addresses the multifactorial nature of CD in SLE.
► Excludes patients with neuropsychiatric SLE (NPSLE) with the aim of examining the milder cognitive deficits reported by the majority of patients to address the concept of ‘brain fog’.
► Indicates that SLE-specific factors (e.g., inflammation, disease damage/duration) are associated with CD as well as chronic disease factors (e.g., fatigue, depression, pain, medication).
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Key messages
How might this impact on clinical practice or future developments?
► Increases awareness that CD in SLE has multiple drivers and therefore treatment should be individually tailored.
► Helps in the development of a biomarker of CD in SLE. ► Aids future clinical trials by highlighting which key factors should be included in the study design.
► Highlights that patients with SLE are likely to employ compensatory brain mechanisms to maintain cognitive performance. As such patients may score similarly to healthy controls of objective measures of cognition but may fatigue quicker. This needs to be taken into consideration in any clinical trials or clinical assessments.
The American College of Rheumatology (ACR) estab-lished a recommended battery of cognitive tests but these require a trained professional to administer. An alternative is the Cambridge Neuropsychological Test Automated Battery (CANTAB), a tool that can assess changes in cognition over time, has been validated in many clinical settings, requires minimally trained administrators and has been successfully used in SLE.7 Another objective approach is functional magnetic resonance imaging (fMRI). fMRI gives a proxy measure of neuronal acti-vation during cognitive testing. To date, only a few articles have reported fMRI findings in SLE but they suggest that patients with SLE may employ compensatory mechanisms within the brain to maintain adequate cognitive performance.8 Even fewer studies9 10 have examined cognition in SLE using a combination of behavioural, functional and structural assessments, although such an approach may better help identify causes and targets for therapy.
We aimed to compare cognitive function between patients with SLE with stable disease and HCs using CANTAB and fMRI. Variables that are known to affect cognition were also examined with differences between the two groups reported.
MeTHOdSPatients with SLE were recruited from Rheumatology depart-ments at Manchester University NHS Foundation Trust Hospi-tals. HCs were recruited via study participants (e.g., friends) and social media. All SLE participants fulfilled ACR 199711 or Systemic Lupus International Collaborating Clinics (SLICC) criteria12 for SLE and were considered clinically stable if no change of treatment was required, and their Systemic Lupus Erythematosus Disease Activity Index-2000 (SLEDAI-2K) score was ≤4.13 Participants with a history of epilepsy, stroke, severe depression/psychiatric conditions or certain central nervous system (CNS)-acting medications were excluded. Severe depres-sion was defined as currently receiving treatment and/or scoring ≥20 on the Montgomery Asberg Depression Rating Scale (MADRS). Participants on low-dose CNS-acting medications or who were taking no more than three such medications (and only if being used to treat conditions other than depression, such as fibromyalgia) were included.
Disease activity was assessed using the British Isles Lupus Assessment Group Index BILAG 200414 and SLEDAI-2K, and organ damage using the SLICC/ACR Damage Index (SDI).15 Specific biomarkers of the inflammatory response activation (B lymphocyte stimulator (BLyS), high sensitivity C reactive protein [hsCRP], interleukin 6 (IL-6)) and vascular/endothelial
(vascular cell adhesion molecule-1 (VCAM-1), vascular endothe-lial growth factor (VEGF)) were measured.
All participants completed validated questionnaires on depres-sion, anxiety and fatigue:
HADS: Hospital Anxiety and Depression Scale.16
BDI-II: Becks Depression Inventory-II.17
MADRS: Montgomery Asberg Depression Rating Scale.18
FSMC: Fatigue Scale for Motor and Cognitive Functions.19
After a literature review, we selected six CANTAB tests20 that assessed:
Visual memory and new learning (PAL: Paired Associates Learning).
Immediate and delayed verbal memory (VRM: Verbal Recog-nition Memory).
Emotional processing (ERT: Emotional Recognition Test).Sustained attention (RVP: Rapid Visual Information
Processing).Executive function (OTS: One Touch Stockings).Spatial working memory (SWM: Spatial Working Memory).Two functional scans were performed using a 3.0 Telsa Philips
Gyroscan ACS NT (Philips, Best, NL) MR scanner while partici-pants completed a WM task (n-back) and a facial emotional recog-nition task (FERT). Two structural scans were also performed: a fluid attenuated inversion recovery and T1-weighted magnetisa-tion-prepared rapid gradient-echo.
All behavioural and assessment data were analysed using inde-pendent t-tests for parametric data, Mann-Whitney U tests for non-parametric data and χ² for proportional data in SPSS 22 and group region of interest analyses were undertaken for the fMRI data using SPM12.
The target number of participants recruited to the study was determined based on fMRI power guidance, where a sample size of between 16 and 32 is considered acceptable.21
To examine any possible associations between SLE and CD exploratory Pearson/Spearman’s correlations and χ²s were undertaken using the SLE group only. These correlations were only conducted using the CANTAB tasks, structural brain abnormalities and fMRI results that were significantly different between the HC and SLE groups. These variables were assessed against factors proposed to affect cognition, including disease duration, disease activity, damage, medication use, aPL/LAC as well as measures of depression and fatigue.
Further details on all methods can be found in the online supplementary data.
reSulTSdemographic and clinical findingsThe SLE group were typical of a stable SLE cohort (table 1) and both groups were matched on age, gender, handedness and ethnicity; patients with SLE had fewer years in education and a lower mean IQ (table 2). The SLE group had higher depres-sion scores (medians within the normal ranges). For each group, the percentages of participants that scored within the mild clin-ical ranges for depression/anxiety were: MADRS 0% HC, 6% SLE, BDI-II 6% HC, 33% SLE, HADS-D 3% HC, 39% SLE and HADS-A 20% HC, 39% SLE, despite excluding for clin-ical depression. The SLE group also had higher levels of motor and cognitive fatigue with median scores in the ‘severe’ (motor) and ‘moderate’ (cognitive) fatigue categories. Several biomarkers of inflammation and endothelial activation showed statistical difference between the two groups (table 2).
All measures of depression positively correlated with both cognitive and motor scores of fatigue (FSMC) and negatively
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Table 1 Clinical and immunological characteristic of the SLE participants (n=36)
Characteristic n (%) or median (lQ, uQ)
Female sex 34 (94%)
Disease duration (years) 10.5 (5, 15)
ANA positive (ever) 34 (94.4)
Elevated IgG anti-dsDNA antibody* 9 (26)
Low C3 or C4* 12 (35)
Anticardiolipin (aCL) antibody-positive* 9 (26)
Lupus anticoagulant positive* 6 (18)
BILAG total score† 1 (0, 2)
SLEDAI-2K 2 (0, 2)
SDI 0 (0, 1)
9/36 (25%) had a score ≥1
Oral corticosteroids (y/n) 12 (33.3)
Average daily corticosteroid dose (mg) (n=12) 8.75 (6.25, 11.25)
Current immunosuppressant use 15 (41.7)
Current antimalarial use 22 (61.1)
Biological medication 3 (8.3)
*At time of study.†Score calculated as stated in Yee et al.22
ANA, antinuclear antibody; BILAG, British Isles Lupus Assessment Group Index; C3, complement component 3; C4, complement component 4; SDI, The Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index; IgG ds-DNA, immunoglobulin G double-stranded DNA.
Table 2 Demographic, psychiatric, fatigue and biomarker characteristics across the participant groups
Variable
Sle (n=36) HC (n=30)
P value Mean (Sd), Median (lQ, uQ) or n (%)
Demographic
Age (years) 40 (32, 48.75) 32 (27, 46.5) P=0.14
Gender (% female) 34 (94) 30 (100) P=0.19
Handedness (% right-handed)
30 (83) 28 (93) P=0.34
Years in education 16.11 (3.51) 17.97 (3.40) P=0.034
WTAR (IQ) 102.5 (98.25, 108) 111 (105, 114) p=0.001
Ethnic origin P=0.132
Caucasian 24 (66.7) 24 (80)
Black Caribbean 4 (11.1) 0
Black African 3 (8.3) 0
Indian 1 (2.8) 0
Bangladeshi 0 1 (3.3)
Chinese 1 (2.8) 1 (3.3)
Other 3 (8.3) 4 (13.3)
Depression
MADRS 4 (1, 8) 1 (0, 3) P=0.012
HADS - D 4 (1, 9) 1 (0, 2) P<0.001
BDI - II 10 (4, 20.25) 3 (0.75, 8) P=0.002
Anxiety
HADS – A 6 (3, 10.5) 5 (2, 7) P=0.08
Fatigue
FSMC – Motor score 36 (22, 40.5) 14 (11.5, 18.5) P<0.001
FSMC – Cognitive score 31 (22, 40) 14 (11.5, 18.5) P<0.001
FSMC – total score 67.5 (44.75, 80.5) 27 (23, 37) P<0.001
Biomarkers of inflammation and endothelial activation
hsCRP (mg/L)1 1.44 (0.66, 5.06) 0.88 (0.39, 1.39) P=0.013
IL-6 (pg/mL)1 1.67 (0.50, 5.33) 0.50 (0.50, 1.32) P=0.003
VCAM-1 (ng/mL)2 474.93 (194.30) 345.66 (53.79) P=0.001
VEGF (pg/mL)1 66.04 (13.93, 139.60) 45.42 (6.04, 114.93) P=0.275
BLyS (ng/mL)1 0.51 (0.35, 0.71) 0.34 (0.27, 0.39) P<0.001
Missing data: WTAR not included for 3 HCs, and 4 SLEs, these participants’ first language was not English and/or they had dyslexia, as such it was felt that the scale would not accurately measure IQ in these participants. MADRS–5 SLE, 2 HC; FSMC–2 SLE, 1 HC hsCRP, IL-6, VEGF, BLyS-2 HC, 2 SLE; VCAM-1–2 SLE.P-values in bold are significant at <0.05.BDI-II, Becks Depression Inventory - II; BLyS, B lymphocyte stimulator; FSMC, Fatigue Scale for Motor and Cognitive Functions; HADS-A, Hospital Anxiety and Depression Scale–Anxiety score; HADS-D, Hospital Anxiety and Depression Scale–Depression score; IL-6, Interleukin 6; MADRS, Montgomery Asberg Depression Rating Scale; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor;WTAR, Weschler Test of Adult Reading; hsCRP, high sensitivity C reactive protein;.
with years in education (see online supplementary table S1). In addition, hsCRP positively correlated with HADS-depression score (rs=0.43, p=0.013).
CAnTAb findingsFrom the 66 participants (36 SLE and 30 HC) who underwent CANTAB testing, 2 SLE participants did not complete all tests due to fatigue.
The SLE group performed less well on the RVP task (a test of sustained attention) compared with the HC group (13 [12, 20] vs 20 [15.75, 22], p=0.002). Compared with the norma-tive data available from CANTAB 33.3% of the SLE participants scored one or more SDs below the RVP mean, whereas only 1% of HC group scored one or more SDs below the mean. The SLE group was also slower to identify emotions from the ERT task and identified more of the emotions incorrectly compared with HCs (p=0.012 and p=0.019, respectively) (table 3).
Structural MrI findingsStructural analysis was conducted on 53 participants (30 HC and 23 SLE). The SLE group had significantly more and larger perivascular spaces (PVS) in the centrum semiovale (CSO-VRS), χ2=15.50, p<0.001. The differences between the SLE and HC group for the PVS in the basal ganglia (BG-VRS) did not reach significance, χ2=8.96, p=0.077 (see online supplementary tables S2-S4).
Functional MrI findingsNot all patients underwent an MRI scan due to scheduling, discomfort and artefact issues. Overall, 23 SLE and 29 HC participants had fMRI scan data available for analysis.
n-back task resultsPatients with SLE performed worse than HCs on the 0-back level (measure of attention, p=0.008) and were also slower to
respond correctly on the 1-back and 2-back levels (measures of WM, p=0.019 and p=0.025, respectively) compared with the HC group (see online supplementary table S5).
Working memory condition (2-0back)ROI analysis revealed significant results for the negative effect of the 2-0back condition. This condition highlights regions where the BOLD signal reduced for both groups during the WM task. Significant results were found in the left transverse temporal gyrus (t=2.12, p=0.039), right superior temporal gyrus (t=2.09, p=0.041) and right caudate (t=−2.45, p=0.018). The left caudate (p=0.058) also showed a similar BOLD response to the right caudate. Results in the left transverse temporal gyrus (LTTG-WM) and right superior temporal gyrus (RSTG-WM) showed a more decreased BOLD response for the HC group compared with the SLE group. In the caudate the reverse was found, the SLE group had a more significant decrease in BOLD
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Table 3 Differences between the SLE and HC groups for each of the CANTAB outcome measures
Variable* Measurement
Sle, n=36 HC, n=30
P value Mean (Sd), Median (lQ, uQ), n (%)
PAL+(visual memory and new learning)
Total errors (adjusted) 29.50 (19.00, 79.75) 24 (10.75, 48.75) P=0.095
VRM(verbal memory)
Free recall – total correct(Max.=18)
10 (8, 13) 10 (8.75, 14) P=0.327
RVP(attention)
Total hits(Max.=27)
13 (12, 20) 20 (15.75, 22) P=0.002
ERT(emotional processing)
Average percentage correct – total (%) 61.49 (8.85) 66.94 (9.36) P=0.019
Overall mean response latency – total(ms)+
1626.10 (1411.71, 2274.22) 1343.15 (1152.27, 1744.23) P=0.012
OTS+(executive function)
Mean choices to correct 1.40 (1.27, 1.73) 1.33 (1.18, 1.62) P=0.484
SWM+(working memory)
Between errors 108.41 (57.96) 94.73 (52.36) P=0.328
Missing data: VRM: 1 SLE, ERT: 1 SLE; RVP: 1 SLE; SWM: 2 SLE; OTS: 1 SLE.P-values in bold are significant at <0.05.*Higher scores indicate better performance except where indicated with a “+”.ERT, emotional recognition task; OTS, one touch stockings; Pal, paired associate learning; RVP, rapid information visual processing; SWM, spatial working memory; VRM, verbal recognition memory.
Figure 1 Significantly different BOLD responses for the SLE and HC groups for the n-back task, 2-0back, negative effect of task. HC, healthy control; SLE, systemic lupus erythematosus.
signal (figure 1). No significant differences between the groups were found for the positive effect of the 2-0back condition.
Attention condition (0back-rest)Using ROI analysis, the SLE group had a greater decrease in signal in the lingual gyrus compared with the HC group (figure 2). No significant differences between the groups were found for the positive effect of the 0back-rest condition.
FERT taskBehaviourally, the SLE group was slower to correctly determine whether a face was female or male when displaying sadness (p=0.035) (see online supplementary table S6). In an ROI anal-ysis, in the sadness-neutral condition, the positive effect of task for this condition showed that the SLE group had an increased
BOLD response in frontal areas compared with the HC group (figure 3). There were no differences in the BOLD responses for the negative effect of task.
exploratory associations between Sle and cognitive functionImproved performance on the attention task negatively correlated with the signal in a default mode network (DMN) region during the WM task (RVP associated with the RSTG-WM, r=−0.60, p=0.003).
Better performance on the emotional processing task nega-tively correlated with the signal in a DMN area during a WM task (ERT average percentage correct associated with the RSTG-WM, r=−0.72, p<0.001). Also, the mean response latency for the identification of the emotions in the emotional processing task negatively correlated with the signal in a cognitive region
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Figure 2 Significantly different BOLD response in the lingual gyrus for the SLE and HC groups, for the n-back task, 0back-rest, negative effect of task. HC, healthy control; SLE, systemic lupus erythematosus.
Figure 3 Significantly different BOLD response for the FERT, sadness-neutral, positive effect of task, SLE vs HC. FERT, facial emotional recognition task; HC, healthy control; SLE, systemic lupus erythematosus.
during the WM task (ERT overall mean response latency asso-ciated with left caudate-WM, r=−0.51, p=0.013), implying a greater response in the DMN when performing better and quicker on an emotional processing task.
Structurally, the enlarged PVS in the centrum semiovale (CSO-VRS) and basal ganglia (BG-VRS) were associated with years in education (rs=−0.48, p=0.022 and rs=−0.45, p=0.035, respectively). Neither was associated with vascular biomarkers including LAC or aCL positivity, or VCAM-1.
The DMN areas (left transverse temporal gyrus and right superior temporal gyrus), areas that are usually attenuated during cognitive tasks, were positively correlated with depres-sion (LTTG-WM with MADRS, r=0.45, p=0.036), VCAM-1 (with RSTG-WM, r=0.53, p=0.01), SDI (with RSTG-WM, rs=0.56, p=0.005) and current use of biological medication (with RSTG-WM, r=0.60, p=0.003). The cognitive areas (right and left caudate) were negatively correlated with IL-6 (with the right, rs=−0.47, p=0.032), VCAM-1 (with the left, r=−0.65, p=0.001), SDI (with the left, rs=−0.57, p=0.005), disease duration (with the left, r=−0.49, p=0.019) and current use of biological medication (r=−0.52, p=0.011). The left caudate was positively correlated with cognitive fatigue as measured by the FSMC-Cognition score (r=0.43, p=0.047). Also, the right caudate was positively correlated with aCL positivity (r=0.51, p=0.015) but not with LAC positivity.
The attention condition of the n-back task, in the visual atten-tion region of the lingual gyrus, positively correlated with the BILAG total score (rs=0.45, p=0.033), IL-6 (rs=0.44, p=0.036), current use of immunosuppressant (r=0.48, p=0.019) and anti-malarial medication (r=0.47, p=0.028) and negatively with BG-VRS score (rs=−0.46, p=0.030) suggesting that higher
inflammatory disease activity increased responses in an atten-tional brain region during an attention task.
The BOLD response to sad faces from the FERT task in the left frontal cluster negatively correlated with the SDI score (rs=−0.57, p=0.005) and disease duration (r=−0.43, 0.047).
dISCuSSIOnWe have identified structural, cognitive and fMRI differences in patients with SLE. While overall cognitive function was compa-rable between groups, the SLE group was less accurate on a test of sustained attention. Anatomically, we found increases in the PVS in the centrum semiovale in 43% of SLE participants and no controls. Using task-based fMRI, there was significant inter-ference in emotional tasks and a reduced ability of patients with SLE to suppress the DMN during cognitive tasks.
Our data support previous work showing that attention is the most common cognitive problem in SLE.4 Such attention deficits can result in problems with other cognitive functions, such as WM although in this study we did not find any other non-emotional cognitive problem in the SLE group. A more detailed assessment of these relationships was limited due to time constraints with testing; however, follow-up studies focusing on these key inter-relationships are planned.
The 0back-rest condition of the fMRI n-back task is a neuronal marker of sustained attention. Patients with SLE had a larger task-negative BOLD response in the lingual gyrus compared with the HC group. This region has been associated with visual attention, visual encoding/processing and WM.23–25 This may explain why our SLE group performed worse on the behavioural (CANTAB) attention task. The few studies published using fMRI in SLE have suggested that patients with SLE employ compen-satory brain mechanisms to maintain cognitive function.8 Our cohort may have failed to recruit compensatory mechanisms on the challenging sustained attention task, resulting in perfor-mance deficits. Exploratory analyses also found that the response to attention in the lingual gyrus negatively correlated with an increase in PVS in the basal ganglia. Previous studies have found patients with SLE to have a greater number and larger PVS in the basal ganglia which did not reach significance in our study; we did however find differences between the HC and SLE in PVS in the centrum semiovale. PVS is an imaging marker for cerebral small vessel disease;26 27 however, we did not find any correla-tions between several serological markers of vascular disease (LAC, aCL antibodies and VCAM-1) and CSO-VRS, although we did exclude patients with severe vascular disease.
On the CANTAB battery, there were no differences between groups for the behavioural WM tasks. However, on fMRI patients with SLE had less task negative BOLD signals in the left transverse temporal gyrus and right superior temporal gyrus. These areas are part of the DMN, which is usually inac-tive during cognitive tasks28 and active during rest and internal processes, such as self-reflective processes and planning.23 28 The limited ability to reduce these signals in SLE implies an inability to inhibit self-reflective processes which can impede perfor-mance on cognitive tasks that do not usually have an emotional component, by allowing emotional interference from self-reflec-tion and worries about task performance.29 In support of this, the FERT fMRI task found that patients with SLE had a greater response to viewing sad faces in frontal regions compared with the HC group. Such increased responses to sad expressions is also associated with depression.30–32 Similarly, patients with SLE were less accurate in correctly identifying emotions on CANTAB and showed evidence of reduced response latency implying a
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level of psychomotor slowing, both of which are associated with depression32 33 and may contribute to some of the differences observed between groups. This is despite our groups scoring within normal ranges on the depression scales and that major depression was an exclusion criteria. We therefore cannot rule out the potential impact mood may have on cognitive perfor-mance in this SLE group even at the subtle end of the scale. Also, we recruited a low disease activity cohort and excluded NPSLE cases, so there may be different subtypes depending on CD severity.
We also noted differences in the caudate between the two groups. Patients with SLE had a larger task negative response compared with the HC group. The caudate, via the network linked to the dorsolateral prefrontal cortex, has been impli-cated in WM34 but usually as an area with an increased BOLD response during WM tasks. The attenuated response we found is in contrast to Mak et al who noted, using a different executive function task, an increased BOLD response in the caudate body from patients with SLE.35 It is therefore unclear if the differences we found are task specific and why our findings were in a task negative direction.
As noted, the SLE group had significantly higher scores on scales for depression and fatigue. While depression scores were still within the ‘normal range’ both motor and cognitive fatigue scores were higher in the SLE group. Both fatigue and mood can affect cognition36 37 and these symptoms are highly prevalent in SLE populations. In our study, mood and fatigue negatively impact neurocognition. It is increasingly recognised that inflam-mation and mood are closely interlinked.38and we found that VCAM-1, IL-6 and BILAG 200414 scores correlated with cogni-tive brain mechanisms, supporting the hypothesis that inflamma-tion in SLE contributes to CD. We also noted associations with the SDI and disease duration, strengthening the suggestion that specific SLE factors directly impact on cognitive function over time.39
We acknowledge some limitations to this study. For the non-fMRI analysis, the participant numbers are small; due to strict exclusion criteria and the use of fMRI as the main outcome. We also made no adjustments for multiple compar-isons. However, many factors were closely correlated and in an exploratory study such as this, a Bonferroni correction would be too conservative. The HC group had a higher IQ and slightly more years in education than the SLE group. IQ can affect performance on cognitive tests but for many of the cognitive measures, no differences were seen so it is unlikely that this was the case. It was also impossible to recruit a SLE patient group on no medication. Patients on low dose psycho-active medications were included as well as those on corti-costeroids. The correlations within the SLE group found no significant associations between corticosteroid dose and cogni-tive measures. Lastly, we chose to use the CANTAB battery as it is a sensitive measure of cognitive function that can assess changes over time and is easy to administer; however, some research has suggested that the tests measure overall cognition but cannot be divided into specific domains, such as executive function, so caution may be needed when interpreting indi-vidual test results.40
In patients with SLE, we have noted impairments in sustained attention while other non-emotional cognitive func-tions remained unaffected. Poor attenuation of the DMN may contribute to CD in SLE, although prospective studies may be needed to confirm this, and our data suggest that in addition to mood and fatigue, inflammatory mechanisms and organ damage impact cognitive functioning in SLE.
Clinically, this study has implications when advising patients about CD in SLE. It has highlighted the multifaceted nature of CD in SLE and that future therapeutic approaches will need to be individually tailored to address the relevant drivers in indi-vidual patients.
Author affiliations1arthritis Research UK Centre for epidemiology, Division of Musculoskeletal and Dermatological sciences, school of Biological sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK2niHR Manchester Biomedical Research Centre, Manchester University nHs Foundation Trust, Manchester academic Health science Centre, Manchester, UK3FBMH Platform sciences, enabling Technologies & infrastructure, FBMH Research & innovation, Manchester academic Health science Centre, The University of Manchester, Manchester, UK4Division of Musculoskeletal and Dermatological sciences, school of Biological sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK5Wolfson Molecular imaging Centre, institute of imaging and Bioinformatics, The University of Manchester, Manchester, UK6Department of Clinical Biochemistry, Manchester University nHs Foundation Trust, Manchester, UK7neuroscience and Psychiatry Unit, Division of neuroscience and experimental Psychology, The University of Manchester, Manchester, UK
Acknowledgements This research was funded by the niHR Manchester Biomedical Research Centre and cofunded by an unrestricted grant from sanofi Genzyme. it was carried out at the niHR Manchester Clinical Research Facility. The work in this manuscript was supported by infrastructure support from the arthritis Research UK Centre for epidemiology (grant reference 20380).
Contributors all authors were involved in some/all of the following: substantial contributions to the conception or design of the work or the acquisition, analysis or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Funding This research was funded by the niHR Manchester Biomedical Research Centre and co-funded by an unrestricted grant from sanofi Genzyme (GZ-2011-10813).
disclaimer The views expressed are those of the author(s) and not necessarily those of the nHs, the niHR or the Department of Health.
Competing interests This study was partially funded by an unrestricted grant from sanofi Genzyme and supported by the Manchester academic Health sciences Centre (MaHsC), niHR Manchester Biomedical Research Centre and niHR Manchester Clinical Research Facility.
Patient consent for publication not required.
ethics approval This study was approved by the national Research ethics service (nRes) Committee north West—Cheshire, 11/nW/0090.
Provenance and peer review not commissioned; externally peer reviewed.
data sharing statement Data are available on request. Data may be available if compliant with ethical and GDPR regulations and our funders and employers policies. For data queries, please contact one of the authors.
Open access This is an open access article distributed in accordance with the Creative Commons attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. see: https:// creativecommons. org/ licenses/ by/ 4. 0/.
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EpidEmiological sciEncE
Risk of severe herpes simplex virus infection in systemic lupus erythematosus: analysis of epidemiology and risk factors analysis in TaiwanTzu-Hao li,1,2,3 chien-chih lai,2,3,4 Wen-Hsiu Wang,5,6 Wei-sheng chen,4 Yen-po Tsao,4 chang-Youh Tsai, 2,4 Yu-sheng chang 7,8,9
To cite: li T-H, lai c-c, Wang W-H, et al. Ann Rheum Dis 2019;78:941–946.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214844).
For numbered affiliations see end of article.
Correspondence todr Yu-sheng chang, division of allergy, immunology, and Rheumatology, department of internal medicine, Taipei medical University-shuang Ho Hospital, new Taipei city 23561, Taiwan; risea65@ yahoo. com. tw
Received 29 november 2018Revised 12 march 2019accepted 13 march 2019published online First 6 april 2019
© author(s) (or their employer(s)) 2019. no commercial re-use. see rights and permissions. published by BmJ.
Key messages
What is already known about this subject? ► Patients with systemic lupus erythematosus (SLE) are susceptible to herpesviridae infection, including herpes simplex virus (HSV) and varicella zoster virus (VZV), which is occasional but often leads to overwhelming diseases.
► However, most of the previous studies have focused on VZV infection, and only few attempts have been made on HSV infection.
What does this study add? ► This nationwide cohort study provided epidemiology data of severe HSV infection in patients with SLE, including viral septicaemia, meningoencephalitis, ocular infection, visceral infection and those with complications after infection, and illustrated significantly higher incidence rate of severe HSV infection in SLE.
► We also investigated the risk factors, and displayed previous skin infection, intravenous steroid pulse therapy and oral daily steroid dose over 7.5 mg prednisolone or equivalent were independent risk factors for severe HSV infection in patients with SLE, while age ≤18 was a protective factor.
How might this impact on clinical practice or future developments?
► This study informed that HSV should be considered a possible differential diagnosis, especially for those with risk factors; early treatment should be applied to prevent severe complications in patients with SLE undergoing steroid therapy.
AbSTrACTObjective patients with systemic lupus erythematosus (slE) are susceptible to herpes simplex virus (HsV) infection, which occasionally leads to severe complications including meningoencephalitis and keratitis. However, few attempts to analyse the associated incidence and risk factors have been made.Methods We enrolled patients with slE recorded between 1997 and 2012 and compared the incidence rate (iR) of severe HsV infection, including meningoencephalitis, septicaemia, ocular and visceral involvement, and other specific complications demanding hospitalisation, with that of a non-slE cohort. a cox multivariate proportional hazards model was applied to analyse the risk factors of severe HsV infection in patients with slE.results a total of 122 520 subjects (24 504 patients with slE and 98 016 age-matched and sex-matched non-slE controls) were included, and a higher iR of severe HsV infection was revealed in the slE group (iR ratio=3.93, p<0.001). in patients with slE, previous oral and genital infection (HR=2.29, p=0.049), intravenous steroid pulse therapy (HR=5.32, p<0.001) and daily oral dose of over 7.5 mg of prednisolone (HR=1.59, p=0.024) were independent risk factors for severe HsV infection, whereas age of ≤18 (HR=0.45, p=0.029) was a protective factor.Conclusions patients with slE are at higher risk of severe HsV infection, and related risk factors include being older than 18 years, having a history of HsV mucocutaneous infection, recent receipt of steroid pulse therapy and a daily oral dose of steroid over 7.5 mg prednisolone.
InTrOduCTIOnSystemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease with multisystem involvement, which primarily affects women of childbearing age. SLE is associated with various comorbidities and impaired quality of life, and sometimes becomes potentially fatal. Among the various comorbidities, infection has been inten-sively explored as a common trigger factor of SLE and a common cause of severe complications or mortality. Patients with SLE are vulnerable to infec-tion by a great variety of typical or opportunistic pathogens, including bacteria, viruses, fungi and parasites, resulting from underlying immunological defects and administration of steroids and other immunosuppressants.1–4
Herpesviridae is a large family of DNA viruses, among which the herpes simplex virus (HSV) and herpes zoster virus (HZV) contribute to many diseases by remaining in the body and recurring in immunosuppressed hosts.5 In addition to oral and genital mucocutaneous infection, HSV may dissem-inate to uncommon sites such as the ocular, pulmo-nary, gastrointestinal, hepatic, genitourinary and central nerve systems; these severe forms of HSV infection spread rapidly and often lead to morbidity and mortality when diagnosis and treatment are delayed.6–11 The frequency and severity of HSV infection are greater in an immunocompromised
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Table 1 Characteristics of SLE and non-SLE matched control cohorts
Variables
Patients with SLE(n=24 504)
Age-matched and sex-matched non-SLE control cohort(n=98 016) P value
Mean age±SD, years 36.25±15.8 36.25±15.8 1
Female, n (%) 21 660 (88.4) 86 640 (88.4) 1
Severe HSV infection, n (%)* 115 (0.5) 161 (0.2) <0.001
Types
Meningoencephalitis 7 (0.029) 4 (0.004) 0.002
Keratitis 82 (0.33) 116 (0.12) <0.001
Non-severe HSV infection, n (%)*
695 (2.8) 2111 (2.2) <0.001
Mean duration of follow-up (SD), years
8.26 (4.40) 12.11 (2.47) <0.001
*Severe HSV infection included meningoencephalitis, eye involvement, keratitis, iridocyclitis, visceral infection, septicaemia and complications demanding hospitalisation; non-severe HSV infection indicated isolated oral and genital mucosal involvement without aforementioned severe HSV infection. The two kinds of diagnoses were mutually exclusive.HSV, herpes simplex virus;SLE, systemic lupus erythematosus.
host; many cases have reported possible high risk and high mortality rates of HSV infection in patients with SLE. Moreover, disseminated HSV infection may mimic acute flares in patients with SLE.12–14
Despite the seriousness of infections, few attempts have been made to examine the association between SLE and HSV, and the incidence, frequency and risk factors of such severe HSV infections in patients with SLE are still unknown. Furthermore, various disease-modifying antirheumatic drugs (DMARDs) and steroids are reportedly associated with the risk of severe infection,15 but few studies focusing on the effects of these immunomodulators in HSV infection have been undertaken. The National Health Insurance Research Database (NHIRD) in Taiwan offers comprehensive medical information on the domestic population, and is thus beneficial in epidemiological research. We used the NHIRD to conduct a retrospective nation-wide cohort study investigating the incidence and risk factors of severe HSV infections in patients with SLE.
PATIEnTS And METHOdSdata sourcesTaiwan’s National Health Insurance (NHI) programme was estab-lished in 1995 and has covered more than 23 million residents of Taiwan by 2012, representing more than 99% of the population and providing an integrated medical database including outpa-tient, inpatient, emergency, dental care and prescription records. Thus, we were able to obtain detailed demographic character-istics from the NHIRD, such as age and sex, as well as medical information such as diagnoses, procedures, dates and times of visits, and prescriptions.
Study populations and endpointsThis retrospective cohort study was conducted by analysing the NHIRD. The diagnosis codes in the data set were based on the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM). In the NHI system, patients with SLE can apply for a catastrophic illness certificate, reviewed by inde-pendent subspecialists according to the 1997 American College of Rheumatology classification criteria for SLE.16 In the present study, patients who had received SLE catastrophic illness certifi-cation (ICD-9-CM 710.0) between January 1997 and December 2012 were selected for inclusion.
The primary endpoint of this study was new incidence of severe HSV infection, which was defined as disseminated HSV infection or HSV infection at uncommon site that could lead to severe morbidity and mortality. Such infections included ocular HSV infection in the hospitalisation or outpatient clinical data sets (ICD-9-CM codes: eye involvement 054.40, 054.49; keratitis 054.2, 054.3; iridocyclitis 054.44), HSV meningoencephalitis (ICD-9-CM codes 054.3 and 054.72), and other complications consisting of myelitis, visceral infection, septicaemia or compli-cations (ICD-9-CM codes 054.74, 054.5, 054.71, 054.79 and 054.8) according to the discharge diagnoses from the hospi-talisation data set. All diagnoses of new severe HSV infection were validated based on usage of anti-HSV treatment, including acyclovir, famciclovir and valacyclovir. The secondary endpoints were non-severe HSV infection during follow-up, which was defined as isolated oral and genital mucosal HSV infection (ICD-9-CM codes 054.0, 054.1x, 054.2, 054.41, 054.6 and 054.73) without the aforementioned diagnosis of severe HSV infection. Subjects with severe HSV infection prior to enrolment were excluded. The non-SLE control group was selected from the 2000 Longitudinal Health Insurance Database, which provides
longitudinally linked anonymised data on 1 000 000 enrollees (nearly 5% of Taiwan’s population) randomly sampled from the NHIRD’s Registry of Beneficiaries in 2000. Each patient with SLE was matched with four non-SLE subjects for age and sex based on the same exclusion criteria. The two cohorts were followed until the development of primary endpoints, discontin-uance of insurance (including death) or end of the study period.
Statistical analysesStudent’s t-test and χ2 test were used for continuous and categor-ical variables, respectively, in the subgroup analyses of the SLE group versus the control group. Incidence rates (IR, per 10 000 person-years) and incidence rate ratios (IRRs) were analysed. A Poisson distribution was used to obtain the CI and compare inci-dence between the groups. The Kaplan-Meier method and the log-rank test were employed to compare the risk of severe HSV infection between the two cohorts. In the SLE cohort, univariate and multivariate Cox proportional hazard models were used to estimate the HRs and 95% CIs for associations among patient characteristics, comorbidities, medication use and severe HSV infection.
Comorbidities leading to secondary immunosuppression status, including diabetes mellitus, end-stage renal disease, inter-stitial lung diseases, stroke, malignancy, cirrhosis and congestive heart failure, and prior mucocutaneous HSV infection were identified using corresponding ICD-9-CM codes before the end of follow-up.17 Medications taken during follow-up, including hydroxychloroquine, azathioprine, mycophenolate mofetil, ciclosporin, cyclophosphamide (CYC), methotrexate and oral and intravenous steroids, were analysed. Because the composi-tion of prescriptions varied with time, we analysed these medica-tions as time-dependent covariates in a Cox regression model.18 The follow-up period of each patient was retrospectively divided into successive 60-daytime blocks; the prescription status of each medication in each 60-day block and its association with the occurrence of the event at the end of each time block were analysed. Oral steroid use was analysed according to the average daily dose in the time block, with a cut-off point of 7.5 mg of prednisolone or an equivalent. All factors with p<0.1 in the univariate analysis were selected for Cox multivariate forward stepwise analysis.19 All statistical analyses were processed using
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Figure 1 Kaplan-Meier curves for severe and non-severe HSV-free survival in the SLE group versus the non-SLE control group. HSV, herpes simplex virus; SLE, systemic lupus erythematosus.
Table 2 IR and IRR of severe HSV infection: overall and subgroup analyses*
SLE group
non-SLE control group Irr (95% CI) P value
Total incidence rate of severe HSV infection
5.36 1.36 3.93 (3.1 to 5.0) <0.001
Meningoencephalitis 0.32 0.03 9.62 (2.4 to 44.8) <0.001
Keratitis 3.82 0.98 3.89 (2.9 to 5.2) <0.001
Male 6.48 1.45 4.48 (2.1 to 9.4) <0.001
Meningoencephalitis 0.46 0.08 6.06 (0.08 to 457.8) 0.283
Keratitis 4.62 0.91 5.06 (2.0 to 12.8) <0.001
Female 5.23 1.35 3.87 (3.0 to 5.0) <0.001
Meningoencephalitis 0.31 0.03 10.86 (2.3 to 67.1) 0.002
Keratitis 3.73 1.00 3.77 (2.7 to 5.1) <0.001
Age 18 or under 2.84 0.83 3.43 (1.1 to 9.5) 0.017
Meningoencephalitis 0 0.07 NA NA
Keratitis 1.62 0.48 3.36 (0.7 to 13.2) 0.077
Age 19–65 5.62 1.27 4.42 (3.3 to 5.6) <0.001
Meningoencephalitis 0.38 0.02 18.66 (3.6 to 184.0) <0.001
Keratitis 4.03 0.94 4.29 (3.1 to 5.9) <0.001
Age 66 and above 7.57 3.66 2.07 (0.6 to 5.6) 0.170
Meningoencephalitis 0 0.35 NA NA
Keratitis 6.05 2.96 2.04 (0.50 to 6.26) 0.224
*The incidence rate represents the number of events per 10 000 person-years.HSV, herpes simplex virus; IR, incidence rate; IRR, incidence rate ratio; NA, not available; SLE, systemic lupus erythematosus.
R V.3.2.2 software. A p value of <0.05 was considered statisti-cally significant.
rESuLTSrisk of severe HSV in the SLE and control cohortsA total of 24 544 patients with SLE from 1997 to 2012 were identified, and 40 of these patients with severe HSV infection prior to enrolment were excluded. In total, 24 504 patients with SLE and 98 016 age-matched and sex-matched control group subjects were included (table 1). In both cohorts, the mean age was 36.3 and the majority of subjects (88.4%) were female. The mean follow-up duration was 8.3 years for the SLE group, which was significantly shorter than that of the control group (12.1 years).
Our investigation found 115 (0.5 %) cases of severe HSV infection in the SLE cohort. This proportion was significantly higher than that in the control group (0.2%, p<0.001). Of the two subtypes, 7 (0.029%) cases of HSV meningoencephalitis and 82 (0.33%) cases of HSV keratitis were identified. Both of these proportions were significantly higher than those in the control group (table 1). Case number of severe HSV infection happened in each year of SLE duration was shown in the online supplementary figure 1. Non-severe HSV infection was also significantly higher in the SLE group (table 1). Kaplan-Meier analysis also revealed a significantly higher cumulative incidence of both severe and non-severe HSV infection in the SLE cohort (figure 1).
Ir and Irr stratified by infection subtypes, sex and ageIn the SLE cohort, the IR of overall severe HSV infection was 5.36 per 10 000 person-years, which was significantly higher than that in the control cohort, with an IRR of 3.93 (95% CI 3.1 to 5.0, p<0.001). Similarly, the IRs for HSV meningoencepha-litis and keratitis were 0.32 and 3.82 per 10 000 person-years, respectively, both of which were significantly higher than those in the control cohort, with IRRs of 9.62 and 3.89, respectively. In analyses stratified by several factors, sex did not modify risk; by contrast, the IR increased with age in both cohorts. Patients with SLE had a higher risk in the 19-65 years old subgroup (IRR=4.42, 95% CI 3.3 to 5.6, p<0.001) and the ≤18 years old subgroup (IRR=3.43, 95% CI 1.1 to 6.5, p=0.017). In the ≥65 years old subgroup, no significant difference in the incidence of
overall severe HSV infection or infection subtypes between the groups was found (table 2).
risk factors of severe HSV infection in patients with SLEA Cox multivariate proportional hazards analysis was employed, with factors at a p value of <0.1 in the univariate Cox regression analysis revealing the following as statistically significant risk factors: previous non-severe infection (HR=2.29, 95% CI 1.01 to 5.23, p=0.049), intravenous steroid (HR=5.32, 95% CI 3.42 to 8.29, p<0.001) and an oral daily prednisolone or equivalent dose over 7.5 mg (HR=1.59, 95% CI 1.06 to 2.36, p=0.024;
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Table 3 Risk factors of severe HSV infection in patients with SLE
Variable
univariate analysis Multivariable analysis*
Hr (95% CI) P value Hr (95% CI) P value
Age
18 and under 0.50 (0.25 to 1.04) 0.062 0.45 (0.22 to 0.92) 0.021
19–65 As reference As reference
66 and above 1.20 (0.49 to 2.96) 0.685 1.23 (0.50 to 3.02) 0.612
Sex 1.26 (0.72 to 2.21) 0.416
Comorbidity
DM 0.64 (0.34 to 1.18) 0.152
ESRD 0.92 (0.56 to 1.50) 0.732
ILD 0.61 (0.22 to 1.65) 0.328
Stroke 0.96 (0.58 to 1.59) 0.875
Malignancy 0.69 (0.10 to 4.94) 0.710
Cirrhosis 0.59 (0.08 to 4.22) 0.599
CHF 0.84 (0.47 to 1.50) 0.564
Previous non-severe HSV infection† 2.34 (1.03 to 5.34) 0.043 2.29 (1.01 to 5.23) 0.049
DMARDs
Hydroxychloroquine 1.17 (0.81 to 1.69) 0.403
Azathioprine 1.57 (1.03 to 2.38) 0.035 – –
Mycophenolate mofetil NA NA
Ciclosporin 1.94 (0.72 to 5.26) 0.193
Cyclophosphamide 1.82 (0.91 to 3.61) 0.086 – –
MTX 1.63 (0.71 to 3.70) 0.246
Steroid
Intravenous 6.19 (4.07 to 9.41) <0.001 5.32 (3.42 to 8.29) <0.001
Oral daily dose over 7.5 mg‡ 2.10 (1.45 to 3.06) <0.001 1.59 (1.06 to 2.36) 0.024
*All factors with p<0.1 in the univariate analysis were selected for Cox multivariate forward stepwise analysis.†Non-severe HSV was defined as isolated oral and genital mucosal involvement without severe HSV infection.‡Average dose was defined as the accumulated prednisolone or equivalent dose during the study period (in milligrams) divided by the time to index (severe HSV infection) or the end of the study (in days).CHF, congestive heart failure; DM, diabetes mellitus; DMARDs, disease-modifying antirheumatic drugs; ESRD, end-stage renal disease; HSV, herpes simplex virus; ILD, interstitial lung diseases; MTX, methotrexate; NA, not available; SLE, systemic lupus erythematosus.
table 3). Conversely, age under 18 appeared to have a protective effect (HR=0.45, 95% CI 0.22 to 0.92, p=0.021; table 3).
dISCuSSIOnTo our knowledge, this was the largest nationwide cohort study to provide an epidemiological analysis of severe HSV infection in patients with SLE, revealing a higher risk of overall severe HSV infection than in the control subjects, with an IRR of 3.93. Most severe HSV infections were cases of keratitis or menin-goencephalitis, the risks of which were significantly higher in the SLE cohort, with IRRs of 3.89 and 9.62, respectively. Risk factors included previous HSV infection and recent use of intra-venous steroid therapy or oral steroids with an average daily dose of over 7.5 mg of prednisolone or an equivalent, whereas age younger than 18 was a protective factor.
The results of this study were validated as follows. First, we believe that our severe HSV infection and SLE diagnoses were accurate. All the diagnoses of severe HSV infection were vali-dated based on usage of anti-HSV agents, and the reimbursement of antiviral drugs required support from reports of HSV-related examinations. Regarding the diagnosis of SLE, catastrophic illness certification for SLE was used. In the NHI system, cata-strophic illness certification for SLE is verified by strict peer review of pertinent medical records and examination reports according to the 1997 American College of Rheumatology classi-fication criteria for SLE.16 Patients with catastrophic illness certi-fication can be exempted from related medical expenses. This
renders SLE diagnosis in this nationwide data set exhaustive, reliable and less vulnerable to selection bias when studying rare complications. Furthermore, the period of observation in this study was up to 15 years, thereby enabling further strata and risk factor analysis. In fact, same analyses using 1:1 matching showed consistent result, but there was insufficient power for the strata analysis (online supplementary tables 1 and 2). Finally, analysing immunosuppressants as time-dependent covariate in the multi-variate Cox regression model was highly effective in identifying the risk of each medication. In clinical practice, prescriptions change over time and medications are often used in combina-tion with one another. Treating medication as a time-dependent covariate fits the model for changes in medication use and served as a method for investigating the contributions of certain medi-cations to subsequent risk during the period before their efficacy diminished. Moreover, the multivariate Cox regression enabled us to analyse the risks of individual medications after the effects of other medications used concurrently had been adjusted.
Among all immunosuppressants, our study found that only steroid use—including intravenous administration and a daily oral dose of over 7.5 mg—represented an independent risk factor for severe HSV infection. Several studies have referred to systemic glucocorticoid as a risk factor for SLE infection through many pathogens, including bacteria, fungi and viruses.20–22 Among viruses, systemic steroid use has been associated with HZV,23 human papillomavirus21 and human cytomegalovirus,24 whereas little attention has been given to HSV. Regarding steroid
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dosage, we investigated the influence of a daily oral dose of over 7.5 mg of prednisolone or an equivalent because a lower dosage is considered a physiological or low-dose steroid—a view supported in the literature.25 One nested case–control study reported a dose-dependent infection risk from a daily oral dose of over 5 mg of a prednisolone equivalent26; however, another study did not support this finding.27 Our study found a risk of severe HSV infection in patients with SLE administered a higher than physiological steroid dose.
Other DMARDs showed no increased risk of severe HSV infection. The possible reason is that, in real-world practice, these immunosuppressants are often used in combination with or after steroid, the strongest risk factor for severe HSV infec-tion in our model. Besides, steroid has faster pharmacological effect than most immunosuppressants. Thus, steroid might already raise the risk in vulnerable patients before other immu-nosuppressants taking effect. In competition with steroid, the most important risk factor, there was no significant risk of other immunosuppressants in the multivariate Cox regression model. Most relevant studies focusing on the relationship between disseminated HSV infection and azathioprine were case reports, and all the cases had concomitant use of steroid.28 29 One study reported that azathioprine use might be associated with higher incidence of skin and genital HSV infection.30 In that study, patients using azathioprine were more likely to use prednisolone of over 10 mg than those without azathioprine. Although steroid use under 10 mg of prednisolone was not analysed in that study, it is still possible that the elevated risk of azathioprine derived from the use of steroid in the azathioprine cohort. According to our results, infection risk increased with a steroid dose as low as 7.5 mg of prednisolone.
To our surprise, infection risk did not increase with CYC use, even in the univariate Cox regression model. CYC is an alkylating immunosuppressive drug notorious for its side effects of infec-tion,3 31 32 including HZV.33 34 The mechanism of severe HSV infection might also differ from HZV reactivation. The studies focused on the relationship between CYC and HSV were mostly conducted on experimental animals and indicated a propensity towards HSV infection and reactivation, but insufficient amounts of epidemiological data have been provided.35 36 One possible explanation for statistical non-significance in the present study may be that Asian physicians are inclined to administer lower doses of CYC.37
Patients with SLE under 18 years of age in our study had a lower IR of severe HSV infection. Together with the associa-tion between previous skin HSV infection and the following severe HSV infection in the study, we believe that the majority of severe HSV infections are due to reactivation from prior exposure. As a result, less exposure to HSV in juveniles led to a significantly lower risk. This was consistent with other studies which have shown that a history mucocutaneous infection, including skin involvement, gingivostomatitis and genitouri-nary involvement,38–40 might be diagnostic clues in susceptible circumstances,38 39 41 such as steroid use in our study. The clin-ical implication of this study is that HSV should be considered a possible differential diagnosis, and early treatment should be applied to prevent severe complications in patients with SLE undergoing steroid therapy.
Despite the meticulous design of our study, several limitations should be noted. First, no examination result was provided in this claim-based data set. Thus, our definition of severe HSV infection was not based on a report of HSV-related data such as serology. However, in addition to ICD-9-CM codes in the data set, all incidents of severe HSV infection were validated based on
usage of antiviral medication, and reimbursement for anti-HSV medication in this claim-based data set required support from reports of related examinations and was subjected to peer review. Thus, we believe that the diagnoses of HSV infections in this study were reliable. For the same reason, the results of SLE-associated examination were also lacking, which precluded us to analyse disease activity and perform subgroup analyses to identify the mechanism of the association. Finally, the NHIRD database lacks lifestyle information, including such psychosocial factors as emotional distress, stress, social support and sexual activity, which have been reported as associated with HSV.42 We were unable to correct this.
In conclusion, this nationwide population-based cohort study identified a higher risk of severe HSV infection in patients with SLE, regardless of sex, with younger age having a protective effect. For patients with SLE who were older, with history of herpes infection, and treatment with intravenous steroids or a high daily oral dose (over 7.5 mg) of prednisolone or an equiva-lent, the risk of severe HSV infection should be considered.
Author affiliations1division of allergy, immunology, and Rheumatology, department of medicine, chiayi Branch, Taichung Veterans general Hospital, chiayi city, Taiwan2Faculty of medicine, school of medicine, national Yang-ming University, Taipei city, Taiwan3institute of clinical medicine, national Yang-ming University, Taipei city, Taiwan4divisionof allergy, immunology, and Rheumatology, department of medicine, Taipei Veterans general Hospital, Taipei, Taiwan5division of allergy, immunology and Rheumatology, mackay memorial Hospital, Taipei, Taiwan6medicine, mackay medical college, sanzhi, Taiwan7division of allergy, immunology, and Rheumatology, department of internal medicine, Taipei medical University shuang Ho Hospital, new Taipei city, Taiwan8division of allergy, immunology, and Rheumatology, department of internal medicine, school of medicine, college of medicine, Taipei medical University, Taipei city, Taiwan9institute of Biomedical informatics, national Yang-ming University, Taipei, Taiwan
Acknowledgements all authors appreciate Wallace academic Editing for their elaborate and professional English editing service.
Contributors T-Hl, c-cl and Y-sc conceived and planned the study. W-HW, W-sc, Y-pT and c-YT contributed to data acquisition and processing. T-Hl, c-cl and Y-sc took the lead in writing the manuscript. all authors provided critical feedback and shaped the research, analysis and the manuscript.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests none declared.
Patient consent for publication not required.
Ethics approval information that potentially identified any individual was encrypted and properly reviewed by the institutional Review Board of Taipei Veterans general Hospital, alongside approval from the national Health Research institutes.
Provenance and peer review not commissioned; externally peer reviewed.
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13 Wakhlu a, nolkha n, srivastava d, et al. What’s your diagnosis?: herpes simplex infection mimicking recurrent acute flares in systemic lupus erythematosus: a case report with review of literature. Indian Journal of Rheumatology 2013;8:134–6.
14 Zink a, manger B, Kaufmann J, et al. Evaluation of the rabbit risk score for serious infections. Ann Rheum Dis 2014;73:1673–6.
15 Ramos-casals m, cuadrado mJ, alba p, et al. acute viral infections in patients with systemic lupus erythematosus: description of 23 cases and review of the literature. Medicine 2008;87:311–8.
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28 Robineau o, Enrico J, lemaire X, et al. Herpes simplex virus meningoencephalitis in a patient with crohn’s disease on azathioprine therapy. Am J Gastroenterol 2010;105:240–1.
29 santos-antunes J, abreu c, magro F, et al. disseminated cutaneous herpes simplex infection in a patient with crohn’s disease under azathioprine and steroids: first case report and literature review. J Crohns Colitis 2014;8:326–30.
30 seksik p, cosnes J, sokol H, et al. incidence of benign upper respiratory tract infections, HsV and HpV cutaneous infections in inflammatory bowel disease patients treated with azathioprine. Aliment Pharmacol Ther 2009;29:1106–13.
31 Bernatsky s, Hudson m, suissa s. anti-rheumatic drug use and risk of serious infections in rheumatoid arthritis. Rheumatology 2007;46:1157–60.
32 pryor Bd, Bologna sg, Kahl lE. Risk factors for serious infection during treatment with cyclophosphamide and high-dose corticosteroids for systemic lupus erythematosus. Arthritis Rheum 1996;39:1475–82.
33 Ferreira Jcoa, marques HH, Ferriani mpl, et al. Herpes zoster infection in childhood-onset systemic lupus erythematosus patients: a large multicenter study. Lupus 2016;25:754–9.
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TranslaTional science
Lupus nephritis is linked to disease-activity associated expansions and immunity to a gut commensalDoua azzouz,1 aidana omarbekova,1 adriana Heguy,2 Dominik schwudke,3 nicolas Gisch, 3 Brad H rovin,4 roberto caricchio,5 Jill P Buyon,1 alexander V alekseyenko,6 Gregg J silverman 1
To cite: azzouz D, omarbekova a, Heguy a, et al. Ann Rheum Dis 2019;78:947–956.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214856).
For numbered affiliations see end of article.
Correspondence toDr Gregg J silverman, new York University school of Medicine, new York, nY 10016, Usa; gregg. silverman@ nyumc. org
Parts of this manuscript were orally presented as a plenary presentation at the 2017 acr/arHP annual Meeting and as such the abstract was published in an online supplementary of Arthritis & Rheumatology.
received 30 november 2018revised 23 January 2019accepted 24 January 2019Published online First 19 February 2019
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2019- 215221
© author(s) (or their employer(s)) 2019. re-use permitted under cc BY-nc. no commercial re-use. see rights and permissions. Published by BMJ.
Key messages
What is already known about this subject? ► Patients with lupus have been reported to have restricted gut microbiome diversity and general patterns of dysbiosis but few patients with active disease have been previously characterised.
What does this study add? ► Patients with lupus were found to have characteristic patterns of gut microbiome dysbiosis that directly paralleled disease activity.
► Patients with systemic lupus erythematosus commonly displayed signs of impaired gut barriers that may result in immune exposure to gut commensal bacteria.
► Intestinal expansions of Ruminococcus gnavus (RG) were directly proportional to overall disease activity and most pronounced in those with lupus nephritis. Lupus faecal samples displayed increases in sIgA-coated RG bacteria. In three independent cohorts, patients with lupus nephritis displayed elevated serum IgG predominantly to RG strain-restricted cell wall lipoglycan antigens.
How might this impact on clinical practice or future developments?
► Identification of R. gnavus as a candidate pathobiont opens new areas of investigation of the mechanistic basis by which these outgrowths may affect the overall pathogenesis of lupus and the immune complex-mediated pathogenesis of lupus nephritis.
► These findings may lead to the development of bioassay(s) with prognostic value for the risk of lupus nephritis.
AbSTrACTbackground/Purpose To search for a transmissible agent involved in lupus pathogenesis, we investigated the faecal microbiota of patients with systemic lupus erythematosus (sle) for candidate pathobiont(s) and evaluated them for special relationships with host immunity.Methods in a cross-sectional discovery cohort, matched blood and faecal samples from 61 female patients with sle were obtained. Faecal 16 s rrna analyses were performed, and sera profiled for antibacterial and autoantibody responses, with findings validated in two independent lupus cohorts.results compared with controls, the microbiome in patients with sle showed decreased species richness diversity, with reductions in taxonomic complexity most pronounced in those with high sle disease activity index (sleDai). notably, patients with sle had an overall 5-fold greater representation of Ruminococcus gnavus (RG) of the Lachnospiraceae family, and individual communities also displayed reciprocal contractions of a species with putative protective properties. Gut RG abundance correlated with serum antibodies to only 1/8 RG strains tested. anti-rG antibodies correlated directly with sleDai score and antinative Dna levels, but inversely with c3 and c4. These antibodies were primarily against antigen(s) in an RG strain-restricted pool of cell wall lipoglycans. novel structural features of these purified lipoglycans were characterised by mass spectrometry and nMr. Highest levels of serum anti-RG strain-restricted antibodies were detected in those with active nephritis (including class iii and iV) in the discovery cohort, with findings validated in two independent cohorts.Conclusion These findings suggest a novel paradigm in which specific strains of a gut commensal may contribute to the immune pathogenesis of lupus nephritis.
InTroduCTIonSystemic lupus erythematosus (SLE) is an inflamma-tory autoimmune disease with hallmarks of B-cell abnormalities, circulating autoantibodies to nuclear antigens and immune-complex formation.1 The heterogeneity of disease presentation and organ involvement in different individuals, and the vari-ability of disease activity from remission to exac-erbations and progression, all contribute to clinical challenges for diagnosis and effective management. Indeed, such heterogeneity suggests that SLE may not represent a single disease but rather several.
Serum autoantibodies to native DNA are a specific diagnostic criterion for SLE,2 and a prog-nostic factor for the development of lupus nephritis (LN) that affects 30%–60% of patients.3 However, the earliest reports of antibody responses to nucleic acids/nucleoproteins were documented in associa-tion with clinically apparent bacterial infections.4–6 Yet two decades later autoantibodies to nuclear antigens were recognised to be a common feature of SLE.7–9 Indeed, some DNA-reactive autoantibodies
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Figure 1 Patients with SLE have altered faecal communities of commensal taxa, elevated faecal immunoglobulin levels and biomarker evidence of altered gut barrier function. (A) Chao1 estimates of alpha diversity represent the total expected number of OTUs that represent quasi-species identified from 16S rRNA amplicon sequencing surveys, with larger values representing higher diversity. Patients with SLE (n=61) have less diverse faecal microbiota than healthy controls (n=17), indicating that SLE commonly have intestinal dysbioses. At far right, the distribution of Chao1 values in patients with SLE with high activity (ie, SLEDAI≥8) was significantly contracted compared with healthy adult controls (HC), with a trend towards more limited diversity in SLE with high disease activity when compared with SLE with low disease activity. We used a cut-point that was associated with greatest statistically significant differences. The SLE low disease activity group had a range of 0–7 SLEDAI scores (n=47), and SLE high disease activity group had scores of 8–18 (n=14). p values were based on the Mann-Whitney test. (B) PcoA showed that the beta diversity within bacterial communities in the faecal microbiomes in healthy controls was less different than the communities from patients with SLE low disease activity (PERMANOVA, p=0.02). Furthermore, healthy subjects were more like one another than were patients with SLE. (C) Results indicate average within group beta diversity differences in the three different groups, with comparisons for Control-SLEDAIhi-SLEDAIlow, p=0.002. The binary comparison, control vs SLE had p=0.02. Both comparisons were done using PERMANOVA test. Examinations of faecal extract demonstrated patients with SLE commonly display: (D) elevated faecal sIgA levels, (E) elevated faecal IgM levels, (F) elevated faecal IgG levels, and (G) elevated faecal calprotectin. Patients with SLE also displayed: (H) elevated serum sCD14 levels and (I) elevated serum α1-acid glycoprotein levels, as measured by commercial assay. Results are for the NYU cohort of adult female patients with SLE, were compared to adult female controls without inflammatory or autoimmune disease (CTL). (D–F) and (H–I) using unpaired two-tailed t test with Welch’s correction, (G) used Mann-Whitney test. Significance for p<0.05. OTUs, operational taxonomic units; PCoA, principal coordinates analysis; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index.
are directly nephritogenic in animal models.10 Conversely, only ~20% of the IgG eluted from lupus kidneys is DNA-reactive,11 suggesting that other antibody reactivities may also contribute to the pathogenesis of LN.12
While a transmissible agent has long been suspected in lupus pathogenesis, only recently has suitable technology become available that enable in-depth consideration of the poten-tial roles of the immense dynamic communities of commensal microorganisms that coevolved with our species. The largest microbiome community resides within our gut, where these microbes play essential roles, including for the early priming of our immune systems13 and subsequent immune regulation.14 Mounting evidence has implicated imbalances within these gut microbial communities, also termed dysbioses, in the autoim-mune pathogenesis of several diseases: inflammatory bowel disease (IBD), type 1 diabetes, multiple sclerosis and rheumatoid arthritis.15 Yet, there have only been a few reports on the human lupus microbiome, in small cohorts that have included only a few active patients.16–18
In the present study, we investigated the gut microbial communities in a cross-sectional cohort of 61 female patients with lupus heterogeneous for ethnicity/race, disease activity and organ involvement and immune profiles. Key findings were then evaluated in two independent lupus cohorts.
MeTHodSethics statementThe study was conducted according to the Declaration of Helsinki. Before study inclusion, written informed consent, approved by the NYU IRB, was obtained from all subjects for research use and publication of their data.
Study designPatients were consecutively recruited from the NYU Langone Medical Center and Bellevue Hospital. All patients fulfilled the American College of Rheumatology Criteria for the diagnosis of SLE.2 Further details on trial-specific inclusion and exclusion criteria, clinical evaluations, sampling and 16S rRNA amplicon analyses are described in the supplementary data. Sections on immunoassays, as well as culture of bacterial strains and puri-fication of R. gnavus lipoglycans (LGs) are also provided (see supplementary materials).
reSulTSPatients with lupus have distinctive patterns of dysbiosis that parallel disease activityIn the discovery phase of our studies, we analysed the faecal microbiota of 61 female patients with lupus in a cross-sectional urban cohort and 17 female healthy controls (online supplemen-tary table 1). Our patients displayed great heterogeneity in their organ involvement, and in disease activity from clinical remis-sion to highly active, that was scored using the composite SLE disease activity index (SLEDAI).19
To define the richness of alpha diversity in gut communities, Chao1 estimates of the total number of operational taxonomic units20 demonstrated that the overall biodiversity of the intes-tinal microbiota in patients with lupus was significantly restricted compared with unaffected female adult controls (Mann-Whitney, p=0.038) (figure 1A). Among patients with lupus, there was a numerical trend towards an inverse correlation of Chao1 esti-mates of alpha diversity with SLEDAI score (Mann-Whitney, p=0.08) (data not shown). Moreover, after empirically consid-ering all possible cut-off values to dichotomise patients based
on SLEDAI score, those with high disease activity (defined as SLEDAI ≥8) displayed significantly restricted microbiota diversity compared with controls (Mann-Whitney, p=0.003) (figure 1A).
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Table 1 Shifts in taxonomic abundance in SLE by disease activity
Taxonomic level Taxonomy Healthy (%) SledAIlow (%) SledAIhigh(%) P value
Class Erysipelotrichi 1.37 1.44 5.92 0.086
Order Erysipelotrichales 1.37 1.44 5.92 0.086
Family Erysipelotrichaceae 1.37 1.44 5.92 0.086
Family Veillonellaceae 1.58 3.41 12.27 0.008
Family Ruminococcaceae 25.66 11.68 15.11 0.045
Genus Blautia 2.23 4.10 3.59 0.077
Genus Ruminococcus 0.53 1.76 3.15 0.007
Species Ruminococcus gnavus 0.25 1.19 2.11 0.013
Species Bacteroides uniformis 2.11 0.87 0.34 0.014
Species Bacteroides fragilis 1.17 2.41 2.20 0.081
Results from univariate Kruskal-Wallis ANOVA analysis of 16S rRNA abundance for all taxa across all subjects for p<0.10 and not FDR corrected. The same bacterial set is identified as Erysipelotrichi, Erysipelotrichales and Erysipelotrichaceae. SLEDAIlow patients have SLEDAI scores of less than 8. For the complete analysis, see online supplementary table 2.ANOVA, analysis of variance; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index.
The human gut microbiome is dominated by four bacterial phyla—Firmicutes, Bacteroidetes, Actinobacteria and Proteo-bacteria—with thousands of different species and countless strains in complex dynamic relationships within individual gut communities.21 22 To visualise community variation, we performed principal coordinates analyses that confirmed that taxonomic distributions within patients with SLE were signifi-cantly different than in controls (PERMANOVA, p=0.02) (figure 1B). When the patients with SLE were stratified by high or low disease activity, the beta diversity differences were even more pronounced (PERMANOVA, p=0.002, data not shown). Furthermore, the variability in beta diversity of healthy subjects was lower than in patients with lupus (figure 1B, C). The within group variability between communities in patients with lupus with low disease activity was also lower than that in patients with high disease activity (figure 1C). However, the severity of the detected disease-associated dysbiosis did not correlate with disease duration (online supplementary figure 1A).
With 16S rRNA amplicon sequence analysis, the taxa in each community with the most highly abundant (>1%) representation at all phylogenetic levels were identified by univariate Kruskal Wallis ANOVA analysis (Table 1 and online supplementary table 2). At the species level, patients with SLE displayed a mean 5-fold overabundance of an anaerobic Gram-positive taxon in the Firmicutes phylum and Lachnospiraceae family, identified as Ruminococcus gnavus23 (RG) (range 0.00%–10.79%, mean±SD 1.35%±2.01%) compared with controls (0.00%–1.27%, 0.25%±0.39%, Mann-Whitney, p=0.01). Strikingly, RG relative abundance correlated with lupus disease activity, as even patients with SLE with low disease activity had a mean 4-fold RG over-representation, while those with high disease activity had >8 fold greater RG abundance (Mann-Whitney, p=0.01) (table 1, complete data in online supplementary table 2). Compared with those without a history of renal disease, patients with a history of renal involvement displayed an even greater abundance of a RG specific amplicon sequence variant (ASV) (Mann-Whitney, p=0.04), as well as several other ASVs that included two assigned to the Veillonella genus (online supplementary table 3). In an individual community these Veillonella species were highly correlated with each other (CLR-transformed, r=0.89, p<10–16) although less strongly with RG (39% V. parvula, 43% V. dispar, p<0.005). Hence, the microbiota in patients with lupus commonly displayed concurrent expansions of both RG and Veillonella species.
As an alternative approach, DADA224 was used to assign ASVs (ie, quasi-species): patients with a history of renal disease displayed increased representation of ASV31 (Mann-Whitney, p<0.05) (online supplementary tables 2-4). In fact, this ASV identifies RG and has the predictive effect size of AUC=0.65 for renal involvement (see Ref. 25 for description of predictive effect size analysis protocol), although this study was not adequately powered for multiple comparison correction. Nonetheless, these findings collectively indicate that patients with LN commonly have intestinal expansions of the RG species.
The lupus microbiome is associated with altered dynamic relationships between speciesWithin microbiome communities, we also looked for evidence of coordinated shifts in the representation of species within indi-vidual lupus communities, and outgrowths of RG were found to be commonly associated with reciprocal reductions of the Bacteroides uniformis species (r=−0.43, Pearson correlation for CLR-transformed abundances, p<0.001). Indeed, lupus patients with the highest disease activity scores also had the lowest abundance of B. uniformis (Mann-Whitney, p=0.01) (table 1), suggesting that these species may be in dynamic reciprocal relationships. Notably, as B. uniformis conveys anti-inflamma-tory properties that can ameliorate metabolic abnormalities in diabetes-prone mice,26 a paucity in active lupus patients could also be relevant to understanding the drivers of autoimmune pathogenesis.
evidence of in vivo gut recognition of RG and altered gut barrier functionTo assess in vivo local recognition of specific gut taxa, we eval-uated the representation of secretory IgA (sIgA)-coated faecal bacteria recovered from 15 unaffected controls and 23 patients with SLE (online supplementary figure 1B). Overall, RG bacteria were prominent among sIgA-coated subcommunities by 16S rRNA analysis, and lupus samples displayed a 2-fold greater abundance of RG among sIgA-coated faecal bacteria than healthy controls, However, in these limited surveys, the numer-ical differences did not attain significance (Wilcoxon rank sum, p=0.14, not significant) (online supplementary figure 1C).
In healthy individuals, an intact intestinal barrier prevents leakage of luminal contents out of, and also systemic IgG from entering into, the gut lumen. In these faecal samples, patients with lupus had 2.6-fold overall sIgA elevations vs controls (Welch
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test, p=0.002) (figure 1D). Furthermore, the elevations of faecal IgM in patients with lupus, as well as the increases in faecal IgG, provide additional circumstantial evidence of an impairment of the epithelial barrier in these patients (figure 1E, F).
To further search for evidence of impaired gut barriers, we measured faecal levels of calprotectin, an accepted biomarker for intestinal barrier defect.27 In these pilot studies, we foud that 12/61 patients with lupus exceeded the level in any of the tested control subjects (Mann-Whitney, p=0.03) (figure 1G). Furthermore, patients with lupus also displayed raised serum soluble CD14 (Welch test, p=0.0002) and raised α1-acid glyco-protein levels (Welch test, p<0.0001) (figure 1H, I), which in other conditions have been attributed to gut bacterial transloca-tion.28 29 Taken together, these findings suggest that patients with lupus may at times experience impaired gut barrier function, which potentially may enable commensals, or their components, to escape the intestinal lumen.
lupus serum IgG antibodies react with a RG2 strain-associated non-protein antigenTo investigate for a special immune relationship in patients with SLE with their candidate pathobionts, we electrophoretically separated the components in extracts of eight monocultured RG strains (online supplementary table 5) after treatment with lysozyme and endonuclease. Immunoblots revealed that many patients with lupus and healthy adult controls had IgG reac-tivity with antigenic bands, common to a range of RG strains (figure 2A–D), and these were protease-sensitive (figure 2E). In contrast, lupus serum antibodies were not generally reac-tive with commensal strains P. copri or B. thetaiotaomicron (figure 2A–C). Notably, serum IgG antibodies of lupus patients with SLEDAI activity scores of ≥8 were often highly reactive with specific conserved protease-resistant bands in the extract of the R. gnavus strain CC55_001C, which is here termed RG2 (online supplementary table 5). These conserved bands in the RG2 strain migrated with ~20–30 kDa protein markers, but such bands were not detected in the seven other RG strains tested (figure 2E).
High antibody responses to the candidate RG pathobiont correlate with high disease activityGiven the common overabundance of RG in patients with active SLE, we investigated for special host-pathobiont relationships within the systemic immune systems of affected individuals. Serum IgA-anti-RG2 antibody responses were significantly higher in patients with SLE than controls, even after normali-sation for total IgA levels (Welch test, p=0.01) (online supple-mentary figure 1D, E). Strikingly, serum IgG anti-RG2 responses were also increased in patients with SLE, and in matched lupus samples we found a significant direct correlation between the relative faecal abundance of the candidate RG pathobiont with the levels of serum IgG anti-RG2 antibodies (Spearman, p=0.002) (figure 2F) but not with the VPI C7-9 strain, here termed RG1 (online supplementary table 5 and data not shown), however such a relationship was also not found in studies of control subjects (not shown).
Based on a cut-off defined by values from unaffected control subjects (see Methods section), significant elevations of serum IgG anti-RG2 antibodies were detected in 22.9% of patients with SLE in the NYU cross-sectional cohort (figure 2G). Further-more, serum levels of IgG anti-RG2 strain antibodies directly correlated with SLEDAI disease score (Spearman, p=0.04) but not with the serum IgG antibodies to the RG1 strain (online
supplementary figure 1F). When patients were dichotomised based on disease activity the statistical differences between these patient subsets were most pronounced, as patients with SLEDAI scores≥8 had significantly higher IgG antibody levels to RG2 than patients who were quiescent or had only low disease activity (Mann-Whitney, p<0.001) (figure 2G), suggesting that the IgG anti-RG2 antibody level could represent a surrogate disease activity marker.
Serum levels of IgG anti-RG2 strain antibodies correlated with anti-native DNA autoantibodies (Spearman, p<0.0001) (figure 2H), which are commonly found in patients with LN.2 30 IgG antibodies to RG2 were also significantly positively correlated with IgG reactivity against human glomerular extract (Spearman, p<0.0001) (figure 2I), which is reported to identify the most nephritogenic anti-DNA antibodies.31 In contrast, there was neither an association with other types of lupus-associated autoantibodies, including anti-Ro52, nor with total IgG levels (online supplementary figure 1G, H). Relevant to clinical disease state, levels of IgG anti-RG2 antibodies also inversely correlated with C3 (Spearman, p<0.01) and C4 levels (Spearman, p<0.007) (figure 2J, K), biomarkers for in vivo activation of the complement cascade implicated in active immune-complex-me-diated pathogenesis in LN.30 Indeed, serum IL-6 displayed a strong direct correlation with IgG anti-RG2 levels (Spearman, p=0.003) (figure 2L), while the relationship with anti-dsDNA was more modest (Spearman, p=0.02) (figure 2M). Further-more, serum α2-interferon (IFN) also correlated with IgG anti-RG2 levels (Spearman, p=0.015), while there was no correlation with anti-dsDNA in the same samples (figure 2N, O). These find-ings provide circumstantial evidence of a connection between host immunity to RG2 antigen(s) and inflammatory cytokine drivers of lupus pathogenesis.
RG2 antigens cross-react with lupus anti-dsdnA antibodiesTo evaluate for relationships between anti-self and anti-RG2 responses in patients with lupus, we assessed the capacity of native DNA and RG2 extract to inhibit lupus serum IgG binding (online supplementary figure 2A, B). As expected, in side-by-side assays, soluble RG2 extract was an efficient inhibitor of the binding of lupus serum IgG to immobilised treated RG2 extract (online supplementary figure 2C). Yet, native DNA only margin-ally inhibited IgG binding to RG2 extract (online supplementary figure 2D). In contrast, RG2 extracts displayed marked dose-de-pendent inhibition of binding of lupus serum IgG to native DNA, whereas RG1 extracts caused little or no inhibition of DNA binding (online supplementary figures 1I, 2B). Strikingly, RG2 displayed high-level (~90% maximal) inhibition of the binding to native mammalian DNA (online supplementary figure 2A) that was nearly as efficient in inhibitory activity as soluble native DNA itself (online supplementary figure 2B).
To further confirm the relevance of RG2 epitopes with antinu-clear antibody responses, we assessed the effect on IgG antibody reactivity with Crithidia luciliae, a protozoan with a kinetoplast organelle used to detect antibodies specific for chromatin.32 As expected, preincubation with native DNA inhibited this anti-body interaction (online supplementary figure 2E-J), and RG2 strain was also inhibitory (online supplementary figure 2J), while a different RG strain (ie, RG1) was not (online supplementary table 5 and online supplementary figure 1I), suggesting that the relevant cross-reactive antigen(s) is strain-restricted (ie, associ-ated with RG2 but not RG1). Collectively, we found that many patients with lupus have anti-dsDNA antibody responses that are cross-reactive with antigens in RG2, which is relevant to the
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Figure 2 Patients with LN have serum IgG that recognise RG2 strain-restricted non-protein antigen(s) and that levels correlate with scores forlupus disease activity. Immunoblot results for IgG from (A) patient with LN (S-134), (B) patients with LN (S-047), (C) patient with non-active lupus (S-096) and (D) healthy control (CTL23). Bacterial extracts were prepared with nuclease and lysozyme treatment; lanes 1–8 represent R. gnavus RG1-RG8 strains (see online supplementary table 4), and lanes 9 and 10 contain extracts from gut commensal species, Bacteroides thetaiotaomicron and Prevotella copri. After electrophoretic transfer, replicate membranes were incubated with different sera at 1:100 dilution, with detection for human IgG deposition. Only patients with highly active lupus had serum IgG that recognised the oligomeric antigenic band in RG2 (red arrow) that was not detected in other RG strains. (E) Immunoblot of human gut isolated strains of R. gnavus, RG1-RG8. For each strain, molecular species were electrophoretically separated side-by-side, after nuclease and lysozyme treatment (left lane) and after additional thorough proteinase K treatment (right lane). Reactivity for serum IgG from patient S-134 with active LN is shown, with the serum tested at 1:100. Inset shows magnified view of non-protein oligomeric bands in RG2 extract, which are also seen in panels A and B. (F) In patients with SLE, RG faecal abundance by 16S rRNA analysis correlates with the levels of serum IgG anti-RG2 antibody. (G) Levels of serum IgG anti-RG2 in individual patients with lupus in the NYU cohort were compared with unaffected adults (see Methods section). Patients with SLE with high disease activity (SLEDAI ≥8) had higher levels of anti-RG2 IgG antibodies than healthy controls and than SLE with low disease activity. (H) Levels of serum IgG antinative DNA directly correlate with IgG anti-RG2 antibodies. Results from multiplex assay using extracts treated with lysozyme and a broad endonuclease, which was separately coupled to a set of paramagnetic beads (Luminex) in parallel with other antigens. (I) Levels of IgG anti-RG2 directly correlate with IgG to an extract of human glomeruli. Results from multiplex assay. (J) Levels of serum C3 inversely correlated with levels of serum lupus IgG anti-RG2 antibodies in patients with SLE. (K) Levels of serum C4 inversely correlated with levels of serum lupus IgG anti-RG2 antibodies in patients with SLE. (L) IgG anti-RG2 levels strongly correlate with serum IL-6 levels. (M) Levels of IgG anti-dsDNA, determined by commercial ELISA (INOVA), have only a modest correlation with serum IL-6 levels. (N) IgG anti-RG2 levels correlate with IFNα2 levels, determined by commercial bead-based assay (Luminex). (O) Levels of IgG anti-dsDNA antibodies do not correlate with serum IFNα2 levels. IgG anti-DNA and RG2 levels were measured by in-house custom bead-based assays. C3 and C4 were measured with commercial ELISA, while cytokines were measured by commercial bead-based assays. Significance was based on Mann-Whitney test or Spearman correlations. LN, lupus nephritis; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index.
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Figure 3 The LG3 pool contains two major series of lipoglycan molecules. (A) The 1H NMR spectrum of native LG3, recorded in D2O at 300 K, shows signals typical for lipoglycans, for example, for methyl groups of N-acetyl sugars, other carbohydrates and aliphatic chains of the lipid anchor. (B) The charge-deconvoluted MS spectrum of negative ion mode analysis of native LG3. (C) The charge-deconvoluted MS spectrum of negative ion mode analysis of de-O-acylated LG3. (Alternate version of this spectrum with additional assigned peaks is shown in online supplementary figure 1L.) (C) The charge-deconvoluted MS spectrum of negative ion mode analysis of de-O-acylated LG3. LG3 contains two major series of lipoglycans (termed series A [red] and B [dark blue]), starting from molecules with molecular weights of 2499.091/2513.107/2527.122 Da (series A) and 3380.395/3394.410/3408.426 Da (series B), respectively. These molecules are present in a series of further hexose-substituted (△m =+162.05 Da) molecular species. All peaks are present with variations in fatty acid composition (△m=14.02 Da is equivalent to CH2) (B). The complexity of the mass spectrum of de-O-acyl LG3 (C) is significantly decreased because of the removal of fatty acids. The overall signal pattern of the two series of lipoglycan species is well preserved. For series B, subspecies with a further alanine-substitution are apparent (light blue). Sodium adducts are represented as △m=21.98 Da, labelled in italic style. Abundances were normalised to the respective base peak of the depicted spectral region.
notion that anti-DNA antibodies with great cross-reactivity more often have nephritogenic potential.10
RG2 contains lipoglycan(s) with antigenic properties recognised by patients with lupusTo isolate the lipoconjugates produced by RG2, we applied a validated Hydrophobic Interaction Chromatography (HIC)-based separation strategy to isolate specific cell wall moieties such as lipoteichoic acids (LTAs), lipoglycans (LGs or lipopro-teins33 from the RG2 strain (online supplementary figure 1J). For further analyses, we combined fractions 23–27 (pool 2) and 28–31 (pool 3) (online supplementary figure 1J) and applied 1H NMR (figure 3A) as well as mass spectrometric (MS) anal-ysis (figure 3B and online supplementary figures 1K, L), We thereby detected significant amounts of LGs, but the absence of 31P NMR signals (data not shown) indicated the absence of LTA molecules. We therefore termed these pools LG2 and LG3,
respectively. Taken together, these analyses documented that LG2 and LG3 from the RG2 strain each represent a series of distinct LGs (online supplementary figure 1K, L).
Further structural analyses were performed after hydrazine treatment that cleaves off all ester-bound residues, such as fatty acids.34 Compared with the MS spectrum of native LG3 (figure 3B and online supplementary Figure 1L), the de-O-acyl LG3 displayed significantly decreased complexity (figure 3C); in native LG3, the signals at 3380.395 Da, 3394.410 Da and 3408.426 Da (start of series Bnative [figure 3B]) shifted to the signal of 2931.966 Da in the spectrum of de-O-acyl LG3 (start of series Bde-O-acyl [figure 3C]), due to the removal of two fatty acids with combined chain length from C30:0 to C32:0. These changes reflected mass differences of 448.43 Da, 462.44 Da or 476.46 Da, respectively. For this series, molecules with varying number of hexoses were also detectable, as the signal pattern displays mass differences of 162.05 Da. A similar pattern was observed in the lower molecular range between 2000 and 2800 Da (figure 3C) for series Ade-O-acyl, for example, signals at 2050.663 Da, 2212.716 Da, 2374.768 Da and 2536.820 Da, representing the corresponding de-O-acylated molecules present in series Anative. Interestingly, LG2 mono-acyl variants of LGs of series A and B were also detected (online supple-mentary figure 1K), and LG2 had a third series (Cnative) of LG molecules that was also observed. Hence, although each of the three series most likely have differing core units, all LG vari-ants display ‘hexose-capping’. The complete structural charac-terisation of these LGs from RG2 is beyond the scope of the current report. An analogous extraction of cell wall moieties from RG1 did not result in any yields of similar LGs, which suggests that RG strains differ in their capacity to produce cell wall LGs.
For investigations of the potential immune epitope features of the RG2 strain, we performed immunoblots with lupus IgG that detected high levels of oligomeric bands in the LG3 pool in the range of ~20–30 kDa protein markers, which resembled the reactivity pattern of protease-resistant antigens in the RG2 extract that were absent in other RG strains (figure 4A–D). In fact, preincubation of a lupus serum with LG3 at 2 µg/mL inhib-ited all immunoblot reactivity with the oligomeric antigenic bands in the LG2 and LG3 pools and with the nuclease-treated RG2 extract, but did not affect the IgG reactivity with the protease-sensitive bands (figure 4E, F). As predicted, lupus IgG immunoreactivity with these oligomeric bands was destroyed by periodate treatment (figure 4G), further supporting the notion that these immunodominant epitopes are on glyco-conjugate molecules. Side-by-side bead-based assays confirmed that the cell wall LG3 pool expresses the strain-restricted immunodominant epitopes in RG2 commonly recognised by lupus sera (figure 4H).
Furthermore, in vitro assays using a reporter cell line trans-fected with the human TLR2 demonstrated dose-dependent stim-ulation for the RG2 extract, and for the purified LG3, which was greater than for the LG2 sample, while the RG1 bacterial extract was devoid of detectable activity under these conditions (online supplementary figure 1M). To confirm the responsible mecha-nism, when studies were repeated in the presence of anti-TLR2 antibodies there was inhibition of NF-κB activation (online supplementary figure 1M). Hence, the LG pools, isolated from the RG2 strain, contain both strain-restricted carbohydrate-asso-ciated antigen(s) recognised by serum IgG in patients with SLE, and component(s) with immunostimulatory properties (online supplementary figure 1M), which might be lipoproteins that commonly copurify with LTA and/or LGs recovered using this HIC isolation protocol.33 34
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Figure 4 Lipoglycans from RG2 represent immunodominant antigens for patients with SLE. Immunoblots of purified LG2 and LG3 pools, and RG2 and RG1 extracts, were performed with sera from (A) SLE-07, (B) SLE-127, (C) SLE-080 that react with the same protease-resistant bands in LG2, LG3 and RG2 (red box), whereas (D) a healthy control reacts with distinct immunodominant antigens also recognised by patients with active SLE that are generally lower MW (blue box). (E) Immune recognition by serum antibodies in SLE-134 with oligomeric bands in LG2 and LG3, and in RG2, (F) are entirely inhibited by preincubation with LG2 at 2 µg/mL. (G) Periodate treatment before blotting destroys antigens in LG2 and LG3. Loading per lane is indicated above at µg/lane. Immunoblots were performed with serum at 1:200, with detection of IgG reactivity. (H) Serum levels of antibodies to LG3 pool and RG2 extract were highly correlated. Levels were determined by bead-based assay side-by-side in sera from 40 patients with SLE with diverse disease activity scores. SLE, systemic lupus erythematosus.
Independent cohorts confirm the association of anti-RG2 responses with lnIn the NYU discovery cohort, patients with active nephritis at the time of blood sampling (based on activity criteria19 had significantly raised levels of IgG anti-RG2 strain-restricted anti-bodies (figure 5A). To confirm this association, we studied a second independent cohort of 27 patients (online supplemen-tary table 1) (Temple University) in which 11/27 had elevated IgG anti-RG2 antibody reactivity, and of the 12 with active LN (based on level of proteinuria and/or concurrent biopsy data), 9 (75%) displayed high anti-RG2 antibody levels (figure 5B). In a third cohort of 17 patients with biopsy-documented active LN (online supplementary table 1) (The Ohio State University), 10 (59%) showed elevations of serum IgG anti-RG2 responses, but by contrast such responses were absent in patients with primary membranous glomerulonephritis and ANCA-associated vasculitis (Welch test, p=0.0025) (figure 5C). Furthermore, for a number
of the patients in the NYU and Temple cohorts the LN diag-nosis was confirmed by renal biopsy (online supplementary table 1). Inhibition studies of pooled sera from each of these three independent geographically separated cohorts confirmed that lupus-associated autoimmune IgG anti-native DNA responses were cross-reactive with epitope(s) in the RG2 strain (online supplementary figure 1N). Taken together, in three independent cohorts high IgG anti-RG2 antibody reactivity with cross-reac-tivity with anti-native DNA autoantibodies (online supplemen-tary figure 1N) was commonly found in patients with LN from proliferative Class III and Class IV histopathology by ISN/RPS criteria35 (online supplementary table 1).
SLE is a condition that overwhelmingly affects women, and due to concerns that gut microbiome can vary based on sex alone,36 our discovery cohort was restricted to female patients. Yet our confirmatory cohorts included several male patients, including individuals with active LN, who also commonly had elevated levels of serum IgG anti-RG2 antibodies (Figure 5, supplemen-tary table 1 and data not shown). Furthermore, raised serum IgG antibody responses to RG2 were found in patients with active LN of diverse race/ethnicity: African-American, Asian, Caucasian, as well as Hispanic-White and Hispanic-Black (online supplemen-tary table 1), which is consistent with the commonness of these immune responses in the three lupus cohorts examined.
dISCuSSIonOur surveys of a cross-sectional discovery cohort documented that the severity of specific patterns of dysbiosis generally correlated with clinical disease activity. The gut microbiome of patients with higher SLEDAI scores had greater restrictions in taxonomic diversity. Indeed, our analyses provide the first evidence that lupus is associated with intestinal outgrowths of RG, an obligate anaerobic species, in the Blautia genus within the Lachnospiraceae family of the class Clostridia.23 RG, which is a common (but generally low-level abundance) commensal in the faecal communities of healthy controls,37 was docu-mented to be overrepresented, with >5-fold outgrowths in patients with lupus, with the greatest expansion in those with high disease activity and especially LN. Patients with lupus in these three geographically dispersed cohorts commonly displayed RG strain-restricted host immune responses that were cross-reactive with anti-DNA immune responses.
Patients with lupus also displayed higher levels of gut secre-tory IgA, a numerical trend towards higher abundance of faecal sIgA-coated RG bacteria and other abnormalities asso-ciated with gut barrier dysfunction. Importantly, there was a significant direct relationship with lupus disease activity and with LN, and the highest levels of serum IgG anti-RG antibody responses primarily directed against a RG strain-restricted cell wall-associated LG-containing antigen. Whereas these RG antigens may leak out of the intestine to induce IgG anti-body responses, conversely IgG cannot enter the gut lumen and reduce the representation of taxa such as RG. Notably, RG2 LG-containing pools included moieties that were potent in vitro NF-κB immune activators via TLR2 (online supple-mentary figure 1M) that has previously been implicated in lupus pathogenesis.38 We speculate that the cross-reactivity of lupus autoreactive B cells to leaked stimulatory RG bacterial components, including TLR ligands(s), may contribute to the initiation and/or flares of lupus disease and especially LN that is a major source of early mortality and morbidity.39
In the human gut, the Lachnospiraceae family members fill a special niche, and degrade complex polysaccharides40
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Figure 5 Active lupus nephritis is associated with elevated IgG anti-RG2 antibodies. (A) Results from individuals in NYU cohort are shown, with comparisons to unaffected controls. (B) Results from individuals in the Temple University cohort with 16 patients with lupus without evidence of renal (ie, non-renal) are compared with 12 with active LN. (C) Results from 17 patients with biopsy-proven active LN from Ohio State University are compared with 6 patients with biopsy-proven idiopathic primary mesnagial glomerulonephritis (MGN) and 20 with IgA nephropathy (IgAN). The cut-off MFI value of 1900 for IgG anti-RG2 (dotted line) was determined by mean for 57 + 2 SD from unaffected 23 healthy controls with gut microbiome data, 15 other healthy individuals, 13 with psoriatic arthritis, and 12 with osteoarthritis). Renal disease was determined by clinical criteria and/or biopsy. Anti-RG2 values were determined by bead-based assay, with significance assigned by Mann-Whitney test.
including those of the intestinal mucus layer, which may lead to a leaky gut.41 Yet RG strains vary greatly in their genomic composition, metabolic features and competitiveness. Indeed some isolates produce Lantibiotic polypeptides that suppress competing anaerobic species,42 and experimental colonisation can cause shifts in T cell balance toward IL-17 production.43 44
RG outgrowths have recently been reported in patients with ankylosing spondylitis, and abundance was reported to correlate with greater disease activity.45 In addition, patients with IBD have also been reported with RG blooms that comprised up to 69% of intestinal communities, and genomic analysis showed that these isolates were generally from a sepa-rate clade of RG than those in healthy individuals.46 In the IBD microbiome, strain-specific genes involved in oxidative stress responses have recently been postulated to confer an advan-tage for RG strains46 which could potentially be relevant to lupus pathogenesis as well.
The design of our observational studies has inherent limita-tions, as we do not currently know if RG expansions are truly causative or if lupus disease activity instead fosters preferen-tial outgrowth of the candidate pathobiont. There is also a potential confounding effect of medications, as most patients were receiving treatment at the time of biosampling, and an enlarging list of medications is postulated to affect the balance of taxa within the gut microbiome community.47 48 However, we discount this potential effect as several of the recruited patients were in-flare while off medication and the same RG outgrowths and immune responses were nonetheless found (online supplementary table 1 and data not shown). The properties of the LGs in RG2, as well as other strains, will also require further investigation. In addition, Gram-positive opportunistic pathogens may have lipoproteins that are activa-tors of innate immunity,49 and such lipoproteins can be copu-rified along with other lipoconjugates using the HIC-based methodology. Hence, future studies will also need to directly assess the specific immunostimulatory properties of the LGs themselves.
Intestinal communities are complex, and emerging data suggest many pathways may be intertwined with lupus patho-genesis. Expansions of the commensal pathobiont species, Segmented Filamentous Bacteria, in susceptible mouse strains can contribute to antinuclear autoantibody production.50
Furthermore, it has recently been reported that translocation and release of Enterococcus gallinarum from the small intes-tine may trigger the Aryl hydrocarbon receptor, resulting in autoantibody production and a type I interferon associated transcript signature in peripheral mononuclear cells.51 Indeed, we speculate that multiple pathobionts, singly or in combi-nation, may contribute to lupus pathogenesis, and hence our finding of concurrent RG outgrowths merit further examina-tion in longitudinal studies, including inception cohorts as well as more in-depth mechanistic studies.
ConCluSIonMounting evidence suggests that overt lupus disease is often preceded by years of a preclinical phase with prominent circu-lating autoantibody responses.52 Lupus disease has also been reported to be intertwined with greater inherent instability and lack of balance within the intestinal community53 which could underlie the restricted taxa diversity and disease-asso-ciated RG outgrowths that we found. Indeed, the abundance of RG outgrowths correlated with lupus disease activity, with the greatest expansions in those with active renal disease, and these immune responses were primarily directed against a strain-restricted cell wall lipoglycan. Notably, individual lupus communities also displayed concordant significant expan-sions of Veillonella species, along with reciprocal contrac-tions of Bacteroides uniformis. Furthermore, our findings also contribute to evidence of a widening range of inflammatory and autoimmune conditions associated with a ‘leaky gut’,54 which may in part be responsible for the lupus-associated RG strain LG-directed antibody responses. We speculate that leakage of RG bacterial components contributes to nephrito-genic autoantibody responses. To pursue these questions, it will be important to characterise the diversity and genetic features of RG strains and to understand when these strains colonise and expand in patients with lupus. In part, these investigations may lead to development of a biomarker assay that aids both earlier diagnosis and better prognostic determinations.
Author affiliations1Department of Medicine, new York University school of Medicine, new York, new York, Usa
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2Department of Pathology, new York University school of Medicine, new York, new York, Usa3Forschungszentrum Borstel, leibniz lungenzentrum, Borstel, Germany4ohio state University, columbus, ohio, Usa5Temple University, Philadelphia, Pennsylvania, Usa6Department of Public Health sciences, Medical University of south carolina, charleston, south carolina, Usa
Acknowledgements We acknowledge the assistance of the nYU immune Monitoring core and the nYU Flow cytometry core Facility, supported by nYU-HHc cTsi Grant Ul1 Tr000038, nYU laura and isaac Perlmutter cancer center support grant, P30ca016087 from the national center for advancing Translational sciences (ncaTs), the nYU Microscopy laboratory and the nYU Genome Technology core. in addition, we thank simone Thomsen, Heiko Käßner and Brigitte Kunz (all at the rc Borstel) for their excellent technical assistance, emma allen-Vercoe for bacterial strains and technical advice, Karsten Zengler for assistance iwith bacterial cultures, and Dan littman, Jeffrey Weiser and Martin Blaser for critical review of data and advice.
Contributors Da, aVa and GJs designed research. Da, ao, Ds and nG performed research. rc, Br and JPB recruited and characterised patients and critiqued the manuscript. Da, aVa and GJs analysed data and wrote the paper.
Funding This work was supported in part by national institutes of Health Grants; r01ai090118 (GJs), r01ai068063 (GJs), r01-ar42455 (GJs), P50 ar070591 (GJs, JPB), niaiD contract for B cell epitope Discovery and Mechanisms of antibody Protection, HHsn272201400019c (GJs), n01-ar-4-2271, an american recovery and reinvestment act supplement (GJs), niH-niaMs r01-ar061569-01a1 (rc), P50 ar070591-01a1/coMPel (GJs and JPB), r01 lM012517 (aVa and GJs), the lupus research institute and the Judith and stewart colton autoimmunity center (GJs). 16s rrna gene sequence determinations and analysis were supported by the P robert Majumder charitable Trust (GJs).
Competing interests nYU has filed intellectual property related to this report.
Patient consent for publication not required.
Provenance and peer review not commissioned; externally peer reviewed.
data sharing statement Data in the text will be freely available. Microbiome community 16s rrna data will be made available on reasonable request.
open access This is an open access article distributed in accordance with the creative commons attribution non commercial (cc BY-nc 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.
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Systemic lupus erythematosus
TranslaTional science
Low-density granulocytes activate T cells and demonstrate a non-suppressive role in systemic lupus erythematosussaifur rahman,1 Divya sagar,1 richard n Hanna,1 Yaima l lightfoot,2 Pragnesh Mistry,2 carolyne K smith,2 Zerai Manna,3 sarfaraz Hasni,3 richard M siegel,4 Miguel a sanjuan,1 roland Kolbeck,1 Mariana J Kaplan,2 Kerry a casey 1
To cite: rahman s, sagar D, Hanna rn, et al. Ann Rheum Dis 2019;78:957–966.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214620).
For numbered affiliations see end of article.
Correspondence toDr Kerry a casey, allen institute for immunology, seattle 98109, Washington, Usa; kerry. casey@ alleninstitute. org and Dr Mariana J Kaplan, systemic autoimmunity Branch, national institute of arthritis and Musculoskeletal and skin Diseases, national institutes of Health, Bethesda 20892, Maryland, Usa; mariana. kaplan@ nih. gov
MJK and Kac are joint senior authors.
received 19 october 2018revised 8 March 2019accepted 12 March 2019Published online First 30 april 2019
© author(s) (or their employer(s)) 2019. re-use permitted under cc BY-nc. no commercial re-use. see rights and permissions. Published by BMJ.
AbSTrACTObjectives The presence of proinflammatory low-density granulocytes (lDG) has been demonstrated in autoimmune and infectious diseases. recently, regulatory neutrophilic polymorphonuclear myeloid-derived suppressor cells (PMn-MDsc) were identified in systemic lupus erythematosus (sle). Because lDG and PMn-MDsc share a similar phenotype with contrasting functional effects, we explored these cells in a cohort of patients with sle.Methods lDG and normal-density granulocytes (nDG) were isolated from fresh blood of healthy donors (HD) and patients with sle. associations between lDG and clinical manifestations were analysed. Multicolor flow cytometry and confocal imaging were performed to immunophenotype the cells. The ability of lDG and nDG to suppress T cell function and induce cytokine production was quantified.results lDG prevalence was elevated in sle versus HD, associated with the interferon (iFn) 21-gene signature and disease activity. also, the lDG-to-lymphocyte ratio associated better with sle disease activity index than neutrophil-to-lymphocyte ratio. sle lDG exhibited significantly heightened surface expression of various activation markers and also of lectin-like oxidised low-density lipoprotein receptor-1, previously described to be associated with PMn-MDsc. supernatants from sle lDG did not restrict HD cD4+ T cell proliferation in an arginase-dependent manner, suggesting lDG are not immunosuppressive. sle lDG supernatants induced proinflammatory cytokine production (iFn gamma, tumour necrosis factor alpha and lymphotoxin alpha) from cD4+ T cells.Conclusions Based on our results, sle lDG display an activated phenotype, exert proinflammatory effects on T cells and do not exhibit MDsc function. These results support the concept that lDG represent a distinct proinflammatory subset in sle with pathogenic potential, at least in part, through their ability to activate type 1 helper responses.
InTrOduCTIOnA low-density neutrophil population was identified in the peripheral blood mononuclear cell population of patients with systemic lupus erythematosus (SLE) in 1986.1 Since then, these cells have been reported in other autoimmune, cancer and infectious diseases
with either proinflammatory (low-density granu-locytes, LDG) or suppressive effects (neutrophilic polymorphonuclear myeloid-derived suppressor cells, PMN-MDSC).2–6 The proinflammatory nature of LDG in SLE was demonstrated by their ability to secrete tumour necrosis factor alpha (TNF-α), interferon gamma (IFN)-γ and type I IFN, cyto-kines frequently implicated in disease pathogen-esis.3 LDG are also potent producers of neutrophil extracellular traps (NETs), which drive type I IFN production by plasmacytoid dendritic cells (pDCs) and directly contribute to endothelial cell dysfunc-tion and vascular damage.7–10 Patients with SLE with increased circulating LDG numbers also demonstrate heightened prevalence of skin involvement, vasculitis, arterial inflammation and coronary plaque.3 11
In contrast, PMN-MDSC were described as immunoregulatory due to their ability to suppress T cell proliferation in infectious, autoimmune, cancer and metabolic diseases.12–16 The immuno-suppressive mechanisms mediated by PMN-MDSC include surface expression of various checkpoint inhibitors (programmed death-ligand 1 [PD-L1], programmed death-ligand 2 [PD-L2] and CD73), along with release of enzymatic or chemical media-tors (arginase-1 [Arg1] and nitric oxide synthase).17 In a lupus nephritis cohort, SLE PMN-MDSC medi-ated suppression in an Arg1-dependent manner, similar to what has been described in multiple cancer settings.14 The multifaceted roles of these
Key messages
► Low-density neutrophil populations may either exhibit proinflammatory (low-density granulocytes, LDG) or suppressive (polymorphonuclear myeloid-derived suppressor cells) properties. Both populations have been reported in systemic lupus erythematosus (SLE).
► SLE LDG demonstrate an activated phenotype, do not suppress T cells and promote Th1 responses.
► Activated SLE LDG also express lectin-like oxidised low-density lipoprotein receptor-1.
► SLE normal-density granulocytes, but not LDG, may suppress T cells in an arginase-dependent manner.
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Table 1 Demographics and clinical characteristics of HD and patients with lupus examined in this study
Hd* SLE‡
Sex (M/F), n M=7, F=73 M=6, F=88
Age, years (median, range) 45 (35–65)† 40 (15–73)
SLEDAI (median, range) – 2 (0–13)
C3, mg/dL (median, range) – 95.5 (44.8–184.9)
C4, mg/dL (median, range) – 16.7 (2.2–43.3)
ESR, mm/hour (median, range) – 23 (2–100)
CRP, mg/L (median, range) – 1.7 (0.15–31.4)
Auto-antibodies (% positive)
Anti-dsDNA – 70
ANA – 95
LAC – 28
ENA – 85
Medications (%)§
Oral corticosteroids – 21
Hydroxychloroquine – 83
Azathioprine – 76
Cyclophosphamide – 19
Mycophenolate mofetil – 18
Methotrexate – 24
Biologics (belimumab/rituxan) – 5
*Human whole blood was collected from HD from the MedImmune Blood Donor programme. †Age of the HD was provided by decade. ‡SLE blood samples were collected from the National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institutes of Health. Clinical parameters for patients with SLE were obtained at the time of visit by routine laboratory test. §Current medications that patients were taking.ANA, anti-nuclear antibodies; C3, complement 3; C4, complement 4; CRP, C-reactive protein; dsDNA, double-stranded DNA; ENA, extractable nuclear antigens; ESR, erythrocyte sedimentation rate; F, female; HD, healthy donors; LAC, lupus anticoagulant; M, male; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index.
low-density neutrophil subsets across a range of diverse indica-tions highlight their importance in human disease.18 19
Despite contrasting functions, LDG and PMN-MDSC have been reported in SLE with strong association to SLE disease activity index (SLEDAI).3 14 Notably, both subsets utilise over-lapping neutrophil surface markers for identification, including CD11b+, CD33+, CD15+ (or CD66b) and human leucocyte antigen-DR isotype (HLA-DR−), after excluding other lineage (LIN) markers (CD3, CD19, CD20 and CD56).19 20 While these subsets are thought of as immature neutrophils with reduced density, most identifying markers are also shared by the mature, terminally differentiated normal-density granulocytes (NDG) counterparts. In view of the shared similarities, a comprehensive and comparative immunophenotyping of activation and regu-latory markers on the low-density and normal-density counter-parts has yet to be performed in the context of linking these cells to SLE disease activity and understanding their relationship to the type I IFN axis and other components of this disease. Additionally, it remains unclear in SLE how the low-density and normal-density counterparts compare functionally in their ability to exert effects on T cells. As a result, there is ambiguity surrounding the identity, nature and role of these low-density subsets in the autoimmune space, particularly SLE.21 22
To address these knowledge gaps, we performed immunophe-notypic, morphological and functional characterisation of this neutrophil subset and compared them with autologous NDG in a well-characterised SLE cohort consisting of both clinically inactive and active patients. We hypothesised that this larger cohort with representation across the disease spectrum would offer enhanced understanding of the functional and pheno-typic features of these cells. Using this approach, we evaluated whether lupus LDG (i) associate with disease activity and, more specifically, the type I IFN pathway, (ii) manifest any of the PMN-MDSC regulatory effects via previously described mech-anisms and (iii) differ from matching NDG on these various phenotypic and functional aspects. As most of the published literature in the SLE field refers to this abnormal neutrophil subset as “LDG,” we will henceforth use the same terminology.
METHOdSMethods are provided in the online supplementary information.
rESuLTSOverview of SLE cohortThe demographic and clinical information for healthy donors (HD) and patients with SLE is summarised in table 1. There was no significant difference in either sex or median age. The median SLEDAI score was 2 with range of 0–13.
Increased LdG in SLE associates with IFn gene signature and disease severityPrevious studies demonstrated the increased presence of LDG in patients with SLE.1 3 14 We examined those findings by eval-uating the prevalence of these cells in our cohort of HD and patients with SLE. Surface markers, previously described in both autoimmune and cancer studies, required for their iden-tification were included.19 20 LDG, purified by density gradient centrifugation, were identified as LIN−(CD3/CD19/CD20/CD56), HLA-DR−, CD11b+, CD33+, CD15+ (figure 1A). LDG prevalence was significantly increased in patients with SLE by 2.9-fold (figure 1B; HD mean±SEM=0.81%±0.16; SLE mean±SEM=2.37%±0.45) and absolute count by 11.5-fold (figure 1C; HD mean±SEM=0.28±0.05; SLE
mean±SEM=3.22±0.53). Additionally, the number of LDG demonstrated significant positive association with SLEDAI score (figure 1D). In line with the strong association with SLEDAI, LDG were observed to be 1.6-fold higher in active versus inactive patients (inactive SLE mean±SEM=2.48±0.71; active SLE mean±SEM=4.05±0.79), suggesting their close relationship with disease activity. To understand whether the observed close association with SLEDAI was due to specific clinical manifestations, LDG numbers were assessed for asso-ciation with various clinical parameters and significantly associated with increased DNA binding (p=0.0165) and low complement levels (p=0.0187) (online supplementary table S1). The observed increase in LDG numbers in SLE versus HD and its association with SLEDAI validate previous observations and demonstrate efficacy of the cohort for the purposes of our study.
The type I IFN pathway plays an important role in disease pathogenesis and exacerbation in SLE and other rheumatic diseases.23–25 LDG that undergo enhanced NET formation lead to externalisation of modified extracellular DNA.26 27 Mate-rial released from NETs stimulates pDCs to release IFN-α and myeloid cells to release type I IFNs.7 28 29 While LDG can lead to the production of type I IFNs in vitro, the in vivo association between LDG and type I IFN pathway remains unknown. To address this, we evaluated the relationship between LDG and type I IFN-regulated genes.30 The LDG frequency was signifi-cantly increased in patients with high versus low type I IFN gene
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Figure 1 Increased prevalence of LDG in SLE associates with disease severity and IFNGS. The frequency of circulating LDG was analysed and correlated with disease severity. (A) Flow cytometry gating strategy for identifying LDG in human PBMC. Prevalence of LDG in HD and patients with SLE (healthy n=80; SLE n=94) as (B) frequency of CD45 (HD mean±SEM=0.81±0.16; SLE mean±SEM=2.37±0.45; p<0.0001) and (C) absolute numbers (HD mean±SEM=0.28±0.05×106/mL; SLE mean±SEM=3.22±0.53×106/mL; p<0.0001). (D) Association between number of LDG with healthy as well as inactive and active patients with SLE (healthy n=80; inactive n=50; active n=44) based on SLEDAI (inactive patients mean±SEM=2.48±0.71×106/mL; active patients with SLE mean±SEM=4.05±0.79×106/mL; p=0.02]. (E) Association between the IFNGS (low n=20; high n=22) and prevalence of LDG in patients with SLE with low (mean±SEM=0.04±0.01) and high (mean±SEM=1.10±0.30; p<0.0001) IFNGS. The relationship between (F) neutrophil:lymphocyte ratio and (G) LDG:lymphocyte ratio with SLEDAI. Individual symbol represents one donor and the mean±SEM is shown. HD, healthy donors; HLA-DR, human leucocyte antigen-DR isotype; IFNGS, interferon 21-gene signature; LDG, low-density granulocytes; LIN, lineage; PBMC, peripheral blood mononuclear cells; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index; SSC, side scatter.
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Figure 2 LDG exhibit activated phenotype in SLE. Both the NDG and LDG from HD and SLE were phenotyped for various activation and regulatory markers (healthy n=20; SLE n=20). Phenotyping of (A) activation and (B) regulatory markers on LDG in HD versus SLE. Phenotyping of (C) activation and (D) regulatory markers on NDG versus LDG from patients with SLE. Individual symbol represents one donor and the mean geometric MFI±SEM is shown. aBar representing geometric MFI value too low to be observed. Arg1, arginase-1; HD, healthy donors; LDG, low-density granulocytes; LOX-1, lectin-like oxidised low-density lipoprotein receptor-1; MFI, mean fluorescence intensity; NDG, normal-density granulocytes; SLE, systemic lupus erythematosus.
signature (IFNGS; figure 1E; low mean±SEM=0.04%±0.0; high mean±SEM=1.10%±0.30; p<0.0001). This demon-strates a strong association between the presence of LDG with both clinical activity and activation of the type I IFN pathway.
While the IFNGS is a strong predictor of type I IFN–driven inflammation, the total neutrophil-to-lymphocyte ratio (NLR) is another commonly used indicator of inflammation that encom-passes many pathways besides type I IFN.31 32 Given the signifi-cantly increased number of LDG in SLE, we compared NLR and evaluated utility of LDG-to-lymphocyte ratio (LLR) as a new and improved predictor of inflammation. In our cohort, while NLR demonstrated a significant difference between active and inactive patients with SLE (p=0.003), it did not signifi-cantly distinguish between inactive patients with SLE and HD (p=0.09) (figure 1F). In contrast, LLR offered better resolution and separation between the same inactive patients with SLE and HD (p<0.0001) (figure 1G). This result suggests that the LLR may be a more sensitive indicator of immune dysregulation than NLR. It would be interesting to further validate the utility of LLR over NLR in other disease areas.
SLE LdG exhibit an activated immunophenotypeThe increased prevalence of LDG across the disease spectrum validated the utility of our cohort for further analysis. Although LDG and PMN-MDSC share a similar immunophenotype, they demonstrate differences in the expression levels of various acti-vation and regulatory markers based on their contrasting func-tions.19 Regulatory markers associated with the suppressive
activity of PMN-MDSC include Arg1, CD73, CD274 (PD-L1) and CD273 (PD-L2),13 15 whereas degranulation markers asso-ciated with the proinflammatory role of neutrophils include CD63 (lysosome-associated membrane glycoprotein (LAMP)-3) and CD107a (LAMP-1).33 The expression of lectin-like oxidised low-density lipoprotein (oxLDL) receptor-1 (LOX-1) was demonstrated to be uniquely expressed by PMN-MDSC in cancer; however, its ability to distinguish MDSC from LDG in SLE remains unexplored.34 It is unknown how the expression levels of these markers compare between LDG and NDG. To address these questions, immunophenotyping was performed on both LDG and matching NDG from HD and patients with SLE. We first examined the immunophenotypic profile of LDG between HD and patients with SLE (figure 2A–B). As LDG share similar phenotypic markers with NDG, we then compared the expression profile of the two cell types in SLE (figure 2C–D) and HD (online supplementary figure S1).
With the exception of intracellular Arg1 and CD63, no signif-icant difference was observed for other markers between LDG from HD and SLE (figure 2A–B). However, when SLE LDG were compared with autologous NDG, significant differences were observed (figure 2C–D). Specifically, LDG expressed signifi-cantly higher (LOX-1, CD63, CD107a and CD274), reduced (intracellular Arg1, CD273 and CD95) or no difference (CD73) in expression levels of markers examined (figure 2C–D and HD in online supplementary figure S1). Apart from CD274, none of the other checkpoint inhibitors (CD273 and CD73) were signifi-cantly elevated on LDG versus NDG (figure 2D). Notably, the
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significantly higher expression of degranulation markers (CD63 and CD107a) and reduced intracellular Arg1 on SLE LDG versus NDG suggest their increased activation status (figure 2C). Of particular interest was the significantly higher surface expres-sion of LOX-1 and CD63 on SLE LDG versus NDG.
These results confirm and expand the notion that SLE LDG and NDG differ immunophenotypically. Based on the higher surface expression of the degranulation markers, SLE LDG display an activated immunophenotype with concomitant expression of LOX-1.
SLE LdG express LOX-1 and demonstrate different morphology compared with ndGThe increased expression of both LOX-1 and CD63 on LDG in SLE had not been previously described. While LOX-1 was recently suggested to be uniquely expressed by suppressive PMN-MDSC, CD63 is a granulocyte activation marker.33 34 LDG and autologous NDG from patients with SLE were anal-ysed by confocal imaging to examine for differences in LOX-1/CD63 staining and morphology. While NDG, identified by char-acteristic multilobed nuclei, demonstrated dim LOX-1/CD63 staining, LDG, with more heterogeneous nuclear morphology including banded nuclei, were observed to encompass four subpopulations based on variable levels of LOX-1 and CD63 expression (figure 3A). The LOX-1 expression on LDG was vali-dated at the messenger level (figure 3B). These data suggest that SLE LDG express LOX-1, and it was co-expressed with CD63 at variable levels.
The LOX-1hi/CD63hi co-expressing population (figure 3A) was selected for further quantitative morphological assessment. Quantitatively, LDG had fewer nuclear lobes compared with autologous NDG (figure 3C; LDG mean±SEM=2.3±0.09; NDG mean±SEM=3.8±0.06). Compared with their NDG counterparts, LDG demonstrated significantly reduced nuclear area, cell area, cell diameter as well as lower nuclear-to-cytoplasm ratio (figure 3D–G). Thus, LDG represent distinct morphology compared with NDG based on the various nuclear and cellular parameters examined.
SLE ndG, but not LdG, restrict Cd4+ T cell proliferation in arginase-dependent mannerBecause SLE LDG express LOX-1, which was recently suggested to be uniquely expressed by suppressive PMN-MDSC, we evalu-ated the cells for their suppressive capacity.34 LDG were assessed for their ability to suppress CD4+ T cell proliferation under both contact-independent and contact-dependent settings. For contact-independent suppression, the arginase mechanism, shown previously to be used by PMN-MDSC and activated neutrophils, was evaluated.14 35 NDG and LDG isolated from patients with SLE were treated overnight either in the absence or presence of argi-nase inhibitor nor-NOHA. The supernatant from such treated cells was then added to freshly isolated healthy naïve CD4+ T cells that were activated with anti-CD3/CD28 beads. As a positive control, the exogenously added arginase was observed to inhibit T cell proliferation in a dose-dependent manner and this was reversed by its inhibitor nor-NOHA (figure 4A–B). While SLE NDG signifi-cantly reduced the proliferative capacity of activated control CD4+ T cells compared with bead control (p=0.0006), an effect reversed by nor-NOHA (p=0.02), SLE LDG did not restrict T cell proliferation (figure 4C). Furthermore, neither NDG nor LDG isolated from HD demonstrated any suppressive effect on T cells (online supplementary figure S2B). Even in a contact-dependent setting, the SLE LDG did not inhibit T cell proliferation (online
supplementary figure S2C). These results were not affected by osmotic stress due to red blood cell lysis treatment (online supple-mentary figure S3).
In line with the increased suppression capacity of lupus NDG in suppressing T cell proliferation by a contact-independent mech-anism, these cells were found to release 3.3-fold higher bioactive arginase than LDG (figure 4D; NDG mean±SEM=27.27±3.2 enzyme units; LDG mean±SEM=8.17±3.3 enzyme units; p=0.008). The SLE NDG arginase synthesis was dampened 3.9-fold after nor-NOHA treatment (NDG − nor-NOHA mean±SEM=27.27±3.2 enzyme units; NDG + nor-NOHA mean±SEM=6.87±2.3 enzyme units). LDG did not show significant difference in amount of arginase synthesised in the absence or presence of nor-NOHA. This also corrobo-rates our observation that SLE NDG have 1.8-fold higher intracellular Arg1 geometric mean fluorescence intensity than LDG (figure 2C; NDG mean±SEM=4996±269; LDG mean±SEM=2817±285). Additionally, no significant difference in levels of released bioactive arginase was observed between HD NDG and autologous LDG (online supplementary figure S2D). These results suggest that SLE LDG do not inhibit T cell prolif-eration under both contact-independent and contact-dependent settings. Additionally, the SLE NDG-driven suppression of T cell proliferation in contact-independent setting is primarily medi-ated by intracellular arginase that is spontaneously released by the cells in its bioactive form.
SLE LdG induce proinflammatory T cell cytokine profileBoth LDG and PMN-MDSC have been demonstrated to drive their respective functions and downstream effects on other cells via the production of key cytokines including IFN-γ and inter-leukin 10.3 17 Given the significant differences between NDG and LDG in their immunophenotypic profile and abilities to suppress T cell proliferation, we examined the effect of these cell types on T cell cytokine production. The supernatant from cell cultures was analysed for different cytokines by Meso Scale Discovery multiplex assay. Only SLE LDG were able to induce significantly higher production of Th1 proinflammatory cyto-kines IFN-γ, TNF-α and lymphotoxin alpha than bead controls (figure 5). These cytokines were not detected in supernatants from LDG or NDG (data not shown). Other cytokines and chemokines examined were either not significantly different or not detected (online supplementary table S2). Overall, func-tional and phenotypic analyses of these cells support that lupus LDG represent a proinflammatory subset that can activate adap-tive immune responses.
dISCuSSIOnThe field of neutrophil biology has evolved significantly over the last decade, and some breakthroughs have focused around the role of low-density neutrophil subsets in autoimmunity, infectious diseases and cancer.15 36 LDG and PMN-MDSC exert contrasting functional effects on T cells, with LDG found to be stimulatory and PMN-MDSC suppressive. In the absence of distinguishing markers, functional assays are further needed in order to char-acterise these subsets as proinflammatory or suppressive. Parallel analysis of low-density and normal-density neutrophils lends important context for interpreting immunophenotypic and func-tional studies. While previous studies in SLE have examined these low-density neutrophil subsets, conflicting reports exist regarding their function and role in disease.3 14 21 22 Although the immuno-phenotypic identification strategy utilised by Wu et al is similar to the approach used in our study (CD11b+CD33+HLA-DR−), cells
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Figure 3 SLE LDG express LOX-1 and demonstrate different morphology compared with autologous NDG. (A) Representative confocal images of autologous SLE NDG and LDG and relative percentage of LDG subgroups. The LOX-1hi/CD63hi population was further characterised for morphology (data representative of five patients with SLE). (B) LOX-1 mRNA expression levels in SLE NDG and LDG (data representative of five patients with SLE). (C–G) Quantitative comparative assessment of various morphological parameters between SLE NDG and LOX-1hi CD63hi LDG population, (C) number of nuclear lobes, (D) nuclear area (NDG mean±SEM=160±7.4 µm2; LDG mean±SEM=101±3.4 µm2; p<0.0001), (E) cell area (NDG mean±SEM=315±14.6 µm2; LDG mean±SEM=245±9 µm2; p<0.0001), (F) nuclear-to-cytoplasm ratio (NDG mean±SEM=0.89±0.07; LDG mean±SEM=0.6±0.03; p<0.0001) and (G) cell diameter (NDG mean±SEM=14±0.72 µm; LDG mean±SEM=11.7±0.41 µm; p<0.0001). Data pooled from five patients with SLE with individual symbol representing one cell and the mean±SEM is shown. Scale bar per image is 10 µm. LDG, low-density granulocytes; LOX-1, lectin-like oxidised low-density lipoprotein receptor-1; NDG, normal-density granulocytes; SLE, systemic lupus erythematosus.
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Figure 4 SLE LDG do not restrict CD4+ T cell proliferation. Both NDG and LDG from patients with SLE were evaluated for their ability to restrict T cell proliferation in an arginase-dependent assay. The relative number of HD proliferating CD4+ T cells was calculated after 72 hours, when co-cultured with supernatant from either SLE NDG or LDG that was cultured overnight in the absence or presence of arginase inhibitor, nor-NOHA. (A) Representative plots and (B) relative number of T cells (normalised to CD3/CD28 bead controls) for different control conditions from three independent experiments. (C) Relative number of T cells (normalised to CD3/CD28 bead controls) when cultured with either NDG or LDG supernatant that was cultured overnight in the absence or presence of nor-NOHA (pooled data from six patients with SLE in three independent experiments). (D) Quantification of bioactive arginase present in the supernatant of the NDG and LDG test conditions (pooled data from six patients with SLE and the mean±SEM is shown). One unit of arginase is the amount of enzyme that will convert 1.0 µmole of L-arginine to ornithine and urea per minute at pH 9.5 and 37°C. HD, healthy donors; LDG, low-density granulocytes; NDG, normal-density granulocytes; NOHA, Nω-hydroxy-nor-arginine; SLE, systemic lupus erythematosus.
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Figure 5 SLE LDG induce proinflammatory T cell cytokine profile. Normalised quantification of (A) IFN-γ, (B) TNF-α and (C) LT-α from the proliferating T cell cultures that were cultured with supernatants derived from either NDG or LDG of patients with SLE (pooled data from six patients with SLE and the mean±SEM is shown). IFN-γ, interferon gamma; LDG, low-density granulocytes; LT-α, lymphotoxin alpha; NDG, normal-density granulocytes; SLE, systemic lupus erythematosus; TNF-α, tumour necrosis factor alpha.
were designated as PMN-MDSC by these investigators because of their observed suppressive functions.14 In our hands, these cells exhibited proinflammatory functions. The cohort studied by Wu et al exhibited high disease activity, and the majority of patients had lupus nephritis, which could underlie the observed differences in function. Furthermore, in our hands, the cell sorting technique used by Wu et al may significantly modify the functional character-istics of granulocytes and myeloid cells (unpublished observations).
In multiple cancers, LOX-1 was demonstrated to be expressed at high levels on suppressive PMN-MDSC as compared with their normal-density counterparts and was thus suggested as a marker for PMN-MDSC.34 Here, we demonstrate for the first time the heightened surface expression of LOX-1 on proinflam-matory LDG in an autoimmune disease. LOX-1 is a class E scav-enger receptor for oxLDL, and in inflammatory diseases such as SLE, elevated oxLDL can induce granulocytic activation and degranulation.37–44 In cancer, LOX-1 has been associated with suppressive activity but is not required for regulatory function.34 LOX-1 expression can be induced by endoplasmic reticulum stress, a common feature of both cancer and autoimmunity.34 45 For these reasons, in autoimmune diseases, LOX-1 should not be used to assess whether a neutrophil is immunosuppressive or proinflammatory.
Indeed, while SLE LDG express LOX-1, they did not display any significant ability to suppress T cells in either contact-independent or contact-dependent assays. In contrast, here we demonstrated suppression mediated by transferred super-natants from overnight cultures of only lupus NDG. We observed that spontaneously released bioactive Arg1 from SLE NDG was 5-fold higher than HD NDG. Metabolism of extracellular argi-nine via Arg1 liberated from PMN-MDSC or neutrophil azurophil
granules is a key mechanism by which these cells are thought to exert their suppressive effect on T cells.46–48 Consistent with the possible involvement of this mechanism in disease, elevated levels of Arg1 have been reported in the serum of patients with autoim-munity, cancer and infectious diseases.49–51 The enhanced suppres-sive ability of SLE NDG may also be due to presence of activated neutrophils in such patients that have increased neutrophil sidero-phore lipocalin-2 (LCN2/NGAL), which can effectively bind and remove iron.52 Sequestration of iron, a key T cell nutrient, from the microenvironment negatively impacts T cell proliferation.53 The ability to release significantly more bioactive arginase coupled with heightened presence of LCN2 allows NDG from patients with SLE to be better suppressors of T cell proliferation than HD NDG. Our data suggest that in SLE, the Arg1-dependent suppres-sion is primarily mediated by NDG and not LDG. While NDG supernatant did not affect CD4 T cell cytokine production, LDG supernatant promoted proinflammatory Th1 cytokine response, further validating their role as drivers of inflammation. Such Th1 cytokine-producing cells have been detected in abundance in the kidneys of patients with lupus nephritis and also correlated with histological disease activity.54–56
In SLE, LDG have been shown to be highly susceptible to form NETs, a process that can lead to immune dysregulation and activation of pDC and myeloid type I IFN axis and induction of IFNGS.7 30 We demonstrated for the first time direct posi-tive correlation between LDG and IFNGS, suggesting a close link between the presence of these cells and activation of type I IFN pathway. As such, future studies should address whether similar correlations are observed in other inflammatory diseases where the type I IFN pathway is considered to play important pathogenic roles. Further understanding the crosstalk between
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aberrant neutrophil subsets and the type I IFN pathway in SLE may provide the identification of additional therapeutic targets in this disease.
In conclusion, we demonstrate that SLE LDG represent a pathogenic subset associated with type I IFN activation and with the development of non-suppressive T cell activation in this disease. The observed proinflammatory role of LDG validates their putative pathogenic role in SLE.
Author affiliations1Department of respiratory, inflammation and autoimmunity, Medimmune llc, Gaithersburg, Maryland, Usa2systemic autoimmunity Branch, national institute of arthritis and Musculoskeletal and skin Diseases, national institutes of Health, Bethesda, Maryland, Usa3lupus clinical research Program, office of the clinical Director, national institute of arthritis and Musculoskeletal and skin Diseases, national institutes of Health, Bethesda, Maryland, Usa4immunoregulation section, autoimmunity Branch, national institute of arthritis and Musculoskeletal and skin Diseases, national institutes of Health, Bethesda, Maryland, Usa
Acknowledgements The data were presented at the neutrophil 2018 conference (http:// theneutrophil. com). This study was supported in part by the intramural research program at niaMs/niH ar041199 and by Medimmune, a member of the astraZeneca Group. currently, Ds is an employee of eli lilly and company (indianapolis, in, Usa), Yll is an employee of University of Texas MD anderson cancer center (Houston, TX, Usa), rMs is an employee of novartis (Basel, switzerland), Mas is an employee of Bristol-Myers squibb company (new York city, nY, Usa) and Kac is an employee of the allen institute for immunology (seattle, Wa, Usa; kerry. casey@ alleninstitute. org). editorial support was provided by JK associates inc., a member of the Fishawack Group of companies. This support was funded by Medimmune.
Contributors sr, rMs, Mas, rK, MJK and Kac conceived and designed the study. sr, Ds, rnH, Yll, PM, cKs, ZM and sH performed the experiments and collected the data. sr, Ds, rnH, Yll, PM, cKs and sH analysed and interpreted the data. all authors were involved in development, review and approval of the manuscript.
Competing interests sr, rnH and rK are employees at Medimmune. Ds, Mas and Kac were employees at Medimmune during the time work was performed on this study. other authors have nothing to declare.
Patient consent for publication not required.
Provenance and peer review not commissioned; externally peer reviewed.
Open access This is an open access article distributed in accordance with the creative commons attribution non commercial (cc BY-nc 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.
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Sjögren’s syndrome
CliniCal sCienCe
Video clip assessment of a salivary gland ultrasound scoring system in Sjögren’s syndrome using consensual definitions: an OMERACT ultrasound working group reliability exercisesandrine Jousse-Joulin, 1 Maria antonietta D’agostino,2 Celine nicolas,3 esperanza naredo,4 sarah Ohrndorf,5 Marina Backhaus,6 Giorgio Tamborrini,7 isabelle Chary-Valckenaere,8 lene Terslev,9 annamaria iagnocco,10 Paz Collado,4 Cristina Hernández-Díaz,11 Frederique Gandjbakhch,12,13 Wolfgang a schmidt,14 Georgios Filippou, 15 Christian Dejaco, 16,17 Martin Helmut stradner,18 Mohamed atia Mortada,19 alojzija Hočevar,20 stavros Chrysidis,21 Ghada el Mardenly,22 Juan José de agustín,23 Ralf Thiele,24 Daryl K MacCarter,25 stephanie Finzel,26 Petra Hanova,27,28 alen Zabotti, 29 Cornelia Glaser,30 Zarrin alavi,3 Daniel sten Hammenfors,31 Florence Gatineau,3 George aW Bruyn32
To cite: Jousse-Joulin s, D’agostino Ma, nicolas C, et al. Ann Rheum Dis 2019;78:967–973.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2019- 215024).
For numbered affiliations see end of article.
Correspondence toDr sandrine Jousse-Joulin, Department of Rheumatology-eRi 29, la Cavale Blanche University Hospital, Brittany University, Brest 29200, France; sandrine. jousse- joulin@ chu- brest. fr
For ’Presented at statement’ see end of article..
Received 4 January 2019Revised 21 March 2019accepted 23 March 2019Published Online First 29 april 2019
© author(s) (or their employer(s)) 2019. no commercial re-use. see rights and permissions. Published by BMJ.
Key messages
What is already known about this subject? ► The current absence of a consensus about definitions and scoring of elementary lesions detected by salivary gland ultrasonography is an obstacle to the diagnosis and monitoring of primary Sjögren’s syndrome.
What does this study add? ► This study substantially advances the validation of a novel, reliable, semiquantitative scoring system for ultrasound abnormalities of the major salivary glands in patients with known or suspected primary Sjögren.
► Video clips shared via the internet proved useful for having multiple experts assess ultrasonography examinations.
How might this impact on clinical practice or future developments?
► Availability of a reliable scoring system for salivary gland ultrasonography abnormalities should assist in the diagnosis and follow-up of primary Sjögren’s syndrome.
► The video clip method used in this study can be expected to improve future reliability assessments.ard.bmj.com
ABSTRACTObjective To develop ultrasound (Us) definitions and a Us novel scoring system for major salivary gland (sG) lesions in patients with primary sjögren’s syndrome (pss) and to test their intrareader and inter-reader reliability using Us video clips.Methods Twenty-five rheumatologists were subjected to a three-round, web-based Delphi process in order to agree on (1) definitions and scanning procedure of salivary gland ultrasonography (sGUs): parotid, submandibular and sublingual glands (PG, sMG and slG); (2) definitions for the elementary sGUs lesions in patients with sjögren’s syndrome; (3) scoring system for grading changes. The experts rated the statements on a 1–5 likert scale. in the second step, sGUs video clips of patients with pss and non-pss sicca cases were collected containing various spectrums of disease severity followed by an intrareader and inter-reader reliability exercise. each video clip was evaluated according to the agreed definitions.Results Consensual definitions were developed after three Delphi rounds. among the three selected sGs, Us assessment of PGs and sMGs was agreed on. agreement was reached to score only greyscale lesions and to focus on anechoic/hypoechoic foci in a semiquantitative matter or, if not possible on a qualitatively (present/absent) evaluation of fatty or fibrous lesions. intrareader reliability for detecting and scoring these lesions was excellent (Cohen’s kappa 0.81) and inter-reader reliability was good (light’s kappa 0.66).Conclusion new definitions for developing a novel semiquantitative Us score in patients with pss were developed and tested on video clips. inter-reader and intrareader reliabilities were good and excellent, respectively.
InTROduCTIOnPrimary Sjögren’s syndrome (pSS) is a systemic autoimmune disease of unclear aetiology that primarily targets the lacrimal and salivary glands
(SG), causing dryness of the eyes and mouth. Mono-nuclear lymphoid cells infiltrate SGs, as well as the lacrimal glands, resulting in structural damage.1 In patients with suspected pSS, establishing the diag-nosis may be challenging, as no single parameter as autoantibody or other biomarker is highly specific and SG histology may be inconclusive.2
The American-European Consensus Group criteria are currently the most widely used
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classification criteria for patients with pSS symptoms.3 Several recent studies suggest that salivary gland ultrasonography (SGUS) may be helpful for evaluating the presence of SG involvement in patients with suspected or established pSS and could be another parameter for diagnosis.4–7 American College of Rheumatolo-gy-European League Against Rheumatism (EULAR) criteria were published more recently without including SGUS.8 Furthermore, two studies support the usefulness of SGUS in monitoring the effect of treatment in clinical trials.7 9
Before SGUS can be used as an outcome measurement instru-ment (OMI), its reliability must be tested. There is an unmet need to standardise and assess SGUS because no international consensus exists on SGUS elementary lesion definitions and scorings. A few efforts have been made to test inter-reliability based on definitions made by few well-designed studies.10–15
To validate the use of SGUS as a possible OMI for SG lesions in patients with pSS, a subtask force of the Outcome Measures in Rheumatology Clinical Trials (OMERACT) working group was created in June 2016. The objectives of the subtask force were as follows: (1) to develop a consensus on definitions of normal ultrasound (US) findings in major SG and a standardised scanning protocol; (2) to develop US definitions on elementary lesions in patients with pSS and a scoring system; (3) to test the reliability of the scoring system on patients with pSS and non-pSS sicca patients using consensual definitions and the protocol using a web-based video clip platform.
MATeRIAlS And MeTHOdSdelphi processA stepwise approach was followed using the OMERACT method-ological framework.16 First, a Delphi survey including questions on elementary SGUS abnormalities was developed based on a systematic literature review.17 The Delphi process is widely used among experts of diverse fields of activity to reach consensus of opinion on a specific topic. This process gathers data on what is known or not known about a fact or a topic from a panel of expert respondents to different surveys. It is a flexible and adapt-able tool. Nevertheless, the expert selection and timing of its conduct should be well defined before its deployment to reach reliable data. Such process has its pitfalls such as low response rates, biased feedback and surveying of the panel of experts who may lack optimal knowledge of the studied topic.18 Thirty-eight statements regarding preliminary definitions were generated by a small steering committee (SJJ, GAWB, EN, MADA) and assem-bled into three sections. Section 1 comprised 27 statements rele-vant to the SGUS normal component definitions and scanning procedure of the parotid glands (PG), submandibular glands (SMG) and sublingual glands (SLG). Section 2 was composed of three statements relevant to definitions of elementary SGUS abnormalities in the PGs, SMGs and SLGs in patients with pSS. Finally, section 3 included eight statements relevant to a new scoring system for grading US abnormalities in the PGs, SMGs and SLGs in patients with pSS.
ParticipantsThe survey was then sent by email to a broader group of 25 rheu-matologists all experts in US and members of the OMERACT US task force in Sjögren disease. The Delphi participants came from 14 countries (Austria, Czech Republic, Denmark, Egypt, France, Germany, Italy, Mexico, Norway, Slovenia, Spain, Swit-zerland, The Netherlands and the USA). The participants were then asked to rate their level of agreement for each statement using a 5-point Likert scale, in which 1 was inappropriate and 5
was completely appropriate. The participants were asked to rate each definition using a level of agreement or disagreement for each statement according to a 1–5 Likert scale with 1=strongly disagree, 2=disagree, 3=neither agree nor disagree, 4=agree and 5=strongly agree. A Likert score of 4 or 5 was considered as agreement. Only when statements achieved a score of >75%, a consensus was considered for appropriately defining the cate-gory. Statements satisfying these requirements were used for the definition and apply for the scoring system during the video clip exercise. Those statements with already achieved agreement, but suggestions for an improved wording in the first Delphi round, were rephrased according to the experts’ comments and reap-praised in the second round.
Statements with a <75% agreement on a score of 4 or 5 on the Likert scale in the first round were not further taken to the second round. Free-comment fields were provided for every statement.
Patient and public involvementOur study advances the development of a reliable consensual semiquantitative scoring system for US abnormalities of the major SG in patients with known or suspected primary Sjögren. This aim was deployed by using and sharing video clips of US-SG among a large panel of experts. There was no patient and public involvement or partnership in the design or conduct of the study.
Video clip collectionThe third phase of the validation process consisted of testing the intrareader and inter-reader reliability of the new scoring system. To this end, the 25 experts were asked to assess SGUS video clips in greyscale. First, a subset of video clips was sent to all experts to assess the image quality and to test the feasibility of the video clip exercise. The experts were instructed to record anonymised video clips of SGUS examinations during their usual clinical practice in subsequent patients with pSS and non-pSS sicca patients seen at their respective institution. Each video clip was limited to a maximum time of 5 s to allow sharing via secure web transfer. Longitudinal and transverse scans were recorded on the same video. In each patient, both PGs and SMGs were exam-ined, yielding 12 video clips per patient. All video clips were sent to the study centre in Brest, France, where they were evaluated for quality requirements for high-resolution image, for example, high-frequency linear US probe at 12–18 MHz, greyscale image only, including longitudinal and transverse planes of left and right PGs and SMGs according to Delphi process, 5 s video clip duration (video clip examples are now available as online supplementary videos). High-quality greyscale videos were thus assembled to create a video clip atlas of normal and abnormal SGUS findings in order to evaluate the diagnostic usefulness of SGUS irrespective of machine type and preset mode. Some of the findings met none of the definitions developed by the Delphi process, as they exhibited fatty replacement or fibrosis but no anechoic or hypoechoic foci. In case the SG scans did not show any anechoic or hypoechoic foci, experts were given the option of scoring such glands qualitatively as fatty or fibrous.
Fatty replacement was defined as fatty infiltration within the gland. Such fatty gland’s surface becomes homogeneously hyperechoic compared with adjacent tissue. Fibrosis was defined as the presence on the gland surface of hyperechoic bands that develop into fibrotic tissue indistinguishable from the adjacent soft tissues.
The video clips that were found to be of high quality were then pooled and sent in random order to the experts who had
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Table 1 Section 1: agreed statements defining the appearance and standardised scanning procedure of the salivary glands examined by ultrasound
Section 1 definitionsAgreement (%)
delphi round
uS appearance
normal PG parenchyma
Uniformly echoic texture (compared with adjacent muscles), with a clear demarcation between the gland and the overlying tissue. The echotexture of the PG is comparable to that of normal thyroid parenchyma.
81.1 1
normal components of PG parenchyma
(a) The normal (non-dilated) excretory duct (Stensen’s duct) is not visible, except sometimes in slim individuals.
81.1 1
(b) The PG vessels are the external carotid artery and retromandibular vein, which are sometimes visible on B-mode images.
81.1 2
(c) The external carotid artery and retromandibular vein divide the PG in a superficial and a deep lobe. These vessels should be used as landmarks for distinguishing between the two lobes.
95.8 1
(d) The larger superficial lobe is clearly visible and may contain typical oval lymph nodes, one in the upper pole and/or one in the middle and/or one in the lower pole of the gland.
90.9 1
(e) The smaller deep lobe may be difficult to assess, depending on gland echogenicity.
90.9 1
normal SMG parenchyma
Uniformly echoic texture (compared with adjacent muscles), with a clear demarcation between the gland and the overlying tissue. SMG echotexture granularity is usually finer compared with the PG and normal thyroid parenchyma.
95.4 2
normal components of SMG parenchyma
(a) The normal (non-dilated) excretory duct (Wharton’s duct) is not visible, except sometimes in slim individuals.
86.3 1
(b) The SMG vessels are the facial artery from the external carotid and the facial vein. The facial artery runs through the parenchyma and is consistently visible in B-mode provided the amount of pressure applied is not excessive.
86.9 2
(c) The facial vein runs along the anterosuperior part of the SMG and may be visible.
77.2 1
(d) Usually, under normal conditions, the SMG lymph nodes are located outside the anterior part of the gland but not within the gland.
86.9 2
uS scanning procedure
PG (a) The PG can be assessed in a transverse plane perpendicular to the mandibular bone, using the mandibular ramus and temporomandibular joint condyle as landmarks.
95.4 2
(b) In the longitudinal plane, the probe is positioned parallel to the mandibular ramus. The PG must be assessed in both the longitudinal and the transverse planes.
90.9 1
SMG (a) The margins of the submandibular triangle are formed by the anterior and posterior bellies of the digastric muscle and the body of the mandible.
90.9 1
(b) The SMG can be assessed in the longitudinal and transverse planes at the posterior part of the submandibular triangle.
87.5 2
(c) The SMG should be assessed in the longitudinal and transverse planes at the posterior part of the submandibular triangle, using the posterior belly of digastric muscle as a landmark.
83.3 1
(d) In some individuals, glandular processes may connect the SMG and PG.
83.3 2
PG, parotid gland; SMG, submandibular gland; US, ultrasound.
participated in all three Delphi rounds. There were two video clip reading rounds, one between January and June 2017, and the second between July and November 2017. Thus, each expert read all the video clips.
Statistical analysisInter-reader reliability was assessed by using kappa statistics, and computing the weighted kappa coefficient (Fleiss-Cohen weights) for each pair of readers for statements with more than two ordinal categories.19 The minimum (min) and maximum (max) kappa values were calculated. Then, Light’s kappa, that is, the mean kappa value, was computed. To assess intrareader reliability, we computed the weighted kappa coefficients between two readings by each expert. We then computed Light’s kappa (mean of intrareader kappa values). The boot-strap percentile method was used to compute the 95% CI of Light’s kappa.20 Kappa values were interpreted according to Landis and Koch.21
In the primary analysis, all findings described as fatty or fibrous lesions were considered as missing data. In a second analysis, we performed a sensitivity analysis in which fatty echostructures were graded 1 (minimal change) and fibrous echostructures were graded 3 (severe change) in the new scoring system.
All statistical analyses were carried out using R language V.3.2.0 (R Foundation for Statistical Computing, Vienna, Austria; https://www. r- project. org).
ReSulTSReaching consensus on definitionsThe 25 experts reached a consensus on definitions after three Delphi rounds. Of the initial 38 candidate statements in the three sections, 28 were finally accepted by consensus. Of the 25 participants, 22 (88%) responded to the first Delphi ques-tionnaire. All these 22 participants responded to the second and third Delphi questionnaires. The first Delphi question-naire was composed of 38 statements divided among the three sections. The participants disagreed on 19 statements pertaining to the following: in section 1, PG assessment and vascularisation, definition and assessment of normal SMG echostructure and vascularisation, and SLG assessment; in section 2, presence of hyperechoic bands in the PG and SMG parenchyma; and in section 3, the scoring system and quanti-tative measurements of each gland. These 19 statements were rephrased for the second Delphi round. The participants then disagreed on nine statements, which pertained to elementary SGUS lesions in section 2 and to the scoring system in section 3. A consensus was reached during this second round on defi-nitions for and the scanning procedure of normal SG. Most of the experts (75%) agreed that the SLGs were too small for a reliable assessment. Table 1 lists the final definitions and the standardised scanning procedure in section 1.
For the third Delphi round, several statements on SLG evalu-ation, the scoring system and measurement of each gland were rewritten. At the end of the third round, a consensus was achieved for elementary lesions (section 2, 95.4% agreement) and the scoring system (section 3, 79.1% agreement). In section 2, PG and SMG abnormalities in pSS were defined as focal or diffuse anechoic/hypoechoic foci (95.4% agreement). No consensus was reached as to the meaning of hyperechoic bands as an elementary lesion (70.8% agreement). However, the experts agreed that the SGs may be difficult to distinguish from the adjacent soft tissues in some patients with SG abnormalities, raising challenges in determining the score (79.1% agreement). In section 3, a novel
four-grade semiquantitative scoring system for the PGs and SMGs in patients with pSS was defined grade 0, normal paren-chyma; grade 1, minimal change: mild inhomogeneity without anechoic/hypoechoic areas; grade 2, moderate change: moderate inhomogeneity with focal anechoic/hypoechoic areas; grade 3, severe change: diffuse inhomogeneity with anechoic/hypoechoic areas occupying the entire gland surface (79.1% agreement).
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Figure 1 Ultrasound (US) images of parotid gland in a four-grade semiquantitative scoring system and two-item qualitative scoring system (fatty and fibrous glands): (A) grade 0, normal parenchyma; (B) grade 1, minimal change: mild inhomogeneity without anechoic/hypoechoic areas; (C) grade 2, moderate change: moderate inhomogeneity with focal anechoic/hypoechoic areas but surrounded with normal tissue; (D) grade 3, severe change: diffuse inhomogeneity with anechoic/hypoechoic areas occupying the entire gland surface but surrounded with no normal tissue; (E) grade 1, parotid gland presents diffuse homogeneity with a hyperechoic gland compared with adjacent tissue; (F) grade 3, parotid gland presents hyperechoic bands that develop into fibrotic tissue indistinguishable from the adjacent soft tissues.
Assessment of scoring system reliability using video clipsWe included 199 high-quality video clips, 104 of them showing the PGs and 95 the SMGs, respectively. Of the 25 experts, 18 read all 199 video clips within the scheduled time frame. Online supplementary file 1 reports their responses. Of the video clips, 33% were graded 0, 20% were graded 1, 23% were graded 2 and 17% were graded 3. Overall, only 3% of video clips were graded as fatty and 4% as fibrous; these percentages ranged
across experts from 0% to 10% and from 0% to 15%, respec-tively. Online supplementary file 2 reports the grades for the PGs and online supplementary file 3 for the SMGs. Of the PG video clips, 37% were graded 0, 18% were graded 1, 19% were graded 2 and 19% were graded 3; 3% were graded as fatty and 3% as fibrous with ranges across experts of 0%–12% and 0%–12%, respectively. Of the SMG video clips, 29% were graded 0, 23% were graded 1, 27% were graded 2 and 16% were graded 3;
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Table 2 Intrareader reliability between 18 experts reading SG video clips
Kappa intraobserver Kappa min Kappa max light’s kappa 95% CI
SMG+PG
DS 0.60 0.97 0.81 0.77 to 0.84
DS+fatty and fibrous
0.60 0.98 0.79 0.76 to 0.83
SMG (n=104)
DS 0.46 0.97 0.75 0.71 to 0.80
DS+fatty and fibrous
0.46 0.97 0.73 0.70 to 0.79
PG (n=95)
DS 0.69 0.98 0.86 0.77 to 0.87
DS+fatty and fibrous
0.64 0.98 0.79 0.76 to 0.83
Primary analysis: according to Delphi scoring (DS), sensitivity analysis: DS+imputing ‘fatty: grade 1, fibrous: grade 3’.PG, parotid gland; SG, salivary gland; SMG, submandibular gland
Table 3 Inter-reader reliability between 18 experts reading SG video clips
Kappa interobserver Kappa min Kappa max light’s kappa 95% CI
SMG+PG
DS 0.35 0.85 0.66 0.61 to 0.70
DS+fatty and fibrous
0.29 0.80 0.62 0.57 to 0.66
SMG (n=104)
DS 0.09 0.81 0.56 0.48 to 0.61
DS+fatty and fibrous
0.09 0.80 0.54 0.48 to 0.60
PG (n=95)
DS 0.48 0.94 0.74 0.68 to 0.78
DS+fatty and fibrous
0.43 0.85 0.68 0.61 to 0.73
Primary analysis: according to Delphi scoring (DS), sensitivity analysis: DS+imputing ‘fatty: grade 1, fibrous: grade 3’.PG, parotid gland; SG, salivary gland; SMG, submandibular gland;
2% were graded as fatty and 4% as fibrous, with ranges across experts of 0%–10% and 0%–18%, respectively. Figure 1 shows the four-grade semiquantitative scoring system with the two qualitative items fatty and fibrous.
Intrareader reliabilityThe primary analysis showed excellent reliability for the PGs and for the SMGs (table 2). Reliability was similar for both the PGs and the SMGs combined (Light’s kappa 0.81; range 0.6–0.97; 95% CI 0.77 to 0.84). The sensitivity analysis in which fatty glands were graded 1 and fibrous glands were graded 3 showed a lower reliability (Light’s kappa 0.79; range 0.6–0.98; 95% CI 0.76 to 0.83).
Inter-reader reliabilityIn the primary analysis, reliability was good for the PGs and SMGs separately (table 3). Similar reliability results were obtained when both glands were combined (Light’s kappa 0.66; range 0.35–0.85; 95% CI 0.61 to 0.70). Reliability was lower
in the sensitivity analysis (Light’s kappa 0.62; range 0.29–0.80; 95% CI 0.57 to 0.66).
dISCuSSIOnIn this first phase of a multiphase process to develop US as an OMI for scoring SG lesions in patients with pSS, the OMERACT task force on Sjögren’s syndrome developed consensual defini-tions through a Delphi process and subsequently tested their reliability in a web-based video clip platform showing dynamic video images of SG. In the Delphi survey, existing and novel criteria were rated by a broad panel of international experts. An agreement on defining SGUS lesions was reached after three rounds of Delphi process and these definitions were finally used to develop a new semiquantitative scoring system for the SGUS assessment of the PGs and SMGs. The results were excellent for the intrareader and good for the inter-reader reliability, respec-tively. The experts agreed that the SLGs should not be evalu-ated by SGUS. To date, there are not enough data concerning morphology and size using US and MRI for these small chal-lenging glands.22
Previous attempts for reaching definitions of SG echostructural lesions were conducted by the EULAR-US pSS group in 2012. This has proven to be difficult as reliability results showed wide variability. Indeed, the reliability was tested on seven elementary lesions either on static and acquisition images on patients with pSS and not on a comprehensive scoring. At the time, an assess-ment on patients with pSS by five experts demonstrated that as to homogeneity of the gland, the inter-reader reliability for PG was moderate and for SMG fair.23 One of the main goals of the OMERACT SGUS subtask force was to develop a standardised SGUS scanning procedure. Training in the implementation of a standardised procedure is instrumental when striving for robust intrareader and inter-reader reliabilities. Studies of SGUS reli-ability that relied instead on static and acquisition images have produced variable results.23–26 Some studies involved the sepa-rate scoring of the items defined by Hocevar et al (echogenicity, homogeneity, hyperechoic bands, posterior border, presence or absence of calcifications and gland size).27–29 Only the homo-geneity item showed good inter-reader reliability.30 Finally, a strong correlation has been demonstrated between homogeneity and the presence of anechoic/hypoechoic foci, indicating that these two items provide similar information.26 This fact should be taken into account when developing a semiquantitative scoring system.31 32
Fatty replacement and fibrosis are not considered in any of the current SGUS scoring systems. Fatty replacement of SGs is seen in healthy elderly individuals and in a small minority of patients with Sjögren’s syndrome. SG fibrosis is common in end-stage pSS and sometimes seen in early-stage pSS. Both fatty replacement and fibrosis were scored in the sensitivity analysis. Intrareader reliability was excellent and inter-reader reliability was good for both items in both the primary and the sensitivity analyses. Our results suggest that both items should be consid-ered for SGUS scoring when the semiquantitative scoring system cannot be applied.
Reliability was lower for the SMGs than for the PGs. This fact may be related to the difference in parenchymal echostruc-ture between these two glands.22 A recent study demonstrated no correlation between findings at the PGs and at the SMGs, whereas correlations were strong between the right and left PGs and between the right and left SMGs.26 Instead of scoring either the PGs or the SMGs we therefore recommend scoring at least one PG and at least one SMG for anechoic/hypoechoic
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foci, hyperechoic (fibrous) bands (grade 3) and fatty replacement (grade 1) as part of an overall semiquantitative scoring system.26
The main study limitation was that we did not include fatty and fibrous gland statements (ie, based on qualitative grading) in our Delphi survey section on US-SG scoring system (ie, current consensus is based only on semiquantitative grading). Only later after the expert reading of the videos that we decided to include fatty and fibrous glands into our US-SG scoring system. We believe that using simplified scoring (only accounting for anechoic/hypoechoic areas) other important data such as fatty and fibrous characteristics of the gland could be omitted. In other words, our study limitation highlights the weakness of current US-SG scoring system and warrants the need for an update of such system for a comprehensive scoring which combines all quantitative and qualitative characteristics of SG.
In conclusion, an international expert consensus was reached using OMERACT methodology for the definitions of normal US appearance and abnormalities seen in patients with suspected or confirmed pSS. In a next step, these definitions were tested in video clips of patients with pSS and non-pSS sicca patients on the PGs and SMGs alone, excluding the SLGs. The sharing of video clips via the internet proved to be a simple and feasible method for having a large number of experts evaluates the reli-ability of an SGUS scoring system. This study constitutes the first step towards a novel, reliable, semiquantitative SGUS scoring system available to clinicians and sonographers assessing the SGs of patients with suspected or confirmed pSS.
Author affiliations1Rheumatology, Brest Medical University Hospital, inseRM esPRi, eRi29, UBO, Brest, France2Rheumatology, aPHP, Hôpital ambroise Paré, Boulogne-Billancourt, France, inseRM U1173, laboratoire d’excellenceinFlaMeX, UFR simone Veil, Versailles-saint-Quentin University, Montigny le Bretonneaux, France3inseRM, CiC 1412, Brest University Hospital, Brest, France4Rheumatology, severo Ochoa University Hospital, Madrid, spain5Rheumatology and Clinical immunology, Charité-Universitätsmedizin, Berlin, Germany6Rheumatology and Clinical immunology, Universital Hospital Charit, Berlin, Germany7Faculty of Medicine, University of Zürich, Zürich, switzerland8Rheumatology, nancy University Hospital and UMR 7365 CnRs-Ul iMoPa (ingéniérieMoléculaire & Physiopathologie articulaire), Université de lorraine, Vandoeuvre-les-nancy, France9Centre for Rheumatology and spinal Diseases, Rigshospitalet, Copenhagen, Denmark10scienze Cliniche e Biologiche, Università degli studi di Torino, Rome, italy11Musculoskeletal Ultrasound laboratory, instituto nacional de Rehabilitacion, Mexico City, Mexico12Rheumatology, CHU Pitie-salpetriere, assistance Publique-Hopitaux de Paris, Paris, France13GRC-UPMC 08, Pierre louis institute of epidemiology and Public Health, Paris 6 University, Paris, France14Rheumatology, Medical Centre for Rheumatology Berlin-Buch, Berlin, Germany15Rheumatology section, Department of Medicine, surgery and neurosciences, University of siena, siena, italy16Rheumatology, Medical University Graz, Graz, austria17Rheumatology, Hospital of Bruneck, Bruneck, italy18Rheumatology and immunology, Medical University Graz, Graz, austria19Rheumatology, Faculty of Medicine Zagazig University, Zagazig, egypt20Rheumatology, University Medical Centre ljubljana, ljubljana, slovenia21Rheumatology, sydvestjysk sygehus esbjerg, esbjerg, Denmark22Rheumatology Department, Cairo University, Cairo, egypt23Rheumatology, Hospital Vall d’Hebron, Barcelona, spain24Division of allergy, immunology and Rheumatology, Department of Medicine, University of Rochester school of Medicine and Dentistry, Rochester, new York, Usa25Rheumatology, north Valley Hospital, Whitefish, Montana, Usa26Rheumatology and Clinical immunology, University Medical Center Freiburg, Freiburg, Germany27Rheumatology, institute of Rheumatology, Prague, Czech Republic28Rheumatology, Hána CB spol s r o, Ceske Budejovice, Czech Republic29Department of Medical and Biological sciences, Rheumatology Clinic, University Hospital santa Maria della Misericordia, Udine, italy
30Department of Rheumatology and immunology, University Medical Center Freiburg, Freiburg, Germany31Department of Rheumatology, Haukeland University Hospital, Bergen, norway32MC Groep Hospitals, lelystad, The netherlands
Presented at The results of this paper were presented at the eUlaR Congress in oral presentation (amsterdam 2018).
Correction notice This article has been corrected since it published Online First. The affiliation for the last author has been corrected.
Acknowledgements The authors thank François Madec and Jacques Bretagnolle for their contribution to the preparation of video analysis and for technical assistance, emmanuel nowak, head of Brest CHRU Data Management Unit as well as antoinette Wolfe for english and Zarrin alavi for critical reviewing of the manuscript.
Contributors Conception and design: sJJ, MaDa, GaWB, en, sO, MB, GT, iCV, lT, ai, PC, CHD, FG, Was, GF, CD, MHs, MaM, aH, sC, GeM, JJa, RT, DKMC, sF, PH, aZ, CG, DsH. statistical analysis: Cn, FG, MaDa. Wrote the manuscript: sJJ, MaDa, GaWB, Za. interpretation of results: sJJ, MaDa, GaWB, Za.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests none declared.
Patient consent for publication not required.
Provenance and peer review not commissioned; externally peer reviewed.
data sharing statement Data will be available upon reasonable request from the corresponding author.
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minor salivary gland infiltrates in sjögren’s syndrome. J Autoimmun 2010;34:400–7.
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3 Vitali C, Bombardieri s, Jonsson R, et al. Classification criteria for sjögren’s syndrome: a revised version of the european criteria proposed by the american-european consensus group. Ann Rheum Dis 2002;61:554–8.
4 Bootsma H, spijkervet FKl, Kroese FGM, et al. Toward new classification criteria for sjögren’s syndrome? Arthritis Rheum 2013;65:21–3.
5 Cornec D, Jousse-Joulin s, Marhadour T, et al. salivary gland ultrasonography improves the diagnostic performance of the 2012 american College of rheumatology classification criteria for sjögren’s syndrome. Rheumatology 2014;53:1604–7.
6 Jonsson MV, Baldini C. Major salivary gland ultrasonography in the diagnosis of sjögren’s syndrome: a place in the diagnostic criteria? Rheum Dis Clin North Am 2016;42:501–17.
7 Jousse-Joulin s, Devauchelle-Pensec V, Cornec D, et al. Brief report: ultrasonographic assessment of salivary gland response to rituximab in primary sjögren’s syndrome. Arthritis Rheumatol 2015;67:1623–8.
8 shiboski CH, shiboski sC, seror R, et al. 2016 american College of Rheumatology/european league against rheumatism classification criteria for primary sjögren’s syndrome: a consensus and data-driven methodology involving three international patient cohorts. Arthritis Rheumatol 2017;69:35–45.
9 Fisher Ba, everett CC, Rout J, et al. effect of rituximab on a salivary gland ultrasound score in primary sjögren’s syndrome: results of the TRaCTiss randomised double-blind multicentre substudy. Ann Rheum Dis 2018;77:412–6.
10 Yoshiura K, Yuasa K, Tabata O, et al. Reliability of ultrasonography and sialography in the diagnosis of sjögren’s syndrome. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997;83:400–7.
11 salaffi F, argalia G, Carotti M, et al. salivary gland ultrasonography in the evaluation of primary sjögren’s syndrome. Comparison with minor salivary gland biopsy. J Rheumatol 2000;27:1229–36.
12 Carotti M, salaffi F, Manganelli P, et al. Ultrasonography and colour Doppler sonography of salivary glands in primary sjögren’s syndrome. Clin Rheumatol 2001;20:213–9.
13 Hocevar a, Rainer s, Rozman B, et al. Ultrasonographic changes of major salivary glands in primary sjögren’s syndrome. evaluation of a novel scoring system. Eur J Radiol 2007;63:379–83.
14 salaffi F, Carotti M, iagnocco a, et al. Ultrasonography of salivary glands in primary sjögren’s syndrome: a comparison with contrast sialography and scintigraphy. Rheumatology 2008;47:1244–9.
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15 Takagi Y, Kimura Y, nakamura H, et al. salivary gland ultrasonography: can it be an alternative to sialography as an imaging modality for sjogren’s syndrome? Ann Rheum Dis 2010;69:1321–4.
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17 Jousse-Joulin s, Milic V, Jonsson MV, et al. is salivary gland ultrasonography a useful tool in sjögren’s syndrome? a systematic review. Rheumatology 2016;55:789–800.
18 Hsu C-C, sandford Ba. The Delphi Technique: Making sense of Consensus. Practical assessment Research & evaluation, 12(10), 2007. available: http:// pareonline. net/ getvn. asp? v= 12& n= 10 [accessed 18 Mar 2019].
19 Fleiss Jl. Measuring nominal scale agreement among many raters. Psychol Bull 1971;76:378–82.
20 efron B, Tibshirani R. An introduction to the bootstrap. Chapman & Hall, 1993. 21 landis JR, Koch GG. The measurement of observer agreement for categorical data.
Biometrics 1977;33:159–74. 22 sumi M, izumi M, Yonetsu K, et al. sublingual gland: Mr features of normal and
diseased states. AJR Am J Roentgenol 1999;172:717–22. 23 Jousse-Joulin s, nowak e, Cornec D, et al. salivary gland ultrasound abnormalities in
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24 Damjanov n, Milic V, nieto-González JC, et al. Multiobserver reliability of ultrasound assessment of salivary glands in patients with established primary sjögren syndrome. J Rheumatol 2016;43:1858–63.
25 Delli K, arends s, van nimwegen JF, et al. Ultrasound of the major salivary glands is a reliable imaging technique in patients with clinically suspected primary sjögren’s syndrome. Ultraschall Med 2018;39:328–33.
26 Mossel e, arends s, van nimwegen JF, et al. scoring hypoechogenic areas in one parotid and one submandibular gland increases feasibility of ultrasound in primary sjögren’s syndrome. Ann Rheum Dis 2018;77:556–62.
27 De Vita s, lorenzon G, Rossi G, et al. salivary gland echography in primary and secondary sjögren’s syndrome. Clin Exp Rheumatol 1992;10:351–6.
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29 Hocevar a, ambrozic a, Rozman B, et al. Ultrasonographic changes of major salivary glands in primary sjogren’s syndrome. Diagnostic value of a novel scoring system. Rheumatology 2005;44:768–72.
30 Milic VD, Petrovic RR, Boricic iV, et al. Major salivary gland sonography in sjögren’s syndrome: diagnostic value of a novel ultrasonography score (0-12) for parenchymal inhomogeneity. Scand J Rheumatol 2010;39:160–6.
31 Theander e, Mandl T. Primary sjögren’s syndrome: diagnostic and prognostic value of salivary gland ultrasonography using a simplified scoring system. Arthritis Care & Research 2014;66:1102–7.
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TranslaTional science
Blockade of CD40–CD154 pathway interactions suppresses ectopic lymphoid structures and inhibits pathology in the NOD/ShiLtJ mouse model of Sjögren’s syndromeGrazyna Wieczorek, Marc Bigaud, sabina Pfister, Melanie ceci, Katriona McMichael, catherine afatsawo, Meike Hamburger, celine Texier, Maurane Henry, celine cojean, Marinette erard, nadja Mamber, James s rush
To cite: Wieczorek G, Bigaud M, Pfister s, et al. Ann Rheum Dis 2019;78:974–978.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213929).
Department of autoimmunity, Transplantation and inflammation, novartis institutes for BioMedical research Basel, Basel, switzerland
Correspondence toDr James s rush, autoimmunity, Transplantation and inflammation, novartis institutes for BioMedical research Basel, Basel 4058, switzerland; james. rush@ novartis. com
received 13 June 2018revised 20 February 2019accepted 25 February 2019Published online First 22 March 2019
© author(s) (or their employer(s)) 2019. no commercial re-use. see rights and permissions. Published by BMJ.
Key messages
What is already known about this subject? ► Previous work has shown evidence of ectopic lymphoid structures and CD40–CD154 ligand expression in salivary glands (SGs) from patients with Sjögren’s syndrome (SS) and mouse models of this disease. Additionally, CD40 knockout mice and prophylactic pathway blockade have been shown to ameliorate sialadenitis in the non-obese diabetic (NOD) mouse model of SS.
What does this study add? ► This study demonstrates evidence of chronic CD40 expression and CD40 pathway activation in SGs from NOD/ShiLtJ mice. Further, it shows evidence of CD40 pathway activation in SG biopsies from patients with SS. Additionally, our results indicate that therapeutic treatment of NOD mice with an anti-CD154 mAb inhibited sialadenitis, reduced numbers of infiltrating lymphocytes and macrophages, and suppressed serum autoantibody levels. Finally, we could demonstrate that CD40–CD154 blockade inhibited expression of genes linked to B-cell activation and affinity maturation in SGs.
How might this impact on clinical practice? ► Collectively, our data suggest that therapeutic blockade of the CD40–CD154 costimulatory pathway could provide a beneficial therapeutic effect in patients with SS.
AbSTrACTObjective To examine the role of cD40–cD154 costimulation and effects of therapeutic pathway blockade in the non-obese diabetic (noD/shiltJ) model of sjögren’s syndrome (ss).Methods We assessed leucocyte infiltration in salivary glands (sGs) from noD/shiltJ mice by immunohistochemistry and examined transcriptomics data of sG tissue from these animals for evidence of a cD40 pathway gene signature. additionally, we dosed Mr1 (anti-cD154 antibody) in noD mice after the onset of ss-like disease and examined the effects of Mr1 treatment on sialadenitis, autoantibody production, sG leucocyte infiltration, gene expression downstream of cD40 and acquaporin 5 (aQP5) expression.results We could detect evidence of cD40 expression and pathway activation in sG tissue from noD mice. additionally, therapeutic treatment with Mr1 suppressed cD40 pathway genes and sialadenitis, inhibited ectopic lymphoid structure formation and autoantibody production, as well as decreased the frequency of antibody-secreting cells in sGs but had minimal effects on aQP5 expression in noD/shiltJ sGs.Conclusion cD40–cD154 interactions play an important role in key pathological processes in a mouse model of ss, suggesting that blockade of this costimulatory pathway in the clinic may have beneficial therapeutic effects in patients suffering from this autoimmune exocrinopathy.
InTrOduCTIOnSjögren’s syndrome (SS) is a chronic, autoimmune disease characterised by sialadenitis and exocrine gland dysfunction.1 2 Hallmarks of the disease in patients with both primary SS (pSS) and secondary SS include the presence of anti-Ro and La autoan-tibodies and mononuclear cell infiltrates in salivary glands (SGs) and lacrimal glands.3 Infiltrates of T and B lymphocytes can form well-organised struc-tures referred to as ectopic lymphoid structures (ELS) that bear morphological and functional simi-larities to germinal centres (GCs).4 Previous work has reported evidence of ELS in SGs from patients with SS and from preclinical models of this disease and evidence of ongoing affinity maturation,5–7 implicating these structures in disease pathology.8
There is relatively little published data on the role of various immune costimulatory pathways in ELS formation and function, despite data supporting the essential role of pathways like CD40–CD154 (CD40 ligand) in GC biology.9 If such interactions were to play a role in ELS formation and function, then pathway blockade would be predicted to eliminate established structures in affected tissue.
To test this hypothesis, we examined the effect of therapeutic administration of an anti-CD154 mono-clonal antibody in the non-obese diabetic (NOD/
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Figure 1 CD40 signature in ELS in SGs and reduction of tertiary lymphoid organs in SGs of NOD mice after 10-week treatment with MR1. A. 14 genes from the B-cell CD40 pathway gene signature were significantly upregulated in microdissected ELS from SGs in 12-week-old animals. These genes included Cd40, Cd80 and Aicda, and they remained elevated in 24-week animals. B. Evidence of CD40 signalling in ELS formation was independently provided by ingenuity pathway analysis, which identified CD40 and CD154 (CD40LG) among top upstream regulators both at weeks 12 and 24. C. Representative in-situ hybridisation images of Cd40, Cd80 and Aicda mRNA expression in inflammatory infiltrates in SGs of mice with NOD (the highest expression of Aicda was noted within ELS as defined by CD21 staining) treated with isotype control or MR1.
ShiLtJ) mouse model of secondary SS,10 and monitored SG ELS, gene expression downstream of CD40, antibody-secreting cell (ASC) frequencies as well as expression of acquaporin 5 (AQP5), a protein essential for secretory cell function.11
MATerIAlS And MeTHOdSCd40 pathway gene signature in SGsCryosections of SGs from untreated NOD/ShiLtJ mice (Charles River, Germany) were used for gene expression profiling by microarray. Laser capture microdissection was applied to enrich for ELS (online supplementary file).
nOd/ShiltJ mouse model of secondary SSTwelve-week-old female NOD/ShiLtJ mice were randomised in two treatment groups (n=15 per group, two experiments): MR1 (Armenian hamster anti-mouse CD154 IgG) and isotype control (IC, hamster anti-mouse IgG, BioXcell) at 15 mg/kg, intraper-itoneally, two times per week for 10 weeks (online Supple-mentary Figure S1). All procedures were performed according to the Swiss law for animal protection and were approved by the Cantonal Veterinary Office in Basel, Switzerland (Animal License No. BS-2482).
Histopathology, immunohistochemistry and in-situ hybridisation of spleen and SGs from nOd/ShiltJ miceParaffin sections of left SGs were stained with haematoxylin and eosin. Automated immunohistochemical stainings for CD3, CD45R, CD138, Iba-1, Ki-67 and AQP5 were performed on Ventana Discovery XT immunostainer (Roche Diagnostics, Swit-zerland) (online Supplementary Table S1). RNAscope Cd40, Cd80 and Aicda probes (Advanced Cell Diagnostics, Newark, CA, USA) were used for in-situ hybridisation.
enzyme-linked immunospot assays for ASCsSingle cell suspensions from SGs and spleens were prepared (online supplementary file), and fresh CD45 +cells were added to precoated enzyme-linked immunospot (ELISPOT) plates and incubated for 20 hours at 37°C in the dark. ASC binding to plates was revealed by adding a Tetremethylbenzidine (TMB substrate solution, and the density of spots was measured with an EliSpot Reader ‘AID classic’.
Anti-ro60 elISAMouse Anti-Y RNA binding protein (SSA/Ro60) was assessed in serum using ELISA kit cat. No. 5710 (Alpha Diag-nostic International, USA) according to the manufacturer’s instructions.
reSulTSupregulation of a b-cell Cd40 gene signature in SGsMicroarray analyses were used to identify genes downstream of CD40 following rCD154 stimulation of primary, human CD19pos B cells (Fisher et al, submitted), 43 of which could be mapped to murine genes. We then examined whether the expression of these genes was modulated in SG tissue from NOD mice (aged 12–24 weeks). In all, 14 genes, including Cd40, Cd80 and Aicda were significantly upregulated in microdissected ELS compared with whole SG tissue at both time points, suggesting chronic pathway activation (figure 1A, online Supplementary Table S2). Pathway analysis also indi-cated that CD40 and CD154 (CD40LG) were among the top upstream regulators both at weeks 12 and 24 (figure 1B), and we were able to demonstrate overlap between CD40 pathway
genes upregulated in SGs from NOD mice and from patients with pSS12 (online Supplementary Figure S2).
Cd40–Cd154 interactions are essential for elS formationNOD mice develop focal cellular infiltrates in SGs at 8–12 weeks of age with 100% incidence that precedes a decline in SG func-tion.13 The above data suggested chronic CD40 pathway signal-ling in NOD SG tissue, and this was supported by evidence of CD40 expression by mononuclear cells in inflammatory foci14 (online Supplementary Figure S3). We therefore opted to test the effects of therapeutic CD40–CD154 blockade by dosing the anti-CD154 mAb MR1 at 15 mg/kg biweekly starting at 12 weeks of age for 10 weeks.
Figure 1B shows that expression of Cd40 and Cd80 was evident in SGs of NOD mice treated with IC. We could also observe expression of Aicda associated with CD21pos cells. Expression of these genes was decreased following chronic dosing of MR1 for 10 weeks, suggesting that CD40–CD154 blockade could reduce expression of genes involved in B-cell differentiation and func-tion at sites of leucocyte accumulation in SG tissue.
Chronic dosing of MR1 also resulted in a significant reduc-tion in ELS in SGs, as defined by clusters of Ki67-positive cells (figure 2A). In addition, there was a strong reduction in the percentage of SG-infiltrating B and T cells, and macrophages. Histological images indicated that MR1 was able to suppress ELS 10 weeks after the start of dosing (figure 2B).
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Figure 2 Reduction of ectopic lymphoid structures in SGs of NOD mice after 10-week treatment with MR1. A. Ten-week treatment with MR1 significantly reduced sialadenitis (quantitative analysis of CD3-positive T cells, CD45R-positive B cells, Iba-1-positive macrophages and Ki-67-positive proliferating cells). Each symbol represents data from an individual animal and square (n=7 for IC and n=4 for MR1 treatment) and round symbols (n=11 for IC and n=14 for MR1 treatment) indicate data obtained from independent experiments. B. Representative images of submandibular SGs in NOD mice at the end of the experiment illustrating sialadenitis in IC-treated animals (left panel) and significantly reduced sialadenitis in anti-CD154-treated animals (right panel).
blockade of Cd40–Cd154 interactions inhibits ASC formation and autoantibody productionMR1 administration resulted in a reduction in total IgG (but not IgM) ASCs in spleen, SGs and bone marrow (figure 3B) and completely suppressed splenic GCs (online Supplementary Figure S4). Anti-CD154-treated mice also displayed a significant reduction in serum anti-Ro IgG levels, although the frequency of Ro-specific ASCs in SGs did not appear to be significantly affected (figure 3C).
blockade of Cd40–Cd154 interactions has minimal effect on AQP-5- expression and localisationAQP5 is a water channel protein involved in regulating produc-tion of saliva and tears,11 and previous data have suggested that levels and distribution of this protein were altered in pSS.15 Following treatment with MR1, we observed minimal change in AQP5 expression in in MR1-treated compared with control animals (online Supplementary Figure S5). In ageing NOD mice, there appeared to be progressive increase in apical AQP5 expres-sion, a process that appeared to be delayed in some but not all animals treated with MR1.
Discussion
Evidence of affinity maturation and presence of autoreactive B cells in SG ELS suggests a potential role of these structures in SS pathology.6 Given the functional similarities between ELS and GCs, we hypothesised that immunological costimulatory
pathways involved in GC biology may play a similar role in ELS. Pharmacological blockade or deficiency in CD40 or CD154 prevents GC formation and this costimulatory pathway has been implicated in ELS biology, as CD40 expression has been observed on infiltrating cells in SGs glands from patients with pSS as well as in NOD/ShiLtJ mice.14 16 Coupled with persistent upregulation of genes downstream of CD40 in ELS from NOD mice and in SG biopsies from patients with pSS, these data suggested that this costimulatory pathway was active in infil-trating SG leukocytes.
We demonstrated that therapeutic blockade with an anti-CD154 mAb blocked both cellular and molecular outcomes of signalling downstream of this costimulatory pathway in SS disease-relevant tissue in NOD mice. These results suggest that CD40–CD154 interactions were essential for sialadenitis, ELS formation, as well as B-cell diversification processes linked to Aicda expression. Furthermore, downstream B-cell differ-entiation into IgG-secreting ASCs was inhibited, and this was reflected in suppression of circulating anti-Ro autoantibodies. In contrast, we observed minimal effects of CD40–CD154 blockade on AQP5 protein levels or cellular localisation SGs, although evidence is equivocal as to whether a decrease in AQP5 expression represents a surrogate marker of SG func-tion in SS.15 17 Assessment of saliva production would be a more direct measure of SG function,13 but unfortunately, our animal licence did not allow the procedure at the time of the experiment.
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Figure 3 Reduction of CD138+ cells and IgG ASCs in SGs, and anti-Ro Ig in serum after 10-week treatment with anti-CD154. A. Anti-CD154 administration resulted in a reduction of CD138-positive plasma cells in SGs of NOD mice at the end of the treatment. B. Significant reduction in total IgG ASC in spleen (i), SGs (II) and bone marrow (III) was observed. Each symbol represents data from an individual animal and square (n=7 for IC and n=4 for MR1 treatment) and round symbols (n=11 for IC and n=14 for MR1 treatment) indicate data obtained from independent experiments. C. Anti-CD154-treated mice showed a significant reduction in serum anti-Ro IgG levels, although the frequency of Ro-specific ASCs in SGs did not appear to be significantly affected (n=11 for IC and n=14 for MR1 treatment).
Previous data indicate that prophylatic administration of a single-dose of MR1 in young NOD mice (4–5 weeks of age; 1–2 months prior to evidence of sialadenitis) could prevent development of sialadenitis and reduce levels of anti-Ro auto-antibodies,18 consistent with results from CD40-deficient NOD mice. However, to our knowledge, this is the first time that therapeutic blockade of CD40–CD154 interactions has been shown to abolish SG inflammation, ELS, expression down-stream of CD40 signalling and reduce autoantibody levels in this model. Our data contrast with earlier work where
adeno-associated viral expression of a soluble CD40-Ig fusion protein in SGs of NOD mice failed to reduce sialadenitis.19 As the authors reported no evidence of fusion protein expression in SGs, it is likely that concentrations of the drug were insuf-ficient to achieve full CD40–CD154 pathway blockade. In contrast, we could clearly demonstrate abrogation of splenic GCs suggesting we had sufficient MR1 exposure for complete pathway blockade in tissue. Collectively, our data suggest that therapeutic blockade of the CD40–CD154 costimula-tory pathway with biologics such as the anti-CD40 antibody
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CFZ533 (Iscalimab) could provide a beneficial therapeutic effect in patients with SS.20
Contributors GW, MB and Jsr planned and reported the study, sB conduted the bioinformatics analysis, KM, ca, MH, cT, MH, cc, Me, nM and Mc conducted and analysed the study. GW and Jsr wrote the paper.
Funding study sponsored by novartis Pharma aG.
Competing interests all authors on the paper are employees of novartis Pharma aG.
Patient consent for publication not required.
Provenance and peer review not commissioned; externally peer reviewed.
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Systemic sclerosis
CliniCal sCienCe
Outcomes of patients with systemic sclerosis treated with rituximab in contemporary practice: a prospective cohort studyMuriel elhai, 1 Marouane Boubaya,2 Oliver Distler, 3 Vanessa smith,4,5 Marco Matucci-Cerinic,6 Juan José alegre sancho,7 Marie-elise Truchetet,8 Yolanda Braun-Moscovici,9 Florenzo iannone,10 Pavel i novikov,11 alain lescoat,12 elise siegert,13 ivan Castellví, 14 Paolo airó,15 serena Vettori,16 ellen De langhe,17 eric Hachulla,18 anne erler,19 lidia ananieva,20 Martin Krusche,21 F J lópez-longo,22 Jörg H W Distler, 23 nicolas Hunzelmann,24 anna-Maria Hoffmann-Vold,25 Valeria Riccieri,26 Vivien M Hsu,27 Maria R Pozzi,28 Codrina ancuta,29 edoardo Rosato,30 Carina Mihai,31 Masataka Kuwana,32 lesley ann saketkoo,33 Carlo Chizzolini,34 Roger Hesselstrand,35 susanne Ullman,36 sule Yavuz,37 simona Rednic,38 Cristian Caimmi,39 Coralie Bloch-Queyrat,40 Yannick allanore,41 for eUsTaR network
To cite: elhai M, Boubaya M, Distler O, et al. Ann Rheum Dis 2019;78:979–987.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214816).
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Correspondence toDr Muriel elhai, Rheumatology a Department, Paris Descartes University, Cochin Hospital, Paris 75014, France; muriel- elhai@ hotmail. fr
Received 24 november 2018Revised 7 February 2019accepted 28 February 2019Published Online First 9 april 2019
© author(s) (or their employer(s)) 2019. no commercial re-use. see rights and permissions. Published by BMJ.
AbSTrACTObjective To assess the safety and efficacy of rituximab in systemic sclerosis (ssc) in clinical practice.Methods We performed a prospective study including patients with ssc from the european scleroderma Trials and Research (eUsTaR) network treated with rituximab and matched with untreated patients with ssc. The main outcomes measures were adverse events, skin fibrosis improvement, lung fibrosis worsening and steroids use among propensity score-matched patients treated or not with rituximab.results 254 patients were treated with rituximab, in 58% for lung and in 32% for skin involvement. after a median follow-up of 2 years, about 70% of the patients had no side effect. Comparison of treated patients with 9575 propensity-score matched patients showed that patients treated with rituximab were more likely to have skin fibrosis improvement (22.7 vs 14.03 events per 100 person-years; OR: 2.79 [1.47–5.32]; p=0.002). Treated patients did not have significantly different rates of decrease in forced vital capacity (FVC)>10% (OR: 1.03 [0.55–1.94]; p=0.93) nor in carbon monoxide diffusing capacity (DlCO) decrease. Patients having received rituximab were more prone to stop or decrease steroids (OR: 2.34 [1.56–3.53], p<0.0001). Patients treated concomitantly with mycophenolate mofetil had a trend for better outcomes as compared with patients receiving rituximab alone (delta FVC: 5.22 [0.83–9.62]; p=0.019 as compared with controls vs 3 [0.66–5.35]; p=0.012).Conclusion Rituximab use was associated with a good safety profile in this large ssc-cohort. significant change was observed on skin fibrosis, but not on lung. However, the limitation is the observational design. The potential stabilisation of lung fibrosis by rituximab has to be addressed by a randomised trial.
InTrOduCTIOnSystemic sclerosis (SSc) is an orphan disease that is characterised by fibrosis of the skin and internal organs, autoimmunity and vasculopathy.1 SSc has the highest cause-specific mortality among connective tissue diseases.2 Progressive interstitial lung disease (ILD) is the leading cause of death in SSc.3 Despite the fatal burden associated with this condition, treatment options for SSc remain limited.4 Preliminary case-reports and series have suggested that rituximab, a chimeric mono-clonal antibody targeting B cells, could improve
Key messages
What is already known about this subject? ► Some efficacy of rituximab in systemic sclerosis (SSc) has been suggested by few small-sized uncontrolled studies. Large controlled studies were lacking.
What does this study add? ► Rituximab is safe in SSc. ► Treatment with rituximab improves skin fibrosis, which is a marker of disease activity and severity as compared with untreated control-patients.
► No significant change was observed on lung fibrosis in the whole cohort.
► Secondary analyses suggest that combination therapy with mycophenolate mofetil might be more effective for treating lung fibrosis.
How might this impact on clinical practice or future developments?
► A clue for the future to get a better impact on SSc outcomes might be combination therapy, which should be further studied.
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Table 1 Baseline characteristics of patients with SSc treated with rituximab and untreated SSc-controls from EUSTAR database
Total(n=9829)
non-treated (n=9575)
Treated (n=254) P value
Sex (Female) 8437 (85.8%) 8256 (86.2%) 181 (71.3%) <0.0001
Age, Mean±SD 54.4±13.8 55 (45–65) 51 (44–58) <0.0001
Diffuse cutaneous form
3022 (31%) 2866 (30.2%) 156 (63.9%) <0.0001
Disease duration (years),Median (Q1–Q3)
5.2 (2:11.1) 5.2 (2–11.1) 4.2 (1.9–9.7) 0.046
ANA 9053 (94.6%) 8813 (94.5%) 240 (98%) 0.026
Anti-Scl70 3123 (33.9%) 2994 (33.4%) 129 (52.9%) <0.0001
ACA 3436 (37.4%) 3410 (38.1%) 26 (10.7%) <0.0001
RNA pol III antibodies
162 (5.3%) 145 (5.2%) 17 (7%) 0.31
Modified Rodnan skin score, Median (Q1–Q3)
(0:86) 7 (3–13) 14 (6–24) <0.0001
Lung fibrosis (on HRCT)
1686 (43.4%) 1505 (41.4%) 181 (71.3%) <0.0001
FVC, Mean±SD 94.5±21.7 96 (81–110) 77 (63–95.2) <0.0001
DLCO, Mean±SD
70.2±23.1 71 (57–84) 57 (43–72.3) <0.0001
Tender joints count, Median (Q1–Q3)
0 (0:0) 0 (0–0) 0 (0–4) <0.0001
Swollen joints count, Median (Q1–Q3)
0 (0:0) 0 (0–0) 0 (0–0.8) <0.0001
CRP, Median (Q1–Q3)
3 (1:7) 2.4 (1–6) 5 (2–14.6) <0.0001
Previous IS or biologics
1798 (18.3%) 1698 (17.7%) 100 (39.4%) <0.0001
Steroids 171 (67.3%)
Prednisone (mg/day), Median (Q1–Q3)
0 (0:5) 0 (0–5) 5 (0–10) <0.0001
Follow-up (months)
25.1 (18.5:38.1) 25.1 (18.9–38) 24.3(13.3–41.4)
0.005
Previous IS (immunosuppressive drugs) include methotrexate, mycophenolate mofetil, azathioprine and cyclophosphamide, whereas biologics include anti-TNF alpha, tocilizumab and abatacept.Values are median (IQR) or mean±SD or numbers (%) of observations.Lung fibrosis was diagnosed on HRCT.ACA, anticentromeres; ANA, antinuclear antibodies; DLCO, diffusing capacity of lung for carbon monoxide; FVC, forced vital capacity; HRCT, high-resolution CT; RNA pol III, RNA polymerases III antibodies; SSc, systemic sclerosis.
skin and lung fibrosis in SSc.5–19 However, these studies were mostly limited by small samples sizes, lack of control arms, single centre designs, discrepancies in study designs and short durations of follow-up. In a previous European Scleroderma Trials and Research (EUSTAR) report on 63 patients with SSc, rituximab treatment was associated with improvement in skin fibrosis, particularly in the diffuse cutaneous form, together with a stabilisation of pulmonary functional testing.20 Though rituximab demonstrated a good safety profile, but this was limited by 6 months follow-up; and only nine of these patients had restrictive SSc-ILD, thus preventing any firm conclusions.
The aim of our study was to investigate larger population with longer follow-up and including more patients with lung involvement to better assess the effect of rituximab on SSc and establish a side effect profile specific for SSc.
MeTHOdSdata sourceThe ongoing EUSTAR is an international, multicentre, prospec-tive registry managed by physicians (list of authors and online supplementary appendix I) and organised centrally by its committee.20
For controls, we interrogated the EUSTAR database at the end of July 2017, providing information on 14 239 patients, from 142 participating centres, fulfilling the 2013 criteria for SSc21 (Strobe Checklist in online supplementary appendix II). Patients without follow-up (n=4664) and patients with previous autol-ogous stem cell transplantation were excluded. The structure of the database, the minimum essential data set and the inclusion criteria have been described in detail previously.22
design and analytic sampleFrom March to September 2017, we queried the database and sent a call for observations in the EUSTAR network regarding rituximab use. Out of 14 239 patients recorded in the data-base, 254 were recruited because of rituximab use (37 centres). We sent a questionnaire to each centre with at least one ritux-imab-treated patient. For each patient, we collected in addi-tion to the database content, the main indication for initiating the treatment, previous treatments received and dosage. The following baseline and follow-up information were also queried: modified Rodnan skin score (mRSS), presence and extent of lung fibrosis on CT, FVC (% predicted), DLCO (% predicted), presence of digital ulcers, overlap disease (defined by co-occur-rence of another autoimmune disease),23 number of tender and swollen joints (/28), DAS-28 ESR/CRP scores, joint contracture and tendon friction rubs, morning stiffness, visual analogic scale for articular pain, HAQ-DI score, presence of lower and upper gastrointestinal symptoms and CRP (mg/L). The number of infu-sions and dosage were recorded. Concomitant treatments and dosage were also collected.
OutcomesThe occurrence of any adverse event (including hypogammaglob-ulinemia,24 infection (and need for hospitalisation), scleroderma renal crisis [SRC]), serious adverse event and discontinuation because of an adverse event was collected. In case of death, we asked the relationship between the death and rituximab: (1) not related, (2) unlikely related, (3) possibly related, (4) probably related and (5) definitely related.
Outcomes were assessed at last observation under treatment (median follow-up [IQR]: 24.3 [13.3–41.4] months) responders for skin fibrosis were defined as patients with ≥5 points and 25% of improvement in mRSS.25 mRSS is frequently used as a primary outcome in clinical trials, therefore we assessed ritux-imab effect also in patients without a ‘skin indication’ and limited the analysis to patients with a mRSS ≥10 (n=131) consistently with distribution of skin involvement in Eustar.3 For lung fibrosis worsening, we used the following cut-offs: ≥10% decrease in FVC and ≥15% decrease in DLCO.26 Evolution of articular involvement was assessed: tender and swollen joints counts (/28), DAS-28 ESR/CRP scores, joint contracture, tendon friction rubs, duration of morning stiffness, articular pain,
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Figure 1 Quality of the matching: Matching 1:4 for skin (A) and for lung involvement (B). Matching allows comparability of cases and controls with less than 10% of difference between all variables studied. Data, presented as mean±SEM, are from the first imputed and matched (ratio 1:4) dataset. DLCO, diffusing capacity of lung for carbon monoxide; FVC, forced vital capacity.
HAQ-DI score and CRP (mg/L). Concomitant steroid use and dosage were collected.
CovariatesCovariates used in the model to calculate propensity score were baseline characteristics: sex, age, cutaneous form, anti-nuclear antibodies (ANA), anticentromere antibodies (ACA), anti-Scl70 antibodies, RNA polymerases III (RNA pol III) anti-bodies, disease duration, region (Western and Eastern Europe, North and South America, Africa and Asia), time between two visits (time between the baseline and final visit under ritux-imab), previous and concomitant use of immunosuppressant (IS) or biologics, mRSS, DLCO, FVC and lung fibrosis. IS drugs included methotrexate, mycophenolate mofetil, azathioprine and cyclophosphamide, whereas biologics included TNF alpha inhibitors, tocilizumab and abatacept.
Statistical analysisCategorical results are presented as counts and percentages. Non-normally distributed variables are presented as median with IQR and normally distributed variables as means±SD. Compar-ison of characteristics of treated and non-treated patients was performed by χ² test and Student’s t-test or the Mann–Whitney test, according to the distribution of the variable. Side effects during treatment with rituximab were summarised by the frequency and percentage. To evaluate efficacy of rituximab for skin fibrosis in SSc, a propensity score approach was used. This score was estimated using a logistic regression with adjustment on regions. The analysis of responders was based on propensity score matching with a ratio 1:4 and a calliper of 0.05 SD of the logit propensity score. To account for missing data, analyses were conducted using multiple imputations by chained equations with 50 imputations obtained after 10 iterations.27 The variables considered in the imputation models were all characteristics used in propensity score. For each complete imputed data set, propensity score was estimated and association between decrease in the mRSS and treatment was evaluated using conditional logistic regression. Results were aggregated by pooling estimates obtained on each imputed dataset according to Rubin’s rules.28 Balance in potentials confounders was assessed by standardised mean differences.29 Sensitivity analyses were performed using
other alternative methods of propensity score analysis: ratio 1:1 within a calliper of 0.05 SD of the logit propensity score, stratification on the quintiles of the propensity score and inverse probability of treatment weighting. The analyses of decrease in the FVC of 10%, decrease in the DLCO of 15% and tapering or stopping steroids were performed according the same method-ology and same covariates as decrease in mRSS. The variation of FVC as a continuous variable was analysed by a linear mixed-ef-fects model. Exploratory analyses of skin fibrosis improvement and lung fibrosis worsening were focused on diffuse cutaneous form and early disease (<5 years of disease duration).
Factors associated with lung improvement in patients treated with rituximab were determined using univariate and multivari-able logistic regression analyses with multiple imputations. Age, disease duration and DLCO were dichotomised according to their median or first quartile for FVC. All variables with p<0.1 in univariate were included in multivariate analysis with adjust-ment on regions. The lack of significant interaction between covariates was checked.
All tests were two-sided at a 0.05 significance level. Analyses were carried out using R statistical software V.3.3.2.
reSulTSbaseline cohort characteristicsFrom March to September 2017, out of 14 239 patients recorded in the database, 254 were recruited because of rituximab use (37 centres): 71% of female, median age: 51 years, 64% of diffuse cutaneous form and 71% displayed lung fibrosis. Median extent of lung fibrosis on CT was 20%.10–29 Anti-Scl70 and ACA antibodies were detected in 53% and 11% of the patients, respectively. An overlap syndrome was diagnosed in 26% of the cohort, consis-tently with previous reports in SSc.23 Previous treatments included DMARDs in 66/252 (32.5%), biologics in 24/252 (9.5%), cyclo-phosphamide in 23/252 (9.1%). Further details are provided in table 1 and online supplementary table I and II.
The leading indication for rituximab was lung involvement in 146/254 (58%) cases, skin in 81/254 (32%) cases and in 108/254 (42%) musculoskeletal involvement (flowchart in online supple-mentary figure 1). Concomitantly to rituximab, patients received steroids for 67.3% (median dose: 10 mg/day [IQR: 5–10]). Total 136 (53.7%) were treated in parallel with DMARDs (methotrexate
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Figure 2 Evolution of skin and lung fibrosis among propensity score-matched patients treated and untreated with rituximab. (A,B) evolution of modified Rodnan skin score between baseline and last visit (under treatment) in patients and propensity score-matched patients from EUSTAR: (A): in patients with skin indication for the treatment; (B) in patients with modified Rodnan skin score at baseline ≥10; (C) evolution of forced vital capacity between baseline and last visit (under treatment) in patients with a lung indication for the treatment and propensity score-matched patients from EUSTAR; (D) evolution of DLCO between baseline and last visit (under treatment) in patients with a lung indication for the treatment and propensity score-matched patients from EUSTAR. Data, presented as mean±SEM, are from the first imputed and matched (ratio 1:4) dataset. DLCO, diffusing capacity of lung for carbon monoxide; EUSTAR, European Scleroderma Trials and Research; FVC, forced vital capacity.
for 75, mycophenolate mofetil for 45, azathioprine for 14 and cyclophosphamide for 6).
Safety analysisAfter a median follow-up of 2 years, nearly 70% of patients (175/254) had no reported side effect, 43 (17%) had minor side effects and 36 (14%) were considered as severe by investigating centre. This led to discontinuation of the treatment in 24 patients (9%). During follow-up, six deaths were recorded: four unrelated to the treatment (two sudden deaths, one sepsis attributed to decu-bitus ulcers, one heart failure) and two possibly related (two respi-ratory insufficiencies in the context of lung carcinoma). In all, five cancers were reported during the follow-up. There was no associa-tion between occurrence of infections and previous or concomitant treatment with IS or hypogammaglobulinemia. Further details are provided in online supplementary table III.
efficacyAmong the 14 239 patients who underwent ≥1 visit, 9575 had ≥1 follow-up (table 1). Comparison of characteristics between patients with and without follow-up showed minor differences (online supplementary table IV). Matching for skin and lung allowed comparability of cases and controls (figure 1).
Skin fibrosisThe mRSS of patients treated for skin fibrosis decreased from mean±SD 22.1 (±9.3) to 14.1 (±8.4) in treated patients (n=74) vs 21.1 (±10.5) to 16.2 (±10.3) (p<0.0001) in untreated patients matched according to propensity score (n=281) (figure 2A). In patients with a mRSS ≥10 (n=131), the mRSS decreased from 21.2 (±8.8) to 13.4 (±7.9) vs 20.4 (±8.2) to 16.0 (±8.2) in controls (p<0.0001) (figure 2B).
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Table 2 Factors associated with lung improvement in patients treated with rituximab
univariate model Multivariate model
Or (95% CI) P value Or (95% CI) P value
Sex 0.84 (0.34 to 2.06) 0.71
Age >55 years 0.9 (0.42 to 1.9) 0.78
ANA 1.07 (0.07 to 16.77) 0.96
Anti-Scl70 0.84 (0.41 to 1.72) 0.63
ACA 0.68 (0.18 to 2.64) 0.58
RNA pol III antibodies 4 (0.45 to 35.38) 0.22
Diffuse cutaneous form 1.57 (0.78 to 3.18) 0.21
Disease duration <5 years 2.02 (0.98 to 4.17) 0.058 1.94 (0.94 to 4.03) 0.077
Previous IS or biologics 0.95 (0.47 to 1.93) 0.89
DLCO <70% 1.68 (0.68 to 4.2) 0.27
FVC <80% 2.05 (0.96 to 4.38) 0.066 1.96 (0.91 to 4.23) 0.089
Variables identified in univariate analysis with p<0.1 were tested in multivariate analysis adjusted on region. Lung fibrosis improvement was defined by increase in forced vital capacity during the follow-up. Previous IS (immunosuppressive drugs) include methotrexate, mycophenolate mofetil, azathioprine and cyclophosphamide, whereas biologics include anti-TNF alpha, tocilizumab and abatacept. Cut-off values to define DLCO and FVC were defined according to distribution.ACA, anticentromeres; ANA, antinuclear antibodies; DLCO, diffusing capacity of lung for carbon monoxide; FVC, forced vital capacity; RNA pol III, RNA polymerases III antibodies.
Figure 3 Adjusted OR for skin fibrosis improvement and lung fibrosis worsening among propensity score-matched patients treated or untreated with rituximab. The analyses of decrease in the modified Rodnan score of 5 points and 25% and decrease in the FVC of 10% and decrease in the DLCO of 15% were based on propensity score matching with a ratio 1:4. For each complete imputed data set, propensity score was estimated and association between decrease in the modified Rodnan score/ in FVC and in DLCO, respectively, and treatment was evaluated using conditional logistic regression. We used other matching method with ratio 1:1 within a calliper of 0.05 SD of the logistic propensity score, stratification on the quintiles of the propensity score and IPTW. Decrease in the modified Rodnan score of 5 points and 25% in patients with (A) skin indication; (B) modified Rodnan skin score >10 at baseline; (C) decrease in the FVC of 10% in patients with lung indication for the treatment; (D) decrease in the DLCO of 15% in patients with lung indication for the treatment. DLCO, diffusing capacity of lung for carbon monoxide; FVC, forced vital capacity; IPTW, inverse probability of treatment weighting.
Rituximab-treated patients had a higher probability of improvement in dermal fibrosis as compared with propensity score matched-controls (22.7 vs 14.03 events per 100 person-years; OR: 2.79 [1.47–5.32]; p=0.002). This was confirmed in sensitivity analyses (figure 3A).
Same results were observed for patients with a mRSS ≥10 (OR: 1.86 [1.25–2.79]; p=0.002) (figure 3B) as well as in subgroup anal-yses (diffuse and early forms; online supplementary figure 2A,B).
lung fibrosisIn lung treated patients (n=146), FVC remained stable during the follow-up (76.3 (±19.3) to 77.7 (±20.2)), similarly to controls (79.1 (±20.6) to 80.7 (±21.1)) (n=497) (figure 2C). DLCO also remained stable both in treated patients (54.4 [±18.8] to 55.5 [±19.5]) and in controls (55.6 [±18.7] to 54.7 [±20.2]) (figure 2D). Extent of fibrosis remained stable (20.0 [±2.1] to 20.0 [±2.5]) (n available=60). Treated patients did not have significantly different rates of decrease in FVC >10% (6.5 vs 6.6 events per 100 person-years; OR: 1.03 [0.55–1.94]; p=0.93) nor in DLCO decrease (figure 3C,D), which was confirmed in all secondary analyses (online supplementary figure 2 C, D and 3). In univariate cox models, lung function had a trend to improve in early disease and in patients with FVC <80%, which was not confirmed in multivariate analysis (table 2).
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Table 3 Evolution of musculoskeletal involvement during treatment with rituximab
Variables baseline last follow-up P value
Tender joints count (/28) (n=85)
7.6±8.6 2.4±4.7 <0.0001
Swollen joints count (/28) (n=81)
3.1±5.2 0.7±2.2 <0.0001
DAS28 ESR (n=25) 5.40 (3.99–6.22) 3.29 (2.02–4.24) <0.0001
DAS28 CRP (n=29) 4.68 (3.55–5.88) 2.60 (1.59–3.87) <0.0001
Joint contracture, n (%) 44/84 (52.4%) 37/84 (44%) 0.35
Tendon friction rubs, n (%) 20/76 (26.3%) 8/76 (10.5%) 0.021
Morning stiffness (min) (n=47)
78.9±72.1 32.1±46.3 <0.0001
Visual analogic scale for articular pain (n=33)
70(50-80) 35 (20–52.5) <0.0001
HAQ-DI (n=12) 1.31 (1.12–1.92) 1.20 (0.45–1.81) 0.10
Values are median (IQR) or mean±SD numbers (%) of observations.P comparing baseline and last follow-up data using Wilcoxon-test or paired t-test for quantitative variables and McNemar or paired χ²-test for proportions, according to distributions.DAS, disease activity score; HAQ, Health Assessment Questionnaire.
In secondary analyses, the concomitant use of mycophenolate to rituximab (n=37 with lung indication) showed a trend for better outcomes (delta FVC with controls: 5.22 [0.83–9.62]; p=0.019 vs delta FVC: 3 [0.66–5.35]; p=0.012 in patients receiving rituximab alone). Furthermore, 9/31 (29%) had an increase >10% in FVC. The difference with patients treated by rituximab alone was not statistically significant (delta: 2.98 [–2.12 to 8.09]; p=0.25). No adjusted analysis could be performed due to the small sample size. DLCO did not change significantly in this subgroup.
There was no difference for skin and lung outcomes according to rheumatoid factor, anti-CCP antibodies or smoking status (online supplementary table V).
Musculoskeletal involvementAll articular parameters, but two (joint contractures and HAQ-DI) significantly decreased with rituximab (table 3). No controlled analysis could be performed because of a high proportion of missing data (eg, 81% for tender and swollen joint counts in control group).
Inflammation and cotreatmentsCRP levels decreased during the treatment from 11.5 (±1.0) to 6.7 (±0.7) mg/L (p<0.0001). About one quarter of the patients treated with steroids at baseline could stop steroids and steroids dosage decreased from 9.5 (±0.5) mg/day to 7.0 (±0.3) mg/day (p<0.0001) in the 130 remaining patients. Patients treated with rituximab were more likely to decrease or stop steroids in controlled analyses (25.3 vs 18.0 events per 100 person-years; OR: 2.34 [1.56–3.53], p<0.0001) (online supplementary figure 3B).
dose-effectRituximab regimens were variable according to centres: for each infusion: dose of 1000 mg for 203 patients, 500 mg for 27 375 mg/m2 for four patients. We observed a dose-effect with a better effect on skin involvement for a total dosage higher than 4000 mg (online supplementary table VI).
dISCuSSIOnIn a large cohort of carefully matched patients treated with ritux-imab for skin, or lung involvement, rituximab was associated
with a good safety profile. The outcomes showed a high efficacy on skin fibrosis, but not on lung. Patients treated with rituximab were 2-fold more likely to stop or decrease steroids.
Following previous promising studies suggesting an ameliora-tion of lung involvement, the raw analyses after propensity score matching clearly show no difference between treated and not treated patients and overall a large proportion of non-progressive patients.7 10 12 14–16 19 Therefore, the results should be interpreted as the lack of effects of rituximab in this sample of patients with SSc. Nevertheless, one may advocate that the lung trajectory of rituximab-treated patients may differ from the one of untreated patients. Indeed, if the treating physician decided to offer ritux-imab therapy, then it is most of the time because of lung activity and severity, which are identified are poor predictive factors. These biases (selection biases, persistence of confounders despite the matching) are limitations to our study inherent to its obser-vational design.30 Therefore, the potential stabilisation of ILD following rituximab therapy has to be addressed in the future but only a randomised trial will provide robust answer.
Some previous small uncontrolled or unadjusted series have suggested some efficacy of rituximab on cutaneous involvement in SSc.5–13 19 29 However, evaluation of drug efficacy in SSc might be difficult because of spontaneous improvement in skin fibrosis during the first years, highlighting the need of control group. In our study, patients treated with rituximab were twofold more likely to improve skin fibrosis in all matched analyses, sensitivity analyses and subgroup approaches. The mRSS is a validated surrogate marker31–34 frequently used as an outcome measure in clinical trials.36 Indeed, worsening in mRSS is asso-ciated with higher mortality, and worsening of internal organ involvement.32 37 38 Conversely, improvement of the skin score is predictive of favourable outcomes, including better survival.38 Therefore, improvement in mRSS observed in rituximab-treated patients might indicate a better prognosis for these patients. Consistently, CRP levels, a marker of disease activity and severity,39 decreased following rituximab treatment.
One may also highlight the different trajectory between skin and lung outcomes in our sample. It must be pointed out that this has already been observed and by example the recent tocili-zumab trial showed some benefit on lung whereas skin was less influenced.40 This highlights the difficulties to design clinical trials in SSc regarding both the choice of the primary outcome measure and also the patients selection criteria.41 A clue for the future to get a better impact on SSc outcomes might be combina-tion therapy which seems to be more effective on lung involve-ment in our study and has shown clear benefit for treatment with rituximab in other diseases.42
Surprisingly, few patients have been previously treated with cyclophosphamide.43 This choice of using first rituximab might be driven by the need of concomitant treatment for musculoskel-etal involvement in almost half of the cohort (online supplemen-tary figure 1). Furthermore, common use of rituximab in other diseases like RA but also connective tissue diseases and vasculitis has given rheumatologists confidence in the management and in the safety of rituximab. Finally, the use of cyclophosphamide and mycophenolate mofetil could be limited by safety data.44
Interestingly, rituximab use doubled the probability of discon-tinuing or decreasing steroids. This is of major importance, since steroids cumulative dose increases the risk of subclinical/clinical atherosclerosis45 and SRC46 in SSc.
In our study, safety profile of rituximab was acceptable with in particular no progressive multifocal leukoencephalopathy or related-death. Furthermore, despite the use of intravenous steroids, only two SRCs were reported.
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The main strengths of our study are (1) large sample size, (2) availability of well-matched controls, (3) standardised collec-tion of data according to a questionnaire and (4) broad spec-trum of patients included corresponding to ‘real-life’ patients and allowing subgroup analyses (diffuse forms, early SSc). Using extensive matching methods, we accounted for many poten-tial cofounders of the associations between rituximab use and outcomes. We also performed several sensitivity and subgroups analyses, which were highly consistent.
Our study has limitations: first, rituximab regimens were variable according to centres. We analysed effect of the dose received and observed a dose-effect for a dosage higher than 4000 mg for skin involvement, suggesting that a dosage of 1000 mg should be used for the first infusions. This is an open label study, which can bias study results towards beneficial effect for the test medication (if the examiner anticipates beneficial effect of the treatment) in an outcome measure that is less objective (ie, mRSS) than FVC, which is more objective and less dependent on the examiner’s bias. There are also limitations inherent to the observational design of this study. However, the importance of disease registries, like EUSTAR, in rare diseases was recently highlighted, as they allowed collecting detailed case studies with standardised information and improving understanding of SSc and its treatment.47 Furthermore, the study reflects clinical practice in this orphaned heterogeneous disease. The duration of exposure to rituximab was relatively short (2 years), which limited precision for safety analyses. We could also not assess safety in control group (no data available) or outcomes that occurred after withdrawal of rituximab. Finally, despite highly matched cohorts across a wide range of characteristics, we cannot rule out residual confounders.
Author affiliations1Rheumatology a Department, Paris Descartes University, Cochin Hospital, Paris, France2Unit of Clinical Research, Paris seine saint Denis University, Bobigny, France3Center of experimental Rheumatology, Zurich, switzerland4Rheumatology and internal Medicine, Ghent University Hospital, Ghent, Belgium5Unit for Molecular immunology and inflammation, ViB inflammation Research Center (iRC), Ghent, Belgium6Department of internal Medicine, University of Florence, Firenze, italy7Rheumatology, Hospital Peset, Valencia, spain8Rheumatology Department, Bordeaux University Hospital, Bordeaux, France9B. shine Department of Rheumatology, Rambam Health Care Campus and Technion, Haifa, israel10interdisciplinary Department of Medicine-Rheumatology Unit, Policlinico, University of Bari, bari, italy11Clinic of nephrology, internal and Occupational Diseases, sechenov First Moscow state Medical University, Moscow, Russian Federation12internal Medicine, CHU south hospital, Rennes, France13Rheumatology, Charit University Hospital, Berlin, Germany14Hospital de la santa Creu i sant Pau, Barcelona, spain, Barcelona, spain15Rheumatology and Clinical immunology service, spedali Civili di Brescia, Brescia, italy16Rheumatology Department, second University of naples, naples, italy17laboratory of Tissue Homeostasis and Disease, skeletal Biology and engineering Research Center, Department of Development and Regeneration, KU leuven, leuven, Belgium18Univ. lille, inserm, CHU lille, U995 - liRiC - lille inflammation Research international Center, service de Médecine interne, Hôpital Claude Huriez, Centre de Référence pour la sclérodermie systémique, FHU iMMinenT, F-59000, lille, France19Division of Rheumatology Department of Medicine iii, Technical University of Dresden, Dresden, Germany20institute of Rheumatology, Russian academy of Medical science, Moscow, Russian Federation21internal Medicine, Rheumatology, immunology and nephrology, asklepios Clinic altona, Hamburg, Germany22Rheumatology, Hospital General Universitario Gregorio Marañón, Madrid, spain23Department of internal Medicine iii, University of erlangen, erlangen, Germany24Department of Dermatology, University of Cologne, Cologne, Germany25Department of Rheumatology, Rikshospitalet University Hospital, Oslo, norway
26Clinical Medicine and Therapy, sapienza University of Rome, Rome, italy27Rutgers-Robert Wood Johnson Medical school scleroderma Program, new Brunswick, new Jersey,Usa28Dipartimento di Medicina, Ospedale san Gerardo, Monza, italy29Rheumatology 2 Department, Universitatea de Medicina si Farmacie Gr T Popa iasi Facultatea de Medicina, iasi, Romania30Centroper la sclerosi sistemica -Dipartimento di Medicina Clinica, Università lasapienza, Rome, italy31internal Medicine and Rheumatology Department, Cantacuzino Hospital, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania32allergy and Rheumatology, nippon Medical school Graduate school of Medicine, Tokyo, Japan33University Medical Center Comprehensive Pulmonary Hypertension Center (UMC-CPHC); Tulane University lung Center Tulane, new Orleans scleroderma and sarcoidosis Patient Care and Research Center new Orleans, new Orleans, louisiana, Usa34Division of immunology and allergy, HCUGe, Geneva, switzerland35Department of Rheumatology, lund University, lund, sweden36University Hospital of Copenhagen, Department of Dermatology, Hospital Bispebjerg, Copenhagen, Denmark37Department of Rheumatology, University of Marmara, istanbul, Turkey38Clinica Reumatologie, Cluj-napoca, Romania39Rheumatology Unit, University of Verona, Verona, italy40URC CRC, Hopital avicenne, Bobigny, France41Rheumatology a Department, Cochin Hospital, aPHP, Paris Descartes University, Paris, France
Correction notice This article has been corrected since it published Online First. affiliation number 11 has been amended.
Collaborators eUsTaR coworkers, serena Guiducci; Ulrich a. Walker; Diego Kyburz; Giovanni lapadula; Britta Maurer; suzana Jordan; Rucsandra Dobrota; Radim Becvar; stanislaw sierakowsky; Otylia Kowal Bielecka; alberto sulli; Maurizio Cutolo; Giovanna Cuomo; ileana nicoara; andré Kahan; Panayiotis G. Vlachoyiannopoulos; Carlo Maurizio Montecucco; Roberto Caporali; Jiri stork; Murat inanc; Patricia e. Carreira; srdan novak; lászló Czirják; Cecilia Varju; eugene J. Kucharz; anna Kotulska; Magdalena Kopec-Medrek; Malgorzata Widuchowska; Franco Cozzi; Blaz Rozman; Carmel Mallia; Bernard Coleiro; armando Gabrielli; Dominique Farge; Chen Wu; Zora Marjanovic; Helene Faivre: Darin Hij; Roza Dhamadi; Frank Wollheim; agneta scheja; Dirk M Wuttge; Kristofer andréasson; Duska Martinovic; alexandra Balbir-Gurman; F. Trotta; andrea lo Monaco; Raffaele Pellerito; Ospedale Mauriziano; Paola Caramaschi ; Jadranka Morovic-Vergles; Carol Black; Christopher Denton; nemanja Damjanov; Jörg Henes; Vera Ortiz santamaria; stefan Heitmann; Dorota Krasowska; Matthias; Paul Hasler; Harald Burkhardt; andrea Himsel; Gianluigi Bajocchi; José antonio Pereira Da silva; Maria João salvador; Bojana stamenkovic; aleksandra stankovic; Carlo Francesco selmi; Maria De santis; Mohammed Tikly; lev n. Denisov; ariane Herrick,Ulf Müller-ladner; Marc Frerix; ingo Tarner; Raffaella scorza; Francesco Puppo; Merete engelhart; Gitte strauss; Henrik nielsen; Kirsten Damgaard; Gabriela szücs; antonio Zea Mendoza; Carlos de la Puente; sifuentes Giraldo Wa; Øyvind Midtvedt; silje Reiseter; Torhild Garen; David launay; Guido Valesini; Ruxandra Maria ionescu; laura Groseanu; Daniela Opris; Roxana sfrent Cornateanu; Razvan ionitescu; ana Maria Gherghe; alina soare; Marilena Gorga; Mihai Bojinca; Mihaela Milicescu; Cord sunderkötter; annegret Kuhn; nora sandorfi; Georg schett; Christian Beyer; Pierluigi Meroni; Francesca ingegnoli; luc Mouthon; Filip De Keyser; Karin Melsens; Francesco P. Cantatore; ada Corrado; line iversen; Carlos alberto von Mühlen; Jussara Marilu Bohn; lilian scussel lonzetti; Kilian eyerich; Rüdiger Hein; elisabeth Knott; Piotr Wiland; Magdalena szmyrka-Kaczmarek; Renata sokolik; ewa Morgiel; Marta Madej; Frédéric a. Houssiau; Brigitte Krummel-lorenz; Petra saar; Martin aringer; Claudia Günther; Rene Westhovens; Jan lenaerts; Branimir anic; Marko Baresic; Miroslav Mayer; Maria Üprus; Kati Otsa; Brigitte Granel; Carolina de souza Müller; sebastião C. Radominski; Valderílio F. azevedo; sergio Jimenez; Joanna Busquets; svetlana agachi; liliana Groppa; lealea Chiaburu; eugen Russu; sergei Popa; Thierry Zenone; Margarita Pileckyte; alessandro Mathieu; alessandra Vacca; Percival D. sampaio-Barros; natalino H. Yoshinari; Roberta G. Marangoni; Patrícia Martin; luiza Fuocco; simon stebbings; John Highton; Peter Chapman; John O’Donnell; lisa stamp; alan Doube; Kamal solanki; Douglas Veale; Marie O’Rourke; esthela loyo; Mengtao li; Walid ahmed abdel atty Mohamed; antonio amoroso; antonietta Gigante; Fahrettin Oksel; Figen Yargucu; Cristina-Mihaela Tanaseanu; Monica Popescu; alina Dumitrascu; isabela Tiglea; Rosario Foti; Rodica Chirieac; Daniel Furst; Peter Villiger; sabine adler; Jacob van laar; Cristiane Kayser; nihal Fathi; Manal Hassanien; Paloma García de la Peña lefebvre; silvia Rodriguez Rubio; Marta Valero exposito; emmanuel Chatelus; Jean sibilia; Jacques eric Gottenberg; Hélène Chifflot; ira litinsky; Paul emery; Maya Buch; Francesco Del Galdo; algirdas Venalis; irena Butrimiene; Paulius Venalis; Rita Rugiene; Diana Karpec; Joseph a. lasky; Vanesa Cosentino; eduardo Kerzberg; Fabiana Montoya; Washington Bianchi; sueli Carneiro; Giselle Baptista Maretti; Dante Valdetaro Bianchi; Massimiliano limonta; antonio luca Brucato; elide lupi; itzhak Rosner; Michael Rozenbaum; Gleb slobodin; nina Boulman; Doron Rimar; Maura Couto; sarah Kahl; Fei Chen; Deborah McCloskey; Halina Malveaux; François spertini;
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Camillo Ribi; Guillaume Buss; Thierry Martin; aurélien Guffroy; Vincent Poindron; Fatima Chotchaeva; nikolay a. Mukhin; sergey Moiseev.
Contributors Me, MB and Ya formulated the study hypotheses and contributed to its design, literature search, composition of the tables and figures and redaction of the first draft and subsequent iterations of the manuscript. Me, MB, CB-Q and Ya: analysis and interpretation of data. Me and MB performed the statistical analyses. OD, Vs, MM-C, JJas, M-eT, YB-M, Fi, Pin, al, es, iC, Pa, sV, eDl, eH, ae, la, MK, FJl-l, JHWD, nH, a-MH-V, VR, VMH, MRP, Ca, eR, CM, MK, las, CC, RH, sU, sY, sR, CC and Ya conceived and launched the eUsTaR database, collected data in their respective countries and offered critical comments regarding the manuscript. all authors have finally approved the submitted version to be published.
Funding eUsTaR database is supported by the World scleroderma Foundation.
Competing interests OD has consultancy relationship with actelion, Bayer, Biogenidec, Boehringer ingelheim, Chemomab, espeRare foundation, Genentech/Roche, GsK, inventiva, italfarmaco, lilly, medac, Medimmune, Mitsubishi Tanabe Pharma, Pharmacyclics, novartis, Pfizer, sanofi, sinoxa and UCB in the area of potential treatments of scleroderma and its complications. OD has received research funding from actelion, Bayer, Boehringer ingelheim, Mitsubishi Tanabe Pharma and Roche in the area of potential treatments of scleroderma and its complications. in addition, OD has a patent mir-29 for the treatment of systemic sclerosis licensed. M-eT has received consulting fees, speaking fees or honoraria from abbvie, BMs, lilly, Medac, MsD, Pfizer, Roche and UCB. Pa has received travel expenses from Roche (<2500€). sV received speaking fees by Pfizer, abbvie, Bristol-Myers squibb, consultant fee from Thermofischer and Boehringer-ingelheim italia and educational support from Pfizer, Roche, BMs. eH has received speaker fees and/or honoraria for consultations from Roche, less than 10 000€. nH received lecture fees from actelion Pharmaceuticals, Pfizer, Roche and grant support from actelion and Bayer Pharmaceuticals. CM has/had consultancy relationship and/or has received honoraria from actelion, abbvie, Roche and Geneva Romfarm in the area of systemic sclerosis and its complications. CC has received travel grants from Roche. Ya has/had consultancy relationship and/or has received grants from actelion, Bayer, BMs, Boehringer-ingelheim, inventiva, Roche, sanofi-aventis in the area of systemic sclerosis.
Patient consent for publication Obtained.
ethics approval each participating centre obtained approval of the local ethics committee and all registered patients granted their informed consent.
Provenance and peer review not commissioned; externally peer reviewed.
data sharing statement no additional unpublished data from the study are available.
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36 Khanna D, Furst De, Clements PJ, et al. standardization of the modified Rodnan skin score for use in clinical trials of systemic sclerosis. J Scleroderma Relat Disord 2017;2:11–18.
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Myositis
CliniCal sCienCe
Anti-Ro52 autoantibodies are associated with interstitial lung disease and more severe disease in patients with juvenile myositissara sabbagh,1 iago Pinal-Fernandez,1,2,3 Takayuki Kishi,4 ira n Targoff,5 Frederick W Miller,4 lisa G Rider,4 andrew lee Mammen 1,2,6
To cite: sabbagh s, Pinal-Fernandez i, Kishi T, et al. Ann Rheum Dis 2019;78:988–995.
Handling editor Josef s smolen
For numbered affiliations see end of article.
Correspondence toDr andrew lee Mammen, niaMs/niH, Bethesda, MD 20892, Usa; andrew. mammen@ nih. gov
ss and iP-F contributed equally.lGR and alM contributed equally.
ss and iP-F are joint first authors.lGR and alM are joint senior authors.
This work was presented in abstract form at aCR 2018 and GCOM 2019.
Received 31 December 2018Revised 8 March 2019accepted 1 april 2019Published Online First 24 april 2019
© author(s) (or their employer(s)) 2019. no commercial re-use. see rights and permissions. Published by BMJ.
AbsTrACT Objectives anti-Ro52 autoantibodies are associated with more severe interstitial lung disease (ilD) in adult myositis patients with antiaminoacyl transfer (t)Rna synthetase autoantibodies. However, few studies have examined anti-Ro52 autoantibodies in juvenile myositis. The purpose of this study was to define the prevalence and clinical features associated with anti-Ro52 autoantibodies in a large cohort of patients with juvenile myositis.Methods We screened sera from 302 patients with juvenile dermatomyositis (JDM), 25 patients with juvenile polymyositis (JPM) and 44 patients with juvenile connective tissue disease–myositis overlap (JCTM) for anti-Ro52 autoantibodies by elisa. Clinical characteristics were compared between myositis patients with and without anti-Ro52 autoantibodies.results anti-Ro52 autoantibodies were found in 14% patients with JDM, 12% with JPM and 18% with JCTM. anti-Ro52 autoantibodies were more frequent in patients with antiaminoacyl tRna synthetase (64%, p<0.001) and anti-MDa5 (31%, p<0.05) autoantibodies. after controlling for the presence of myositis-specific autoantibodies, anti-Ro52 autoantibodies were associated with the presence of ilD (36% vs 4%, p<0.001). Disease course was more frequently chronic, remission was less common, and an increased number of medications was received in anti-Ro52 positive patients.Conclusions anti-Ro52 autoantibodies are present in 14% of patients with juvenile myositis and are strongly associated with anti-MDa5 and antiaminoacyl tRna synthetase autoantibodies. in all patients with juvenile myositis, those with anti-Ro52 autoantibodies were more likely to have ilD. Furthermore, patients with anti-Ro52 autoantibodies have more severe disease and a poorer prognosis.
InTrOduCTIOnIdiopathic inflammatory myopathies (IIMs) are a heterogeneous group of systemic autoimmune diseases characterised by weakness, chronic inflam-mation of skeletal muscles and elevated serum muscle enzyme levels.1 Many patients also have extramuscular manifestations, including involve-ment of the skin, lungs and/or joints. Most patients with IIM have a myositis-specific autoantibody (MSA), defined as an autoantibody found only in patients with IIM, which are typically mutually exclusive.2 In contrast, myositis-associated autoan-tibodies (MAAs) are found in IIM but may also be present in patients with other autoimmune diseases
and may be seen in association with an MSA or other MAAs.
MSAs are associated with specific phenotypes.2 3 For instance, antimelanoma differentiation-associ-ated gene 5 (MDA5) autoantibodies are associated with cutaneous ulceration and palmar papules, minimal muscle involvement, arthritis, intersti-tial lung disease (ILD) and high fatality rate.4–7 In contrast, patients with autoantibodies recognising histidyl-transfer (t)RNA synthetase (ie, Jo1) have antisynthetase syndrome, a unique multisystem autoimmune disease characterised by a combination of myositis, ILD, arthritis, Raynaud’s phenomenon, fever and/or mechanic’s hands.8 Of note, while many phenotypic features are similar between juve-nile and adult IIM with the same MSAs, there are some important differences. For example, adults with anti-p155/140 (transcription intermediary factor 1; TIF-1) autoantibodies have an increased risk of malignancy, whereas anti-p155/140 (TIF-1) autoantibody-positive children do not.2 9
In adult patients with IIM, the most common MAA is anti-Ro52.10 Interestingly, anti-Ro52
Key messages
What is already known about this subject? ► The clinical features and prognosis of patients with juvenile myositis and anti-Ro52 autoantibodies were poorly defined.
What does this study add? ► Approximately 15% of a large North American cohort of patients with juvenile myositis have anti-Ro52 autoantibodies.
► Patients with juvenile myositis and anti-Ro52 autoantibodies are more likely to develop interstitial lung disease (ILD).
► Anti-Ro52 autoantibodies are more common in patients with juvenile myositis and anti-MDA5 and antisynthetase autoantibodies.
► Patients with juvenile myositis and anti-Ro52 autoantibodies more often have a chronic disease course and require more medications.
How might this impact on clinical practice or future developments?
► Anti-Ro52 autoantibodies are useful prognostic markers for ILD and severe disease in patients with juvenile myositis.
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Myositis
Figure 1 Swarm plot of anti-Ro52 autoantibody ELISA results for juvenile healthy controls and patients with JIIM divided into JDM, JPM and JCTM. The dashed line of 20 units indicates the cut-off value for anti-Ro52 autoantibody positivity. Out of 371 patients with JIIM, 53 (14%) were positive for anti-Ro52 autoantibodies by ELISA. Of these patients, 42 had JDM, 3 had JPM and 8 had JCTM. Out of 90 juvenile healthy controls, one patient (1.1%) was positive for anti-Ro52 autoantibodies by ELISA. JCTM, juvenile connective tissue myositis; JDM, juvenile dermatomyositis; JIIM, juvenile idiopathic inflammatory myopathy; JPM, juvenile polymyositis.
autoantibodies often co-occur with anti-Jo1 autoantibodies11 and adult patients with both autoantibodies have more severe ILD and more frequently develop lung fibrosis than those with anti-Jo1 autoantibodies alone.12 13 In addition, higher anti-Ro52 autoantibody titres are associated with the development of more severe ILD,14 myositis and joint impairment in adult patients positive with anti-Jo1.15 Patients with both anti-Jo1 and anti-Ro52 autoantibodies have a poorer response to various immu-nosuppressive drugs and a decrease in survival.13 15
A recent analysis of 22 children with myositis revealed that 23% had anti-Ro52 autoantibodies, although specific clinical associations were not examined.16 The purpose of this study was to define the prevalence and clinical features associated with anti-Ro52 autoantibodies in a large cohort of patients with juve-nile myositis.
PATIenTs And MeTHOdsPatients and serum samplesOf the 543 patients from the Childhood Myositis Heterogeneity Collaborative Study who were enrolled between 1989 and 2016, with probable or definite myositis by Bohan and Peter criteria,17 those with a serum sample available for autoantibody testing at the time of enrolment were included in the study. Among the 371 patients with juvenile myositis included, 302 (81.4%) had juvenile dermatomyositis (JDM), 25 (6.7%) had juvenile poly-myositis (JPM) and 44 (11.9%) had juvenile connective tissue disease–myositis (JCTM) overlap. The JCTM subgroup included patients meeting the criteria for myositis and another autoim-mune disease, including 13 with juvenile systemic lupus erythe-matosus, 11 with juvenile systemic sclerosis, 7 with juvenile idiopathic arthritis and 13 with other autoimmune conditions including autoimmune hepatitis, eosinophilic fasciitis, diabetes mellitus, lichen sclerosis, linear morphea, psoriasis, Sjögren’s syndrome and ulcerative colitis. Sera from 90 healthy control children enrolled in the same studies were available.
All subjects were enrolled in institutional review board-ap-proved natural history studies as previously described,18 and all provided informed consent. A standardised physician question-naire captured demographics, clinical and laboratory features, environmental exposures at illness onset or diagnosis, as well as therapeutic usage and responses.18 Seven organ system symptom scores at diagnosis, defined as the number of symptoms present divided by the number of symptoms assessed, and an overall clin-ical symptom score as the average of the seven individual organ symptom scores were calculated as previously described.19–21 In 7 of 33 patients, the presence of ILD was diagnosed by high-res-olution CT (HRCT) and lung biopsy. In 11 of 33 patients, ILD was diagnosed by HRCT alone and in 5 patients, ILD was diag-nosed by biopsy alone. Seven patients were diagnosed with ILD by chest radiographic imaging combined with pulmonary function testing and did not undergo HRCT or lung biopsy. Three patients did not have imaging records available, and the diagnosis of ILD was based on physician documentation in the medical record. Complete clinical response and remission were defined as at least 6 months of inactive disease on or off therapy, respectively.20 A course of treatment was defined as a single episode from beginning of administration of a given medication to the termination of treatment with that medica-tion, or combination of medications, in each patient. Medical record review, conducted in >75% of patients, verified the clin-ical, demographic, laboratory and therapeutic data contained in the physician questionnaires. Follow-up visits occurred in 55% of patients, with an average time from enrolment date to final
evaluation of 4.3 years. Patient characteristics in our cohort are comparable with other registry-based JDM cohorts in terms of demographics and disease manifestations.22–25
Autoantibody assaysAnti-Ro52 autoantibodies were detected using an enhanced performance Ro52 ELISA (SS-A 52 ELISA, Quanta Lite, INOVA Diagnostics, San Diego, CA) according to the manufacturer’s instructions. Other myositis autoantibodies were detected as previously described.18 26
AnalysisDichotomous variables were expressed as percentages and absolute frequencies, and continuous features were reported as means and SD. Pairwise comparisons for categorical variables between groups were made using χ2 test or Fisher’s exact test, as appropriate, while continuous variables were compared using Student’s t-test. Logistic and linear regression were used to adjust the comparisons for possible confounding variables, including the year of diagnosis, length of follow-up and MSAs. Creatine kinase, a highly positively skewed variable, was expressed as median, first and third quartile for descriptive purposes and transformed through a base-10 logarithm for analysis. All statis-tical analyses were performed using Stata/MP V.14.1 (StataCorp). As this was an exploratory study, a two-sided p value of ≤0.05 was considered statistically significant.
resulTsPrevalence and demographics of patients with anti-ro52 autoantibodiesAnti-Ro52 autoantibodies were more prevalent in patients with juvenile IIM (JIIM) than in healthy control children (14% vs 1%). Sera from 14% of patients with JDM, 12% with JPM and 18% with JCTM had anti-Ro52 autoantibodies (figure 1, table 1). There were no significant differences in gender, race, age at diagnosis or delay to diagnosis between patients with juvenile myositis, with and without anti-Ro52 autoantibodies (table 2).
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Table 1 Prevalence of anti-Ro52 autoantibodies among patients with juvenile myositis
Clinical subgroupAnti-ro52 autoantibody positive % (n/n)
Juvenile myositis (N=371) 14% (n=53) ***
Juvenile dermatomyositis (N=302) 14% (n=42) ***
Juvenile polymyositis (N=25) 12% (n=3) *
Juvenile connective tissue-disease myositis (N=44):
18% (n=8) ***
Juvenile lupus erythematosus (N=13) 23% (n=3) **
Juvenile systemic sclerosis (N=11) 0% (n=0)
Juvenile idiopathic arthritis (N=7) 29% (n=2) *
Other autoimmune diseases† (N=13) 23% (n=3) **
Myositis-specific autoantibody subgroup
Anti-p155/140 (TIF-1) (N=119) 11% (n=13)
Anti-NXP2 (N=77) 14% (n=11)
Anti-MDA5 (N=32) 31% (n=10) *
Antiaminoacyl tRNA synthetase (N=14) 64% (n=9) ***
Anti-SRP (N=7) 0% (n=0)
Anti-Mi2 (N=13) 15% (n=2)
Anti-HMGCR (N=4) 50% (n=2)
MSA negative (N=96) 5% (n=5) **
Juvenile healthy controls (N=90) 1% (n=1)
x² or Fisher’s exact tests were used to compare the percentage of positive patients compared with the percentage of negative patients within each myositis clinical and autoantibody subgroup.*p<0.05, **p<0.01, ***p<0.001.†autoimmune hepatitis, eosinophilic fasciitis, fasciitis, juvenile diabetes mellitus, lichen sclerosis, linear morphea, psoriasis, Sjögren’s syndrome, ulcerative colitis.HMGCR, 3-Hydroxy-3-Methylglutaryl-CoA Reductase; MDA5, melanoma differentiation associated protein-5; MSA, myositis-specific autoantibody; NXP2, nuclear matrix protein-2; SRP, signal recognition particle;tRNA, transfer RNA;TIF-1, transcription intermediary factor 1.
Table 2 General features of patients with juvenile myositis, with and without anti-Ro52 autoantibodies
Total(n=371)% (n/n) orMean (sd)
Anti-ro52 autoantibody positive (n=53)% (n/n) orMean (sd)
Anti-ro52 autoantibody negative (n=318)% (n/n) orMean (sd) p value
Age at diagnosis 9.0 (4.4) 9.5 (4.7) 8.9 (4.3) 0.3
Age at enrolment 12.5 (7.1) 12.6 (7.7) 12.5 (7.0) 1.0
Delay to diagnosis (years)
0.7 (1.2) 0.55 (0.56) 0.75 (1.27) 0.3
Follow-up (years) 5.8 (6.4) 4.3 (6.4) 6.0 (6.4) 0.09
Female 71% (263/371) 74% (39/53) 70% (224/318) 0.6
Race
White 65% (240/371) 57% (30/53) 66% (210/318) 0.2
Black 16% (59/371) 21% (11/53) 15% (48/318) 0.3
Hispanic 6% (24/371) 6% (3/53) 7% (21/318) 1.0
Other races * 13% (48/371) 17% (9/53) 12% (39/318) 0.3
Myositis-specific autoantibodies
Anti-p155/140 (TIF-1)
33% (119/359) 26% (13/50)† 34% (106/309)‡ 0.2
Anti-NXP2 21% (77/366) 21% (11/52)† 21% (66/314)‡ 1.0
Anti-MDA5 9% (32/368) 19% (10/53) 7% (22/315)‡ 0.01
Anti-aminoacyl tRNA synthetase
4% (14/360) 18% (9/49)† 2% (5/311)‡ <0.001
Anti-SRP 2% (7/360) 0% (0/49)† 2% (7/311)‡ 0.6
Anti-Mi2 4% (13/354) 4% (2/49)† 4% (11/305)‡ 0.7
Anti-HMGCR 1% (4/371) 4% (2/53) 1% (2/318) 0.10
MSA negative 27% (96/362) 9% (5/53) 29% (91/309)‡ 0.002
Dichotomous variables were represented as percentage (count/total) and continuous variables as mean (SD). χ2 or Fisher's exact tests were used to compare dichotomous variables, as appropriate, while continuous variables were compared using Student's t-test.*Asian (Korean, Japanese, Chinese, Indian, Filipino), Pacific Islands, American Indian.†N≠53 due to missing data.‡N≠318 due to missing data.HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; MDA5, melanoma differentiation associated protein-5; MSA, myositis-specific autoantibody; NXP2, nuclear matrix protein-2; SRP, signal recognition particle;TIF-1, transcription intermediary factor 1; tRNA, transfer RNA.
Prevalence of anti-ro52 autoantibodies among myositis-specific autoantibody subgroupsOf those patients positive for anti-Ro52 autoantibodies, 26% had coexisting anti-p155/140 (TIF-1) autoantibodies, 21% had antinuclear matrix protein 2 (NXP2) autoantibodies, 19% had anti-MDA5 autoantibodies, 18% had antiaminoacyl tRNA synthetase autoantibodies, 4% had anti-Mi2 autoantibodies, 4% had anti-3-hydroxy-3-methylglutaryl-CoA reductase autoanti-bodies and 9% were MSA negative (table 2). Anti Ro52 auto-antibodies were significantly increased in the anti-MDA5 and antiaminoacyl tRNA synthetase autoantibody subgroups than in other MSA subgroups (table 1). For instance, anti-Ro52 autoan-tibodies coexisted in 31% of juvenile IIM sera with anti-MDA5 autoantibodies and 64% of those with antiaminoacyl tRNA synthetase autoantibodies (table 1). Similarly, anti-MDA5 auto-antibodies coexisted in 19% of anti-Ro52 autoantibody positive sera and 7% of anti-Ro52 autoantibody negative sera. Antia-minoacyl tRNA synthetase autoantibodies coexisted in 18% of anti-Ro52 autoantibody-positive sera and 2% of anti-Ro52 auto-antibody-negative sera (table 2). Less than 15% of those with anti-p155/140 (TIF-1), NXP2, antisignal recognition particle or anti-Mi2 autoantibodies, and only 5% of those without an MSA were anti-Ro52 positive (table 1).
Pulmonary manifestations among patients with anti-ro52 autoantibodiesAfter controlling for the presence of MSAs (including antia-minoacyl tRNA synthetase and anti-MDA5 autoantibodies),
a multivariate analysis showed anti-Ro52 autoantibodies were highly associated with pulmonary involvement. Overall, patients with anti-Ro52 autoantibodies more often had ILD (36% vs 4%), dyspnoea on exertion (59% vs 25%) and a higher early pulmonary score (mean 0.18 vs 0.08) than those without these autoantibodies (table 3). Within the anti-MDA5 autoantibody positive subgroup, Ro52 reactivity was even more strongly asso-ciated with ILD: 70% of those with coexisting anti-Ro52 auto-antibodies had ILD compared with only 9% of those who were anti-Ro52 negative (table 4). Similarly, among the antiaminoacyl tRNA synthetase autoantibody subgroup, 100% of anti-Ro52 autoantibody-positive and 40% of anti-Ro52-negative patients had ILD (table 4). Other pulmonary manifestations were also associated with Ro52 reactivity within the anti-MDA5 and antia-minoacyl tRNA synthetase autoantibody subgroups. Specifically, among those patients with anti-MDA5 autoantibodies, patients who also were positive for anti-Ro52 autoantibodies more often had dyspnoea on exertion (90% vs 27%) and higher early pulmonary scores than those who were anti-Ro52 autoantibody negative. Only 1 of 33 patients with ILD in our JIIM cohort had rapidly progressive ILD, and this patient was positive for both anti-MDA5 and anti-Ro52 autoantibodies. In patients with
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Table 3 Clinical features of patients with juvenile myositis, with and without anti-Ro52 autoantibodies
signs/symptoms ever present Total(n=371)% (n/n) orMean (sd)
Anti-ro52 autoantibody positive (n=53) % (n/n) or Mean (sd)
Anti-ro52 autoantibody negative (n=318) % (n/n) or Mean (sd)
univariate p value Multivariate p value
Muscle involvement
Proximal weakness 99% (369/371) 98% (52/53) 100% (317/318) 0.3 0.3
Myalgia 64% (234/363) 62% (32/52)* 65% (202/311)† 0.6 0.1
Distal weakness 47% (170/363) 46% (24/52)* 47% (146/311)† 0.9 0.9
Muscle atrophy 37% (136/367) 44% (23/52)* 36% (113/315)† 0.2 0.3
Falling episodes 45% (164/367) 44% (23/52)* 45% (141/315)† 0.9 1.0
Lung involvement
Dyspnoea on exertion 30% (109/366) 59% (30/51)* 25% (79/315)† <0.001 <0.001
Interstitial lung disease 9% (33/369) 36% (19/53) 4% (14/316)† <0.001 <0.001
Dysphonia 32% (118/367) 32% (17/53) 32% (101/314)† 1.0 0.7
Joint involvement
Arthralgia 64% (236/369) 70% (37/53) 63% (199/316)† 0.3 0.4
Joint contractures 61% (224/370) 63% (33/52)* 60% (191/318) 0.6 0.7
Arthritis 51% (189/370) 60% (31/52)* 50% (158/318) 0.2 0.7
Skin involvement
Heliotrope 79% (293/369) 87% (46/53) 78% (247/316)† 0.2 0.2
Gottron´s papules 82% (305/370) 77% (41/53) 83% (264/317)† 0.3 0.3
Malar rash 70% (259/371) 68% (36/53) 70% (223/318) 0.7 0.6
Photosensitivity 48% (172/362) 49% (25/51)* 47% (147/311)† 0.8 0.9
V or Shawl sign rash 31% (113/369) 42% (22/53) 29% (91/316)† 0.06 0.07
Linear extensor erythema 36% (130/363) 31% (16/52)* 37% (114/311)† 0.4 0.3
Calcinosis 29% (109/371) 28% (15/53) 30% (94/318) 0.9 0.1
Raynaud´s phenomenon 15% (55/369) 23% (12/53) 14% (43/316)† 0.09 0.04
Mechanic´s hands 7% (27/366) 9% (5/53) 7% (22/313)† 0.6 0.5
Gastrointestinal involvement
Dysphagia 41% (151/370) 38% (20/53) 41% (131/317)† 0.6 1.0
Regurgitation 21% (77/370) 26% (14/53) 20% (63/317)† 0.3 0.5
Systemic involvement
Weight loss 42% (155/369) 52% (27/52)* 40% (128/317)† 0.1 0.8
Fever 31% (112/358) 41% (21/51)* 30% (91/307)† 0.1 0.8
Muscle Enzymes
Peak creatine kinase, IU/L 781 (252–5142) 1121 (225–3971) 750 (256–5249) 0.7 0.9
Peak aldolase, IU/L 20.0 (34.5) 18.0 (22.5) 20.3 (36.1) 0.6 0.3
Severity at onset 2.2 (1.1) 2.2 (1.7) 2.2 (0.9) 0.9 0.4
Early total symptom score 0.2 (0.1) 0.27 (0.14) 0.23 (0.11) 0.03 0.8
Early muscle score 0.4 (0.2) 0.37 (0.18) 0.38 (0.20) 0.7 0.5
Early joint score 0.5 (0.4) 0.48 (0.38) 0.45 (0.43) 0.6 0.1
Early cutaneous score 0.3 (0.1) 0.26 (0.15) 0.25 (0.14) 0.6 0.4
Early gastrointestinal score 0.1 (0.1) 0.08 (0.11) 0.07 (0.11) 0.6 1.0
Early pulmonary score 0.1 (0.2) 0.18 (0.23) 0.08 (0.14) <0.001 0.002
Early cardiac score 0.0 (0.1) 0.05 (0.12) 0.02 (0.07) 0.04 0.05
Early constitutional symptoms score
0.4 (0.3) 0.48 (0.34) 0.38 (0.26) 0.02 1.0
Dichotomous variables were represented as percentage (count/total), continuous variables as mean (SD) and the creatine kinase was presented as median (Q1-Q3). For the univariate analysis, dichotomous variables were compared using χ2 or Fisher's exact tests, as appropriate while continuous variables were compared using Student's t-test. Multivariate analysis used linear or logistic regression adjusted for length of follow-up, year of onset and autoantibodies. Creatine kinase was log-transformed prior to statistical analysis.*N≠53 due to missing data.†N≠318 due to missing data.
antiaminoacyl tRNA synthetase autoantibodies, anti-Ro52 auto-antibody-positive patients had increased frequency of dyspnoea on exertion (89% vs 40%), although this did not reach statis-tical significance. Patients with coexisting anti-p155/140 (TIF-1) and anti-Ro52 autoantibodies also had an increased frequency of ILD (15% vs 1%) and dyspnoea on exertion (50% vs 16%)
compared with anti-p155/140 (TIF-1) autoantibody-positive patients who were anti-Ro52 autoantibody negative (table 4). Of note, in the MSA negative subgroup, none of the 5 anti-Ro52 autoantibody-positive patients had ILD (table 4). The association of anti-Ro52 autoantibodies with ILD was signif-icant within the JDM clinical subgroup: 33% of anti-Ro52
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Table 4 Pulmonary features of patients with juvenile myositis, with and without anti-Ro52 autoantibodies within juvenile myositis clinical and autoantibody subgroups
Anti-ro52 autoantibody positive % (n/n) orMean (sd)
Anti-ro52 autoantibody negative % (n/n) orMean (sd) p value
JDM subgroup (N=302)
Interstitial lung disease 33% (14/42) 1% (3/258)* <0.001
Dyspnoea on exertion 62% (26/42) 19% (50/258)* <0.001
Early pulmonary score 0.20 (0.22) 0.07 (0.13) <0.001
JPM subgroup (N=25)
Interstitial lung disease 33% (1/3) 18% (4/22) 0.5
Dyspnoea on exertion 50% (1/2)* 67% (14/21)* 1.0
Early pulmonary score 0.17 (0.29) 0.19 (0.20) 0.8
JCTM subgroup (N=44)
Interstitial lung disease 50% (4/8) 19% (7/36) 0.09
Dyspnoea on exertion 43% (3/7)† 42% (15/36) 1.0
Early pulmonary score 0.12 (0.25) 0.09 (0.16) 0.7
Anti-MDA5 autoantibody subgroup (N=32)
Interstitial lung disease 70% (7/10) 9% (2/22) 0.001
Dyspnoea on exertion 90% (9/10) 27% (6/22) 0.002
Early pulmonary score 0.29 (0.19) 0.02 (0.06) <0.001
Anti-aminoacyl tRNA synthetase autoantibody subgroup (N=14)
Interstitial lung disease 100% (9/9) 40% (2/5) 0.03
Dyspnoea on exertion 89% (8/9) 40% (2/5) 0.09
Early pulmonary score 0.31 (0.31) 0.27 (0.30) 0.8
Anti-p155/140 (TIF-1) autoantibody subgroup (N=119)
Interstitial lung disease 15% (2/13) 1% (1/106) 0.03
Dyspnea on exertion 50% (6/12)* 16% (17/105)* 0.01
Early pulmonary score 0.16 (0.24) 0.06 (0.12) 0.01
Anti-NXP2 autoantibody subgroup (N=77)
Interstitial lung disease 9% (1/11) 0% (0/65)† 0.1
Dyspnoea on exertion 45% (5/11) 27% (18/66) 0.3
Early pulmonary score 0.16 (0.19) 0.10 (0.14) 0.2
MSA-negative subgroup (N=96)
Interstitial lung disease 0% (0/5)† 10% (9/90) 1.0
Dyspnoea on exertion 25% (1/4)* 33% (30/90) 1.0
Early pulmonary score 0.04 (0.09) 0.08 (0.15) 0.5
Dichotomous variables were represented as percentage (count/total), continuous variables as mean (SD). For the univariate analysis, dichotomous variables were compared using χ2 or Fisher's exact tests, as appropriate while continuous variables were compared using Student's t-test.*Data missing for two patients within juvenile myositis clinical or autoantibody subgroup.†Data missing for one patient within juvenile myositis clinical or autoantibody subgroup.JCTM, juvenile connective tissue myositis; MDA5: melanoma differentiation associated protein-5;JDM, juvenile dermatomyositis; JPM, juvenile polymyositis; NXP2, nuclear matrix protein-2; SRP, signal recognition particle; TIF-1, transcription intermediary factor 1;tRNA, transfer RNA.
autoantibodies-positive patients with JDM had ILD compared with 1% of anti-Ro52-negative patients with JDM (table 4).
Other clinical manifestations among patients with anti-ro52 autoantibodiesIndependent of MSA status, anti-Ro52 autoantibodies were also associated with Raynaud’s phenomenon (23% vs 14%) (table 3). Furthermore, within the anti-NXP2 subgroup, Ro52 reactivity was associated with more cutaneous involvement: patients with
both anti-NXP2 and anti-Ro52 autoantibodies had a higher prevalence of V or Shawl-sign rashes (55% vs 17%) and linear extensor erythema (64% vs 20%) than anti-NXP2 autoanti-body-positive patients without anti-Ro52 autoantibodies. Those with both anti-NXP2 and anti-Ro52 autoantibodies also had more frequent gastro-oesophageal regurgitation (55% vs 17%). Within the anti-MDA5 subgroup, however, anti-Ro52 auto-antibodies were associated with less frequent linear extensor erythema (11% vs 50%). Patients with anti-Ro52 autoantibodies also had a higher mean early cardiac score, defined by the pres-ence of cardiac symptoms at diagnosis.19 There were no other significant differences in the prevalence of the muscle, lung, joint, cutaneous, gastrointestinal or constitutional manifestations between patients with and without anti-Ro52 autoantibodies in univariate or multivariate analysis or in examining these features in anti-Ro52 autoantibody-positive patients in the presence of another MSA.
disease severity among patients with anti-ro52 autoantibodiesSeveral other differences in outcomes and medications received between patients positive and negative for anti-Ro52 autoanti-bodies suggested that anti-Ro52 autoantibodies are associated with more severe disease (table 5). The disease course in patients with anti-Ro52 autoantibodies was more often chronic contin-uous (78% vs 52%) and less often monocyclic (3% vs 25%). Anti-Ro52-positive patients were more often American College of Rheumatology (ACR) functional class 4 (11% vs 4%) at the last clinical evaluation and had a higher mean ACR functional class score at that assessment. Anti-Ro52 autoantibodies were also associated with an increased total number of medications received (mean 4.8 vs 3.8). Anti-Ro52 autoantibody-positive patients more often received intravenous pulse steroids (79% vs 52%). Anti-Ro52 autoantibody-positive patients less often achieved clinical remission (5% vs 27%). Lastly, on univar-iate analysis, but not multivariable analysis, patients with anti-Ro52 autoantibodies less often experienced a complete clinical response (17% vs 32%) and had more medication treatment trials per year (mean 3.5 vs 2.2).
Those with both anti-NXP2 and anti-Ro52 autoantibodies also more often had a severe (class IV) ACR functional class (27% vs 3%) and more frequent wheelchair use (60% vs 20%) as compared with patients positive for anti-NXP2 who were anti-Ro52 autoantibody negative. There was no other association of coexisting MSAs and anti-Ro52 autoantibodies on clinical outcomes or medications received.
Anti-ro52 autoantibody titresAnti-Ro52 autoantibody titres did not significantly differ between JDM, JPM and JCTM groups. Overall, we found that higher anti-Ro52 titres are associated with shorter follow-up time, more treatment trials per year, higher early total symptom score, more total number of medications used, higher total func-tional class, higher severity at onset, higher early pulmonary score, higher early constitutional symptoms score and higher total functional class in patients with juvenile IIM (all p<0.05; data not shown). However, as the Spearman correlation coeffi-cients were ≤0.2 for each association, the clinical significance of high autoantibody titres is modest.
dIsCussIOnHere, we used a large cohort of patients with juvenile myositis to study the prevalence and clinical significance of anti-Ro52
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Table 5 Disease outcomes and medications used in patients with juvenile myositis, with and without anti-Ro52 autoantibodies
Total(n=371)% (n/n) orMean (sd)
Anti-ro52 autoantibody positive (n=53)% (n/n) orMean (sd)
Anti-ro52 autoantibody negative (n=318)% (n/n) orMean (sd) univariate p value Multivariate p value
Disease
Monocyclic course 22% (65/297) 3% (1/37)* 25% (64/260)† 0.003 0.02
Polycyclic course 23% (68/297) 19% (7/37)* 23% (61/260)† 0.5 0.9
Chronic continuous course 55% (164/297) 78% (29/37)* 52% (135/260)† 0.002 0.05
Steinbrocker functional class at final assessment
Mean functional class 1.4 (0.8) 1.7 (1.0) 1.4 (0.8) 0.007 0.007
Functional class 1 70% (257/367) 53% (28/53) 73% (229/314)† 0.003 0.09
Functional class 2 21% (77/367) 34% (18/53) 19% (59/314)† 0.01 0.3
Functional class 3 4% (13/367) 2% (1/53) 4% (12/314)† 0.7 0.2
Functional class 4 5% (20/367) 11% (6/53) 4% (14/314)† 0.05 0.008
Mortality 4% (13/371) 6% (3/53) 3% (10/318) 0.4 0.4
Hospitalised 58% (206/355) 66% (35/53) 57% (171/302)† 0.2 0.4
Mean number of hospitalizations 1.3 (1.9) 1.3 (1.4) 1.3 (2.0) 0.9 0.8
Wheelchair use 19% (68/360) 24% (12/50)* 18% (56/310)† 0.3 0.2
Response to treatment
Complete clinical response 30% (91/304) 17% (7/42)* 32% (84/262)† 0.04 0.4
Remission 24% (74/312) 5% (2/43)* 27% (72/269)† 0.002 0.05
Total number of medications used 3.9 (2.1) 4.8 (2.5) 3.8 (2.0) 0.003 0.05
Treatment trials per year 2.3 (2.8) 3.5 (3.0) 2.2 (2.7) 0.004 0.1
Medications received
Oral steroids 99% (309/312) 100% (43/43)* 99% (266/269)† 1.0 .
Intravenous pulsed steroids 56% (174/312) 79% (34/43)* 52% (140/269)† <0.001 0.03
Methotrexate 74% (230/312) 86% (37/43)* 72% (193/269)† 0.05 0.4
Intravenous immunoglobulin 36% (112/312) 49% (21/43)* 34% (91/269)† 0.06 0.08
Other DMARDs‡ 23% (73/312) 35% (15/43)* 22% (58/269)† 0.06 0.3
Dichotomous variables were represented as percentage (count/total), continuous variables as mean (SD). For the univariate analysis, dichotomous variables were compared using χ2 or Fisher’s exact tests, as appropriate while continuous variables were compared using Student's t-test. Multivariate analysis used linear or logistic regression adjusted for length of follow-up, year of onset and autoantibodies.*N≠53 due to missing data†N≠318 due to missing data‡Azathioprine, Chlorambucil, Chloroquine, Colchicine, Cyclophosphamide, Cyclosporine, Dapsone, Hydroxychloroquine, Intravenous Immunoglobulin, Lefluonmide, Methotrexate, Mycophenolate mofetil, Sodium thiosulfate, QuinacrineACR, American College of Rheumatology; DMARDs, disease-modifying anti-rheumatic agents.
autoantibodies in children with IIM. We found anti-Ro52 auto-antibodies to be strongly associated with ILD and other pulmo-nary manifestations in patients with juvenile myositis. We also found that children with anti-Ro52 autoantibodies have more severe disease, underwent more intense treatment regimens and had lower rates of disease remission than those without anti-Ro52 autoantibodies. In children with myositis, anti-Ro52 auto-antibodies were associated with antiaminoacyl tRNA synthetase autoantibodies, as previously described in adults.11 We also found that anti-Ro52 autoantibodies were associated with anti-MDA5 autoantibodies in paediatric patients with myositis, which has not been reported previously.
Importantly, our analyses indicate that the presence of anti-Ro52 autoantibodies is strongly associated with ILD, even after adjusting for the presence of MSAs such as anti-MDA5 and anti-aminoacyl tRNA synthetase autoantibodies. Indeed, the associa-tion of Ro52 reactivity with ILD is not limited to the anti-MDA5 and antiaminoacyl tRNA synthetase autoantibody subgroups but extends to other MSA subgroups that are not classically asso-ciated with ILD, such as children with anti-p155/140 (TIF-1) autoantibodies. However, none of the five patients who are anti-Ro52 autoantibody positive and MSA negative had ILD.
Current practice encourages screening patients with juvenile myositis for MSAs such as anti-MDA5 and antiaminoacyl tRNA synthetase autoantibodies, as these autoantibodies confer risk for developing ILD, and their presence is a determinant of clin-ical management and patient prognosis. In light of the current findings demonstrating that anti-Ro52 autoantibodies are an independent predictor of ILD, screening patients with juvenile myositis for these autoantibodies may also be prudent.
In adult patients with IIM, anti-Ro52 autoantibodies have been associated with poorer response to immunosuppressive drugs and decreased survival.13 15 Similarly, in our juvenile cohort, anti-Ro52 autoantibodies are associated with more severe disease and poorer outcomes. Of note, the presence of anti-Ro52 autoanti-bodies was associated with a higher early cardiac score which is a measure of patient-reported cardiac symptoms including palpitations, chest pain and syncope. However, among the nine anti-Ro52-positive patients with one or more of these symptoms, only three had EKG changes or echocardiogram abnormali-ties. As the severity of other clinical manifestations, including muscle, joint, skin, gastrointestinal and systemic features were not associated with Ro52 reactivity, it seems likely that disease severity seen in the anti-Ro52-positive patients is a consequence
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of pulmonary disease. Additional studies are required to clarify this point. Nonetheless, our findings highlight the potential use of anti-Ro52 autoantibodies as a predictor of disease severity and poor prognosis in juvenile myositis, which underscores the potential use of screening patients with juvenile IIM for anti-Ro52 autoantibodies.
Of particular significance is the novel association of anti-Ro52 autoantibodies and anti-MDA5 autoantibodies in our JIIM cohort. In adult patients with IIM, anti-Ro52 autoanti-bodies often co-occur with anti-Jo1 autoantibodies, and in adult anti-Jo1-positive patients, Ro52 reactivity is associated with more severe ILD. A small case series reported coexisting anti-Ro52 autoantibodies in 6 of 13 anti-MDA5 autoantibody-pos-itive patients, 5 of whom had rapidly progressive ILD.27 Interestingly, only 1 of 33 patients with ILD in our JIIM cohort had rapidly progressive ILD, and this patient was positive for both anti-MDA5 and anti-Ro52 autoantibodies.
Although we have now established an association between antiaminoacyl tRNA synthetase and anti-Ro52 autoantibodies in adults and children, it remains unclear why these autoantibodies co-occur. It has been proposed that local autoantibody produc-tion induced by type I interferon (IFN)28 could be a driving force behind the production of both anti-Jo1 and anti-Ro52 autoan-tibodies, given the increase in B-cell activating factor receptors in the sera of patients with IIM with these autoantibodies.29 In the current study of juvenile IIM, we now also demonstrate an association between anti-MDA5 and anti-Ro52 autoantibodies. Interestingly, both MDA5 and Ro52 are cytosolic, IFN-induced proteins; perhaps concurrent overexpression of these proteins in patients with juvenile IIM leads to the development of autoim-munity against both. However, we do not have adequate type I IFN measurements to further examine this hypothesis.
This current study has several limitations. First, this cohort of patients with juvenile myositis had some data collected retro-spectively, resulting in some missing data and were collected over more than 20 years, with potential chronology bias. However, we adjusted the variables of this study for the year of diagnosis and tested the distribution of missing values across groups and did not find evidence of a significant bias. Second, although imaging studies were available to confirm the diagnosis of ILD in more than 90% of patients who had ILD, pulmonary function testing data were not available for many of the patients, as a number of the children were of young age when such testing is unreliable in children. Thus, we were not able to study whether patients with ILD and anti-Ro52 autoantibodies had more severe pulmonary dysfunction than those without these autoantibodies. Also, we cannot confirm the absence of ILD as many of the chil-dren without clinical suspicion of ILD did not have imaging and/or pulmonary function testing. This, however, is a limitation of standard clinical care in paediatric patients who have challenges to undergo such testing.
Overall, this study shows that anti-Ro52 autoantibodies are present in 14% of patients with juvenile myositis and are strongly associated with ILD, more severe illness and poorer outcomes, even when correcting for the coexistence of MSAs. In patients with juvenile myositis, anti-Ro52 autoantibodies are associated with the presence of antisynthetase autoantibodies, as previously reported in patients with adult myositis, and with anti-MDA5 autoantibodies, and the coexistence of these MSAs increases the likelihood of ILD and poor outcome. The current standard of care in patients with juvenile myositis who have reactivity to MSAs associated with pulmonary manifestations (such as anti-MDA5 and antiaminoacyl tRNA synthetase autoan-tibodies) is to have a high index of suspicion for the development
of ILD and modify management accordingly. Our data suggest that testing for anti-Ro52 autoantibodies may also have a role in disease monitoring, management and patient prognosis in patients with juvenile myositis.
Author affiliations1Muscle Disease Unit, laboratory of Muscle stem Cells and Gene Regulation, national institute of arthritis and Musculoskeletal and skin Diseases,national institutes of Health (niH), Bethesda, MD, United states2Department of neurology, Johns Hopkins University school of Medicine, Baltimore, MD, United states3Faculty of Health sciences, Universitat Oberta de Catalunya, Barcelona, spain4environmental autoimmunity Group, national institute of environmentalHealth sciences, national institutes of Health (niH), Bethesda, MD, United states5Va Medical Center, Oklahoma Medical Research Foundation, Oklahoma City, OK, United states6Department of Medicine, Johns Hopkins University school of Medicine, Baltimore, MD, Usa
Collaborators *Members of the Childhood Myositis HeterogeneityCollaborative study Group who contributed to this project:Bita arabshahi, lilliana Barillas-arias, Mara Becker, aprilBingham, Ruy Carrasco, Victoria Cartwright, Rodolfo Curiel, Marietta M.DeGuzman, Barbara anne eberhard, Barbara s. edelheit, Terri Finkel, stephen W.George, ellen a. Goldmuntz, William Hannan, Michael Henrickson, adam M. Huber,anna Jansen, James Jarvis, lawrence Jung, ildy M. Katona, steven J. Klein, WPatrick Knibbe, Bianca a. lang, Carol B. lindsley, Gulnara Mamyrova, lindaMyers, stephen R. Mitchell, Kabita nanda, Terrance P. O’Hanlon, Murray H.Passo, Maria D. Perez, Donald a. Person, linda i. Ray, Rafael F. Rivas-Chacon,Tova Ronis, Deborah Rothman, adam schiffenbauer, Bracha shaham, David sherry,abigail smukler, Matthew l. stoll, sangeeta H. sule, scott a. Vogelgesang, Rita Volochayev, Jennifer C. Wargula, Pamela Weiss.
Contributors ss, iPF, lGR and alM conceived the work. lGR and inT acquired, analysed and interpreted the data. ss, iPF and TK analysed and interpreted the data. FWM interpreted data. ss, iPF and alM drafted the work and revised it critically for important intellectual content. TK, iPF, FWM and lGR revised the work for critically important intellectual content. all authors approved the final version of the manuscript. all members of the Childhood Myositis Heterogeneity Collaborative study Group contributed by (1) providing substantial contributions to the acquisition of data, (2) revising the work critically for important intellectual content and (3) providing final approval of the version published.
Funding This research was supported by the intramural Research Programs of the national institute of arthritis and Musculoskeletal and skin Diseases (Zia aR041203) and the national institute of environmental Health sciences (Z01 es101074 and Z01 es101081) of the national institutes of Health.
Competing interests none declared.
Patient and public involvement statement Patients were involved in the research from the time they provided consent to join this natural history study. The research questions were not explicitly developed nor informed by their priorities, experience and preferences. The patients/public were not involved in the design of this study. Patients were not involved in the recruitment to and conduct of the study. Patients were not asked to assess the burden of the intervention and time required to participate in the research. Patients have not and will not be involved in choosing the methods and agreeing plans for dissemination of the study results to participants and wider relevant communities.
Patient consent for publication not required.
ethics approval all subjects were enrolled in natural history study approved by the national institutes of Health institutional institutional Review Board, and all patients provided informed consent.
Provenance and peer review not commissioned; externally peer reviewed.
data sharing statement all data relevant to the study are included in the article or uploaded as supplementary information.
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9 Targoff in, Mamyrova G, Trieu eP, et al. a novel autoantibody to a 155-kd protein is associated with dermatomyositis. Arthritis Rheum 2006;54:3682–9.
10 Kobayashi i, Okura Y, Yamada M, et al. anti-melanoma differentiation-associated gene 5 antibody is a diagnostic and predictive marker for interstitial lung diseases associated with juvenile dermatomyositis. J Pediatr 2011;158:675–7.
11 Pinal-Fernandez i, Casal-Dominguez M, Huapaya Ja, et al. a longitudinal cohort study of the anti-synthetase syndrome: increased severity of interstitial lung disease in black patients and patients with anti-Pl7 and anti-Pl12 autoantibodies. Rheumatology 2017;56:999–1007.
12 la Corte R, lo Mo naco a, locaputo a, et al. in patients with antisynthetase syndrome the occurrence of anti-Ro/ssa antibodies causes a more severe interstitial lung disease. Autoimmunity 2006;39:249–53.
13 Váncsa a, Csípo i, németh J, et al. Characteristics of interstitial lung disease in ss-a positive/Jo-1 positive inflammatory myopathy patients. Rheumatol Int 2009;29:989–94.
14 Bauhammer J, Blank n, Max R, et al. Rituximab in the treatment of Jo1 antibody-associated antisynthetase syndrome: anti-Ro52 positivity as a marker for severity and treatment response. J Rheumatol 2016;43:1566–74.
15 Marie i, Hatron PY, Dominique s, et al. short-term and long-term outcome of anti-Jo1-positive patients with anti-Ro52 antibody. Semin Arthritis Rheum 2012;41:890–9.
16 srivastava P, Dwivedi s, Misra R. Myositis-specific and myositis-associated autoantibodies in indian patients with inflammatory myositis. Rheumatol Int 2016;36:935–43.
17 Bohan a, Peter JB. Polymyositis and dermatomyositis (second of two parts). N Engl J Med 1975;292:403–7.
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19 Habers Gea, Huber aM, Mamyrova G, et al. Brief report: association of myositis autoantibodies, clinical features, and environmental exposures at illness onset with disease course in juvenile myositis. Arthritis Rheumatol 2016;68:761–8.
20 Oddis CV, Rider lG, Reed aM, et al. international consensus guidelines for trials of therapies in the idiopathic inflammatory myopathies. Arthritis Rheum 2005;52:2607–15.
21 Kishi T, Bayat n, Ward MM, et al. Medications received by patients with juvenile dermatomyositis. Semin Arthritis Rheum 2018;48:513–22.
22 McCann lJ, Juggins aD, Maillard sM, et al. The Juvenile Dermatomyositis national Registry and Repository (UK and ireland)--clinical characteristics of children recruited within the first 5 yr. Rheumatology 2006;45:1255–60.
23 Pachman lM, abbott K, sinacore JM, et al. Duration of illness is an important variable for untreated children with juvenile dermatomyositis. J Pediatr 2006;148:247–53.
24 Pachman lM, Hayford JR, Chung a, et al. Juvenile dermatomyositis at diagnosis: clinical characteristics of 79 children. J Rheumatol 1998;25:1198–204.
25 Constantin T, Ponyi a, Orbán i, et al. national registry of patients with juvenile idiopathic inflammatory myopathies in Hungary--clinical characteristics and disease course of 44 patients with juvenile dermatomyositis. Autoimmunity 2006;39:223–32.
26 Kishi T, Rider lG, Pak K, et al. association of anti-3-Hydroxy-3-Methylglutaryl-Coenzyme a reductase autoantibodies with DRB1*07:01 and severe myositis in juvenile myositis patients. Arthritis Care Res 2017;69:1088–94.
27 Huang K, shojania K, Yeung J, et al. FRi0448 MDa5 antibody positive clinical amyopathic dermatomyositis (CaDM): a single tertiary centre case series of 13 patients. Ann Rheum Dis 2018;77 suppl 2:753–53.
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29 Kryštůfková O, Barbasso Helmers s, Venalis P, et al. expression of BaFF receptors in muscle tissue of myositis patients with anti-Jo-1 or anti-Ro52/anti-Ro60 autoantibodies. Arthritis Res Ther 2014;16.
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TranslaTional science
Focused HLA analysis in Caucasians with myositis identifies significant associations with autoantibody subgroupssimon rothwell, 1 Hector chinoy, 2,3 Janine a lamb,4 Frederick W Miller,5 lisa G rider,5 lucy r Wedderburn,6,7 neil J McHugh, 8 andrew l Mammen, 9,10 Zoe e Betteridge,8 sarah l Tansley,8,11 John Bowes, 12 Jiří Vencovský,13 claire T Deakin,6,7 Katalin Dankó,14 limaye Vidya,15 albert selva-o’callaghan,16 lauren M Pachman,17 ann M reed,18 Øyvind Molberg,19 olivier Benveniste,20 Pernille r Mathiesen,21 Timothy r D J radstake,22 andrea Doria, 23 Jan de Bleecker,24 annette T lee,25 Michael G Hanna,26 Pedro M Machado,27,28 William e ollier,4,29 Peter K Gregersen,30 leonid Padyukov,31 Terrance P o’Hanlon,5 robert G cooper,32 ingrid e lundberg,31 on behalf of Myositis Genetics consortium (MYoGen)
To cite: rothwell s, chinoy H, lamb Ja, et al. Ann Rheum Dis 2019;78:996–1002.
Handling editor Josef s smolen
► additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2019- 215046)
For numbered affiliations see end of article.
Correspondence tosimon rothwell, centre for Genetics and Genomics, arthritis research UK, University of Manchester, Manchester M13 9Pl, UK; s. rothwell@ manchester. ac. uk
received 9 January 2019revised 14 March 2019accepted 30 March 2019Published online First 28 May 2019
© author(s) (or their employer(s)) 2019. re-use permitted under cc BY-nc. no commercial re-use. see rights and permissions. Published by BMJ.
AbsTrACTObjectives idiopathic inflammatory myopathies (iiM) are a spectrum of rare autoimmune diseases characterised clinically by muscle weakness and heterogeneous systemic organ involvement. The strongest genetic risk is within the major histocompatibility complex (MHc). since autoantibody presence defines specific clinical subgroups of iiM, we aimed to correlate serotype and genotype, to identify novel risk variants in the MHc region that co-occur with iiM autoantibodies.Methods We collected available autoantibody data in our cohort of 2582 caucasian patients with iiM. High resolution human leucocyte antigen (Hla) alleles and corresponding amino acid sequences were imputed using snP2Hla from existing genotyping data and tested for association with 12 autoantibody subgroups.results We report associations with eight autoantibodies reaching our study-wide significance level of p<2.9×10–5. associations with the 8.1 ancestral haplotype were found with anti-Jo-1 (Hla-B*08:01, p=2.28×10–53 and Hla-DrB1*03:01, p=3.25×10–9), anti-PM/scl (Hla-DQB1*02:01, p=1.47×10–26) and anti-cn1a autoantibodies (Hla-DrB1*03:01, p=1.40×10–11). associations independent of this haplotype were found with anti-Mi-2 (Hla-DrB1*07:01, p=4.92×10–13) and anti-HMGcr autoantibodies (Hla-DrB1*11, p=5.09×10–6). amino acid positions may be more strongly associated than classical Hla associations; for example with anti-Jo-1 autoantibodies and position 74 of Hla-DrB1 (p=3.47×10–64) and position 9 of Hla-B (p=7.03×10–11). We report novel genetic associations with Hla-DQB1 anti-TiF1 autoantibodies and identify haplotypes that may differ between adult-onset and juvenile-onset patients with these autoantibodies.Conclusions These findings provide new insights regarding the functional consequences of genetic polymorphisms within the MHc. as autoantibodies in iiM correlate with specific clinical features of disease, understanding genetic risk underlying development of autoantibody profiles has implications for future research.
InTrOduCTIOnThe idiopathic inflammatory myopathies (IIM) are a spectrum of rare autoimmune diseases charac-terised clinically by muscle weakness and systemic organ involvement. Clinically, IIM are heteroge-neous and may be subclassified as dermatomyositis (DM), inclusion body myositis (IBM), immune-me-diated necrotising myopathy, polymyositis (PM) and antisynthetase syndrome.1 2
Key messages
What is already known about this subject? ► The strongest genetic risk among patients with idiopathic inflammatory myopathies (IIM) is thought to be within the human leucocyte antigen (HLA) region in autoantibody defined subgroups.
What does this study add? ► This study shows for the first time genetic differences between adult-onset and juvenile-onset patients with anti-TIF1 autoantibodies.
► Amino acid imputation identifies novel associations with autoantibodies that are stronger than classical HLA associations, indicating key positions within HLA molecules that may confer risk.
How might this impact on clinical practice or future developments?
► Differing associations in adult and juvenile onset disease with the same autoantibody suggests distinct aetiologies and disease mechanisms.
► As autoantibodies in myositis correlate with specific clinical features of disease, understanding genetic risk underlying development of certain autoantibody profiles will have implications for future research in IIM.
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Myositis autoantibodies can be detected in approximately 60%–70% of children and adults with IIM.3 4 Autoantibodies unique to IIM are known as myositis-specific autoantibodies (MSA), with patients rarely possessing more than one MSA.5 Autoantibodies that present in patients with myositis in associa-tion with another connective tissue disease are known as myosi-tis-associated autoantibodies (MAA).
Clinical classification of IIM can lead to heterogeneous subgroups, however, some MSA positive subgroups have specific clinical features and may respond differently to treatment. Therefore, autoantibody status may be a more meaningful way to characterise patients to understand pathogenesis and predict prognosis.3 For example, patients with anti-histidyl–tRNA-syn-thetase (anti-Jo-1) commonly present with myositis, Raynaud’s phenomenon, polyarthritis and a high frequency of interstitial lung disease.6 Patients with autoantibodies directed against anti-Mi-2 present with hallmark cutaneous manifestations of DM, generally milder myositis and a favourable response to immuno-suppressive treatment.7
IIM are thought to be complex genetic diseases, initiated by immune activation following specific environmental events in genetically predisposed individuals. The major histocompat-ibility complex (MHC), also known as the human leucocyte antigen (HLA) region, has consistently been identified as the strongest risk factor for IIM and clinical subgroups.8 Studies have shown that the strongest HLA associations among patients with IIM are found when stratifying by autoanti-body status.9–11 In particular, the strongest risk is with the 8.1 ancestral haplotype (8.1 AH), a common haplotype of exten-sive linkage disequilibrium (LD) in Caucasian populations that confers susceptibility to IIM and many other autoimmune or immune-mediated diseases.12 Associations with alleles inde-pendent of the 8.1 AH have also been reported.9 New MSAs and MAAs have since been discovered, and small studies have identified potential HLA associations with these autoantibodies, for example, HLA-DRB1*11:01 in patients with anti-3-hy-droxy-3-methylglutaryl-coenzyme A reductase (anti-HMGCR) autoantibodies in adult-onset disease.13
Here, we aimed to correlate serotype with genotype in patients with IIM, with sufficient statistical power, to identify novel risk variants in the MHC region that confer susceptibility to IIM autoantibodies and to provide a definitive replication cohort for previously reported associations.
MeTHOdsstudy populationThrough the Myositis Genetics Consortium (MYOGEN), 2582 Caucasian cases from 14 countries were recruited (online supple-mentary table 1).8 Written informed consent was obtained from all patients with approval from research ethics committees of institutional review boards at each participating centre. Patients were enrolled in to MYOGEN if they fulfilled Bohan and Peter criteria for PM and adult and juvenile DM,8 or Griggs, Medical Research Council (MRC) or European Neuromuscular Centre (ENMC) criteria for patients with IBM.14 Shared control geno-types were drawn from a pool of 15 651 individuals from 12 countries as described previously.8 Genotypes from an additional 19 UK juvenile-onset patients were included from the Juvenile Dermatomyositis Cohort Biomarker Study and Repository. Patients were classified as having juvenile IIM if age of onset was <16 years old, except in the USA, where 18 years old was considered the cut-off.
Autoantibody testingMyositis relevant autoantibodies were detected using immu-noprecipitation (IP), line blot or ELISA, as detailed in online supplementary table 2. Further details are described in online supplementary method.
GenotypingGenotyping was performed in accordance with UK-based and US-based Illumina protocols (Centre for Genetics and Genomics Arthritis Research UK, University of Manchester; Feinstein Institute, New York; University College London, London, UK). Standard quality control (QC) was performed as described previously.8
HLA imputationClassical HLA alleles and corresponding amino acid sequences were imputed using SNP2HLA with reference data collected by the Type 1 Diabetes Genetics Consortium.15 Cases and controls were imputed together. Postimputation QC removed variants with a minor allele frequency <0.01 and variants with an infor-mation score <0.8.
statistical analysisWe employed a case-control study design in each autoanti-body subgroup analysis. Controls were matched for ethnicity using principal components analysis (PCA), due to absence of geographically matched controls for each IIM cohort. Four healthy controls for each case were matched for ethnicity using (PCA) coordinates, using a method described previously.16 Analysis was performed in R (V.3.1.0) on the dosage of vari-ants (genotype probabilities) which takes into account imputa-tion uncertainty. Single-test association was performed for HLA alleles and omnibus tests for multiallelic sites. For multiallelic sites, such as amino acids, we identified the most common residue or allele in the control population to be selected as the reference. A logistic regression assuming an additive model was used to test for association, with gender and the top 10 principal components included as covariates. Forward stepwise logistic regression was used to test for independent effects conditional on the variant of interest. ORs ratios were generated with multivar-iate logistic regression. Significance was defined as p<2.9×10–5 based on a Bonferroni correction of the pre-QC 1700 variants imputed by SNP2HLA.
resuLTsAutoantibody frequencies in the study populationGenetic data were available for 2582 patients with IIM, the majority of whom were tested for at least one autoantibody. The number of autoantibody positive cases present in the cohort is shown in table 1, for groups where n>10. Autoantibody frequencies are as a proportion of those in tested individuals and do not necessarily represent the prevalence of autoantibodies in an IIM population.
strong classical HLA associations are observed in IIM autoantibody subgroupsData quality was evaluated after imputation by determining the concordance between imputed alleles and existing HLA typing from next generation sequencing technology (HistoGenetics, New York, USA) for 162 individuals. There was a high level of concordance across all loci for both 2-digit (96.6%–100%) and 4-digit (94.8%–100%) alleles (online supplementary table
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Table 1 Number of patients with myositis with myositis-specific or associated autoantibodies
Antibody
Adult-onset IIM Juvenile-onset IIM Total number positive(Adult+juvenile-onset)number tested number antibody positive number tested number antibody positive
Jo-1 1937 325 (16.8%) 447 7 (1.6%) 332
TIF1 1697 91 (5.4%) 356 106 (29.8%) 197
PM/Scl 1883 120 (6.4%) 457 16 (3.5%) 136
Mi-2 1884 87 (4.6%) 456 17 (3.7%) 104
NXP2 1502 28 (1.9%) 360 65 (18.1%) 93
cN1A 232 46 (19.8%) 81 18 (22.2%) 64
SRP 1822 45 (2.5%) 457 0 45
HMGCR 1264 51 (4.0%) 130 1 (0.8%) 52
MDA5 1674 18 (1.1%) 402 17 (4.2%) 35
SAE 1518 31 (2.0%) 397 0 31
PL7 1927 23 (1.2%) 457 0 23
PL12 1927 11 (0.6%) 457 0 11
Myositis-specific/associated autoantibodies n>10 in cohort, stratified by adult-onset and juvenile-onset disease. Frequency is of those patients tested for each autoantibody within the cohort, where testing was not necessarily random.IIM, idiopathic inflammatory myopathies.
Table 2 Classical HLA allele associations
Autoantibody number of cases
snP2HLA classical HLA association
Gene Allele P value Or (95% CI)
Jo-1 332 HLA-B 08:01 2.28×10–53 3.37 (2.37 to 4.83)
HLA-DRB1 03:01 3.25×10–9 3.09 (2.08 to 4.30)
TIF1 197 HLA-DQB1 02 2.34×10–10 2.49 (1.88 to 3.31)
PM/Scl 136 HLA-DQB1 02:01 1.47×10–26 17.50 (10.63 to 30.59)
Mi-2 104 HLA-DRB1 07:01 4.92×10–13 5.47 (3.48 to 8.77)
cN1A 64 HLA-DRB1 03:01 1.40×10–11 9.23 (4.95 to 18.01)
HMGCR 52 HLA-DRB1 11 5.09×10–6 4.92 (2.52 to 9.97)
4-digit alleles are preferentially reported unless a 2-digit allele is significantly more associated. P values (uncorrected), OR and 95% CI were calculated in a regression including the most significantly associated independent variants. Associations reaching study-wide significance are reported (p<2.9×10–5).HLA, human leucocyte antigen.
3). After stringent QC removing variants with low frequency (<0.01) and poor imputation accuracy (r2<0.8), our anal-ysis included 509 classical HLA alleles and amino acids. In the primary analysis, adult-onset and juvenile-onset cases were combined and analysed together against matched healthy controls. Autoantibody associations reaching study-wide signifi-cance of p<2.9×10–5 are reported in table 2.
The strongest HLA association was found with anti-Jo-1 auto-antibodies and HLA-B*08:01 (p=2.28×10–53, OR=3.37, 95% CI 2.37 to 4.83). Conditioning on HLA-B*08:01 in a stepwise logistic regression model revealed an independent association with HLA-DRB1*03:01 (p=3.25×10–9, OR=3.09, 95% CI 2.08 to 4.30). We analysed antisynthetase autoantibody posi-tive patients as a group (n=381), and the strength of association became stronger (HLA-B*08:01, p=1.79×10–56, OR=3.14, 95% CI 2.27 to 4.38 and HLA-DRB1*03:01, p=3.31×10–9, OR=2.73, 95% CI 1.96 to 3.80). We did not find associations with rarer aminoacyl-tRNA-synthetases individually, likely due to small sample sizes. However, in a small sample size of 23 patients, only 30.4% of patients with anti-PL7 antibodies carried HLA-DRB1*03:01, compared with 79.2% of patients with anti-Jo-1 autoantibodies, and 23% of healthy controls (online supple-mentary table 4). This may suggest that not all antisynthetase autoantibodies have the same associations with 8.1 AH. The ‘haplotype phased’ raw HLA genotypes for patients with rarer
antisynthetase autoantibodies are included in online supplemen-tary tables 5-10.
A strong HLA association was observed in patients with anti-TIF1 autoantibodies with the HLA-allele group DQB1*02 (p=2.34×10–10, OR=2.49, 95% CI 1.88 to 3.31). Within this allele group, there were associations with HLA-DQB1*02:01 (p=1.69×10–5) and HLA-DQB1*02:02 (p=1.04×10–4).
Associations with class II alleles of the 8.1 AH were observed for patients with anti-Jo-1, anti-PM/Scl and anti-cN1A. Anti-HMGCR and anti-Mi-2 were the only autoantibodies tested that were associated with HLA alleles not forming part of the 8.1 AH. Although the strong association with anti-HMGCR auto-antibodies and HLA-DRB1*11 was confirmed in adult-onset patients, a stronger association was observed with the 4-digit allele HLA-DRB1*11:01 (p=3.22×10–7, OR=11.90, 95% CI 4.72 to 31.85) before the allele was removed from the initial analysis due to low imputation accuracy (r2=0.75).
Using a more conservative threshold correcting for the number of antibodies analysed (p<2.4×10–6), the associa-tion with HLA-DRB*11 and anti-HMGCR autoantibodies did not reach our significance threshold. There were no signifi-cant associations with classical HLA alleles and anti-NXP2, anti-SRP, anti-MDA5 or anti-SAE autoantibodies.
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Table 3 The strongest amino acid associations in autoantibody subgroups of IIM
Autoantibody number of cases
Amino acid association
Gene Variant P value Or (95% CI)
Jo-1 332 HLA-DRB1 Position 74 Omnibus 3.47×10–64
Alanine (ref)
Arginine 1.24×10--8 2.94 (2.03 to 4.26)
Glutamine 0.02 0.59 (0.38 to 0.89)
Leucine 6.21×10–5 2.78 (1.67 to 4.55)
Glutamic acid 0.5 1.20 (0.70 to 1.96)
HLA-B Position 9 Omnibus 7.03×10–11
Tyrosine (ref)
Aspartic acid 2.10×10–10 3.33 (2.30 to 4.84)
Histidine 0.77 0.96 (0.73 to 1.26)
TIF1 197 HLA-DQB1 Position 37 Omnibus 1.49×10–10
Tyrosine (ref)
Isoleucine 2.34×10–10 2.49 (1.88 to 3.31)
PM/Scl 136 HLA-DRB1 Position 74 Omnibus 2.48×10–40
Alanine (ref)
Arginine 5.06×10–25 18.37 (10.88 to 32.98)
Glutamine 0.43 1.26 (0.70 to 2.21)
LeucineGlutamic acid
0.580.58
0.70 (0.17 to 2.32)1.34 (0.44 to 3.56)
Mi-2 104 HLA-DRB1 Position 4 Omnibus 3.69×10–13
Arginine (ref)
Glutamine 3.26×10–12 4.93 (3.17 to 7.79)
cN1A 64 HLA-DRB1 Position 74 Omnibus 6.15×10–14
Alanine (ref)
Arginine 1.37×10–9 8.68 (4.43 to 18.1)
Glutamine 0.02 0.17 (0.03 to 0.61)
Leucine 0.05 3.31 (0.93 to 11.02)
Glutamic acid 0.43 0.45 (0.03 to 2.30)
SRP 45 HLA-DRB1 Position 74 Omnibus 1.91×10–5
Alanine (ref)
Arginine 9.47×10–4 3.51 (1.69 to 7.57)
Glutamine 0.13 0.38 (0.08 to 1.18)
Leucine 0.63 1.36 (0.32 to 4.34)
Glutamic Acid 5.69×10–4 5.07 (2.05 to 13.17)
HMGCR 42 HLA-DRB1 Position 58 Omnibus 2.42×10–6
Alanine (ref)
Glutamic acid 5.13×10–6 4.91 (2.52 to 9.97)
SAE 31 HLA-DQB1 Position 57 Omnibus 2.66×10–6
Aspartic acid (ref)
Alanine 1.71×10–5 8.52 (3.42 to 24.47)
Valine 0.41 1.61 (0.49 to 4.92)
Serine 0.94 0.92 (0.04 to 7.66)
OR and 95% CI were calculated in a regression model including significant independent variants, using the most common amino acid in the population as the reference. Associations reaching study-wide significance are reported (p<2.9×10–5).IIM, idiopathic inflammatory myopathies.
Amino acid positions may be more strongly associated than classical HLA allelesSNP2HLA was used to impute amino acid positions. We employed a case-control study design in each autoantibody subgroup analysis, and autoantibodies with significant amino acid associations are reported in table 3.
The omnibus p value reports the significance of the amino acid position, and the effect sizes of the potential amino acids at this location were calculated in a regression model. The loca-tion of associated amino acids within the 3D structure of HLA molecules are shown in online supplementary figure 1. For some
autoantibodies, such as anti-Jo-1, anti-PM/Scl and anti-cN1A, amino acid associations were markedly stronger than the clas-sical HLA associations reported in table 2. Regional association plots for autoantibodies with significant associations are shown in online supplementary figure 2A-H. Using a more conserva-tive threshold correcting for the number of antibodies analysed (p<2.4×10–6), associations with amino acids and anti-SRP, anti-HMGCR and anti-SAE autoantibodies did not reach our signif-icance threshold.
For anti-Jo-1, anti-PM/Scl, anti-cN1A and anti-SRP auto-antibodies, the strongest association was with position 74 of
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Table 4 Differences in association at the HLA-DQB1*02 locus in adult-onset and juvenile-onset patients with anti-TIF1 autoantibodies
dQb1*02:01 dQb1*02:02
Adult-onset TIF1, n=91 P=0.06OR=1.54 (0.97 to 2.44)
P=2.96×10–5
OR=3.31 (1.89–5.84)
Juvenile-onset TIF1, n=106 P=3.70×10–5
OR=2.47 (1.61 to 3.80)P=0.13OR=1.50 (0.87 to 2.51)
OR with 95% CI..
HLA-DRB1 (table 3). For anti-Jo-1, anti-PM/Scl and anti-cN1A autoantibodies, an arginine at position 74 of HLA-DRB1 conferred the strongest risk. In patients with anti-PM/Scl and anti-cN1A autoantibodies, an arginine conferred all of the risk at this position, whereas in patients with anti-Jo-1 autoantibodies there was also evidence of risk attributable to other amino acids.
Stepwise conditional analysis showed evidence of multiple independent effects in the HLA region with anti-Jo-1; posi-tion 74 of HLA-DRB1 and position 9 of HLA-B. These were associated at a similar level of significance (p=3.47×10–64 vs p=5.25×10–64) and remained significant after conditioning on either position. Analysing antisynthetase autoantibody positive patients as a group increased the strength of association with these independent amino acid positions (HLA-DRB1 Position 74, p=5.69×10–67 and HLA-B Position 9, p=8.63×10–11).
HLA alleles may differentiate adult-onset and juvenile-onset patients with anti-TIF1 autoantibodiesFor autoantibodies that occur at high frequencies in both adult-onset and juvenile-onset patients with IIM, the cohort was strat-ified to investigate potential differences in HLA associations between these populations. Patients with anti-TIF1 autoanti-bodies were stratified into adult-onset (n=91) and juvenile-onset (n=106) disease. Of these, all but six adult-onset patients were recorded as having DM. The association at the HLA-DQB1 locus differed between adult-onset and juvenile-onset patients (table 4). In adult-onset IIM, the strongest 4-digit HLA asso-ciation was with HLA-DQB1*02:02 (p=2.96×10–5, OR=3.31, 95% CI 1.89 to 5.84). In contrast, in juvenile-onset patients, a strong association with HLA-DQB1*02:01 was observed, but the strongest association was with HLA-DRB1*03:01 (p=6.21×10–
6, OR=2.69, 95% CI 1.75 to 4.15), which is on the same haplo-type as HLA-DQB1*02:01.
To ensure that this difference was not due to the effects of different methods of autoantibody detection, analysis was restricted to UK patients serotyped using IP in the same centre at the University of Bath. Nineteen additional juvenile-onset patients were included, recently genotyped through the Juvenile Dermatomyositis Cohort Biomarker Study and Repository. This subanalysis included UK adult-onset (n=40) and juvenile-onset patients (n=48) with anti-TIF1 autoantibodies. The strongest associations for juvenile-onset patients were with alleles of the 8.1 AH, which were not significant in the adult-onset cohort. For example, HLA-DQB1*02:01 had a stronger association in the juvenile-onset cohort (p=0.004, OR=2.48, 95% CI 1.33 to 4.61) in comparison to the adult-onset cohort (p=0.11, OR=1.69, 95% CI 0.87 to 3.20). Conversely, HLA-DQB1*02:02 was more strongly associated in adult-onset (p=0.01, OR=2.38, 95% CI 1.16 to 4.76) than in juvenile-onset disease (p=0.09, OR=1.85, 95% CI 0.89 to 3.64), thus replicating trends from the original cohort. However, due to the reduced sample size, these associations did not reach study-wide significance. These
trends were also seen when restricting the analysis to US juve-nile-onset patients with anti-TIF1 autoantibodies (n=70); the association with HLA-DQB1*02:01 (p=0.01, OR=2.05, 95% CI 1.18 to 3.54) was stronger than with HLA-DQB1*02:02 (p=0.76, OR=0.89, 95% CI 0.40 to 1.81).
There were no significant classical HLA or amino acid asso-ciations in either adult-onset or juvenile-onset patients with anti-NXP2 or anti-MDA5 autoantibodies. For anti-Jo-1, anti-PM/Scl, anti-Mi-2 and anti-cN1A, the numbers in the juvenile-onset cohorts were too small to reach our study-wide significance. However, for alleles associated in the total cohort, the trends for association were in the same direction in juve-nile-onset IIM (online supplementary table 11).
dIsCussIOnWe used HLA imputation to find associations with IIM autoanti-bodies and both classical HLA alleles and amino acid positions that may confer risk. By applying this novel approach, we report for the first genetic differences between adult-onset and juvenile-onset anti-TIF1-positive patients. There are independent associations of HLA-DRB1 and HLA-B for the development of anti-Jo-1 autoanti-bodies, implicating multiple genetic features of the 8.1 AH. Amino acid imputation identified novel associations with autoantibodies that are stronger than the classical HLA allelic association, indi-cating key positions within HLA molecules that may confer risk for various antibodies.
For certain autoantibodies, the strongest amino acid associations were at a similar level of significance as the strongest specific HLA association (online supplementary figure 2A-H). These amino acids may act as a proxy for the risk allele due to carriage on risk haplotypes. In other instances, amino acid associations were markedly more significant than any HLA allele. For example, in patients with anti-PM/Scl autoantibodies, amino acid position 74 of HLA-DRB1 had a stronger association (p=2.48×10–40) than any allelic association (p=1.47×10–26). For patients with anti-Jo-1 autoantibodies, forward stepwise conditional analysis suggested independent associations within the 8.1 AH of HLA-B*08:01 and HLA-DRB1*03:01. Other MSAs may have multiple independent associations within the HLA region that we were underpowered to detect, in line with other seropositive autoimmune diseases.17
Imputation of classical HLA alleles confirmed strong associ-ations between alleles of the 8.1 AH and anti-Jo-1, anti-PM/Scl and anti-cN1A autoantibodies. Specific HLA alleles are thought to target particular autoantigens, resulting in a breakdown in immu-nological tolerance to self-antigens. However, studies have shown that there are additional genetic features of the 8.1 AH that predis-pose individuals to immune-mediated diseases, such as NF-kappaB and TNF-alpha polymorphisms,18 19 and gene copy number vari-ants of complement genes.20 The association of alleles independent of the 8.1 AH with anti-Mi-2 and anti-HMGCR, as well as finding that some autoantibodies do not have strong HLA associations, suggests that not all patients with IIM share a common genetic risk. The 8.1 AH may be more critical for generation of a particular autoantibody repertoire rather than for the development of IIM. Environmental triggers including viruses, infections, UV radiation, drugs and additional genetic risk factors may contribute to disease heterogeneity.
The position of amino acids may give insight into whether these associations are functionally important. For example, amino acid position 74 faces inwards of the peptide binding groove in HLA DR molecules and may change the structure to accommodate autoantigenic peptides, whereas amino acid position 4 lies outside of the peptide binding groove (online supplementary figure 1).
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An arginine at position 74 of HLA-DRB1 was the most strongly associated amino acid position in patients with anti-Jo-1, anti-PM/Scl and anti-cN1A autoantibodies and has been implicated in other autoimmune diseases in Caucasian individuals.21 Arg-74 almost exclusively resides on HLA-DRB1*03 alleles, which may explain the strong association with many IIM autoantibodies and HLA-DRB1*03:01. It will be interesting to examine whether risk alleles in other ethnic populations share the same amino acids as in Caucasian populations.22 Our method of analysis was unable to distinguish whether risk was attributable to amino acid motifs or to individual amino acid positions due to the high level of LD within these molecules. Previous studies have identified HLA-DRB1 motif 9EYSTS13 as risk for IIM,23 and the HLA-DRB1 motif 70QKXXR74 for patients with anti-Jo-1 and anti-PM/Scl autoantibodies.9 In both of these cases, however, the motifs are no stronger than the classical association with HLA-DRB1*03:01, or indeed an arginine at position 74 of HLA-DRB1.
We found evidence of different haplotypic associations for adult-onset and juvenile-onset patients with anti-TIF1 autoanti-bodies. After stratifying by age, there was an association between anti-TIF1 autoantibodies and the 8.1 AH in juvenile-onset patients that was absent in adult-onset patients. Differences in association could be due to distinct aetiologies or to epitope spreading. An association of anti-TIF1 autoantibodies with cancer in adult-onset disease is not present in juvenile-onset disease.24 25 Cancer-associ-ated myositis may develop as a paraneoplastic process, with neoan-tigens in the cancer triggering autoimmunity in adult disease.26 In juvenile-onset patients with anti-TIF1 autoantibodies, however, an association with the 8.1 AH may suggest a different aetiology, where environmental risk factors such as infections,27 28 UV expo-sure29 or others,30 may initiate disease. Differences in association with anti-HMGCR autoantibodies in adult-onset patients (HLA-DRB1*11:01) and juvenile onset-patients (HLA*DRB1*07:01) have recently been described.13
There are some limitations with our study. Due to the nature of this large multicentre collection, not all patients were tested using gold standard IP. Alternatively, patients were included if they tested positive for an autoantibody by a commercially available line blot. Although concordance between these different methods has not been comprehensively studied, there is evidence that these assays are robust.31 32 A subanalysis of patients with anti-Jo-1 and anti-PM/Scl autoantibodies serotyped using EUROIMMUN line blots resulted in the same associations as those serotyped by IP (data not shown). Another limitation is the potential mischarac-terisation of patients as antibody negative. Patients may have rare autoantibodies not currently included on commercially available line-blots, undiscovered antibodies not detectable in cell lines used for IP or low/negative titres of antibody during disease remission. Mischaracterising patients as antibody negative would result in a type-2 error and should not affect our primary findings. For these reasons, we did not compare antibody positive against antibody negative-patient groups.
In conclusion, these results confirm strong associations between HLA alleles and autoantibody specificities present in Caucasian patients with IIM. As well as strong associations with the 8.1 AH, we show that there are associations independent of this haplotype and that risk within this region may differ between adult-onset and juvenile-onset patients with anti-TIF1 autoantibodies. As autoantibodies in myositis correlate with specific clinical features of disease, understanding the mecha-nisms behind development of different autoantibodies should be a focus of ongoing research.
Author affiliations1centre for Genetics and Genomics, arthritis research UK, University of Manchester, Manchester, UK2national institute for Health research Manchester Biomedical research centre, central Manchester University Hospitals nHs Foundation Trust, Manchester, UK3Manchester academic Health science centre, salford royal nHs Foundation Trust, salford, UK4centre for epidemiology, The University of Manchester, Manchester, UK5environmental autoimmunity Group, national institute of environmental Health sciences, Bethesda, Maryland, Usa6niHr Great ormond street Biomedical research centre, University college london, london, UK7arthritis research UK centre for adolescent rheumatology, University college london, london, UK8Pharmacy and Pharmacology, University of Bath, Bath, UK9Muscle Disease Unit, laboratory of Muscle stem cells and Gene regulation, national institute of Musculoskeletal and skin Diseases, national institutes of Health, Bethesda, Maryland, Usa10Departments of neurology and Medicine, Johns Hopkins University school of Medicine, Baltimore, Maryland, Usa11royal national Hospital for rheumatic Diseases, royal United Hospitals Bath nHs Foundation Trust, Bath, UK12arthritis research UK centre for Genetics and Genomics, The University of Manchester, Manchester, UK13institute of rheumatology and Department of rheumatology, charles University, Prague, czech republic14internal Medicine, University of Debrecen, Debrecen, Hungary15rheumatology Unit, royal adelaide Hospital, University of adelaide, adelaide, south australia, australia16internal Medicine Department, Vall d’Hebron General Hospital, Universitat autonoma de Barcelona, Barcelona, spain17ann & robert H. lurie children’s Hospital of chicago, northwestern University, Feinberg school of Medicine, chicago, illinois, Usa18Pediatrics, Duke University, Durham, north carolina, Usa19Department of rheumatology, University of oslo, oslo, norway20Department of internal Medicine and clinical immunology, Pitié-salpêtrière University Hospital, France, France21Department of Paediatrics and adolescent Medicine, rigshospitalet, copenhagen, Denmark22Department of rheumatology and clinical immunology, Utrecht Medical center, Utrecht, The netherlands23Division of rheumatology, University of Padova, Padova, italy24Department of neurology, Ghent University, Ghent, Belgium25robert s. Boas center for Genomics and Human Genetics, The Feinstein institute for Medical research, Manhasset, new York, Usa26Mrc centre for neuromuscular Diseases, Department of neuromuscular Diseases, University college london institute of neurology, london, UK27Department of rheumatology, University college london Hospital nHs Foundation Trust, london, UK28Department of rheumatology, northwick Park Hospital, london north West University Healthcare nHs Trust, london, UK29school of Healthcare sciences, Manchester Metropolitan University, Manchester, , UK30center for Genomics and Human Genetics, The Feinstein institute for Medical research, Manhasset, new York, Usa31Division of rheumatology, Department of Medicine, Karolinska University Hospital, stockholm, sweden32Mrc/arUK centre for integrated research into Musculoskeletal ageing, University of liverpool, liverpool, , UK
Acknowledgements We thank Mr. Paul new (salford royal Foundation Trust) for ethical and technical support. We thank all of the patients and their families who contributed to this study. Thanks to Douglas Bell (niH/nieHs) and elaine remmers (niH/nHGri) for critical review of the manuscript. The authors would like to acknowledge the assistance given by iT services at The University of Manchester. a full list of acknowledgments is included in the supplementary materials.
Collaborators The following members of the Us childhood Myositis Heterogeneity study Group contributed to this study: Drs Barbara s adams (University of Michigan, ann arbor, Mi), catherine a Bingham (Hershey Medical center, Hershey, Pa), Gail D cawkwell (all children’s Hospital, st. Petersburg, Fl), Terri H Finkel (children’s Hospital of Philadelphia, Philadelphia, Pa), steven W George (ellicott city, MD), Harry l Gewanter (richmond, Va), ellen a. Goldmuntz (children’s national Medical center, Washington, Dc), Donald P Goldsmith (st christopher’s Hospital for children, Philadelphia, Pa), Michael Henrickson (children’s Hospital, Madera, ca), lisa imundo (columbia University, new York, nY), ildy M Katona (Uniformed services University, Bethesda, MD), carol B lindsley (University of Kansas, Kansas city), chester P oddis (University of Pittsburgh, Pittsburgh, Pa), Judyann c olson (Medical college of Wisconsin, Milwaukee), David sherry (children’s Hospital of
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Philadelphia, Philadelphia, Pa), scott a Vogelgesang (Walter reed army Medical center, Washington, Dc), carol a Wallace (children’s Medical center, seattle, Wa), Patience H White (George Washington University, Washington, Dc) and lawrence s Zemel (connecticut children’s Hospital, Hartford).
Contributors Jal, rGc, iel, FWM, lGr, nJMcH and Hc devised the study concept and design and obtained funding. sr, Hc and Jal wrote the manuscript. sr performed the statistical analysis. JB contributed statistical support. Jal, rGc, iel, FWM, nJMcH, Hc, lGr, lP and lrW contributed to the interpretations of findings. other authors contributed samples and/or data and all authors contributed to and approved the manuscript.
Funding This study was supported in part by: association Francaise contre les Myopathies (aFM), The european Union sixth Framework Programme (project autocure; lsH-018661), european science Foundation (esF) in the framework of the research networking Programme european Myositis network (eUMYoneT), The swedish research council and grants provided by the stockholm county council (alF project), the swedish rheumatism association, King Gustaf V 80 year Foundation, the intramural research programs of the national institute of environmental Health sciences (nieHs) and the national institute of arthritis and Musculoskeletal and skin Diseases (niaMs), the national institutes of Health (niH), european community’s FP6, autocure lsHB cT-2006-018661, The UK Myositis support Group, arthritis research UK (18474 and 20380), Medical research council (Mr/n003322/1), The cure JM Foundation, the Wellcome Trust, the Henry smith charity UK, action Medical UK. The czech cohort was supported by Project for conceptual Development of research organization 00023728 from Ministry of Health in the czech republic. antibody testing in the UK JDrG cohort was supported by a BMa Dorris Hillier Grant 2012 and the Bath institute of rheumatic Diseases. Thank you to the research Fund of region Zeeland, Denmark. Pedro M. Machado is supported by the national institute for Health research (niHr) University college london Hospitals (Ucl) Biomedical research centre (Brc). lrW is supported by the national institute for Health research (niHr) Great ormond street Hospital (GosH) Biomedical research centre (Brc). This report includes independent research supported by the niHr Biomedical research centre Funding scheme.
disclaimer The views expressed in this publication are those of the authors and not necessarily those of the nHs, the national institute for Health research or the Department of Health.
Competing interests JV reports grants from Ministry of Health in the czech republic, grants from european community’s FP6, autocure lsHB cT-2006-01866, grants from european science Foundation, during the conduct of the study. iel reports grants from swedish research council, grants from european science Foundation, grants from association Francaise contre les Myopathies (aFM), grants from stockholm county council, grants from The european Union sixth Framework Programme, during the conduct of the study.
Patient consent for publication not required.
ethics approval Written informed consent was obtained from all cases with approval from research ethics boards at each participating centre.
Provenance and peer review not commissioned; externally peer reviewed.
data sharing statement no data are available.
Open access This is an open access article distributed in accordance with the creative commons attribution non commercial (cc BY-nc 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.
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13 Mammen al, Gaudet D, Brisson D, et al. increased Frequency of DrB1 * 11 : 01 in anti – Hydroxymethylglutaryl-coenzyme a reductase – associated autoimmune Myopathy. Arthritis Care Res. 2012;64:1233–7.
14 rothwell s, cooper rG, lundberg ie, et al. immune-array analysis in sporadic inclusion body myositis reveals Hla-DrB1 amino acid heterogeneity across the myositis spectrum. Arthritis Rheumatol 2017;69:1090–9.
15 Jia X, Han B, onengut-Gumuscu s, et al. imputing amino acid polymorphisms in human leukocyte antigens. PLoS One 2013;8:e64683.
16 Gregersen PK, Kosoy r, lee aT, et al. risk for myasthenia gravis maps to a 151 Pro→ala change in TniP1 and to human leukocyte antigen-B*08. Ann Neurol. 2012;72:927–35.
17 Betteridge Ze, chinoy H, cooper rG, et al. 175 myositis-specific autoantibodies rarely coexist with each other: an analysis of the Ukmyonet and eumyonet cohorts. Rheumatology 2016;55.
18 chinoy H, li cK-c, Platt H, et al. Genetic association study of nF-κB genes in UK caucasian adult and juvenile onset idiopathic inflammatory myopathy. Rheumatology 2012;51:794–9.
19 chinoy H, salway F, John s, et al. Tumour necrosis factor-alpha single nucleotide polymorphisms are not independent of Hla class i in UK caucasians with adult onset idiopathic inflammatory myopathies. Rheumatology 2007;46:1411–6.
20 lintner Ke, Patwardhan a, rider lG, et al. Gene copy-number variations (cnVs) of complement C4 and C4A deficiency in genetic risk and pathogenesis of juvenile dermatomyositis. Ann Rheum Dis 2016;75:1599–606.
21 Menconi F, osman r, Monti Mc, et al. shared molecular amino acid signature in the Hla-Dr peptide binding pocket predisposes to both autoimmune diabetes and thyroiditis. Proceedings of the National Academy of Sciences 2010;107:16899–903.
22 okada Y, Kim K, Han B, et al. risk for acPa-positive rheumatoid arthritis is driven by shared Hla amino acid polymorphisms in asian and european populations. Hum Mol Genet 2014;23:6916–26.
23 rider lG, shamim e, okada s, et al. Genetic risk and protective factors for idiopathic inflammatory myopathy in Koreans and american whites: a tale of two loci. Arthritis Rheum 1999;42:1285–90.
24 Trallero-araguás e, rodrigo-Pendás Jose Ángel, selva-o’callaghan a, et al. Usefulness of anti-p155 autoantibody for diagnosing cancer-associated dermatomyositis: a systematic review and meta-analysis. Arthritis Rheum 2012;64:523–32.
25 Fiorentino DF, chung ls, christopher-stine l, et al. Most patients with cancer-associated dermatomyositis have antibodies to nuclear matrix protein nXP-2 or transcription intermediary factor 1γ. Arthritis Rheum 2013;65:2954–62.
26 Pinal-Fernandez i, Ferrer-Fabregas B, Trallero-araguas e, et al. Tumour TiF1 mutations and loss of heterozygosity related to cancer-associated myositis. Rheumatology 2018;57:388–96.
27 Manlhiot c, liang l, Tran D, et al. assessment of an infectious disease history preceding juvenile dermatomyositis symptom onset. Rheumatology 2008;47:526–9.
28 Pachman lM, lipton r, ramsey-Goldman r, et al. History of infection before the onset of juvenile dermatomyositis: results from the national institute of arthritis and musculoskeletal and skin diseases research registry. Arthritis Rheum 2005;53:166–72.
29 shah M, Targoff in, rice MM, et al. Brief report: ultraviolet radiation exposure is associated with clinical and autoantibody phenotypes in juvenile myositis. Arthritis Rheum 2013;65:1934–41.
30 Miller FW, lamb Ja, schmidt J, et al. risk factors and disease mechanisms in myositis. Nat Rev Rheumatol 2018;14:255–68.
31 Betteridge Z, chinoy H, new P. Validation of commercial myositis line blots. Global conference on Myositis, Washington, 2017:14.
32 Ghirardello a, rampudda M, ekholm l, et al. Diagnostic performance and validation of autoantibody testing in myositis by a commercial line blot assay. Rheumatology 2010;49:2370–4.
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Table 1 Performance of different back pain features for the diagnosis of established AxPsA and active AxPsA
Sensitivity (%)
Specificity (%)
Positive LR
Negative LR
Established AxPsA (n=109)*
CBP 68 60 1.76 0.51
IBP 59 84 3.75 0.48
Modified IBP (≥3 out of 5) 84 80 4.27 0.18
Active AxPsA (n=72)†
CBP 100 66 2.97 0.00
IBP 82 `88 7.25 0.20
Modified IBP (≥3 out of 5) 91 75 3.61 0.13
Established AxPsA, n=109; active AxPsA, n=72; CBP, n=191; IBP, n=99; modified IBP, n=145.*Established AxPsA group comprises all patients in the cohort with a diagnosis of AxPsA.†Active AxPsA is a subset of the established AxPsA.AxPsA, axial psoriatic arthritis; CBP, chronic back pain; IBP, inflammatory back pain; LR, likelihood ratio.
Inflammatory back pain criteria perform well in subset of patients with active axial psoriatic arthritis but not among patients with established axial disease
Psoriatic arthritis (PsA) is a progressive, potentially destructive and disabling immune-mediated inflammatory joint disease and is characterised by involvement of both the appendicular and axial skeleton. Inflammatory spinal disease is one of three inflamma-tory musculoskeletal (MSK) manifestations that frequently occur in PsA. The reported prevalence of axial disease in patients with PsA is quite variable, and has been reported to be as high as up to 78%.1 Axial involvement is typically characterised by chronic lower back pain, and a significant proportion of such patients develop disabilities due to spinal inflammation. The early identi-fication of patients with axial involvement among patients with psoriasis (PsO) therefore assumes considerable importance.
There are limited data about the utility of inflammatory back pain (IBP) criteria among patients with axial PsA (AxPsA). Previous studies have described low sensitivity of IBP criteria among AxPsA (around 68%).2 We read with interest the article of Yap et al, where authors report that IBP criteria may not perform well for axial involvement in PsA.3 The authors also report the low sensitivity and high specificity of IBP criteria among AxPsA. We also aimed to assess the diagnostic performance of back pain features among a much larger cohort of patients with PsA.
We have previously reported clinical and genetic association of sacroiliitis (SI) among 283 patients with PsA.4 Now, we further extend this analysis by examining the data of 406 patients with PsA (fulfilling the ClASsification criteria for Psoriatic ARthritis (CASPAR) criteria) in a cross-sectional assessment. All patients were assessed in a dedicated research clinic, where clinical, labo-ratory and radiographic details were collected. Dedicated radio-graphic sacroiliac joint views were obtained in all patients. We defined the criteria for identifying SI if ≥grade 2 radiographic changes were present (unilateral or bilateral). All radiographs were scored by a consultant MSK radiologist and two trained rheumatologists.4 It was considered only those radiographs as showing SI that were scored positively by at least two readers (MSK radiologist and at least one rheumatologist).
IBP features as suggested by Assessment of SpondyloArthritis international Society (ASAS)5 were obtained from all patients who reported back pain. Moreover, we also inquired about back pain and IBP features in all those patients who had a docu-mented rheumatologist-diagnosed AxPsA, which was based on appropriate clinical symptoms and radiographic evidence of SI; this was considered as a gold standard for diagnosing AxPsA for this particular study. In our study, none of the patients with AxPsA had unilateral grade 2 SI.
Clinically active axial disease was defined as patients with spinal pain score (numerical rating scale 0–10) of ≥4 and The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score ≥4 despite taking non-steroidal anti-inflammatory drugs; and all these patients had radiographic evidence of SI. The diag-nostic label of ‘established AxPsA’ was used for all patients in total, diagnosed with AxPsA, regardless whether axial disease in remission or there were ongoing active symptoms.
A cohort of 406 patients with PsA (mean age 52.3±12 years; 54% female; mean PsA duration of 16±7 years) was studied. Twenty-seven per cent (109/406) of the cohort had rheuma-tologist-diagnosed AxPsA. All such patients with AxPsA had
radiographic SI, and had either present or past history of chronic backache. The mean age of patients with AxPsA was 51±10 years, and 53% were male. In patients with a diagnosis of estab-lished AxPsA, we found that chronic back pain (CBP) (presence or absence), regardless of the characteristics of back pain, had very low sensitivity and specificity (68% and 60%, respectively, table 1). Interestingly, IBP criteria had poor sensitivity but quite good specificity (59% and 84%, respectively) among established AxPsA. Furthermore, we checked the performance of CBP and IBP among patients with active AxPsA. We noted that CBP had 100% sensitivity and suboptimal specificity of 66%; however, fulfilling the IBP criteria led to significant improvement in detecting active AxPsA (sensitivity and specificity of 82% and 88%, respectively). Additionally, we also looked at the sensitivity and specificity of lower cut-off points for the IBP questionnaire, as the suggested standard cut-off points may be too high for screening for early disease or in patients already using systemic therapies for PsO and PsA.6 By using the lower cut-off points of 3 out of 5 for ASAS IBP criteria, the sensitivity and specificity to detect established AxPsA improved from 59% and 84% to 84% and 80%, respectively; similarly, lower cut-offs had quite high sensitivity and acceptable specificity among patients with active AxPsA.
Our study findings are clinically important in two ways. First, we found that IBP typically identifies patients with active AxPsA symptoms compared with patients with quiescent or partially treated AxPsA symptoms. Hence, IBP criteria are perhaps not helpful for screening patients with PsA for axial involvement in a cross-sectional analysis. It is possible that earlier studies have examined the performance of IBP criteria for AxPsA, regardless of whether the axial disease in remission or having active symp-toms, which resulted in its suboptimal performance.3 Second, we found that reducing the IBP cut-offs in our study significantly improved its performance, without losing much of the specificity among AxPsA. Since this screening questionnaire has been devel-oped in ankylosing spondylitis (AS), further work is required to investigate whether there is any differential performance of IBP criteria among patients with AxPsA and AS. The number of studies has suggested that there are clinical, radiologic and genetic differences between AxPsA and AS, suggesting that these are distinct entities. Moreover, contrary to AS, peripheral joint disease is much more prevalent among PsA, and the severity of
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peripheral joint disease is a potential predictor of axial involve-ment among PsA.
In conclusion, it would appear that the ASAS suggested IBP questionnaire (which was originally developed for AS) performed poorly in identifying patients with inactive AxPsA symptoms. We suggest that the standard cut-offs for this questionnaire be used for patients with active AxPsA, and the lower cut-offs should be used among patients with established AxPsA, where patients can potentially be in remission or partial remission. This study results have important implications for the use of this instru-ment for screening purposes. Further work is required to vali-date these observations.
Muhammad Haroon,1 Phil Gallagher,2 Oliver FitzGerald2
1Division of Rheumatology, Department of Medicine, University Hospital Kerry, Tralee, Ireland2Department of Rheumatology, St Vincent’s University Hospital, Dublin, Ireland
Correspondence to Dr Muhammad Haroon, Division of Rheumatology, Department of Medicine, University Hospital Kerry, Tralee V92 NX94, Ireland; mharoon301@ hotmail. com
Handling editor Josef S Smolen
Contributors MH, PG and OFG conceived the study, its design, coordination, data interpretation and manuscript drafting and editing.
Competing interests MH: unrestricted educational grant from AbbVie and Pfizer, and member of advisory boards for AbbVie and Celgene.
Patient consent for publication Obtained.
Ethics approval St Vincent’s Healthcare Group Ethics and Medical Research Committee.
Provenance and peer review Not commissioned; externally peer reviewed.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
To cite Haroon M, Gallagher P, FitzGerald O. Ann Rheum Dis 2019;78:1003–1004.
Received 15 October 2018Revised 24 November 2018Accepted 3 December 2018Published Online First 14 December 2018
Ann Rheum Dis 2019;78:1003–1004. doi:10.1136/annrheumdis-2018-214583
RefeRences 1 Battistone MJ, Manaster BJ, Reda DJ, et al. The prevalence of sacroilitis in psoriatic
arthritis: new perspectives from a large, multicenter cohort. A department of veterans affairs cooperative study. Skeletal Radiol 1999;28:196–201.
2 Aydin SZ, Kilic L, Kucuksahin O, et al. Performances of inflammatory back pain criteria in axial psoriatic arthritis. Rheumatology 2017;56:2031–2.
3 Yap KS, Ye JY, Li S, et al. Back pain in psoriatic arthritis: defining prevalence, characteristics and performance of inflammatory back pain criteria in psoriatic arthritis. Ann Rheum Dis 2018;77:1573–7.
4 Haroon M, Winchester R, Giles JT, et al. Clinical and genetic associations of radiographic sacroiliitis and its different patterns in psoriatic arthritis. Clin Exp Rheumatol 2017;35:270–6.
5 Sieper J, van der Heijde D, Landewé R, et al. New criteria for inflammatory back pain in patients with chronic back pain: a real patient exercise by experts from the Assessment of SpondyloArthritis international Society (ASAS). Ann Rheum Dis 2009;68:784–8.
6 Haroon M, Ahmad M, Baig MN, et al. Inflammatory back pain in psoriatic arthritis is significantly more responsive to corticosteroids compared to back pain in ankylosing spondylitis: a prospective, open-labelled, controlled pilot study. Arthritis Res Ther 2018;20:73.
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Table 1 Univariate analysis of patient characteristics by kidney outcome
Characteristics Total %Anyresponse % CR % % CR PR % % PR NR % % NR P value P value*
Female 85 87.6 52 53.6 30 30,9 81.1 22 22.68041 100 33 34.02 86.84 0.2924 0.85
Clinical features
APS* 13 13.4 10 10.3 5 5.2 13.5 5 5.2 22.7 3 3.1 7.9 0.2629 0.22
aPL antibody positivity 61 62.9 26 26.8 15 15.5 40.5 11 11.3 50.0 35 36.1 92.1 <0.001 <0.001
LAC 37 38.1 17 17.5 7 7.2 18.9 10 10.3 45.5 20 20.6 52.6 0.0079 0.018
aCL 35 36.1 20 20.6 8 8.2 21.6 12 12.4 54.5 15 15.5 39.5 0.0334 0.57
anti-beta2 GPI 22 22.7 12 12.4 7 7.2 18.9 5 5.2 22.7 10 10.3 26.3 0.7464 0.49
Triple positivity 15 15.5 8 8.2 5 5.2 13.5 3 3.1 13.6 7 7.2 18.4 0.811 0.51
Anti-dsDNA 43 44.3 22 22.7 13 13.4 35.1 9 9.3 40.9 24 24.7 63.2 0.041 0.0214
Low C3 levels 77 79.3 36 37.1 25 25.8 67.6 11 11.3 50.0 38 39.2 100.0 0.001 <0.001
Low C4 levels 27 27.8 15 15.5 8 8.2 21.6 7 7.2 31.8 12 12.4 31.6 0.5627 0.64
Arterial hypertension 63 64.9 36 37.1 23 23.7 62.2 13 13.4 59.1 28 28.9 73.7 0.4252 0.27
Hyperlipidaemia 45 46.3 27 27.8 17 17.5 45.9 10 10.3 45.5 18 18.6 47.4 0.98 1
aGAPPS ≥ 12 24 24.7 10 10.3 6 6.2 16.2 4 4.1 18.2 14 14.4 36.8 0.0846 0.0267
LN class IV 82 84.5 50 51.5 32 33.0 86.5 18 18.6 81.8 32 33.0 84.2 0.8891 1
Therapy
Mycophenolate mofetil 53 54.6 32 33.0 21 21.6 56.8 11 11.3 50.0 21 21.6 55.3 0.8764 1
Cyclophosphamide 38 39.2 22 22.7 14 14.4 37.8 8 8.2 36.4 16 16.5 42.1 0.888 0.67
EUROLUPUS regimen 28 28.9 16 16.5 11 11.3 29.7 5 5.2 22.7 12 12.4 31.6 0.7583 0.65
Other immunosuppresants 6 6.2 4 4.1 2 2.1 5.4 2 2.1 9.1 2 2.1 5.3 0.8128 1
Anticoagulation 37 38.1 29 29.9 22 22.7 59.5 7 7.2 31.8 8 8.2 21.1 0.0022 00059
Statisticall significant p-values (p < 0.05) are marked in bold.*Computing together any response, CR+PR.aGAPPS, adjusted global antiphospholipid score; CR, complete response; NR, no response; PR, partial response.
Anticoagulation in patients with concomitant lupus nephritis and thrombotic microangiopathy: a multicentre cohort study
The management of lupus nephritis (LN) and concomitant thrombotic microangiopathy (TMA), with or without antiphos-pholipid antibodies (aPL), remains controversial, and few studies are available to inform clinical management.1–4
The purpose of this multicentre retrospective study was to analyse the impact of anticoagulation (vitamin K antag-onists (VKAs) and/or heparins) in addition to conventional
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Figure 1 Comparison of kidney outcomes between patients receiving anticoagulation and those without ((A) all 97 patients; (B), limiting to patients positive for antiphospholipid antibodies). aPL, antiphospholipid antibodies; CR, complete response; LN, lupus nephritis; NR, no response; PR, partial response;TMA thrombotic microangiopathy.
immunosuppression on kidney outcomes (assessed at 12 months, according to the Kidney Disease: Improving Global Outcomes-KDIGOguidelines5) in patients with biopsy-proven LN and concomitant TMA.
Data source, population and statistical analysis are detailed in the online supplementary material 1. Anticoagulation was considered if given for at least three consecutive months after TMA diagnosis.
We retrospectively identified 97 patients with biopsy-proven LN and TMA (2007–2017). See online supplementary table 1 for clinical and demographic characteristics. Laboratory param-eters were collected at the time of the biopsy. The mean age of patients was 38.9±15.2 years (13–69) and 85 females (87.6%). Most had proliferative LN (class IV in 84.5%). In total, 42 (43%) patients presented with acute and 55 (57%) with features of chronic TMA. All patients had received treatment with stan-dard immunosuppressants (55% mycophenolate, 39% cyclo-phosphamide, 6% other regimen) and steroids. At 12 months, complete response (CR) was observed in 37 patients (38.1%), partial response (PR) in 22 (22.6%) and no response (NR) in 38 (39.1%). Sixty-one patients (62.9%) were aPL positive and 37 (38.1%) of these patients received anticoagulation with a VKA and/or heparins. Mean duration of anticoagulation therapy after TMA and LN diagnosis was 7.7 months (3-12).
We observed a higher rate of clinical response (CR/PR, together or computed separately) in patients who received anti-coagulation (CR in 22 (59.46%), PR in 7 (18.91%); NR in 8 (21.62%)) compared with those without (CR in 15 (25.0%), PR in 15 (25.0%); NR in 30 (50%)), p<0.01) (table 1).
When limiting the analysis on the 61 patients with aPL, we observed a higher rate of complete response in those receiving anticoagulation (patients receiving anticoagulant therapy: CR in 22 (59.46%), PR in 7 (18.91%); NR in 8 (21.62%)) versus patients not receiving VKA/heparins (CR in 8 (30.77%), PR in 7 (26.92%); NR in 8 (34.62%),p=0.046) (figure 1).
After multivariate analysis, aPL positivity (any) (β=1.23,OR 2.4;95% CI 1.2 to 7.3;p=0.03), anti-dsDNA positivity (β=1.98,OR 12.8; 95% CI 3.0 to 71.3; p=0.002) and chronic features of TMA (β=1.31,OR 3.0; 95% CI 1.2 to 17.5; p=0.04) were all associated with no kidney response.
When limiting the analysis to aPL-positive patients, after adjusting for type of immunosuppressant therapy and LN class, variables that were significantly associated with CR+PR were features of acute TMA rather than chronic (β=1.95, OR 8.62; 95% CI 1.4 to 97.1; p=0.03) and the use of VKA/heparins (β=1.21 OR 2.1; 95% CI 1.02 to 16.2; p=0.046).
In summary, in our study the use of anticoagulation was asso-ciated with any response to treatment at 1 year, in line with the fact that about 60% of the patients with CR received VKA or heparins. Similarly, when limiting the analysis to patients with aPL, we observed a rate of any response (either CR+PR) as high as 66% in patients receiving anti-coagulant treatment compared with those receiving immunosuppression alone (34%).
Despite its limitations (the relatively short duration of follow-up to gauge the relapse rate; lack of standardised protocol for LN treatment; the use of anticoagulation agents was not randomised but based on the treating physicians’ judge-ment), this study represents the largest available multicentre cohort of real-life systemic lupus erythematosus patients with biopsy-proven LN and concomitant TMA.
To conclude, in patients with concomitant LN and TMA, the use of anticoagulation appeared protective and warrants further investigation as a therapeutic tool; the presence of aPL, anti-dsDNA antibodies and chronic features of TMA was associated with poorer kidney outcomes.
Savino Sciascia,1,2 Jinoos Yazdany,3 Maria Dall'Era,3 Roberta Fenoglio,2 Massimo Radin,1 Ishita Aggarwal,3 Maria J Cuadrado,4,5 Karen Schreiber,5,6,7 Antonella Barreca,8 Mauro Papotti,8 Dario Roccatello1,2
1Center of Research of Immunopathology and Rare Diseases- Coordinating Center of the Network for Rare Diseases of Piedmont and Aosta Valley, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy2SCU Nephrology and Dialysis(ERKnet member), S. Giovanni Bosco Hospital, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy3Division of Rheumatology, Russell/Engleman Research Center, University of California, San Francisco, California, USA4Lupus Unit, Department of Rheumatology, Guy’s and St Thomas’ Hospital, London, UK5Department of Thrombosis and Haemophilia, Guy’s and St Thomas’ Hospital, London, UK6King Christian X’s Rheumatology Hospital, Graasten, Denmark
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7Copenhagen Lupus and Vasculitis Clinic, Center for Rheumatology and Spine Diseases, Copenhagen, Denmark8Division of Pathology, Department of Medical Sciences, University of Turin, Turin, Italy
Correspondence to Dr Savino Sciascia, Center of Research of Immunopathology and Rare Diseases- Coordinating Center of the Network for Rare Diseases of Piedmont and Aosta Valley, Department of Clinical and Biological Sciences, University of Turin, Turin 10154, Italy; savino. sciascia@ unito. it
Handling editor Josef S Smolen
Contributors All the authors made substantial contributions to the conception and design of the work, the acquisition, analysis and interpretation of data. They all parteciapted to drafting the work or revising it critically for important intellectual content. All the authors gave the final approval of the version submitted.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
► Additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214559).
Manuscripts based on work previously presented at the following conferences and published as a conference abstract.ACR 2018: https:// acrabstracts. org/ abstract/ clinical- outcomes- and- response- to- anti- thrombotic- treatment- among- patients- with- concomitant- lupus- nephritis- and- thrombotic- microangiopathy- a- multicenter- cohort- study/? msg= fail& shared= email EULAR 2018: https:// web. eular. org/ EULAR_ Production/ 2018_ Amsterdam. nsf/ fmWebPGMbyDayPublic? OpenForm https:// web. eular. org/ EULAR_ Production/ 2018_ Amsterdam. nsf/ fmWebPGMbyDayPublic? OpenForm.
To cite Sciascia S, Yazdany J, Dall’Era M, et al. Ann Rheum Dis 2019;78:1004–1006.
Received 10 October 2018Revised 22 November 2018Accepted 25 November 2018Published Online First 14 December 2018
Ann Rheum Dis 2019;78:1004–1006. doi:10.1136/annrheumdis-2018-214559
RefeRences 1 Mejía-Vilet JM, Córdova-Sánchez BM, Uribe-Uribe NO, et al. Prognostic significance of
renal vascular pathology in lupus nephritis. Lupus 2017;26:1042–50. 2 Pattanashetti N, Anakutti H, Ramachandran R, et al. Effect of thrombotic
microangiopathy on clinical outcomes in indian patients with lupus nephritis. Kidney Int Rep 2017;2:844–9.
3 Sciascia S, Cuadrado MJ, Khamashta M, et al. Renal involvement in antiphospholipid syndrome. Nat Rev Nephrol 2014;10:279–89.
4 Moroni G, Ventura D, Riva P, et al. Antiphospholipid antibodies are associated with an increased risk for chronic renal insufficiency in patients with lupus nephritis. Am J Kidney Dis 2004;43:28–36.
5 KDIGO Clinical Practice Guideline for Glomerulonephritis KDIGO Clinical Practice Guideline for Glomerulonephritis KDIGO Clinical Practice Guideline for Glomerulonephritis. 2012. Available: http://www. kdigo. org/ clinical_ practice_ guidelines/ pdf/ KDIGO- GN- Guideline. pdf [Accessed 4 Jun 2018].
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Figure 1 Impact of organ damage on belimumab efficacy. The forest plots illustrate the performance of baseline variables in predicting SRI-4 response to standard-of-care therapy with and without the addition of belimumab in patients with active SLE, based on results from logistic regression analyses. Following simple logistic regression analyses, multiple logistic regression models were constructed for selected baseline variables in order to assess independence, priority and confounding potentiality. P values <0.05 were considered statistically significant.Level of statistical significance: *p<0.05; **p<0.01; ***p<0.001.Anti-dsDNA, anti–double-stranded DNA; CS, corticosteroid; SDI, Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index; SELENA-SLEDAI, Safety of Estrogens in Lupus Erythematosus National Assessment–SLE Disease Activity Index; SLE, systemic lupus erythematosus; SRI-4, SLE responder index 4.
Established organ damage reduces belimumab efficacy in systemic lupus erythematosus
Post hoc analyses of the phase III trials of belimumab BLISS-521 and BLISS-762 revealed superiority of belimumab over placebo in patients with systemic lupus erythematosus (SLE) with high baseline disease activity, positive anti–double-stranded (ds)DNA titres and low complement levels, as well as in patients receiving corticosteroids.3 Later, real-life observations demonstrated that established organ damage prior to treatment initiation predicted reduced belimumab efficacy.4 We aimed at validating this finding in the BLISS-52 and BLISS-76 SLE populations. Access to data was granted by GlaxoSmithKline (Uxbridge, UK).
In total, 1684 patients were included in the analysis. The BLISS-52 trial comprised 865 and the BLISS-76 trial 819 patients with SLE who were randomised to receive belimumab 1 mg/kg, belimumab 10 mg/kg or placebo, along with standard-of-care treatment.
Organ damage was assessed using the Systemic Lupus Inter-national Collaborating Clinics/American College of Rheuma-tology Damage Index (SDI).5 Response to treatment was defined as fulfilment of the SLE responder index 4 (SRI-4) conditions
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at week 52 (primary endpoint of the BLISS trials).1 2 Patients who withdrew or required changes in the background therapy other than those permitted by protocol were considered non-responders.
Logistic regression was used to evaluate the association of SDI scores with treatment response. Adjustments for previ-ously reported predictors of belimumab efficacy3 were made as appropriate.
Baseline characteristics are presented in online supplementary table S1, and results in figure 1. In patients who received belim-umab, baseline SDI >1 was a strong predictor of reduced treat-ment efficacy. This was more prominent in the belimumab 10 mg/kg arm (OR 0.48, 95% CI 0.29 to 0.77; p=0.003), where SLE duration showed no significant association. High disease activity and corticosteroid use at baseline did not confound the result. Venous thrombosis (OR 0.19, 95% CI 0.04 to 0.87; p=0.033) and cognitive impairment/psychosis (OR 0.27, 95% CI 0.11 to 0.63; p=0.003) were found to be the SDI items mostly driving this association (see online supplementary tables S2–S5). In the placebo arm, baseline SDI scores >1 did not predict the SRI-4 status at week 52 (OR 0.73, 95% CI 0.44 to 1.20; p=0.210). Importantly, anti-dsDNA positivity and low C3/C4 levels were not found to impact belimumab efficacy at the dose of 10 mg/kg.
We herein demonstrated that established organ damage was associated with a substantial reduction in belimumab treatment efficacy, irrespective of baseline SLE activity or serological status. This was more prominent in patients with baseline SDI scores >1 versus SDI scores >0, and mainly driven by venous thrombosis, as previously implicated,4 and cognitive impairment/psychosis.
Organ damage in SLE is associated with unfavourable disease course and premature mortality,6 7 which may partly account for the findings, and is known to accumulate with disease dura-tion.6 It is therefore worth noting that the association between organ damage and belimumab efficacy was not confounded by SLE duration. The mechanisms underlying the observed effect of previous thrombotic events remain to be elucidated; exploration of possible roles for the antiphospholipid antibodies/syndrome and/or use of anticoagulants is merited.
Previous comparative analysis revealed superiority of belim-umab over placebo in patients with positive anti-dsDNA titres and low C3/C4 levels.3 In our analysis of non-comparative nature, no association was found between SRI-4 achievement and anti-dsDNA positivity or hypocomplementaemia in the belimumab 10 mg/kg arm, and low C3/C4 levels even predicted non-response in the belimumab 1 mg/kg and placebo arms.
Patients with active nephritis and neuropsychiatric SLE were excluded from the trials. Implications derived from the study may therefore not be applicable to these SLE subsets.
In agreement with previous literature, our results suggest that belimumab may be expected to be more efficacious in patients with SLE with limited or no organ damage accrued prior to treatment initiation, irrespective of disease duration or activity grade, and provide additional clinical guidance on the use of belimumab therapy.
Ioannis Parodis,1,2 Alvaro Gomez,1,2 Sharzad Emamikia,1,2 Katerina Chatzidionysiou1,2
1Division of Rheumatology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden2Rheumatology, Karolinska University Hospital, Stockholm, Sweden
Correspondence to Dr Ioannis Parodis, Division of Rheumatology, Department of Medicine, Karolinska Institutet, Stockholm 17176, Sweden; ioannis. parodis@ ki. se
Handling editor Josef S Smolen
Acknowledgements The authors would like to thank GlaxoSmithKline (Uxbridge, UK) for granting access to the data from the BLISS-52 and BLISS-76 trials ( ClinicalTrials. gov identifiers NCT00424476 and NCT00410384, respectively), as well as all participating patients.
Contributors Study conception, design and coordination: IP, KC. Acquisition of data: IP, AG, SE. Statistics: IP, AG, SE. Interpretation of the results: IP, AG, SE, KC. Manuscript draft: IP, AG. All authors read and critically revised the manuscript for intellectual content, approved its final version prior to submission and agree to be accountable for all aspects of the work.
Funding The study was supported by grants from the Swedish Research Council, Professor Nanna Svartz Foundation (reference no. 2017-00213), Swedish Rheumatism Association, King Gustaf V’s 80-year Foundation, Ingegerd Johansson’s Fund, Stockholm County Council and Karolinska Institutet Foundations, and was independent of pharmaceutical sponsors.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement The datasets used and analysed during the current study are available from the corresponding author on reasonable request.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
► Additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214880).
To cite Parodis I, Gomez A, Emamikia S, et al. Ann Rheum Dis 2019;78:1006–1007.
Received 6 December 2018Revised 20 December 2018Accepted 20 December 2018Published Online First 4 January 2019
Ann Rheum Dis 2019;78:1006–1007. doi:10.1136/annrheumdis-2018-214880
RefeRences 1 Navarra SV, Guzmán RM, Gallacher AE, et al. Efficacy and safety of belimumab in
patients with active systemic lupus erythematosus: a randomised, placebo-controlled, phase 3 trial. Lancet 2011;377:721–31.
2 Furie R, Petri M, Zamani O, et al. A phase III, randomized, placebo-controlled study of belimumab, a monoclonal antibody that inhibits B lymphocyte stimulator, in patients with systemic lupus erythematosus. Arthritis Rheum 2011;63:3918–30.
3 van Vollenhoven RF, Petri MA, Cervera R, et al. Belimumab in the treatment of systemic lupus erythematosus: high disease activity predictors of response. Ann Rheum Dis 2012;71:1343–9.
4 Parodis I, Sjöwall C, Jönsen A, et al. Smoking and pre-existing organ damage reduce the efficacy of belimumab in systemic lupus erythematosus. Autoimmun Rev 2017;16:343–51.
5 Gladman D, Ginzler E, Goldsmith C, et al. The development and initial validation of the Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index for systemic lupus erythematosus. Arthritis Rheum 1996;39:363–9.
6 Gladman DD, Goldsmith CH, Urowitz MB, et al. The Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Index for Systemic Lupus Erythematosus International Comparison. J Rheumatol 2000;27:373–6.
7 Rahman P, Gladman DD, Urowitz MB, et al. Early damage as measured by the SLICC/ACR damage index is a predictor of mortality in systemic lupus erythematosus. Lupus 2001;10:93–6.
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Rituximab in moderate to severe non-renal systemic lupus erythematosus: a reanalysis of the EXPLORER study
Many targeted therapies have been developed for systemic lupus erythematosus (SLE) in recent years and almost all have failed to meet their predesigned endpoints.1 Among these ther-apies, rituximab (RTX) has reported good results in several open-label studies,2 3 but failed to meet its primary endpoints
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Figure 1 Response rate using new criteria. (A) Response rate for the four criteria used in the reanalysis in the rituximab and placebo group. (B) Response rate for the more stringent SRI-5, SRI-6, SRI-7 and SRI-4 in the rituximab and placebo group. mBICLA, modified BILAG-based Composite Lupus Assessment; mLLDAS, modified Lupus Low Disease Activity Score; SRI-4, Systemic Lupus Erythematosus Response Index-4; SRI-4 with OCS, SRI-4 with oral corticosteroid tapering.
in two randomised controlled trials (RCTs), EXPLORER and LUNAR.4 5
Among the proposed explanations for these failures, the primary outcome criteria’s lack of sensitivity has been suggested.6 The availability of new SLE response criteria led us to reanalyse the EXPLORER study which assessed RTX efficacy in severe non-renal SLE, using four newly available scores: SLE Response Index-4 (SRI-4), a modified SRI-4 (including reduction of glucocorticoids ≤10 mg/day between days 169 and 365), a modified BILAG-based Clinical Lupus Assessment (mBICLA) and a modified Lupus Low Disease Activity Score (mLLDAS), (online supplementary file 1 for details on criteria).7 8
The EXPLORER trial was a 52-week, –multicentre, randomised, double-blind placebo-controlled trial of RTX in 257 patients with moderately to severely active non-renal SLE (online supplementary table 1). Patients were randomised at a 2:1 ratio to receive intravenous RTX (1000 mg) or placebo on days 1, 15, 168 and 182, which was added to the baseline immunosuppressive regimen and to additional daily oral pred-nisone (0.5 mg/kg to 1.0 mg/kg based on initial BILAG score) (online supplementary table 2). Raw data from the original EXPLORER study were retrieved from Roche Pharmaceuticals on presentation of a prespecified reanalysis plan. Reanalysis was conducted in the total intention to treat population.
At 52 weeks, RTX failed to show a statistically significant efficacy over placebo for all four newly described response criteria (p>0.05 for all comparisons, figure 1A). More strin-gent SRI-5, SRI-6, SRI-7 and SRI-8 did not favour RTX treatment neither (figure 1B). There was no difference in the cumulative prednisone dose (4223±2831 mg in the RTX group vs 4390±2678 mg in the placebo group, p=0.65 using bilateral Student t-test).
Subgroup analyses were performed for all four response criteria using EXPLORER’s prespecified subgroups and other clinically relevant subgroups such as hydroxychloroquine or tobacco use, baseline immunosuppressive treatment and the organ-domain involvement at baseline (figure 2A–C). In terms of SRI-4 response, patients without mucocutaneous involve-ment were more likely to benefit from RTX (OR 10.2, 95% CI 1.25 to 82.8, p=0.03; figure 1C). There was a statistical trend for people with baseline haematological involvement to have a better SRI-4 response to RTX (OR 7.20, 95% CI 0.86 to 60.1, p=0.07; figure 1C). Considering patients with vasculitis at baseline, an OR calculation was not possible because none of these patients treated with placebo had an SRI-4 response. However, 39.3% of patients with vasculitis at baseline (n=28) treated by RTX had a SRI-4 response at week 52 (p=0.04 using Fisher’s exact test). The same observation was made with the other outcome measures (SRI-4 with corticosteroid dose reduction, mBICLA, and mLLDAS responses) but the difference was not significant (online supplementary figure 1).
Several explanations have been raised to explain SLE RCT failures, such as high-dose corticosteroids, associated immuno-suppressive medication, inappropriate endpoints and patient heterogeneity.9 In the current reanalysis, we retrospectively took into account these factors but failed to demonstrate RTX superiority over placebo. However, it should be noted that there were significant agreeability between old and new response criteria (online supplementary figure 2). Future SLE trials might need to better select their patients (eg, using tran-scriptomic signature), reduce or avoid steroiden studies report the excellent efficacy of RTX in SLE, our reanalysis does not allow us to show that RTX could be effective in SLE, at least
in non-renal moderate to severe SLE. bursts at inclusion and concomitant immunosuppressive medications to overcome the difficulties of demonstrating efficacy in SLE. Although many open studies report the excellent efficacy of RTX in SLE, our reanalysis does not allow us to show that RTX could be effec-tive in SLE, at least in non-renal moderate to severe SLE.
Marc Scherlinger,1,2,3 Claire Carcaud,1,2 Marie-Elise Truchetet,1,2,3 Thomas Barnetche,1 Pierre Duffau,1,2,3 Lionel Couzi,1,2,3 Julien Seneschal,1,2 Patrick Blanco,1,2,3 Estibaliz Lazaro,1,2,3 Christophe Richez1,2,3
1Centre Hospitalier Universitaire de Bordeaux, FHU ACRONIM, Bordeaux, France2Université de Bordeaux, Bordeaux, France3CNRS-UMR 5164 Immuno ConcEpT, Bordeaux, France
Correspondence to Dr Estibaliz Lazaro; estibaliz. lazaro@ chu- bordeaux. fr and Dr Christophe Richez, CNRS-UMR, 5164 Immuno ConcEpT, 146 rue Léo Saignat, 33076 Bordeaux, France; christophe. richez@ chu- bordeaux. fr
Handling editor Josef S Smolen
Contributors MS, CC and M-ET did the figures. MS, EL and CR wrote the manuscript. MS and TB conducted the data analysis (statistics). PD, LC, JS, PB, EL and CR took part in the design of the study and of the reanalysis. All authors approved the last version of the manuscript.
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Figure 2 Forest plot representing OR of treatment response in different subgroups. (A) Baseline demographic and disease characteristics. (B) Baseline organ BILAG involvement. (C) Baseline treatment characteristics. AZA, azathioprine; CS, corticosteroids; dsDNA, double-stranded DNA; mBICLA, modified British Isles Lupus Assessment Group (BILAG)-based Composite Lupus Assessment; mLLDAS, modified Lupus Low Disease Activity Score; MMF, mycophenolate mofetil; MTX, methotrexate; NA, non assessable; SRI-4, Systemic Lupus Erythematosus Response Index-4; SRI-4 with OCS, SRI-4 with oral corticosteroid tapering.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests MS, TB, PD, LC, JS, PB and CC declare no conflict of interest. CR has received consultancy and speaking fees from AstraZeneca, Roche, Glenmark, BMS, Lilly, UCB and Janssen. EL has received consultancy and speaking fees from GSK and UCB. M-ET has received consultancy fees and/or research funding from BMS, Roche, Lilly and UCB. Roche Pharmaceuticals provided freely EXPLORER raw data but did not take part in neither the analysis nor the writing of the manuscript.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
MS, CC, EL and CR contributed equally.
To cite Scherlinger M, Carcaud C, Truchetet M-E, et al. Ann Rheum Dis 2019;78:1007–1010.
Received 27 November 2018Revised 20 December 2018Accepted 20 December 2018
Published Online First 4 January 2019
Ann Rheum Dis 2019;78:1007–1010. doi:10.1136/annrheumdis-2018-214833
RefeRences 1 Lazaro E, Scherlinger M, Truchetet ME, et al. Biotherapies in systemic lupus
erythematosus: new targets. Joint Bone Spine 2017;84:267–74. 2 Terrier B, Amoura Z, Ravaud P, et al. Safety and efficacy of rituximab in systemic
lupus erythematosus: results from 136 patients from the French AutoImmunity and Rituximab registry. Arthritis Rheum 2010;62:2458–66.
3 Contis A, Vanquaethem H, Truchetet ME, et al. Analysis of the effectiveness and safety of rituximab in patients with refractory lupus nephritis: a chart review. Clin Rheumatol 2016;35:517–22.
4 Merrill JT, Neuwelt CM, Wallace DJ, et al. Efficacy and safety of rituximab in moderately-to-severely active systemic lupus erythematosus: the randomized, double-blind, phase II/III systemic lupus erythematosus evaluation of rituximab trial. Arthritis Rheum 2010;62:222–33.
5 Rovin BH, Furie R, Latinis K, et al. Efficacy and safety of rituximab in patients with active proliferative lupus nephritis: the Lupus Nephritis Assessment with Rituximab study. Arthritis Rheum 2012;64:1215–26.
6 Bruce IN, Gordon C, Merrill JT, et al. Clinical trials in lupus: what have we learned so far? Rheumatology 2010;49:1025–7.
7 Rao V, Gordon C. Advances in the assessment of lupus disease activity and damage. Curr Opin Rheumatol 2014;26:510–9.
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8 Franklyn K, Lau CS, Navarra SV, et al. Definition and initial validation of a Lupus Low Disease Activity State (LLDAS). Ann Rheum Dis 2016;75:1615–21.
9 Reddy V, Jayne D, Close D, et al. B-cell depletion in SLE: clinical and trial experience with rituximab and ocrelizumab and implications for study design. Arthritis Res Ther 2013;15(Suppl 1):S2.
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Table 1 Characteristics of SLE pregnancies overall and according to aspirin use
Characteristic
All pregnant visits (n=475)*
Pregnant visits with aspirin (n=121)
Pregnant visits without aspirin (n=354)
Patient characteristic
Age, mean (SD) 31.0 (4.9) 30.5 (4.6) 31.2 (5.0)
Ethnicity, n (%)
Asian 66 (14) 7/66 (11) 59/66 (89)
Native North American 3 (1) 2/3 (67) 1/3 (33)
Black 88 (19) 9/88 (10) 79/88 (90)
Caucasian 209 (44) 71/209 (34) 138/209 (66)
Hispanic 62 (13) 20/62 (32) 42/62 (68)
Indian subcontinent 25 (5) 8/25 (32) 17/25 (68)
Other 22 (5) 4/22 (18) 18/22 (82)
Country, n (%)
Canada 121 (25) 27/121 (22) 94/121 (78)
USA 105 (22) 20/105 (19) 85/105 (81)
Mexico 52 (11) 19/52 (37) 33/52 (63)
Europe 146 (31) 49/146 (34) 97/146 (66)
South Korea 51 (11) 6/51 (12) 45/51 (88)
Any postsecondary education, n (%) 310/452 (69) 69/310 (22) 241/310 (78)
BMI, mean (SD) 25.8 (5.9) 26.3 (5.2) 25.6 (6.1)
Obstetrical history
Parity, mean (SD) 1.1 (1.0) 1.1 (1.0) 1.2 (1.0)
Nulliparous, n (%) 134/461 (29) 37/134 (28) 97/134 (72)
Previous fetal loss <24 weeks, n (%)
84/456 (18) 22/84 (26) 62/84 (74)
SLE characteristics
Disease duration (years), mean (SD) 5.6 (3.3) 5.6 (3.3) 5.6 (3.3)
SLEDAI, mean (SD) 3.3 (3.8) 3.0 (3.6) 3.4 (3.9)
SLICC damage score, mean (SD) 0.5 (1.0) 0.6 (1.0) 0.5 (1.0)
Any positive aPL, n (%) 34/104 (33) 13/34 (38) 21/34 (62)
LAC, n (%) 19/104 (18) 6/19 (32) 13/19 (68)
ACL, n (%) 12/104 (12) 3/12 (25) 9/12 (75)
GP1 IgG, n (%) 18/104 (17) 9/18 (50) 9/18 (50)
Nephritis, n (%) 53(11) 11/53(21) 42/53 (79)
Comorbidities
Any renal disease†, n (%) 83 (17) 17/83 (20) 66/83 (80)
CKD (eGFR≤90 mL/min/1.73 m²), n (%)
43/459 (9) 6/43 (14) 37/43 (86)
CKD stage ≤3 (eGFR≤60 mL/min/1.73 m²), n (%)
11/459 (2) 5/11 (45) 6/11 (55)
Hypertension, n (%) 79 (17) 24/79 (30) 55/79 (70)
Taking anticoagulation, n (%) 28 (6) 12/28 (43) 15/28 (54)
Year of pregnancy visit
2000– 2004, n (%) 39 (8) 11/39 (28) 28 (72)
2005– 2009, n (%) 157 (33) 46/157 (29) 111/157 (71)
2010– 2014, n (%) 218 (46) 52/218 (24) 166/218 (76)
2015– 2017, n (%) 61 (13) 12/61 (20) 49/61 (80)
*Denominator=475 unless otherwise stated.†Includes chronic kidney disease, active nephritis and/or nephrotic syndrome within the last year.ACL, anticardiolipin antibody; aPL, antiphospholipid antibody; BMI, body mass index; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate; GP1, anti-B2-glycoprotein-1; LAC, lupus anticoagulant; SLE, systemic lupus erythematosus; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index; SLICC, Systemic Lupus International Collaborating Clinics.
Low aspirin use and high prevalence of pre-eclampsia risk factors among pregnant women in a multinational SLE inception cohort
Women with systemic lupus erythematosus (SLE) carry a substantially higher risk for pre-eclampsia compared with the general population.1 Aspirin reduces the risk of pre-eclampsia in high-risk pregnancies by more than half2 and thus is recom-mended in SLE.3–5 The European League Against Rheumatism recommends aspirin in SLE pregnancies, particularly in those with nephritis or positive antiphospholipid antibodies (aPL).5 Despite this, little is known about current practice. Therefore, we assessed the prevalence of aspirin use in SLE pregnancies within the Systemic Lupus International Collaborating Clinics inception cohort, which has been described elsewhere.6
SLE women aged 18–45 with a pregnancy documented at one or more annual study visits (spanning 2000–2017) were included. For each pregnant visit, aspirin use, traditional pre-eclampsia risk factors (hypertension, chronic kidney disease, diabetes, nulliparity, body mass index ≥35, age >40), aPL and active lupus nephritis were assessed (see variable defi-nitions in online supplementary material). Aspirin use was compared among those with and without each/any risk factor, and over time.
We identified 475 pregnancies among 300 women. Mean SLE duration at the time of pregnancy was 5.6 years (SD 3.1). Half (51%) of pregnancies had ≥1 traditional pre-eclampsia risk factor, 34/104 (33%) had positive aPL and 53/475 (11%) had nephritis (table 1). Aspirin was used in 121 (25%) preg-nancies. While a third of pregnancies in Caucasians (71/209, 34%, 95% CI 28% to 41%) and Hispanics (20/62, 32%, 95% CI 22% to 45%) were aspirin exposed, only 9/88 (10%, 95% CI 5% to 18%) and 7/66 (11%, 95% CI 5% to 20%) of preg-nancies in Black and Asian subjects were respectively aspirin exposed. Aspirin use did not differ among pregnancies with or without ≥1 traditional risk factor (58/234, 25% (95% CI 20% to 31%) vs 63/241, 26% (95% CI 21% to 32%)), any tradi-tional risk factor individually, or nephritis (see online supple-mentary table 1). There was a potential trend for increased aspirin use among pregnancies with positive aPL (13/34, 38%, 95% CI 24% to 55%) compared with those without aPL (16/70, 23%, 95% CI 15% to 34%), although CI overlapped. Sensitivity analyses excluding multiple pregnancies within the same women yielded similar results. Aspirin use did not increase from 2000 to 2017 (χ2 test for trend in proportions, p=0.13).
Our study is the first to assess aspirin use in SLE pregnancies according to the presence of pre-eclampsia risk factors. Among the 475 SLE pregnancies in this prospective, multinational inception cohort, additional pre-eclampsia risk factors were present in half, while aspirin was taken in only one-quarter and did not differ from background aspirin use among the same women at non-pregnant visits (see online supplemen-tary material). Even without considering SLE itself as a major risk factor, aspirin use was no more prevalent among those
with other traditional indications for aspirin in pregnancy, and the majority of those with aPL and nephritis were not taking aspirin. The low aspirin use among Black SLE subjects is note-worthy given the worse reproductive outcomes observed in this population.7
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Study limitations include lack of data on gestational age and pregnancy outcomes. In addition, aspirin could have been introduced at/or following the study visit when the pregnancy was documented, highlighting the importance of the rheuma-tologist in reviewing aspirin use and initiating it, if not already done, in pregnant SLE women. However, assuming either a somewhat normal or a left-skewed distribution of gestational ages at the pregnant visits, a substantial proportion of visits would have taken place after 12–16 weeks’ gestation, by which time aspirin should have been initiated.2 3
In conclusion, we have potentially identified an important gap between practices and current recommendations for the care of pregnant SLE women, and call for further studies of factors contributing to aspirin use in lupus pregnancies.
Arielle Mendel,1 Sasha B Bernatsky,1,2 John G Hanly,3 Murray B Urowitz,4 Ann Elaine Clarke,5 Juanita Romero-Diaz,6 Caroline Gordon,7,8 Sang-Cheol Bae,9 Daniel J Wallace,10 Joan T Merrill,11 Jill P Buyon,12 David A Isenberg,13 Anisur Rahman,13 Ellen M Ginzler,14 Michelle Petri,15 Mary Anne Dooley,16 Paul R Fortin,17 Dafna D Gladman,4 Kristján Steinsson,18 Rosalind Ramsey-Goldman,19 Munther A Khamashta,20 Cynthia Aranow,21 Meggan Mackay,21 Graciela S Alarcón,22 Susan Manzi,23 Ola Nived,24 Andreas Jönsen,24 Asad A Zoma,25 Ronald F van Vollenhoven,26 Manuel Ramos-Casals,27 Guillermo Ruiz-Irastorza,28 Sam Lim,29 Ken C Kalunian,30 Murat Inanc,31 Diane L Kamen,32 Christine A Peschken,33 Søren Jacobsen,34 Anca Askanase,35 Jorge Sanchez-Guerrero,36 Ian N Bruce,37,38 Nathalie Costedoat-Chalumeau,39 Évelyne Vinet1,2
1Division of Rheumatology, McGill University Health Centre, Montreal, Quebec, Canada2Division of Clinical Epidemiology, Research Institute of the McGill University Health Center, Montreal, Quebec, Canada3Division of Rheumatology, Department of Medicine and Department of Pathology, Queen Elizabeth II Health Sciences Centre and Dalhousie University, Halifax, Nova Scotia, Canada4Lupus Program, Centre for Prognosis Studies in the Rheumatic Disease and Krembil Research Institute, Toronto Western Hospital, University of Toronto, Toronto, Ontario, Canada5Division of Rheumatology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada6Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición, Tlalpan, Mexico7Rheumatology Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, UK8Rheumatology Department, City Hospital, Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, UK9Department of Rheumatology, Hanyang University Hospital for Rheumatic Diseases, Seoul, Republic of Korea10Cedars-Sinai Medical Centre, David Geffen School of Medicine at UCLA, Los Angeles, California, USA11Department of Clinical Pharmacology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA12Division of Rheumatology, Department of Medicine, New York School of Medicine, New York City, New York, USA13Department of Medicine, Centre for Rheumatology, University College London, London, UK14Department of Medicine, SUNY Downstate Medical Center, Brooklyn, New York, USA15Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA16Thurston Arthritis Research Center, University of North Carolina, Chapel Hill, North Carolina, USA17Division of Rheumatology, Centre Hospitalier Universitaire de Québec et Université Laval, Québec City, Quebec, Canada18Center for Rheumatology Research, Landspitali University Hospital, Reykjavik, Iceland19Division of Rheumatology, Feinberg School of Medicine, Northwestern University Chicago, Chicago, Illinois, USA20Lupus Research Unit, The Rayne Institute, St Thomas’ Hospital, King’s College London School of Medicine, London, UK21Lupus Center of Excellence, Feinstein Institute for Medical Research, Manhasset, New York, USA22Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
23Lupus Center of Excellence, Allegheny Health Network, Pittsburgh, Pennsylvania, USA24Department of Clinical Sciences and Rheumatology, Lund University, Lund, Sweden25Lanarkshire Centre for Rheumatology, Hairmyres Hospital, East Kilbride, UK26Unit for Clinical Therapy Research (CliTRID), Karolinska Institute, Stockholm, Sweden27Josep Font Autoimmune Diseases Laboratory, IDIBAPS, Department of Autoimmune Diseases, Hospital Clínic, Barcelona, Spain28Autoimmune Diseases Research Unit, Department of Internal Medicine, BioCruces Health Research Institute, Hospital Universitario Cruces, University of the Basque Country, Barakaldo, Spain29Division of Rheumatology, Emory University School of Medicine, Atlanta, Georgia, USA30University of California San Diego School of Medicine, La Jolla, California, USA31Division of Rheumatology, Department of Internal Medicine, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey32Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, USA33Department of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada34Copenhagen Lupus and Vasculitis Clinic, Section 4242, Center for Rheumatology and Spine Diseases, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark35Hospital for Joint Diseases, Seligman Centre for Advanced Therapeutics, New York University, New York City, New York, USA36Department of Rheumatology, Mount Sinai Hospital and University Health Network, University of Toronto, Toronto, Ontario, Canada37NIHR Manchester Biomedical Research Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK38Arthritis Research UK Centre for Epidemiology, Centre for Musculoskeletal Research, Faculty of Biology, Medicine and Health, School of Biological Sciences, The University of Manchester, Manchester, UK39Centre de Reference Maladies Auto-immunes et Systemiques Rares, Service de Medecine Interne, Hospital Cochin, Paris, France
Correspondence to Dr Évelyne Vinet, Research Institute of the McGill University Health Centre, Montréal, QC H4A 3S5, Canada; evelyne. vinet@ mcgill. ca
Handling editor Josef S Smolen
Contributors EV had full access to all the data in this study and takes full responsibility as a guarantor for the integrity of the data and the accuracy of the data analysis. EV, AM, SBB, JGH, MBU, AEC, JRD, CG, SCB, DJW, JTM, JPB, DAI, AR, EMG, MP, MAD, PRF, DDG, KS, RRG, MAK, CA, MM, GSA, SM, ON, AJ, AAZ, RFV, MRC, GRI, SL, KCK, MI, DLK, CAP, SJ, AA, JSG, INB and NCC conceived and designed the study. EV, AM, SBB, JGH, MBU, AEC, JRD, CG, SCB, DJW, JTM, JPB, DAI, AR, EMG, MP, MAD, PRF, DDG, KS, RRG, MAK, CA, MM, GSA, SM, ON, AJ, AAZ, RFV, MRC, GRI, SL, KCK, MI, DLK, CAP, SJ, AA, JSG, INB and NCC analysed the data. EV, AM, SBB, JGH, MBU, AEC, JRD, CG, SCB, DJW, JTM, JPB, DAI, AR, EMG, MP, MAD, PRF, DDG, KS, RRG, MAK, CA, MM, GSA, SM, ON, AJ, AAZ, RFV, MRC, GRI, SL, KCK, MI, DLK, CAP, SJ, AA, JSG, INB and NCC interpreted the data and drafted the manuscript.
Funding This study was funded through a McGill University Health Centre Research Award. EV receives a salary support from a Fonds de Recherche Québec Santé Clinical Research Scholar-Junior 1 Award. SCB is supported by the Bio & Medical Technology Development Program of the National Research Foundation funded by the Ministry of Science and ICT (NRF-2017M3A9B4050335). SJ is supported by The Danish Rheumatism Association (A-3865). AEC is supported by an Arthritis Society Chair in Rheumatic Diseases. The Hopkins Lupus Cohort is supported by a National Institutes of Health grant (R01 AR069572) awarded to MP. The Birmingham SLICC cohort was funded by a Lupus UK grant awarded to CG.
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval McGill University Health Centre.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement No additional data available.
Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially,
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and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.
© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
► Additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 214434).
To cite Mendel A, Bernatsky SB, Hanly JG, et al. Ann Rheum Dis 2019;78:1010–1012.
Received 12 September 2018Revised 21 November 2018Accepted 3 December 2018Published Online First 20 December 2018
Ann Rheum Dis 2019;78:1010–1012. doi:10.1136/annrheumdis-2018-214434
RefeRences 1 Clowse MEB, Jamison M, Myers E, et al. A national study of the complications of lupus
in pregnancy. Am J Obstet Gynecol 2008;199:127.e1–127.e6. 2 Bujold E, Roberge S, Lacasse Y, et al. Prevention of preeclampsia and intrauterine
growth restriction with aspirin started in early pregnancy: a meta-analysis. Obstet Gynecol 2010;116(2 Pt 1):402–14.
3 Visintin C, Mugglestone MA, Almerie MQ, et al. Management of hypertensive disorders during pregnancy: summary of NICE guidance. BMJ 2010;341:c2207.
4 LeFevre ML, U.S. Preventive Services Task Force. Low-dose aspirin use for the prevention of morbidity and mortality from preeclampsia: U.S. Preventive Services Task Force recommendation statement. Ann Intern Med 2014;161:819–26.
5 Andreoli L, Bertsias GK, Agmon-Levin N, et al. EULAR recommendations for women’s health and the management of family planning, assisted reproduction, pregnancy and menopause in patients with systemic lupus erythematosus and/or antiphospholipid syndrome. Ann Rheum Dis 2017;76:476–85.
6 Urowitz MB, Gladman D, Ibañez D, et al. Clinical manifestations and coronary artery disease risk factors at diagnosis of systemic lupus erythematosus: data from an international inception cohort. Lupus 2007;16:731–5.
7 Buyon JP, Kim MY, Guerra MM. Predictors of Pregnancy Outcome in a Prospective, Multiethnic Cohort of Lupus Patients. Ann Intern Med 2015;163:153–63.
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Table 1 Patient characteristics of ‘relapser’ and ‘non-relapser’
All patientsEarly relapser(<90 days post-TCZ)
Late relapser(>90 days post-TCZ) Non-relapser P value
No of patients 26 7/26 (27%) 8/26 (31%) 11/26 (42%)
Days to relapse after TCZ stop n/a 56 (36–88) 140 (109–196) n/a 0.0003#
Sex F:M ratio (% F) 13:13 (50%) 5:2 (71%) 4:4 (50%) 4:7 (36%) 0.35
Age at TCZ start (years) 70.0 (43.8–89) 61.9 (55.6–89.0) 71.7 (43.8–80.4) 69.4 (54.3–81.9) 0.88
GCA/PMR†/TAK (n) 23/2/1 7/0/0 6/1/1 10/1/0
Polymyalgia at diagnosis 17/26 (65%) 5/7 (71%) 5/8 (63%) 7/11 (64%) 0.67
Cranial ischaemic symptoms at diagnosis* 11/26 (42%) 2/7 (28%) 5/8 (71%) 5/11 (46%) 0.42
Hb (g/L) at diagnosis 117 (91–162) 113 (91–115) 126 (108–162) 120 (95–154) 0.11
CRP (mg/L) at diagnosis 105 (9.2–295) 143 (56–285) 65 (32.5–204) 46 (9.2–295) 0.11
ESR (mm/h) at diagnosis 78 (8–110) 80 (70–98) 80 (8–110) 63 (10–92) 0.15
Platelet count (×10^9/L) at diagnosis 378 (223–697) 398 (277–602) 399 (236–697) 287(223–457) 0.08
Biopsy-proven GCA (n (%)) 12/18 (67%) 4/6 (67%) 4/5 (80%) 4/7 (57%) 0.71
LVV in CDU or PET 21/26 (81%) 6/7 (86%) 6/8 (75%) 9/11 (82%) 0.54
Disease duration before current TCZ (days) 225.5 (8–1582) 247 (8–1081) 198 (42–1582) 222 (25–1008) 0.96
Relapses (n) before TCZ 1 (0–4) 1 (0–3) 1 (0–4) 1 (1–2) 0.46
TCZ infusions received before stop (n) 10 (4–25) 9 (5–15) 9.5 (6–24) 13 (4–25) 0.80
PDN (mg/day) at TCZ start 17.5 (0–100) 15 (0–50) 40 (5–80) 20 (0–100) 0.26
PDN (mg/day) at TCZ stop 5 (0–20) 5 (0–7.5) 5 (0–20) 2.5 (0–5) 0.12
Previous therapy
Previously having had a TCZ cycle 5/26 (19%) 1/7 (14%) 2/8 (25%) 2/11 (18%) 0.87
Previously having had MTX therapy 8/26 (31%) 3/7 (43%) 3/8 (38%) 2/11 (18%) 0.48
The three outcome groups were compared by Kruskal-Wallis with Dunn’s correction, except for (#) where a non-parametric t-test was used to compare the two relapse groups.*Amaurosis fugax (2-2-0), anterior ischemic optic neuropathy (AION) (2-1-1), stroke (0-0-0) or jaw claudication (4-2-5).†PMR=isolated PMR without GCA.CDU, color Doppler ultrasound; CRP, C reactive protein; ESR, erythrocyte sedimentation rate; GCA, giant cell arteritis; LVV, large-vessel vasculitis; MTX, methotrexate; PDN, prednisone; PMR, polymyalgia rheumatica; TAK, Takayasu arteritis; TCZ, tocilizumab; n/a, not available.
Serial IL-6 measurements in patients with tocilizumab-treated large-vessel vasculitis detect infections and may predict early relapses
Tocilizumab (TCZ) has been approved for giant cell arteritis (GCA). Interleukin-6 (IL-6) receptor blockade suppresses clin-ical disease and is steroid sparing.1 2 Since IL-6 induces the acute-phase response, the clinically used inflammation markers (C reactive protein (CRP), erythrocyte sedimentation rate (ESR)) are suppressed during TCZ treatment. Whether serum IL-6 is useful in monitoring disease activity and detecting infections in TCZ-treated GCA is unknown.
We longitudinally measured IL-6 in 23 patients with intrave-nous TCZ–treated GCA, two patients with polymyalgia rheu-matica and one patient with Takayasu arteritis of our GCA cohort (EKBB-239/09), and in 13/26 patients additionally before TCZ treatment. Patient characteristics are shown in table 1. At each visit, clinical and laboratory parameters (white blood cell (WBC), CRP, ESR) were assessed. Relapse was defined as the need for treatment intensification following new or increasing symptoms, or rising CRP/ESR not otherwise explained.2
During TCZ therapy, CRP, ESR and WBC rapidly declined and were sustainably suppressed. IL-6 levels rose and remained elevated throughout TCZ treatment (figure 1). Daily predni-sone doses were successfully tapered after TCZ was initiated (figure 1C). During 23.8 patient-years of TCZ therapy, eight
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0 90 180
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Figure 1 Longitudinal interleukin-6 (IL-6) levels during tocilizumab (TCZ) therapy. IL-6 serum levels were measured immediately before TCZ infusion and at the time of a clinically suspected infection or giant cell arteritis (GCA) relapse during therapy. IL-6 was determined using an electrochemiluminescence method (Elecsys). (A) IL-6, C reactive protein (CRP), erythrocyte sedimentation rate (ESR) and white blood cell (WBC) levels are shown immediately before the first versus the second dose of TCZ (n=13 for IL-6, n=26 for CRP, ESR and WBC). (B) IL-6 levels depending on the therapy duration is shown. The TCZ doses are grouped as indicated. (C) TCZ therapy allowed rapid tapering of the daily prednisone dose. (D) during documented infections (‘@infection’), IL-6 increased in all subjects compared with the mean of the previous two IL-6 measurements in the same patient (‘before’). CRP and ESR remained suppressed in infections. WBC increased variably. (E) PET/CT image showing pleuritis (arrows) in the patient with viral pleuropericarditis. (F) Subjects who displayed a clear decline (slope ≤−0.1) relapsed later (146 vs 67 days to relapse). Wilcoxon signed-rank (A and D), Kruskall-Wallis with Dunn’s correction (B and C), and linear regression was used, respectively. **p<0.01, ***p<0.001, ****p<0.0001. FU, follow-up.
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infections were documented with available IL-6 levels (online supplementary table S1). IL-6, but not CRP, ESR or WBC, was significantly higher in all infections compared with the mean of the last two previous measurements (p=0.0078; median 3.1-fold increase (1.6–16.6-fold)) (figure 1D). The highest measured IL-6 was 1409 pg/mL in a suspected viral pleuropericarditis (figure 1E).
We observed eight relapses during therapy. IL-6 levels were inconsistently elevated, and CRP was normal with one excep-tion (online supplementary table S2 and online supplementary figure S2). In addition to IL-6 increases during infections and some relapses, we observed ‘IL-6 spikes’ associated with a spinal synovial cyst (n=1), influenza vaccination (n=1), and in four asymptomatic patients with GCA without any specific event. Hence, in total, 14/18 (78%) of patients with IL-6 increases had a clinically plausible cause (online supplementary figure S3). In the remaining, subclinical disease or masked viral infections are potential explanations.3
We next tested if longitudinal IL-6 dynamics during therapy (median 11.5 IL-6 measurements per patient) associate with relapses after TCZ stop. TCZ was stopped in all patients. Follow-up was available for a median of 7.1 months (range 1–20.1 months). At 90 days and 180 days of follow-up after TCZ stop, 73% and 42% patients remained relapse-free, respec-tively (figure 1F). No severe, ischaemic relapse occurred, and remission could be achieved by re-installation of TCZ or pred-nisone treatment (online supplementary table S3). Women were slightly over-represented in the group with early relapses (table 1). The last measured IL-6 value was comparable between the groups (median 38.3 vs 36.1 vs 39.8 pg/mL for no, early and late relapse). In 13/26 subjects, serum IL-6 declined during treat-ment (online supplementary figure S4), which was associated with low risk for early relapses (<90 days after last infusion). Consequently, relapse-free survival was longer in those with an IL-6 decline (median 146 vs 67 days, p=0.02) (figure 1F).
In rheumatoid arthritis, IL-6 <30 pg/mL after 12 weeks predicted remission after 52 weeks.4 5 In contrast, others reported a link between lower serum IL-6 at TCZ stop and subsequent GCA flares.3 Our study suggests that longitudinal IL-6 measure-ments rather than absolute IL-6 levels before treatment cessa-tion may predict remission in TCZ-treated GCA. Limitations to our study include the lack of IL-6 measurements in all patients before TCZ start, the lack of TCZ level measurements due to sample availability, the lack of consequent imaging at relapse or IL-6 ‘spikes’, the focus on medically attended infections only and the inclusion of predominantly relapsing patients.
An increase in IL-6 during TCZ therapy should trigger a careful assessment for infection or disease activity. Absence of a longitudinal IL-6 decline during TCZ therapy may be indicative of ongoing subclinical disease activity and a risk for early relapse after treatment cessation. The specificity and sensitivity of IL-6 for these outcomes need to be defined in a prospective study.
Christoph T Berger,1,2 Birke Rebholz-Chaves,3 Mike Recher,1,4 Tobias Manigold,3 Thomas Daikeler3
1Clinical Immunology, Medical Outpatient Clinic, University Hospital Basel, Basel, Switzerland2Translational Immunology, Department of Biomedicine, University of Basel, Basel, Switzerland3Department of Rheumatology, University Hospital Basel, Basel, Switzerland4Immunodeficiency Lab, Department of Biomedicine, Basel University Hospital, Basel, Switzerland
Correspondence to Dr Christoph T Berger, Departmentof Internal Medicine, University Hospital Basel, Basel 4031, Switzerland; Christoph. Berger@ usb. ch and Dr Thomas Daikeler, Department of Rheumatology, University Hospital Basel, Basel, Switzerland; thomas. daikeler@ usb. ch
Handling editor Josef S Smolen
Acknowledgements We thank Christian Schindler from the Swiss Tropical and Public Health Institute, Basel, Switzerland for support with statistical analysis, the nursing staff from the medical outpatient clinic, and Silke Purschke and the Clinical Trial Unit team for administrative support.
Contributors CTB collected the data, analysed data and drafted the manuscript. BR-C collected the data, analysed data and drafted the manuscript. MR has analysed data and contributed to the manuscript writing. TM recruited patients and contributed to the manuscript writing. TD designed the study, analysed data and drafted the manuscript.
Funding This work was funded by the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (grant no. SNSF PZ00P3-173517) and the ’Margot und Erich Goldschmidt & Peter René Jacobson-Stiftung’ (both to CTB). MR is supported by a professorship of the Swiss National Science Foundation (PP00P3_173186).
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement All data are available as online supplementary data.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
CTB and BR-C contributed equally.
To cite Berger CT, Rebholz-Chaves B, Recher M, et al. Ann Rheum Dis 2019;78:1012–1014.
Received 6 November 2018Revised 4 December 2018Accepted 10 December 2018Published Online First 22 January 2019
Ann Rheum Dis 2019;78:1012–1014. doi:10.1136/annrheumdis-2018-214704
RefeRences 1 Villiger PM, Adler S, Kuchen S, et al. Tocilizumab for induction and maintenance
of remission in giant cell arteritis: a phase 2, randomised, double-blind, placebo-controlled trial. The Lancet 2016;387:1921–7.
2 Stone JH, Tuckwell K, Dimonaco S, et al. Trial of tocilizumab in giant-cell arteritis. N Engl J Med 2017;377:317–28.
3 Gloor AD, Yerly D, Adler S, et al. Immuno-monitoring reveals an extended subclinical disease activity in tocilizumab-treated giant cell arteritis. Rheumatology 2018;57:1795–801.
4 Aizu M, Mizushima I, Nakazaki S. Changes in serum interleukin-6 levels as possible predictor of efficacy of tocilizumab treatment in rheumatoid arthritis. Mod Rheumatol 2017;24:592–8. doi:.
5 Nishimoto N, Amano K, Hirabayashi Y, et al. Drug free REmission/low disease activity after cessation of tocilizumab (Actemra) Monotherapy (DREAM) study. Mod Rheumatol 2014;24:17–25.
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To DAPSA or not to DAPSA? That is not the question
In a recent publication in the Annals of the Rheumatic Diseases, van Mens et al have provided an analysis of different criteria for assessment of remission and low disease activity (LDA) in psori-atic arthritis (PsA).1 We would like to congratulate the authors for a thoughtful and thorough analysis of various disease activity measures in PsA and for providing the readers with interesting data. As so often, interpretations of data and conclusions may be seen diversely from different angles and we take the liberty to interpreting the data differently than the authors themselves.
The authors have evaluated remission (REM) and LDA by the disease activity in psoriatic arthritis (DAPSA), clinical disease activity in psoriatic arthritis (cDAPSA), minimal disease activity (MDA), psoriatic arthritis disease activity score (PASDAS) and very low disease activity (VLDA) for residual disease activity. These scores differ on two accounts: first, MDA and VLDA are solely state measures and, therefore, do not provide information on the actual level of disease activity, while DAPSA, cDAPSA and PASDAS are continuous measures that allow following patients’ disease activity through their whole disease history and define disease activity states, including remission and LDA; second, the philosophy behind DAPSA and cDAPSA on the one hand and MDA, PASDAS and VLDA on the other hand differs, since the latter scores comprise many aspects of psoriatic disease (PsoD) and thus include skin involvement, enthesitis, dactylitis and others, while the former focuses solely on the joint involvement of PsA, leaving the other variables of PsoD to being assessed by respective, widely validated separate scoring systems rather than lumping them together into one score. Interestingly, during the development of composite measures for PsoD, the authors them-selves apparently disagree on how these should be constructed, since the components included differ among them, as can be seen in a recently summative presentation of these scores.2
Given that MDA, PASDAS and VLDA have in common that they comprise various aspects of PsoD, it was surprising to see that MDA and VLDA correlated better with DAPSA/cDAPSA REM and LDA than with the more closely related PASDAS-REM. Of note, while a LDA state for PASDAS has been defined,3 the authors did not show these data in relation to DAPSA and MDA. It would be important for the readers of the Journal if the authors could convey these data in their response. Of note, PASDAS was already affected with stringency issues in remis-sion, allowing for almost 10% of the patients to have 4–7 tender joints, a situation hardly compatible with remission of arthritis. These data confirm and support the decision provided in recent recommendations to apply primarily DAPSA as a continuous and state measure of disease activity and MDA as a state measure.2
As elaborated in previous discussions,2 4 the difference between a holistic, multidimensional measure of disease activity, such as the PASDAS and MDA, and unidimensional measures, such as DAPSA or psoriasis area and severity index (PASI), has nothing to do with neglecting important aspects of disease. The authors imply this by stating that ‘residual skin disease was highest in patients achieving DAPSA or cDAPSA remission…’. However, this is a misinterpretation of the DAPSA construct. The DAPSA is a measure for PsA and thus arthritis, not for PsoD. Since one has to use the PASI or body surface area (BSA) anyway to calculate a score like PASDAS or a pure state like MDA/VLDA, one can refrain from throwing it into a composite tool and such vali-dated skin score separately to see if psoriasis is still active or not, irrespective of whether patients are in DAPSA remission or
not. Physicians will interpret results from psoriasis and arthritis separately, in fact, they are forced to do this, as the therapeutic implications of disease activity in the two domains are clearly different.5 6 While we are permanently discussing how proper outcomes assessment is limited by physician time, the ‘first lump, then separate’-strategy is not apt, at least in clinical practice. In trials, although trial endpoints should also be useful to clinical practice, we may care less.
Do we really? In fact, this multidimensional approach has already been proven to be unreliable in a previous analysis. In the PRESTA trial, the efficacy of 50 mg vs 100 mg etanercept weekly was compared in patients with PsA who also had significant psoriatic skin involvement.7 8 In the original trial,7 the 100 mg dose showed better efficacy in reducing skin involvement, but not even a hint of higher efficacy in arthritis, enthesitis and dactylitis. The data on musculoskeletal disease were obtained by the traditional unidi-mensional measures, namely ACR response rates, dactylitis scores and enthesitis scores. However, when a multidimensional measure for PsA was used,8 there was a significant difference between the doses, which was not seen when DAPSA was employed. If regula-tors would have allowed such measure to be used as an outcome for PsA, the implication would have been to use 100 mg etanercept weekly in all patients with PsA, while the difference observed was only based on different response of cutaneous involvement. Thus, the use of unidimensional scores, as done in the original PRESTA trial, saved society lots of costs and individual patients were spared from the risk of more adverse events that might have been seen at the higher dose.7 In contrast, the use of multidimensional measures that assess PsoD, not PsA, would have led to wrong conclusions. To only assume that lower proportions, like for VLDA compared with DAPSA-REM are more ‘stringent’ might be a semantically wrong conclusion— stringency may relate to external types of validity, such as progression of joint damage here (which was not assessed in the study, but non-progression was seen by us in DAPSA-REM).9
Finally, for rheumatologists, the main focus is arthritis and for that the DAPSA serves them well in daily practice. Indeed, in DAPSA remission all other musculoskeletal complaints are gone or almost gone, as also shown by the authors (no dactylitis in 100% and no enthesitis in 96% of patients in DAPSA REM), confirming our own results.10
Thus, in summary, the data presented showing the very low proportion of patients achieving VLDA imply that treatment for PsA would have to be adapted because of residual skin disease, which might put patients at risk and society at increased costs. In contrast, the high frequency of DAPSA remission reveals that the patients do very well for their PsA under the treatment provided by the authors and that some may be in need for addi-tional local or systemic therapy for other components of their PsoD.
Monika M Schoels, Josef S Smolen, Daniel Aletaha
Department of Rheumatology, Medical University of Vienna, Vienna, Austria
Correspondence to Dr Monika M Schoels, Medical University of Vienna, Vienna 1090, Austria; monika. schoels@ meduniwien. ac. at
Contributors All authors have equally contributed to this e-Letter.
Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent Not required.
Provenance and peer review Not commissioned; internally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
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To cite Schoels MM, Smolen JS, Aletaha D. Ann Rheum Dis 2019;78:e61.
Received 9 April 2018Accepted 19 April 2018Published Online First 5 May 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213618
Ann Rheum Dis 2019;78:e61. doi:10.1136/annrheumdis-2018-213548
RefeRences 1 van Mens LJJ, van de Sande MGH, van Kuijk AWR, et al. Ideal target for psoriatic
arthritis? Comparison of remission and low disease activity states in a real-life cohort. Ann Rheum Dis 2018;77:251–7.
2 Smolen JS, Schöls M, Braun J, et al. Treating axial spondyloarthritis and peripheral spondyloarthritis, especially psoriatic arthritis, to target: 2017 update of recommendations by an international task force. Ann Rheum Dis 2018;77:3–17.
3 Helliwell PS, FitzGerald O, Fransen J. Composite disease activity and responder indices for psoriatic arthritis: a report from the GRAPPA 2013 meeting on development of cutoffs for both disease activity states and response. J Rheumatol 2014;41:1212–7.
4 Schoels MM, Aletaha D, Smolen JS. Defining remission and treatment success using the DAPSA score: response to letter by Helliwell and Coates. Ann Rheum Dis 2015;74:e67.
5 Coates LC, Kavanaugh A, Mease PJ, et al. Group for Research and Assessment of Psoriasis and Psoriatic Arthritis 2015 Treatment Recommendations for Psoriatic Arthritis. Arthritis Rheumatol 2016;68:1060–71.
6 Gossec L, Smolen JS, Ramiro S, et al. European League Against Rheumatism (EULAR) recommendations for the management of psoriatic arthritis with pharmacological therapies: 2015 update. Ann Rheum Dis 2016;75:499–510.
7 Sterry W, Ortonne JP, Kirkham B, et al. Comparison of two etanercept regimens for treatment of psoriasis and psoriatic arthritis: PRESTA randomised double blind multicentre trial. BMJ 2010;340:c147.
8 FitzGerald O, Helliwell P, Mease P, et al. Application of composite disease activity scores in psoriatic arthritis to the PRESTA data set. Ann Rheum Dis 2012;71:358–62.
9 Aletaha D, Alasti F, Smolen JS. Disease activity states of the DAPSA, a psoriatic arthritis specific instrument, are valid against functional status and structural progression. Ann Rheum Dis 2017;76:418–21.
10 Smolen J, Aletaha D, Gladman D, et al. Outcomes associatded with achievement of various treatment targets in patients with psoriatic arthritis receiving adalimumab. Ann Rheum Dis 2017;76(Suppl 2):677.
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Response to: 'To DAPSA or not to DAPSA? That is not the question' by Schoels et al
We thank the authors for the interest in our paper and are grateful for the opportunity to respond to the points raised.1 We agree that there is a clear distinction between composite measures of psoriatic arthritis such as disease activity in PsA (DAPSA) and composite measures of psoriatic disease such as minimal or very low disease activity (MDA/VLDA) and the PsA disease activity score (PASDAS). As the Vienna group rightly point out, these measures differ in terms of the components included, but not due to disagreement within the outcome measure community as suggested in the letter. The choice of components for each composite measure was decided using different methodology in the development of each one, thus resulting in different measures. We believe that this variation in scores is one reason for the need to compare such scores in different populations to establish the optimal measure or measures for psoriatic arthritis (PsA). Indeed when the DAPSA was originally suggested, it was because the same components used in the disease activity in reac-tive arthritis (DAREA) were identified in a principal component analysis (PCA) in PsA. Interestingly in this analysis, the variables tested were taken from the Outcome mesaures in Rheumatology (OMERACT) PsA domains and therefore DAPSA was not, a priori, designed specifically to be a unidimensional composite measure. The fourth component identified in the PCA was the psoriasis area and severity index (PASI) highlighting the impor-tance of skin in PsA despite the fact that patients had a low base-line mean PASI of only 3.3. While PASI was not included in the DAPSA as the eigenvalue was 0.949 and therefore just under the threshold of 1,2 it is interesting to imagine how the results may have differed if it were developed in a group with slightly more active skin disease.
We agree with and highlighted the issues of residual disease activity in PASDAS remission within the paper. This perhaps relates to the number of measures included allowing one component to
be high and the overall score to be in the remission range. It seems unlikely that PASDAS in its current form would be adopted into routine clinical practice but it has shown excellent discrimination in clinical trials with consistently superior effect sizes highlighting its potential use in this setting. As requested, we have performed further analysis on the PASDAS low disease activity (LDA) (≤3.2) state in our dataset. A total of 151/227 or 66.5% of patients met the criteria (23 had missing data prohibiting calculation). This is a similar proportion to MDA where the agreement between defini-tions was highest (kappa 0.638). However, agreement was reason-able with both c/DAPSA LDA (0.517 and 0.504, respectively). For example, 143 patients were in DAPSA and PASDAS LDA, 7 were in PASDAS LDA but not DAPSA and 38 were in DAPSA LDA but not PASDAS showing overlapping but slightly different definitions. While PASDAS includes different domains of PsA, it is relatively specific to musculoskeletal disease and was designed as a unidimensional measure based on treatment changes in PsA (using similar methodology to the disease activity score (DAS) in rheumatoid arthritis). It does not include a measure of psoriasis. The levels of residual disease activity were similar or slightly higher than the other definitions (we attach an updated table 1 from the paper here), particularly for skin psoriasis where 46% of patients had a PASI>1. This further highlights the need to assess psoriasis either within (MDA) or additional to (PASDAS/DAPSA) any target chosen.
We also agree with the point that stringency does not just relate to proportions achieving the outcomes but to the external validity of these outcomes. MDA/VLDA, PASDAS and DAPSA have all been shown to be associated with radiographic damage in previous studies.3–6 Unfortunately it was not possible to confirm in this cross-sectional study without radiographs. We did, however, use quality of life as an external validity marker which again high-lighted the need to assess skin disease with poorer quality of life seen in patients in DAPSA remission with a PASI>1.7
As regards the PRESTA trial, we believe that this study highlights the need to choose an appropriate outcome measure to answer
Correspondence response
Table 1 Residual disease activity in patient reaching low disease activity or MDA
DAPSA LDA (195)
cDAPSA LDA (195)
MDA5/7 (162)
MDA joints (117)
MDA skin (120)
MDA skin and joints (83)
PASDAS LDA (151)
Swollen joint count, N (%)
0 143 (74) 143 (73) 126 (78) 101 (86) 92 (77) 78 (94) 115 (75)
1–3 48 (25) 48 (25) 33 (20) 16 (14) 26 (22) 5 (6) 32 (21)
4–6 4 (2) 4 (2) 3 (2) 0 (0) 2 (2) 0 (0) 6 (4)
Tender joint count, N (%)
0 110 (56) 111 (56) 103 (64) 106 (91) 76 (63) 75 (90) 90 (59)
1–3 56 (29) 56 (29) 39 (24) 11 (9) 28 (23) 8 (10) 36 (24)
4–7 19 (10) 19 (10) 12 (7) 0 (0) 12 (10) 0 (0) 15 (10)
8+ 6 (3) 5 (3) 6 (4) 0 (0) 4 (3) 0 (0) 10 (7)
Enthesitis count, N (%)
0 186 (95) 187 (96) 157 (97) 112 (96) 116 (97) 79 (95) 148 (98)
1–2 7 (4) 6 (3) 4 (2) 4 (3) 3 (2) 3 (4) 0 (0)
3–4 2 (1) 2 (1) 1 (1) 1 (1) 1 (1) 1 (1) 3 (2)
Dactylitis count
0 195 (100) 195 (100) 162 (100) 117 (100) 120 (100) 83 (100) 151 (100)
PASI, N (%)
0–1 136 (70) 136 (70) 120 (74) 83 (71) 120 (100) 83 (100) 108 (54)
>1 59 (30) 59 (30) 42 (26) 34 (29) 0 (0) 0 (0) 43 (46)
CRP (Normal<5 mg/dL), N (%)
Raised 22 (11) 22 (11) 18 (11) 12 (10) 15 (13) 10 (12) 16 (11)
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Correspondence response
the question set by each individual study. The PRESTA trial was unusual as it recruited patients with severe psoriasis and PsA. In such a population, a composite measure such as the composite psoriatic disease activity index (CPDAI) may be appropriate to establish the overall improvement in psoriatic disease. While there was no difference in musculoskeletal outcomes, which would have been shown by secondary unidimensional outcomes, the CPDAI results would have highlighted the importance of considering a dose or treatment that is optimal in all domains. Thus, it may have allowed regulators to support the use of a higher dose in patients that were similar to the target population of the trial, that is, patients with severe skin and joint involvement. This more person-alised and variable approach to treatment is now seen in the use of secukinumab where patients with PsA are routinely treated with 150 mg but for those with active skin disease, the 300 mg dose is recommended by both Food and Drug Administration (FDA) and European Medicines Agency (EMA).
We also agree that for rheumatologists the principal focus of treatment is the peripheral arthritis; however, we remain concerned that this is to the detriment of optimising therapy for patients. Many studies, in addition to ours, have high-lighted the importance of active skin disease and its associa-tion with poorer quality of life for the patient. While we do not expect rheumatologists to specialise in the management of psoriasis, an awareness of skin disease and basic assessment using tools such as the body surface as included in MDA would support appropriate comanagement of patients with PsA to optimise their quality of life.
We also recognise the pressure on clinicians’ time but do not see how this differs between the measures proposed in the new T2T recommendations. Whether the VLDA/MDA criteria are assessed, or the DAPSA plus a separate assessment of skin and enthesitis are performed, the time taken is the same. MDA is a modular measure where the items are not ‘lumped’ into one numerical score but assessed individually with a cut point to achieve for each thus allowing appropriate treatment choices. With DAPSA plus individual measures, the DAPSA could be calculated and then the skin/enthesitis would have to be assessed separately although clear guidelines on measures or targets for this are not defined. Thus, the time for clinician assessment is identical and both scores are quick and feasible to calculate/assess. To further support busy clinicians, the Group for Research and Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) have recently developed a free ‘app’ for both Apple and Android that includes an MDA calculator allowing entry of patient reported outcomes and physician assessments to produce an easy to inter-pret summary of disease activity by domain (figure 1).
Providing additional domains are assessed alongside the DAPSA, opinion of disease activity should be similar and appropriate treatment is likely to be the same. The modular approach of VLDA/MDA allows clear visualisation of which domains are active to guide therapy. If patients do not achieve VLDA due to ongoing psoriasis activity, this does not mandate immediate escalation of biological therapy to risk patient safety or societal costs. This could simply suggest the need for concomitant topical therapy as highlighted by the authors with the DAPSA example given.
While there have been strong views on the positives and negatives of each target suggested in the recommendations, it is important to remember that our shared goal is to ensure that patients are assessed more appropriately, both in clinical trials and in routine practice allowing optimisation of treat-ment using a treat to target approach. We believe that encour-aging physicians to briefly assess skin disease and other MSK domains in addition to the focus on peripheral arthritis is key to therapeutic optimisation in PsA given our goal to ‘to opti-mise long-term health-related quality of life and social partici-pation through control of signs and symptoms’.8
Leonieke J J van Mens,1 Marleen G H van de Sande,1 Arno W R van Kuijk,2 Dominique L P Baeten,1 Laura C Coates3
1Clinical Immunology and Rheumatology, Amsterdam Rheumatology and Immunology Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands2Rheumatology, Amsterdam Rheumatology and immunology Center, Reade, Amsterdam, The Netherlands3Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK
Correspondence to Dr Laura C Coates, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX1 2JD, UK; lauraccoates@ gmail. com
Handling editor Gerd R Burmester
Contributors This reply was written by LCC and LJJvM and reviewed by the other authors prior to submission.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent Not required.
Provenance and peer review Commissioned; internally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
Figure 1 Screenshot from the GRAPPA app MDA calculator. GRAPPA, Group for Research and Assessment of Psoriasis and Psoriatic Arthritis; HAQ, health assessment questionnaire; VAS, visual analogue scale.
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To cite van Mens LJJ, van de Sande MGH, van Kuijk AWR, et al. Ann Rheum Dis 2019;78:e62.
Received 30 April 2018Accepted 1 May 2018Published Online First 18 May 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213548
Ann Rheum Dis 2019;78:e62. doi:10.1136/annrheumdis-2018-213618
RefeRences 1 Schoels M, Smolen J, Aletaha D. To DAPSA or not to DAPSA? That is not the question.
Ann Rheum Dis 2019;78:e61.
2. Nell-Duxneuner VP, Stamm TA, Machold KP, et al. Evaluation of the appropriateness of composite disease activity measures for assessment of psoriatic arthritis. Ann Rheum Dis 2010;69:546–9.
3 Coates LC, Cook R, Lee KA, et al. Frequency, predictors, and prognosis of sustained minimal disease activity in an observational psoriatic arthritis cohort. Arthritis Care Res 2010;62:970–6.
4 Coates LC, Helliwell PS. Validation of minimal disease activity criteria for psoriatic arthritis using interventional trial data. Arthritis Care Res 2010;62:965–9.
5 Helliwell PS, Kavanaugh A. Radiographic progression in psoriatic arthritis achieving a good response to treatment: data using newer composite indices of disease activity. Arthritis Care Res 2018;70:797–800.
6 Aletaha D, Alasti F, Smolen JS. Disease activity states of the DAPSA, a psoriatic arthritis specific instrument, are valid against functional status and structural progression. Ann Rheum Dis 2017;76:418–21.
7 van Mens LJJ, van de Sande MGH, van Kuijk AWR, et al. Ideal target for psoriatic arthritis? Comparison of remission and low disease activity states in a real-life cohort. Ann Rheum Dis 2018;77:251–7.
8 Smolen JS, Schöls M, Braun J, et al. Treating axial spondyloarthritis and peripheral spondyloarthritis, especially psoriatic arthritis, to target: 2017 update of recommendations by an international task force. Ann Rheum Dis 2018;77:3–17.
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Infrapatellar fat pad resection during total knee arthroplasty: yet another reason for?
There has been an interesting series of commentaries based on the initial paper by Pan et al.1–4 I note with interest the suggestion by Binks et al4 that to preserve the infrapatellar fat pad (IPFP) and perform later arthroscopic resection on a case by case basis may be a viable option for management of anterior knee pain (AKP) after total knee arthroplasty (TKA). This is contrary to the solution offered by Han et al3 to use a prospective MRI to screen patients prior to TKA with subsequent resection based on signal changes within the IPFP. This approach would however, prove difficult in the resource-con-strained environment of most primary care facilities.
Current arguments in support of preserving the IPFP are largely based around the incidence of postoperative AKP.5 6 One area of conflicting evidence is the incidence of patella tendon shortening, also known as patella baja, after resection. Recent studies have shown no significant effect on postresection patella tendon length.7 8 This suggests that postresection patella tendon length is unlikely to be a major factor when considering to resect the IPFP or not.
The most common argument for IPFP resection is the increased surgical access to the knee. It is widely accepted that the most important aspect of knee arthroplasty is the correct fitting of components.9 Only with correctly fitting components can normal function be attained. In order to achieve this ‘correct fit’ the IPFP is often resected. Although this is a logical argument, there is no objective evidence in support of this.
The study by Sekiya et al10 showed that in patients with AKP following resection there was a significant proportion with scar tissue deep to the tendon. So perhaps it is insufficient resection and not the decision to remove the IPFP that results in AKP. And with recent studies showing a lack of patella tendon shortening post IPFP resection, limiting one argument for its preservation, this may be more accepted in the future.
Thomas J Ryan
Correspondence to Dr Thomas J Ryan, Department of Orthopaedics, John Hunter Hospital, Newcastle, NSW 2305, Australia; Tom- ryan@ hotmail. com
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent Not required.
Provenance and peer review Not commissioned; internally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
To cite Ryan TJ. Ann Rheum Dis 2019;78:e63.
Accepted 2 May 2018Published Online First 15 May 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213701
Ann Rheum Dis 2019;78:e63. doi:10.1136/annrheumdis-2018-213684
RefeRences 1 Pan F, Han W, Wang X, et al. A longitudinal study of the association between
infrapatellar fat pad maximal area and changes in knee symptoms and structure in older adults. Ann Rheum Dis 2015;74:1818–24.
2 White LD, Melhuish TM. The role of infrapatellar fat pad resection in total knee arthroplasty. Ann Rheum Dis 2016;75:e66.
3 Han W, Pan F, Liu Z, et al. Response to: ’The role of infrapatellar fat pad resection in total knee arthroplasty’ by White et al. Ann Rheum Dis 2016;75:e67.
4 Binks MJ, Vlok R, Holyoak R, et al. Preoperative MRI to plan infrapatellar fat pad resection during total knee arthroplasty. Ann Rheum Dis 2017;76:e34.
5 White L, Holyoak R, Sant J, et al. The effect of infrapatellar fat pad resection on outcomes post-total knee arthroplasty: a systematic review. Arch Orthop Trauma Surg 2016;136:701–8.
6 Duan G, Liu C, Lin W, et al. Different factors conduct anterior knee pain following primary total knee arthroplasty: a systematic review and meta-analysis. J Arthroplasty 2018.
7 Sellars H, Yewlett A, Trickett R, et al. Should we resect hoffa’s fat pad during total knee replacement? J Knee Surg 2017;30:894–7.
8 İmren Y, Dedeoğlu SS, Çakar M, et al. Infrapatellar fat pad excision during total knee arthroplasty did not alter the patellar tendon length: a 5-year follow-up study. J Knee Surg 2017;30:479–83.
9 White L, Stockwell T, Hartnell N, et al. Factors preventing kneeling in a group of pre-educated patients post total knee arthroplasty. J Orthop Traumatol 2016;17:333–8.
10 Sekiya H, Takatoku K, Takada H, et al. Painful knee after total knee arthroplasty is not a frequent complication and could be treated by arthroscopic debridement. Bone Joint J 2016;98(Suppl 3):152.
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Response to: ‘Infrapatellar fat pad resection during total knee replacement: yet another reason?’ by Ryan
We thank Ryan1 for the interest in our original paper2 and response3 regarding to the associations between infrapatellar fat pad (IPFP) maximal area and knee osteoarthritic measures including symptoms and structural changes.
Our previous studies revealed that IPFP maximal area and volume were associated with reduced knee pain, decreased loss of cartilage volume and reduced risks of cartilage defect progression, indicating a beneficial effect of IPFP size.2 4 On the other hand, our further investigation demonstrated that IPFP signal intensity alteration was negatively associated with maximum area of IPFP and, moreover, associated with increased knee cartilage defects, subchondral bone marrow lesions and knee pain, suggesting IPFP with abnormal quality may play a detrimental role in knee osteoarthritis (OA).5
Based on these findings, we proposed that IPFP with normal qualities, rather than abnormal quality, should be preserved or not damaged during total knee arthroplasty (TKA), while IPFP with abnormal quality should be resected rather than preserved. Because of the biphasic effects of IPFP on OA, a proposed solution for improving patient outcomes after TKA involves using screening MRIs to identify IPFP signal inten-sity alteration, which is a subsequent indication for resec-tion during TKA surgeries.3 This approach is not ‘contrary’ to approach offered by Sekiya,6 which suggested the use of postarthroplasty arthroscopic IPFP debridement. Moreover, the underlying rationales of two solutions are similar, despite different timing. Han et al5 also suggested that IPFP screening has the potential to reduce reoperation and perhaps reduce morbidity of severe knee pain post-TKA beyond 2%.
Patellar tendon shortening could be one of the reasons for post-TKA anterior knee pain as findings from Gwyn et al’s retrospective study7 reported that complete IPFP resec-tion was associated with patellar tendon shortening 1 year after TKA. The authors had noted that studies also reporting IPFP excision during TKA did not alter the patellar tendon length.8 9 Despite the inconclusive effect of IPFP resection on patellar tendon shortening, the protective effect of normal IPFP on knee may be largely attributed to buffering and lubri-cating functions in knee joints, which absorb shocks from anterior knee and reduce friction between patellar tendon and tibia.10 11
The authors agree that correct fitting of components is critical for favourable TKA outcomes. Traditionally, the IPFP has been removed in order to improve surgical exposure and to prevent interposition during baseplate implantation.12 Despite the significant evolution of TKA technology that no longer requires the resection of IPFP for better surgical access, IPFP is still partially or totally resected in around 88% of TKAs13 regardless of IPFP quality. Thus, evidence-based changes of practice towards preoperative IPFP evaluation and subsequent indication for resection or preservation are eagerly needed.
As for whether insufficient debridement of IPFP can cause anterior knee pain, our original cohort study was not able to provide an answer. We acknowledge that our findings, along with findings from our recent articles,4 5 were observational and there is not enough evidence to support preservation or resection of IPFP during TKA for patients with OA at very
late stage. So far, there is no evidence to support the assump-tion that it is insufficient debridement, not the decision to debride the IPFP, that results in anterior knee pain.
In summary, our previous observational studies have suggested biphasic effects of IPFP on OA. In order to imple-ment evidence-based changes of practice, well-designed randomised controlled trials are urgently required to test this hypothesis: preserve IPFP with normal quality and resect IPFP with pathological changes showing on MRI (signal intensity alterations) during TKA may have beneficial effects on patients’ postsurgical outcomes.
Zhaohua Zhu,1 Weiyu Han,2 Guangfeng Ruan,1,3 Shuang Zheng,4 Changhai Ding1,3,4
1Clinical Research Center, Zhujiang Hospital, Southern Medical University, Guangzhou, China2Department of Orthopedics, Zhujiang Hospital, Southern Medical University, Guangzhou, China3Department of Rheumatology and Immunology, Arthritis Research Institute, The First Affiliated Hospital of Anhui Medical University, Hefei, China4Menzies Institute for Medical Research, University of Tasmania, Hobart, Australia
Correspondence to Dr Changhai Ding, Clinical Research Centre, Zhujiang Hospital, Southern Medical University, Guangzhou, China; Changhai. Ding@ utas. edu. au
Handling editor Josef S Smolen
Contributors ZZ and CD had full access to all of the data in the study and take responsibility for the integrity and accuracy of the content. Study design: ZZ, WH, CD. Acquisition of data: ZZ, WH, GR, SZ and CD. Analysis and interpretation of data: ZZ, WH, GR, SZ and CD. Manuscript preparation: ZZ, WH, GR, SZ and CD.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Commissioned; internally peer reviewed.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
To cite Zhu Z, Han W, Ruan G, et al. Ann Rheum Dis 2019;78:e64.
Received 11 June 2018Accepted 11 June 2018Published Online First 27 June 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213684
Ann Rheum Dis 2019;78:e64. doi:10.1136/annrheumdis-2018-213701
RefeRences 1 Ryan TJ. Infrapatellar fat pad resection during total knee arthroplasty: yet another
reason for? Ann Rheum Dis 2019;78:e63. 2 Pan F, Han W, Wang X, et al. A longitudinal study of the association between
infrapatellar fat pad maximal area and changes in knee symptoms and structure in older adults. Ann Rheum Dis 2015;74:1818–24.
3 Han W, Pan F, Liu Z, et al. Response to: ’The role of infrapatellar fat pad resection in total knee arthroplasty’ by White et al. Ann Rheum Dis 2016;75:e67–E67.
4 Cai J, Xu J, Wang K, et al. Association between infrapatellar fat pad volume and knee structural changes in patients with knee osteoarthritis. J Rheumatol 2015;42:1878–84.
5 Han W, Aitken D, Zhu Z, et al. Signal intensity alteration in the infrapatellar fat pad at baseline for the prediction of knee symptoms and structure in older adults: a cohort study. Ann Rheum Dis 2016;75:1783–8.
6 Sekiya H. Painful knee is not uncommon after total knee arthroplasty and can be treated by arthroscopic debridement. Open Orthop J 2017;11:1147–53.
7 Gwyn R, Kotwal RS, Holt MD, et al. Complete excision of the infrapatellar fat pad is associated with patellar tendon shortening after primary total knee arthroplasty. Eur J Orthop Surg Traumatol 2016;26:545–9.
8 İmren Y, Dedeoğlu SS, Çakar M, et al. Infrapatellar fat pad excision during total knee arthroplasty did not alter the patellar tendon length: a 5-year follow-up study. J Knee Surg 2017;30:479–83.
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9 Sellars H, Yewlett A, Trickett R, et al. Should we resect Hoffa’s fat pad during total knee replacement? J Knee Surg 2017;30:894–7.
10 Yusuf E, Nelissen RG, Ioan-Facsinay A, et al. Association between weight or body mass index and hand osteoarthritis: a systematic review. Ann Rheum Dis 2010;69:761–5.
11 Chang J, Liao Z, Lu M, et al. Systemic and local adipose tissue in knee osteoarthritis. Osteoarthritis Cartilage 2018:864–71.
12 Van Beeck A, Clockaerts S, Somville J, et al. Does infrapatellar fat pad resection in total knee arthroplasty impair clinical outcome? A systematic review. Knee 2013;20:226–31.
13 Baker PN, van der Meulen JH, Lewsey J, et al. The role of pain and function in determining patient satisfaction after total knee replacement. Data from the National Joint Registry for England and Wales. J Bone Joint Surg Br 2007;89:893.
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Serum complement factor C5a in IgG4-related disease
We read with great interest the correspondence by Umehara et al1 which discussed the difficulties in the diagnosis of IgG4-re-lated disease (IgG4-RD). They mention that increased IgG4 concentration is not a specific marker for IgG4-RD. There-fore, the markers for the diagnosis and the disease activity of IgG4-RD are demanded.
Approximately a quarter of patients with active IgG4-RD have hypocomplementaemia defined by the lower normal limit of C3 or C4 levels.2 Complement pathways consist of three pathways: clas-sical, alternative and lectin pathways.3 Here, we focus on comple-ment factors as makers of IgG4-RD and attempt to evaluate the entire complement system in IgG4-RD.
We enrolled 28 patients with active and untreated IgG4-RD, diagnosed based on the 2011 comprehensive diagnostic criteria4 and 28 sex-matched and age-matched healthy donors. We evaluated the characteristics of patients with IgG4-RD, the number of affected organs and the IgG4-RD Responder Index (IgG4-RD RI).5 None of the healthy donors had a history of inflammatory disease.
In the sera of the patients with IgG4-RD and the healthy donors, we measured the levels of complement factors, C1q, C2, C3, C3b/iC3b, C4, C4b, C5, C5a, C9, factor D, factor I, mannose-binding lectin (MBL), factor B, factor H and properdin, using the MILLIPLEX MAP Human Comple-ment Panel 1 and Panel 2 (Merck Millipore, Darmstadt, Germany). Written informed consent was obtained from all patients and donors.
Table 1 summarises the characteristics of patients with IgG4-RD. The patients with IgG4-RD at diagnosis had significantly higher levels of C5 and C5a (figure 1A) and significantly lower levels of C4, C4b and factor D compared with the healthy donors (online supplementary table 1). C5a had a significant correlation with C3 (figure 1B) but not with C4 (figure 1C).
The levels of C5a in remission were significantly changed compared with the levels of C5a at diagnosis (16 305 pg/mL to 10 030 pg/mL, p=0.0043) (figure 1H). Other complement factors showed no significant change between the levels at diagnosis and the levels in remission (figure 1D–G,1I).
We divided the patients with IgG4-RD into two groups by the median of C5a: high-C5a and low-C5a groups. The high-C5a group had significantly higher IgG (figure 1J) and IgG4 (figure 1K) levels compared with the low-C5a group, but the IgG4/IgG ratio was not significantly different (figure 1L). The high-C5a group also had significantly higher levels of soluble IL-2 receptor (figure 1M). There were no significant differences in the number of affected organs or in the IgG4-RD RI (figure 1N,O).
Our finding that C5a is negatively correlated with C3 may indicate that patients with hypocomplementaemia as shown in a clinical study2 have higher C5a levels. In addition, the absence of a correlation between C4 and C5a levels suggests that it is not appropriate to bracket C3 and C4 as the same component of hypocomplementaemia.
C5a has a strong inflammatory potency through binding to C5a receptor (C5aR).6 Considering the reduced C5a levels observed in patients with IgG4-RD in remission, C5a-C5aR-targeted drugs may be effective against IgG4-RD. With the success of the C5 inhibitor eculizumab for parox-ysmal nocturnal haemoglobinuria7 and the progression of
a number of novel C5a-C5aR1-targeted drugs including the C5aR1 antagonist avacopan for antineutrophilic cyto-plasmic antibody (ANCA)-associated vasculitis,8 comple-ment therapeutics have shown great promise.9
Based on the high serum circulating immune complex in autoimmune pancreatitis, the classical pathway is thought to be involved in IgG4-RD.10 Our findings of decreased C4 and C4b and normal MBL levels in the patients are compatible with this supposition. However, because IgG4 is ineffective at activating complement (in contrast to IgG1 and IgG3),
11 the mechanisms underlying complement activation remain a mystery. A limitation of our study is that we are unable to answer this question based on our results.
In conclusion, the serum C5a level has been high in active disease and low in remission in IgG4-RD. Our data suggest that C5a may be a therapeutic target for IgG4-RD.
Correspondence
Table 1 Demographic, clinical and laboratory characteristics of patients with IgG4-RD
Clinical parameter Value
Age at diagnosis, years,median (IQR)
64 (56–73)
Females, n (%) 7 (25)
BMI, kg/m2 (IQR) 20.9 (19.3–23.0)
History
Allergy, n (%) 11 (39)
Asthma, n (%) 3 (11)
Organ involvement,n (%)
Lacrimal gland and orbit 10 (36)
Salivary gland 15 (54)
Lymph node 18 (64)
Thyroid 2 (7)
Lung 5 (18)
Pancreas 4 (14)
Bile duct 1 (4)
Kidney 4 (14)
Aorta 4 (14)
Prostate 2 (7)
Retroperitoneum 5 (18)
No of affected organs,median (IQR)
2 (1–3)
IgG4-RD Responder Index,median (IQR)
9 (6–15)
Laboratory test results,median (IQR)
White cells, ×109/μL 6.4 (5.4–7.4)
Eosinophils, % 5.0 (2.0–6.4)
Haemoglobin, g/dL 13.1 (12.1–14.5)
Platelets, ×109/μL 230 (195–260)
C-reactive protein, mg/dL 0.19 (0.05–0.77)
Erythrocyte sedimentation rate, mm/hour
41 (10–63)
Creatinine, mg/dL 0.83 (0.61–0.95)
Estimated GFR, mL/min/1.73 m2 71.9 (60.2–81.0)
Soluble IL-2 receptor, U/mL 584 (454–1261)
IgG, mg/dL 1985 (1494–3039)
IgG4, mg/dL 393 (175–876)
IgG4/IgG ratio, % 18.3 (11.1–34.0)
IgE, IU/mL 632 (203–1435)
BMI, body mass index; GFR, glomerular filtration rate; IgG4-RD: IgG4-related disease; IL-2, interleukin-2.
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Correspondence
Shoichi Fukui,1,2 Yuya Fujita,1 Tomoki Origuchi,1,3 Takahiro Maeda,2,4 Atsushi Kawakami1
1Department of Immunology and Rheumatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan2Department of Community Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan3Department of Rehabilitation Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan4Department of General Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
Correspondence to Dr Shoichi Fukui, Department of Community Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 8528501, Japan; fukui- ngs@ umin. ac. jp
Contributors All authors were involved in drafting this letter or revising it critically for important intellectual content, and all authors approved the final version for publication. SF had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent Obtained.
Provenance and peer review Not commissioned; internally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
Figure 1 The patients with IgG4-RD at diagnosis had significantly higher levels of C5a compared with the sex-matched and age-matched healthy donors (A). C5a had a significant correlation with C3 (B) but not with C4 (C). The level of C5a in remission was significantly changed compared with the level of C5a at diagnosis (H), but other complement factors showed no significant change (D–G, I). The high-C5a group had significantly higher IgG (J) and IgG4 (K) levels compared with the low-C5a group, but the IgG4/IgG ratio was not significantly different (L). The high-C5a group had significantly higher levels of soluble IL-2 receptor (M). There were no significant differences in the number of affected organs or in the IgG4-RD RI (N, O). IgG4-RD, IgG4-related disease; IgG4-RD RI, IgG4-RD Responder Index; IL-2, interleukin-2.
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Correspondence
► Additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213705).
To cite Fukui S, Fujita Y, Origuchi T, et al. Ann Rheum Dis 2019;78:e65.
Received 3 May 2018Accepted 6 May 2018Published Online First 6 June 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213729
Ann Rheum Dis 2019;78:e65. doi:10.1136/annrheumdis-2018-213705
RefeRences 1 Umehara H, Okazaki K, Kawano M, et al. How to diagnose IgG4-related disease. Ann
Rheum Dis 2017;76:e46.
2 Wallace ZS, Deshpande V, Mattoo H, et al. IgG4-related disease: clinical and laboratory features in one hundred twenty-five patients. Arthritis Rheumatol 2015;67:2466–75.
3 Holers VM. The complement system as a therapeutic target in autoimmunity. Clin Immunol 2003;107:140–51.
4 Umehara H, Okazaki K, Masaki Y, et al. Comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD), 2011. Mod Rheumatol 2012;22:21–30.
5 Carruthers MN, Stone JH, Deshpande V, et al. Development of an IgG4-RD responder index. Int J Rheumatol 2012;2012:1–7.
6 Guo RF, Ward PA. Role of C5a in inflammatory responses. Annu Rev Immunol 2005;23:821–52.
7 Brodsky RA, Young NS, Antonioli E, et al. Multicenter phase 3 study of the complement inhibitor eculizumab for the treatment of patients with paroxysmal nocturnal hemoglobinuria. Blood 2008;111:1840–7.
8 Jayne DRW, Bruchfeld AN, Harper L, et al. Randomized Trial of C5a Receptor Inhibitor Avacopan in ANCA-Associated Vasculitis. J Am Soc Nephrol 2017;28:2756–67.
9 Hawksworth OA, Li XX, Coulthard LG, et al. New concepts on the therapeutic control of complement anaphylatoxin receptors. Mol Immunol 2017;89:36–43.
10 Muraki T, Hamano H, Ochi Y, et al. Autoimmune pancreatitis and complement activation system. Pancreas 2006;32:16–21.
11 Kamisawa T, Zen Y, Pillai S, et al. IgG4-related disease. Lancet 2015;385:1460–71.
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Response to: ‘Serum complement factor C5a in IgG4-related disease’ by Fukui et al
We appreciate the interest shown by Dr S Fukui and colleagues1 in our recent paper, ‘How to diagnose IgG4-related disease (IgG4-RD)’.2
IgG4-RD is characterised by increased serum IgG4 concen-trations and number of IgG4-positive plasma cells in affected lesions.3 However, its diagnosis can be difficult, because clinical signs vary depending on the organs affected.
A Japanese team established comprehensive diagnostic criteria for IgG4-RD, 2011 focusing on the concentration of serum IgG4 (>135 mg/dL), numbers of IgG4-positive cells (>10 cells/HPF) and ratio of IgG4-positive to IgG-positive cells (>40%) in affected tissues.4
However, many cases of IgG4-RD with low levels of IgG4 and mimickers of IgG4-RD with increased serum IgG4 have been reported and the concept of IgG4-RD has become blurred. Therefore, new specific markers or new criteria for IgG4-RD are required.5
Human IgG4 has a unique structure distinguished from the other IgG subclasses by lack of interaction with the comple-ment system and inability to activate the classical complement pathway.6 7 However, hypocomplementaemia has been observed in IgG4-RD ever since the concept was first established8 and is in particular more frequent and sometimes severe in IgG4-related kidney disease.9 A recent study showed that non-IgG4 IgG such as IgG1, which can activate complement, might be involved in the activation of complement in IgG4-RD.10
Fukui et al reported the significant increase of C5a in IgG4-RD and suggested it for a therapeutic target.1 However, serum C5a elevation is also a very important feature of active systemic lupus erythematosus (SLE). Since elevated C5a levels in IgG4-RD are much lower than in active lupus cases11 and only some groups of patients with IgG4-RD have hypocomplementaemia, the production of C5a in SLE and IgG4-RD likely exhibits signifi-cant differences in both underlying mechanism and pathogenetic role.
Although the details of the pathogenesis of IgG4-RD are unclear at present, recent basic studies have implicated the toll-like receptor (TLR) signalling pathway.12 Activation of C5aR in macrophages inhibits TLR-4 signals resulting in reduction of IL-12, IL-23 and IL-27 production by inflammatory macro-phages.13 Thus, the role of cross-talk between innate and acquired immunity through the complement pathway and TLR system is attracting attention in the pathogenesis of IgG4-RD.14
Hisanori Umehara,1 Mitsuhiro Kawano2
1Division of RA and Autoimmune Diseases, Nagahama City Hospital, Shiga, Japan2Division of Rheumatology, Department of Internal Medicine, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan
Correspondence to Professor Hisanori Umehara, Division of RA and Autoimmune Diseases, Nagahama City Hospital, Shiga 526-8580, Japan; umehara606@ gmail. com
Handling editor Josef S Smolen
Competing interests None declared.
Patient consent Not required.
Provenance and peer review Commissioned; internally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
To cite Umehara H, Kawano M. Ann Rheum Dis 2019;78:e66.
Received 17 May 2018Accepted 17 May 2018Published Online First 6 June 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213705
Ann Rheum Dis 2019;78:e66. doi:10.1136/annrheumdis-2018-213729
RefeRences 1 Fukui S, Fujita Y, Origuchi T, et al. Serum complement factor C5a in IgG4-related
disease. Ann Rheum Dis 2018;78:e65. 2 Umehara H, Okazaki K, Kawano M, et al. How to diagnose IgG4-related disease. Ann
Rheum Dis 2017;76:e46. 3 Umehara H, Okazaki K, Masaki Y, et al. A novel clinical entity, IgG4-related disease
(IgG4RD): general concept and details. Mod Rheumatol 2012;22:1–14. 4 Umehara H, Okazaki K, Masaki Y, et al. Comprehensive diagnostic criteria for IgG4-
related disease (IgG4-RD), 2011. Mod Rheumatol 2012;22:21–30. 5 Umehara H, Okazaki K, Nakamura T, et al. Current approach to the diagnosis of
IgG4-related disease - Combination of comprehensive diagnostic and organ-specific criteria. Mod Rheumatol 2017;27:381–91.
6 Schuurman J, Perdok GJ, Gorter AD, et al. The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds. Mol Immunol 2001;38:1–8.
7 van der Neut Kolfschoten M, Schuurman J, Losen M, et al. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange. Science 2007;317:1554–7.
8 Masaki Y, Dong L, Kurose N, et al. Proposal for a new clinical entity, IgG4-positive multiorgan lymphoproliferative syndrome: analysis of 64 cases of IgG4-related disorders. Ann Rheum Dis 2009;68:1310–5.
9 Kawano M, Saeki T, Nakashima H, et al. Proposal for diagnostic criteria for IgG4-related kidney disease. Clin Exp Nephrol 2011;15:615–26.
10 Yamada K, Yamamoto M, Saeki T, et al. New clues to the nature of immunoglobulin G4-related disease: a retrospective Japanese multicenter study of baseline clinical features of 334 cases. Arthritis Res Ther 2017;19:262.
11 Sakuma Y, Nagai T, Yoshio T, et al. Differential activation mechanisms of serum C5a in lupus nephritis and neuropsychiatric systemic lupus erythematosus. Mod Rheumatol 2017;27:292–7.
12 Watanabe T, Yamashita K, Sakurai T, et al. Toll-like receptor activation in basophils contributes to the development of IgG4-related disease. J Gastroenterol 2013;48:247–53.
13 Hawlisch H, Belkaid Y, Baelder R, et al. C5a negatively regulates toll-like receptor 4-induced immune responses. Immunity 2005;22:415–26.
14 Umehara H, Nakajima A, Nakamura T, et al. IgG4-related disease and its pathogenesis-cross-talk between innate and acquired immunity. Int Immunol 2014;26:585–95.
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Drug-induced lupus erythematosus following immunotherapy with anti-programmed death-(ligand) 1
We read with interest the study of Kostine et al describing rheumatic immune-related adverse events (irAE), which occur in 6.6% of patients treated for cancer by anti-programmed death-(ligand) 1 (PDL1).1 These new adverse effects pose signif-icant challenges to patient care in terms of optimal management of these autoimmune damaging toxicities, while allowing effec-tive antitumor therapy to continue.
The PD(L)1 pathway is involved in the maintenance of immune tolerance, and the blockage of this axis by anticancer immunotherapy could trigger autoimmune diseases and espe-cially lupus.2 3 We then searched in the pharmacovigilance register of our institution—the ‘Registre des Effets Indésirables Sévères des Anticorps Monoclonaux Immunomodulateurs en Cancérologie (REISAMIC)’—whether cases of drug-induced lupus erythematosus (DI-LE) were reported following anti-PD(L)1 immunotherapies.
Between October 2013 and July 2017, five cases of DI-LE were recorded in REISAMIC. Given the number of patients having received anti-PD(L)1 during the same period (n=1044), the estimated incidence of DI-LE was 0.48%. All patients gave their written informed consent for the use of their data in this report. The patients’ characteristics are summarised in table 1. The patients had developed DI-LE at a median (range) age of 63 (48–80) years. None of the patients had a history of autoim-mune disease before starting anti-PD(L)1. The most specific sign of DI-LE was subacute cutaneous lupus erythematous (SCLE) in four patients and chilblain lupus in the remaining patient. One patient having SCLE had also declared a systemic lupus erythematosus (SLE) according to the Systemic Lupus Interna-tional Collaborating Clinics criteria.4 The DI-LE was revealed by a frank maculopapular rash in the four patients with SCLE (figure 1). The median time of DI-LE occurrence was 10 (range: 4–22) weeks after the initiation of immunotherapy. Antinuclear antibodies in serum were found positive for two (40%) out of the five patients and were specifically positive for anti-Sjögren’s syndrome-related antigen A (SSA). These two SSA-positive patients had SCLE but no eye or mouth dryness symptoms suggestive of Sjögren’s disease. A skin biopsy was performed in all cases except the chilblain lupus. The skin biopsies revealed a lymphocytic infiltrate of the dermis, predominantly around adnexal sites. Alcian blue staining revealed mucin deposits in all patients. Direct immunofluorescence assays for IgG or C3 in skin biopsy were positive in two of the four patients tested (50%). The treatment of DI-LE was based on topical cortico-steroids in all cases, with the antimalarial hydroxychloroquine added in the SLE case, and the outcome was favourable with a resolution in all cases.
This report is the first series of cases of lupus erythema-tosus induced by anti-PD(L)1 immunotherapy. A recent similar case report of pembrolizumab-related subacute cutaneous lupus erythematosus was provided.5 The DI-LE has been variously reported after drug exposure such as hydralazine, procainamide, quinidine, oestrogen, tumour necrosis factor inhibitors, chlor-promazine, isoniazid, practolol, penicillamine and minocycline.6 We believe that anti-PD(L)1 immunotherapies should also now be added to this list.
Based on our experience and the present case series, DI-LE induced by anti-PD(L)1 was characterised by an extensive,
non-itchy and frankly macular or papular erythematous rash. The DI-LE diagnosis relies on the combination of the dermato-logical presentation associated with pathological features char-acterised by a lymphocytic dermal infiltration predominantly located at periadnexal sites, and mucin deposits.7 The confron-tation between the clinical appearance and the pathological aspects is often useful to differentiate between DI-LE and other non-specific cutaneous irAEs, or other specific autoimmune skin diseases that can be induced by anti-PD(L)1 such as psoriasis, toxic epidermal necrolysis, lichen planus, bullous dermatitis and dermatomyositis.8
These new cases of lupus induced by anti-PD(L)1 should incite rheumatologist and internists to dedicate further prospective study for irAE. Investigation of potential biomarkers of irAEs such as the genetic background, serum
Correspondence
Figure 1 Photographs and histologic assessment of skin biopsies of cutaneous lupus erythematosus lesions induced by treatment with anti-PD(L)1. (A) Patient 4, erythematous papules and plaques with an annular, polycyclic configuration: generalised subacute lupus erythematous. (B) Patient 1, erythematous macules on the neck: subacute cutaneous lupus erythematosus. (C) Patient 2, symmetric papulosquamous erythematous rashes on the lower limbs. (D) Patient 3, erythematous macules and plaques on the back: subacute cutaneous lupus erythematosus. (E) Skin biopsy from patient 1, haematoxylin eosin saffron (HES) staining, ×2.5: peripheral and periadnexal monomorphic lymphocytic inflammatory infiltrate over the entire dermis. (F) Skin biopsy from patient 4, HES staining, ×5: lichenoid dermatosis with staged apoptotic bodies in the epidermis. Peripheral inflammatory mononuclear infiltrate in the upper dermis. (G) Skin biopsy from patient 3, Alcian blue staining, ×10: mucin deposits in the dermis.
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Tabl
e 1
Char
acte
ristic
s of
the
patie
nts
havi
ng d
evel
oped
DI-L
E fo
llow
ing
trea
tmen
t with
ant
i-PD(
L)1
imm
unot
hera
py
Gen
der,
age,
can
cer
hist
olog
yPr
evio
us c
ance
r tr
eatm
ents
Dru
gCa
usal
rel
atio
nshi
p
Tim
e to
oc
curr
ence
of
DI-L
E*
(in w
eeks
)D
I-LE
form
†Se
veri
ty
grad
e
His
topa
thol
ogic
al
char
acte
rist
ics
of a
ski
n bi
opsy
Dir
ect
imm
unofl
uore
scen
ce in
sk
in b
iops
yA
utoi
mm
une
biol
ogy
in s
erum
‡
Seru
m c
reat
ine
kina
se (n
orm
al
valu
e <
145
IU/L
)O
ther
irA
Es
T rea
tmen
t fo
r cu
tane
ous
lupu
s, an
d ou
tcom
eBe
st o
vera
ll an
titu
mou
r re
spon
se, a
nd r
eint
rodu
ctio
n of
PD
(L)1
(or
reas
on fo
r w
ithd
raw
al)
Patie
nt 1
, wom
an, 4
8 ye
ars
old,
trip
le-
nega
tive
brea
st
carc
inom
a
Farm
orub
icin
e-en
doxa
n-5-
fluor
oura
cil;
erib
ulin
, cap
ecita
bine
; ge
mci
tabi
ne
Atez
oliz
umab
Like
ly6
SCLE
Clin
ical
asp
ect a
nd
loca
tion:
ery
them
atos
us, n
eckl
ine
1In
flam
mat
ory
mon
omor
phic
ly
mph
ocyt
e in
filtr
ate
in
periv
ascu
lar a
nd p
eria
dnex
al
site
s th
roug
hout
the
derm
is.
Alci
an b
lue
stai
ning
reve
aled
m
ucin
dep
osits
in th
e de
rmis.
Posi
tive
for I
gG, l
inea
r, m
oder
ate,
ep
ider
mal
der
mal
junc
tion
Neg
ativ
e fo
r C3d
Neg
ativ
eTi
tre
for A
NA
nega
tive
(<1/
80),
aDN
A<10
, EN
A ne
gativ
e co
mpl
emen
t in
norm
al ra
nge
(C3=
1.73
; C4
=0.
26)
Nor
mal
(75
IU/L
)N
oTo
pica
l ste
roid
s, re
solu
tion
in
2 w
eeks
PR No
inte
rrup
tion
of a
tezo
lizum
ab
Patie
nt 2
, wom
an, 8
0 ye
ars
old,
diff
use
larg
e B-
cell
lym
phom
a
R-CH
OP;
R-G
EMOX
; R-
lena
lidom
ide
Niv
olum
abLi
kely
14SC
LECl
inic
al a
spec
t and
lo
catio
n: p
apul
osqu
amou
s er
ythe
mat
ous
in th
e lo
wer
lim
bs, s
ymm
etric
al
2In
flam
mat
ory
periv
ascu
lar
lym
phoc
ytic
infil
trat
e of
the
uppe
r and
mid
dle
derm
is.
Alci
an b
lue
stai
ning
reve
aled
m
ucin
dep
osits
.
Neg
ativ
eN
egat
ive
Titr
e fo
r AN
A<1/
80;
aDN
A<10
; EN
A ne
gativ
eLo
w c
ompl
emen
t C4
and
CH50
but
nor
mal
C3
(C3=
1.53
; C4<
0.02
; CH
50<
10)
Nor
mal
(28
IU/L
)Ye
s: he
patit
is
grad
e 2
Topi
cal s
tero
ids,
reso
lutio
n in
3
wee
ks
PD T em
pora
ry w
ithdr
awal
of i
mm
unot
hera
py d
ue to
lupu
s, an
d th
en d
efini
tive
with
draw
al o
f niv
olum
ab d
ue to
di
seas
e pr
ogre
ssio
n
Patie
nt 3
, wom
an,
66 y
ears
old
, ca
rcin
oma
epid
erm
oid
Radi
othe
rapy
; ca
rbop
latin
-cet
uxim
ab-
5-flu
orou
raci
l; pa
clita
xel-c
etux
imab
; m
etho
trex
ate-
carb
opla
tine
Niv
olum
abLi
kely
4SC
LECl
inic
al a
spec
t and
loca
tion:
er
ythe
mat
osus
on
the
trun
k, b
ack
and
face
2Pe
rivas
cula
r lym
phoc
ytic
in
filtr
ate
of th
e up
per d
erm
is
with
dis
cret
e va
cuol
isat
ion
of th
e ep
ider
mal
bas
al la
yer.
Alci
an b
lue
stai
ning
reve
aled
m
ucin
dep
osits
in th
e de
rmis.
Neg
ativ
ePo
sitiv
e w
ith
ANA+
and
SSA+
Titr
e fo
r AN
A=1/
640;
aD
NA<
10;
ENA=
1.5
wea
kly
posi
tive;
SSA
=94
U/
mL,
com
plem
ent i
n no
rmal
rang
e (C
3=1.
42;
C4=
0.2;
CH5
0 no
t do
ne)
Nor
mal
(30
IU/L
)N
oTo
pica
l ste
roid
s, re
solu
tion
in
2 w
eeks
SD Tem
pora
ry w
ithdr
awal
of i
mm
unot
hera
py d
ue to
lupu
s
Patie
nt 4
, man
, 63
year
s ol
d, m
elan
oma
Non
ePe
mbr
oliz
umab
Cert
ain
22SC
LECl
inic
al a
spec
t and
loca
tion:
gen
eral
ised
, cl
early
ery
them
atos
us, p
apul
ar a
nd
mac
ular
asp
ects
on
the
trun
k, b
ack,
ab
dom
en a
nd th
orax
Syst
emic
lupu
s er
ythe
mat
osus
(SLI
CC
crite
ria4 w
ere
SCLE
+ar
thra
lgia
+po
sitiv
e se
rum
ant
ibod
ies)
3Li
chen
oid
derm
atos
is w
ith
stag
ed a
popt
otic
bod
ies
in
the
epid
erm
is. P
erip
hera
l in
flam
mat
ory
mon
onuc
lear
in
filtr
ate
in th
e up
per d
erm
is.
Alci
an b
lue
stai
ning
did
not
re
veal
any
muc
in d
epos
its in
th
e de
rmis.
Posi
tive
for I
gG: d
isco
ntin
uous
lo
w-in
tens
ity b
and
and
epid
erm
al
derm
al ju
nctio
n. C
3d p
ositi
ve:
roug
h ba
nd
Posi
tive
with
SSA
+/
SSB+
Titr
e fo
r AN
A>1/
1280
(m
ottle
d as
pect
); aD
NA<
10;
ENA>
28 h
ighl
y po
sitiv
e w
ith S
SA>
241
U/m
L an
d SS
B=86
U/m
L (n
<7)
, com
plem
ent i
n no
rmal
rang
e (C
3=1.
07;
C4=
0.21
; CH5
0>60
)
Elev
ated
:23
8 IU
/L w
hen
lupu
s ap
pear
ed,
then
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Correspondence
levels of autoimmune factors and cytokines may help better understand these immunological adverse events and autoim-mune conditions in general.
Jean-Marie Michot,1,2 Mathilde Fusellier,1 Stephane Champiat,1,3 Charles Velter,4 Capucine Baldini,1,3 Anne-Laure Voisin,5 Francois-Xavier Danlos,1 Yolla El Dakdouki,1 Maxime Annereau,6 Xavier Mariette,7 Caroline Robert,4 Khadija Cherif,8 Aurélien Marabelle,1 Christine Mateus,4 Olivier Lambotte2,3,9,10
1Medical Oncology and Drug Development Department, Institut Gustave Roussy, Villejuif, France2Department of Internal Medicine and Clinical Immunology, Hôpital Bicêtre, Le Kremlin-Bicêtre, France3University of Paris Sud, Le Kremlin-Bicêtre, France4Department of Medical Oncology, Institut Gustave Roussy, Villejuif, France5Department of Pharmacovigilance, Institut Gustave Roussy, Villejuif, France6Department of Pharmacy, Institut Gustave Roussy, Villejuif, France7Department of Rheumatology, Hôpital Bicêtre, Le Kremlin-Bicêtre, France8Department of Biopathology, Institut Gustave Roussy, Villejuif, France9INSERM U1184, Immunology of Viral Infections and Autoimmune Diseases, Le Kremlin- Bicêtre, France10Commissariat à l’Energie Atomique (CEA), Fontenay-aux-Roses, France
Correspondence to Dr. Jean-Marie Michot, Drug Development Department, Institut Gustave Roussy, Villejuif F-94805, France; jean- marie. michot@ gustaveroussy. fr
Handling editor Josef S Smolen
Acknowledgements The authors thank David Fraser (Biotech Communication SARL, Ploudalmézeau, France) for copy-editing assistance.
Contributors All authors contributed to the patient care management and manuscript writing. All authors approved the manuscript submitted.
Competing interests None declared.
Patient consent Obtained.
Ethics approval Ethics Board Committee and Institutional Board of Institut Gustave Roussy.
Provenance and peer review Not commissioned; internally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
To cite Michot J-M, Fusellier M, Champiat S, et al. Ann Rheum Dis 2019;78:e67.
Received 29 April 2018Accepted 1 May 2018Published Online First 1 June 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213691
Ann Rheum Dis 2019;78:e67. doi:10.1136/annrheumdis-2018-213677
RefeRences 1 Kostine M, Rouxel L, Barnetche T, et al. Rheumatic disorders associated with immune
checkpoint inhibitors in patients with cancer-clinical aspects and relationship with tumour response: a single-centre prospective cohort study. Ann Rheum Dis 2018;77:393–8.
2 Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999;11:141–51.
3 Prokunina L, Castillejo-López C, Oberg F, et al. A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans. Nat Genet 2002;32:666–9.
4 Petri M, Orbai AM, Alarcón GS, et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 2012;64:2677–86.
5 Shao K, McGettigan S, Elenitsas R, et al. Lupus-like cutaneous reaction following pembrolizumab: An immune-related adverse event associated with anti-PD-1 therapy. J Cutan Pathol 2018;45:74–7.
6 Niklas K, Niklas AA, Majewski D, et al. Rheumatic diseases induced by drugs and environmental factors: the state-of-the-art - part two. Reumatologia 2016;54:165–9.
7 Obermoser G, Sontheimer RD, Zelger B. Overview of common, rare and atypical manifestations of cutaneous lupus erythematosus and histopathological correlates. Lupus 2010;19:1050–70.
8 Hofmann L, Forschner A, Loquai C, et al. Cutaneous, gastrointestinal, hepatic, endocrine, and renal side-effects of anti-PD-1 therapy. Eur J Cancer 2016;60:190–209.
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Checkpoint inhibitor-associated immune arthritis
We read with interest the paper by Kostine et al.1 Immune checkpoint inhibitors (ICIs) have dramatically improved clin-ical outcomes in various cancer types and are increasingly being used in earlier disease settings and in combination.2 The main targets of ICIs, programmed cell death 1 (PD-1) and its ligand PD-L1 as well as cytotoxic T-lymphocyte-as-sociated protein 4 (CTLA-4) are involved in downregulating autoimmunity, with PD-1-deficient C57BL/6 mice exhibiting lupus-like features and CTLA-4 enabling Treg suppression of autoreactive T cell activation.3 It was therefore no surprise that immune-related adverse events (irAEs) such as immune arthritis, rheumatoid arthritis or systemic lupus erythema-tosus have occurred from ICIs use. However, the characteris-tics, timing and outcomes of those irAEs in the setting of ICIs remain poorly known.4
Here, we used VigiBase (http://www. vigiaccess. org/), the WHO database of individual safety case reports, to identify 86 cases of immune arthritis (searching the preferred terms among the 36 973 adverse events reported following treatment with ICIs.5
Patients had a wide spectrum of age (median: 63 years (range: 40–90)), cancer types (mostly lung cancer (39.7%) and mela-noma (36.8%)) and geographic locations (table 1). Most patients (82.6%) received anti-PD-1 monotherapy, whereas 10.5% received a combination of anti-PD-1/PD-L1 and anti-CTLA-4.
Reported irAEs (table 1) included rheumatoid arthritis (68%), autoimmune arthritis (16%) or lupus (15%).
The median timing of irAEs onset in relation to ICI initiation was 2.7 (IQR: 0.5–7.3) months (available in 18 patients), with 50% occurring in the first 8 weeks of treatment. Further, several other irAEs occurred in 22 cases (25.6%), most commonly muscle disorder and colitis.
In general, grade 3 toxicities generally warrant6 suspension of ICIs and initiation of high-dose corticosteroids, while discon-tinuation of ICIs is recommended with grade 4 toxicities (with the exception of endocrinopathies). The detailed management of individual cases was not available in Vigibase. However, the main outcome of irAEs was available in 36 (41.9%) patients, with 26 (72.2%) patients recovering or having recovered at the time of the last evaluation. Among all cases, death occurred in 3 (4.1%).
Notably, we observed a dramatic increase in reporting inci-dence over time (n=45 (52.3%) in 2017 and 14 additional cases until 16 February 2018). We speculate that this is due to an increased usage of ICIs as well as a better recognition of these new associations with ICIs.
While irAEs have been reported as individual cases or short series, this analysis is the first comprehensive series to report its detailed clinical features on a larger scale, including the median delay between ICI treatment and irAEs.
The supplied data come from a variety of sources. The likelihood of a causal relationship is not the same in all reports. The informa-tion does not represent the opinion of the WHO.
Laurent Arnaud,1 Bénédicte Lebrun-Vignes,2 Joe-Elie Salem2
1Department of Rheumatology, Hôpitaux Universitaires de Strasbourg & INSERM UMRS-1109, Strasbourg, France2AP-HP, Pitié-Salpêtrière Hospital, Department ofPharmacology, CIC-1421, Pharmacovigilance Unit; INSERM, UMR ICAN 1166;Sorbonne Universités, UPMC Univ Paris 06, Faculty of Medicine; Institute ofCardiometabolism and Nutrition (ICAN), F-75013, Paris, France
Correspondence to Professor Laurent Arnaud, Department of Rheumatology Hôpitaux Universitaires de Strasbourg & INSERM UMRS-1109 Strasbourg France; laurent. arnaud@ chru- strasbourg. fr
Correspondence
Table 1 Detailed characteristics of the 86 casesCharacteristics N (%) Data availability, n (%)
Reporting year 86 (100%)
2018 (through 16 February 2018) 14 (16.3)
2017 45 (52.3)
2016 18 (20.9)
2015 6 (7.0)
2012–2014 3 (3.6)
Region reporting, n (%) 86 (100)
Americas 50 (58.1)
Europe 27 (31.4)
Australia 5 (5.8)
Asia 4 (4.6)
Africa 0 (0)
Reporters 81 (94.2)
Health professional 63 (77.7)
Non-health professional 18 (22.3)
Gender 77 (89.5)
Male 41 (53.2)
Female 36 (46.8)
Age at onset, median (IQR) 63.5 (55–76) 52 (60.4)
Drugs 86 (100)
Monotherapy with anti-PD1/PD-L1 71 (82.6)
Nivolumab 46 (53.5)
Pembrolizumab 20 (23.3)
Durvalumab 3 (3.5)
Atezolizumab 2 (2.3)
Monotherapy with anti-CTLA4 6 (7.0)
Ipilimumab 6 (7.0)
Combination therapy 9 (10.5)
Nivolumab+Ipilimumab 6 (7.0)
Pembrolizumab+Ipilimumab 3 (3.5)
Indications 68 (79.1)
Lung cancer 27 (39.7)
Malignant melanoma 25 (36.8)
Urothelial carcinoma 5 (7.4)
Renal carcinoma 4 (5.9)
Neoplasm malignant (tissue not specified) 3 (4.4)
Ductal adenocarcinoma of pancreas 1 (1.5)
Melanoma+Renal cell carcinoma 1 (1.5)
Oesophageal carcinoma 1 (1.5)
Squamous cell carcinoma of head and neck 1 (1.5)
Reported autoimmune diseases 86 (100)
Rheumatoid arthritis 59 (68.6)*
Autoimmune arthritis 14 (16.3)
Cutaneous lupus erythematosus 7 (8.1)
Systemic lupus erythematosus 5 (5.8)
Lupus erythematosus (unspecified type) 1 (1.2)
Median delay between treatment and autoimmune AE, median (IQR)
2.7 (0.5–7.3) months 18 (22.1)
Severity of AEs (severe) 73 (84.9) 86 (100)
Death 3 (4.1) 73 (84.9)
Concurrent immune-related adverse events 86 (100)
Muscle weakness/disorder 6 (7.0)
Colitis 5 (5.8)
Thyroid disease 4 (4.6)
Sjögren’s syndrome 1 (1.2)
Mixed connective tissue disease 1 (1.2)
Duodenitis 1 (1.2)
Oesophagitis 1 (1.2)
Enteritis 1 (1.2)
Hepatitis 1 (1.2)
Cold agglutinin disease 1 (1.2)
Autoimmune haemolytic anaemia 1 (1.2)
Myocarditis 1 (1.2)
Interstitial lung disease 1 (1.2)
Raynaud's phenomenon 1 (1.2)
Uveitis 1 (1.2)
Pleural effusion 1 (1.2)
Vitiligo 1 (1.2)
*Including 13 cases with aggravated rheumatoid arthritis.
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Contributors All authors analysed the data and wrote the manuscript.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent Not required.
Provenance and peer review Not commissioned; internally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
To cite Arnaud L, Lebrun-Vignes B, Salem J-E. Ann Rheum Dis 2019;78:e68.
Received 26 March 2018Revised 18 April 2018Accepted 19 April 2018Published Online First 3 May 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213691
Ann Rheum Dis 2019;78:e68. doi:10.1136/annrheumdis-2018-213470
REFERENCES 1 Kostine M, Rouxel L, Barnetche T, et al. Rheumatic disorders associated with immune
checkpoint inhibitors in patients with cancer-clinical aspects and relationship with tumour response: a single-centre prospective cohort study. Ann Rheum Dis 2018;77:393–8.
2 Weber J, Mandala M, Del Vecchio M, et al. Adjuvant Nivolumab versus Ipilimumab in Resected Stage III or IV Melanoma. N Engl J Med 2017; 377:1824–35.
3 Kong YC, Flynn JC. Opportunistic Autoimmune Disorders Potentiated by Immune-Checkpoint Inhibitors Anti-CTLA-4 and Anti-PD-1. Front Immunol 2014;5:206.
4 Moslehi JJ, Salem JE, Sosman JA, et al. Increased reporting of fatal immune checkpoint inhibitor-associated myocarditis. Lancet 2018;391:933.
5 Lindquist M. VigiBase, the WHO Global ICSR Database system: basic facts. Drug Inf J 2008;42:409–19.
6 Brahmer JR, Lacchetti C, Schneider BJ, et al. Management of immune-related adverse events in patients treated with immune checkpoint inhibitor therapy: American Society of Clinical Oncology clinical practice guideline. J Clin Oncol 2018:JCO.2017.77.638.
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Response to: ‘Drug-induced lupus erythematosus following immunotherapy with anti-programmed death-(ligand) 1’ by Michot et al and ‘Checkpoint inhibitor-associated immune arthritis’ by Arnaud et al
We thank Michot et al for their comments1 on our original paper entitled ‘Rheumatic disorders associated with immune checkpoint inhibitors in patients with cancer-clinical aspects and relationship with tumour response: a single-centre prospective cohort study’.2 We also appreciate the information added in the letter by Arnaud et al also reporting data from the WHO database.3
By adding this series of programmed death-(ligand) 1 (PD-(L)1)-induced lupus erythematosus, the authors contribute to our better knowledge of the wide spectrum of immune-re-lated adverse events (irAEs).
Indeed, since PD-1-deficient mice spontaneously devel-oped lupus-like autoimmune diseases with arthritis and glomerulonephritis,4 such clinical phenotypes could be expected in patients treated with anti PD-(L)1 agents. However, only a few cases of lupus-like cutaneous reaction have yet been reported with anti-PD-1 agents and, more surprisingly, one lupus-like nephritis has been attributed to anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) treatment.5 While the role of immune checkpoints is actively being investigated in murine lupus models,6 7 there is still a long road ahead to understand why some patients will experience lupus-like irAE with cancer immunotherapy. We must keep in mind that PD-1 knockout models do not reproduce an anti-PD-1 treatment as PD-1 knockout mice performed their thymic selection without PD-1 expression while patients receive anti-PD-1 treatment many years later, a long time after the major period of thymic selection. This reinforced the hypothesis of a possible genetic background (PD-1 gene polymorphisms) highlighted by Michot et al, to be investigated in patients presenting with irAEs in order to elucidate whether checkpoint inhibitors trigger specific disease on host genetic susceptibility or not. Furthermore, it would be worthwhile to know the baseline status for the two patients with antinuclear antibodies as the presence of anti-cyclic citrullinated peptide antibodies has been detected before immunotherapy in some patients experiencing PD-1-induced rheumatoid arthritis.8 Another hot research area concerns the role of commensal gut microbiota on the outcome of cancer immunotherapies, focusing mainly on efficacy. The concept that gut microbiota may modulate systemic autoimmunity is not new, and there is emerging evidence that dysbiosis participates in the complex patho-genesis of lupus.9 Thus, if microbiota can shape tumour response, its involvement (including viruses) in the patho-genesis of irAEs should be further investigated, as suggested by Chaput et al for colitis.10
PD-(L1)-induced autoimmune diseases have strongly renewed interest in assessing the role of immune checkpoints in classical autoimmune diseases, although we observed some differences in terms of demographics and an autoim-mune biology often negative. Co-stimulatory molecules have been tested in the treatment of systemic lupus erythematosus (SLE) such as CD154 and CTLA-4 Ig but trials failed to achieve their primary outcome.11 Despite disappointing results, the central role of co-stimulation between antigen-presenting cells
and T cells in the pathogenesis of lupus, as well as efficacy data of abatacept on articular and cutaneous manifestations,12 called for further evaluation. Recently, a fully human anti-body directed against the inducible T-cell co-stimulator ligand showed safety and potential efficacy in patients with SLE with arthritis.13
The next wave of immuno-oncology will include agonists of co-stimulatory signals, such as OX40 antibodies already being tested in clinical trials, which might also trigger lupus-like disease and other autoimmune conditions. As highlighted by Arnaud et al, the expanding use of cancer immunotherapies and the better recognition of irAEs result in higher reporting incidence of irAEs. Registers remain the best way to collect more accurate data on characteristics, management and outcomes of irAEs. Such as the European League Against Rheumatism study group for Registers and Observational Drug Studies on bDMARDs,14 European-wide collaboration across several local/nationwide registers will likely help to improve our management of rheumatic irAEs.
Marie Kostine, Christophe Richez, Thierry Schaeverbeke, on behalf of the FHU- ACRONIM
Department of Rheumatology, Centre Hospitalier Universitaire, Bordeaux, France
Correspondence to Dr Marie Kostine, Department of Rheumatology, Centre Hospitalier Universitaire, Bordeaux 33000, France; marie. kostine@ chu- bordeaux. fr
Handling editor Josef S Smolen
Collaborators FHU-ACRONIM: Aquitaine’s Care And Research Organisation For Inflammatory And Immune-Mediated Diseases.
Contributors MK, CR and TS: conception. MK drafted the manuscript. CR and TS critically reviewed and approved the final version of the manuscript.
Competing interests None declared.
Patient consent Not required.
Provenance and peer review Commissioned; internally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
To cite Kostine M, Richez C, Schaeverbeke T, et al. Ann Rheum Dis 2019;78:e69.
Received 26 May 2018Accepted 29 May 2018Published Online First 22 June 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213470 ► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213677
Ann Rheum Dis 2019;78:e69. doi:10.1136/annrheumdis-2018-213691
RefeRences 1 Michot JM, Fusellier M, Champiat S, et al. Drug-induced lupus erythematosus
following immunotherapy with anti-programmed death-(ligand) 1. Ann Rheum Dis 2019;78:e67.
2 Kostine M, Rouxel L, Barnetche T, et al. Rheumatic disorders associated with immune checkpoint inhibitors in patients with cancer-clinical aspects and relationship with tumour response: a single-centre prospective cohort study. Ann Rheum Dis 2018;77:393–8.
3 Arnaud L, Lebrun-Vignes B, Salem JE. Checkpoint inhibitor-associated immune arthritis. Ann Rheum Dis 2019;78:e68.
4 Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999;11:141–51.
5 Fadel F, El Karoui K, Knebelmann B. Anti-CTLA4 antibody-induced lupus nephritis. N Engl J Med 2009;361:211–2.
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6 Liao W, Zheng H, Wu S, et al. The systemic activation of programmed death 1-PD-L1 axis protects systemic lupus erythematosus model from nephritis. Am J Nephrol 2017;46:371–9.
7 Ceeraz S, Sergent PA, Plummer SF, et al. VISTA deficiency accelerates the development of fatal murine lupus nephritis. Arthritis Rheumatol 2017;69:814–25.
8 Belkhir R, Burel SL, Dunogeant L, et al. Rheumatoid arthritis and polymyalgia rheumatica occurring after immune checkpoint inhibitor treatment. Ann Rheum Dis 2017;76:1747–50.
9 Talotta R, Atzeni F, Ditto MC, et al. The microbiome in connective tissue diseases and vasculitides: an updated narrative review. J Immunol Res 2017;2017:1–11.
10 Chaput N, Lepage P, Coutzac C, et al. Baseline gut microbiota predicts clinical response and colitis in metastatic melanoma patients treated with ipilimumab. Ann Oncol 2017;28:1368–79.
11 Ceeraz S, Nowak EC, Burns CM, et al. Immune checkpoint receptors in regulating immune reactivity in rheumatic disease. Arthritis Res Ther 2014;16:469.
12 Danion F, Rosine N, Belkhir R, et al. Efficacy of abatacept in systemic lupus erythematosus: a retrospective analysis of 11 patients with refractory disease. Lupus 2016;25:1440–7.
13 Cheng LE, Amoura Z, Cheah B, et al. A randomized, double-blind, parallel-group, placebo-controlled, multiple-dose study to evaluate AMG 557 in patients with systemic lupus erythematosus and active lupus arthritis. Arthritis Rheumatol 2018.
14 Kearsley-Fleet L, Závada J, Hetland ML, et al. The EULAR study group for registers and observational drug studies: comparability of the patient case mix in the European biologic disease modifying anti-rheumatic drug registers. Rheumatology 2015;54:1074–9.
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Checkpoint inhibitor-induced polymyalgia rheumatica controlled by cobimetinib, a MEK 1/2 inhibitor
We read with great interest the recently published paper by Kostine et al1 reporting on musculoskeletal immune-related adverse events (IRAE) related to cancer immunotherapy. We applaud the authors for being able to identify the incidence of these side effects and of their relation to tumour response.
Of particular interest to us is the fact that most of the muscu-loskeletal IRAE were polymyalgia-like. We are eager to add to that discussion by presenting this case of checkpoint inhibitor-in-duced polymyalgia rheumatica (PMR) that was partially treated by targeted therapy with a MEK 1/2 inhibitor.
A 70-year-old woman presented in November 2015 with rectal cancer that was locally invasive and metastatic to peritoneum and lung. She failed chemotherapy (5-fluorouracil, leucovorin and irinotecan) and in September 2016 was enrolled in a trial of atezolizumab (anti-PD-L1) every 2 weeks plus cobimetinib (a MEK 1/2 inhibitor). Cobimetinib was given daily for 3 weeks each month followed by a 1-week break.
In March 2017, the patient developed shoulder and hip girdle pain accompanied by prolonged morning stiffness. A reduction in the dose of cobimetinib made her feel worse. Lyrica and Oxyco-done were added to her regimen without benefit. The patient noted that her symptoms were episodic, coming only during the week each month that she was off cobimetinib. Her symptoms would resolve promptly 2 days after she resumed cobimetinib.
The patient was referred to rheumatology in September 2017 and was thought to have checkpoint inhibitor-induced PMR. Laboratory evaluation performed when she was symptomatic (at the end of a week off of cobimetinib) revealed an erythrocyte sedimentation rate (ESR) 72. Because it appeared that her PMR was controlled by the MEK 1/2 inhibitor, prednisone 10 mg daily was added for only 5 days each month, starting on day 3 of the off-cobimetinib week. The patient did very well until January 2018 when her malignancy progressed. Atezolizumab and cobimetinib were discontinued; she was started on a different regimen. Following discontinuation of cobimetinib, her PMR symptoms recurred and she required daily prednisone.
Autoimmune diseases induced by checkpoint inhibitors may give insight into the pathogenesis of native autoimmune diseases such as PMR. This case of a patient with checkpoint inhibitor-in-duced PMR responding to a MEK 1/2 inhibitor suggests that there could be a role for MEK inhibition in the management of PMR.
The protein kinase cascade Raf/MEK/ERK regulates gene expression in response to stimuli such as extracellular mitogens, growth factors and cytokines. Downstream effects are important in cellular proliferation and apoptosis. Disordered activity of this pathway leads to uncontrolled tumour cell proliferation. The pathway has therefore become a useful therapeutic target in KRAS-mutated and BRAF-mutated cancers.
In addition to its role in cellular proliferation, the MEK/ERK cascade also plays a role in inflammatory responses. A number of studies have shown that the MEK/ERK cascade promotes the production of a variety of proinflammatory cytokines, and MEK inhibitors have been reported to impair T-lymphocyte function. For example, Vella et al demonstrated that the MEK inhibitor trame-tinib suppresses T-cell proliferation and cytokine production by CD4+ and CD8+ T cells.2 Dumont et al showed that the MEK-in-hibitor PD98059 inhibits, among other things, IL-6 production.3
In rheumatoid arthritis, the MEK/ERK pathway is upregu-lated in the synovial tissues.4 5 The MEK inhibitor PD184352 inhibited paw oedema and clinical arthritis scores and attenu-ated disease-induced weight loss in a collagen-induced arthritis mouse model.5 The MEK inhibitor JTP-74057 reduced TNF-α and interleukin 6 (IL-6) production in human, mouse and rat peripheral blood mononuclear cells and was equal to lefluno-mide in suppressing the development of both adjuvant-induced arthritis and collagen-induced arthritis mouse models.6
Patients with PMR have elevated circulating levels of IL-6; the anti-IL-6 receptor antibody tocilizumab has been used effectively in this condition. In our patient with checkpoint inhibitor-as-sociated PMR, treatment with the MEK inhibitor cobimetinib suppressed inflammation, perhaps via suppression of IL-6. This case suggests that MEK inhibitors could be a promising steroid-sparing agent for PMR.
Karmela Kim Chan, Anne R Bass
Department of Medicine, Division of Rheumatology, Hospital for Special Surgery, New York, New York, USA
Correspondence to Dr Karmela Kim Chan, Department of Medicine, Division of Rheumatology, Hospital for Special Surgery, New York, NY 10021, USA; chanka@ hss. edu
Contributors Both KKC and ARB have a research interest in checkpoint inhibitor-associated rheumatic adverse events. This particular patient is followed by KKC. KKC identified the material and wrote the first draft of the case. ARB reviewed the material and provided editing support.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent Not required.
Ethics approval HSS IRB.
Provenance and peer review Not commissioned; internally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
To cite Chan KK, Bass AR. Ann Rheum Dis 2019;78:e70.
Received 28 April 2018Accepted 30 April 2018Published Online First 14 May 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213682
Ann Rheum Dis 2019;78:e70. doi:10.1136/annrheumdis-2018-213672
RefeRences 1 Kostine M, Rouxel L, Barnetche T, et al. Rheumatic disorders associated with immune
checkpoint inhibitors in patients with cancer—clinical aspects and relationship with tumour response: a single-centre prospective cohort study. Ann Rheum Dis 2018;77:393–8.
2 Vella LJ, Pasam A, Dimopoulos N, et al. MEK inhibition, alone or in combination with BRAF inhibition, impairs multiple functions of isolated normal human lymphocytes and dendritic cells. J Immunother Cancer 2013;1(Suppl 1):P93.
3 Dumont FJ, Staruch MJ, Fischer P, et al. Inhibition of T cell activation by pharmacologic disruption of the MEK1/ERK MAP kinase or calcineurin signaling pathways results in differential modulation of cytokine production. J Immunol 1998;160:2579–89.
4 Schett G, Tohidast-Akrad M, Smolen JS, et al. Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum 2000;43:2501–12.
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5 Thiel MJ, Schaefer CJ, Lesch ME, et al. Central role of the MEK/ERK MAP kinase pathway in a mouse model of rheumatoid arthritis: potential proinflammatory mechanisms. Arthritis Rheum 2007;56:3347–57.
6 Yamaguchi T, Kakefuda R, Tanimoto A, et al. Suppressive effect of an orally active MEK1/2 inhibitor in two different animal models for rheumatoid arthritis: a comparison with leflunomide. Inflamm Res 2012;61:445–54.
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Response to: 'Checkpoint inhibitor-induced polymyalgia rheumatica controlled by cobimetinib, a MEK 1/2 inhibitor' by Chan and Bass
We read with genuine interest the original case reported by Chan and Bass1 in response to our paper describing musculo-skeletal immune-related adverse events (irAE) related to cancer immunotherapy.2
Immune checkpoint inhibitors (ICI) represent an exciting new standard of care in selected advanced cancers and are being actively investigated in others. Unfortunately, only a subset of patients benefit from ICI and the current challenge is to trans-form the majority of non-responders to responders.3 This will likely be improved by combining ICI with other treatment strat-egies such as small molecules.
Two MEK inhibitors, trametinib and cobimetinib, are already used in the clinical setting, combined with BRAF inhibitors.4 Toxicity (mainly rash, peripheral oedema, cardiac and ocular complications) is one of the obstacles for the development of MEK inhibitors as single agent in patients with cancer but the dual inhi-bition of MEK and BRAF minimises toxicity. Initial attempts of combining vemurafenib or dabrafenib (anti-BRAF) plus cobime-tinib or trametinib (anti-MEK) with ipilimumab (anti CTLA-4) failed due to toxicity. Combination or sequential therapy with anti-PD-(L)1 agents represents a more promising approach and several clinical trials are ongoing (ie, NCT03178851; NCT02908672; NCT02130466; NCT03201458). Therefore, the number of patients exposed to both MEK and PD-(L)1 inhibitors is likely to increase and we are looking forward to see whether more observa-tions of ICI-induced polymyalgia rheumatica (PMR) improved by MEK inhibitors will be reported in the future.
Considering MEK inhibitors as a promising steroid-sparing agent for PMR is a bit provocative, however behind every success story is a first enthusiastic clinical observation and the authors provided solid underlying pathogenesis. Furthermore, there will be always concern of using an anticancer therapy for a non-can-cerous disease but one good example comes from methotrexate, as illustrated with the historical perspective of this drug by Weinblatt.5 While the rheumatology community was first reluc-tant, it has now become the standard of care therapy in patients with rheumatoid arthritis. The observation reported by Chan and Bass suggests that, in ICI-induced PMR, MEK inhibitors could both reinforce the antitumour treatment and control the irAE. As oncologists have wondered whether immunosuppres-sive therapies given to treat irAEs may reduce the antitumour efficacy of ICI, MEK inhibitors could offer an attractive thera-peutic option for such adverse events. On the other hand, this particular situation should be a first step indicating that MEK
inhibitors might also represent a possible alternative in case of corticoresistant or corticodependent PMR. Let us see whether or not MEK inhibitors will also become part of our therapeutic arsenal in rheumatology.
Marie Kostine,1 Léa Dousset,2 Thierry Schaeverbeke,1 on behalf of the FHU-ACRONIM1Department of Rheumatology, Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France2Department of Dermatology, Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France
Correspondence to Dr Marie Kostine, Department of Rheumatology, Centre Hospitalier Universitaire de Bordeaux, Bordeaux 33000, France; marie. kostine@ chu- bordeaux. fr
Handling editor Josef S Smolen
Contributor FHU-ACRONIM: AQUITAINE’S CARE AND RESEARCH ORGANISATION FOR INFLAMMATORY AND IMMUNE-MEDIATED DISEASES
Contributors MK drafted the manuscript. LD and TS critically reviewed and approved the manuscript.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent Not required.
Provenance and peer review Commissioned; internally peer reviewed.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
To cite Kostine M, Dousset L, Schaeverbeke T, et al. Ann Rheum Dis 2019;78:e71.
Received 7 June 2018Accepted 8 June 2018Published Online First 20 June 2018
► http:// dx. doi. org/ 10. 1136/ annrheumdis- 2018- 213672
Ann Rheum Dis 2019;78:e71. doi:10.1136/annrheumdis-2018-213682
RefeRences 1 Chan KK, Bass AR. Checkpoint inhibitor-induced polymyalgia rheumatica controlled by
cobimetinib, a MEK 1/2 inhibitor. Ann Rheum Dis 2019;78:e70. 2 Kostine M, Rouxel L, Barnetche T, et al. Rheumatic disorders associated with immune
checkpoint inhibitors in patients with cancer-clinical aspects and relationship with tumour response: a single-centre prospective cohort study. Ann Rheum Dis 2018;77:393–8.
3 Sharma P, Allison JP. The future of immune checkpoint therapy. Science 2015;348:56–61.
4 Grimaldi AM, Simeone E, Festino L, et al. MEK inhibitors in the treatment of metastatic melanoma and solid tumors. Am J Clin Dermatol 2017;18:745–54.
5 Weinblatt ME. Methotrexate in rheumatoid arthritis: a quarter century of development. Trans Am Clin Climatol Assoc 2013;124:16–25.
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Tofacitinib in steroid-dependent relapsing polychondritis
Relapsing polychondritis (RP) is a rare progressive inflamma-tory condition involving cartilaginous structures, predominantly those of the ears, nose and laryngotracheobronchial tree. The McAdam’s diagnostic criteria require meeting three out of six of the following: recurrent chondritis of both auricles, noner-osive inflammatory polyarthritis, chondritis of nasal cartilages, ocular inflammation (conjunctivitis, keratitis, scleritis and/or uveitis), chondritis of respiratory tract, cochlear and/or vestib-ular damage (neurosensory hearing loss, tinnitus or vertigo).1 Systemic corticosteroids remain the mainstay of treatment for RP. Immunosuppressive agents (eg, methotrexate, azathioprine, dapsone, cyclophosphamide, etc) have been frequently used for the treatment of more severe and steroid-resistant manifesta-tions of RP. The administration of biologics in patients with RP has been also described.2 3
In the recently published French multicentre retrospec-tive cohort study, Moulis et al reported 41 patients with RP treated with 105 biological agents, including tumour necrosis factor (TNF) inhibitors, tocilizumab, anakinra, rituximab and abatacept.4 The reasons for initiating biological therapy were corticosteroid dependence, corticosteroid resistance or intol-erance to methotrexate. Within the first six months of treat-ment, overall response (ie, at least partial clinical improvement) rate was 62.9%. However, only 19.0% of patients achieved a complete response defined by no clinical activity. Response rates for TNF inhibitors, tocilizumab and rituximab (63.3%–71.4%) were numerically higher compared with those for abatacept and anakinra (50.0%–53.3%). The results of the retrospective study suggested differences in efficacies depending on the organ involvement, that is, tocilizumab and TNF inhibitors, particularly adalimumab, were the most effective biologics for nasal/auric-ular chondritides and joint inflammation, respectively. Notably, steroid-sparing effect was modest (median daily corticosteroid dose was reduced by 5 mg of prednisone equivalent), and nearly three-quarters of patients finally discontinued biologics due to insufficient efficacy (34.1%), loss of efficacy (18.1%) or adverse drug reactions (20.9%).
Tofacitinib belongs to a new class of the Janus kinase inhibitors. It is approved for the treatment of rheumatoid arthritis, psoriasis and psoriatic arthritis. Current evidence suggests that tofacitinib may be also effective in patients with the other inflammatory diseases, such as ulcerative colitis, atopic dermatitis and alopecia. We observed a 39-year-old female patient with a 6-year history of RP that manifested by progressive nasal chondritis with saddle nose
deformity, recurrent arthritis and scleritis, involvement of laryn-geal and tracheal cartilages (figure 1), low-grade fever and labo-ratory signs of inflammation (high erythrocyte sedimentation rate, C-reactive protein and rheumatoid factor). Diagnosis of RP was established according to the presence of four out of six McAdam’s criteria. Treatment with medium-dose (30 mg/day) prednisone was initially effective. For steroid tapering, methotrexate, azathioprine, mycophenolate mofetil and dapsone were administered sequen-tially. However, all immunomodulators were discontinued within a relatively short period of time as a result of adverse drug reac-tions (azathioprine and dapsone were stopped within 1 month due to anaemia and leucopenia) or persistent clinical and laboratory activity (methotrexate and mycophenolate mofetil were discon-tinued within 3–4 months due to clinical deterioration after pred-nisone tapering to 15 mg/day). Despite medium-dose to high-dose (up to 40 mg/day) corticosteroids, the disease showed a progressive course with recurrent episodes of acute respiratory distress and a persistent laboratory activity. The patient rejected treatment with any parenteral biologics. However, she gave an informed consent for an off-label oral tofacitinib administration. This agent was chosen given its action on the intracellular pathways responsible for triggering the inflammatory response. Treatment with tofacitinib 10 mg daily rapidly induced a clinical decrease in disease activity with corticosteroid tapering. At 12 months of treatment, she was in stable clinical remission and free from corticosteroids, while CT scanning showed an improvement of laryngeal wall thickening.
RP is an immune-mediated disease with poorly understood pathogenesis. Human and animal studies suggest that a complex cytokine network orchestrates the recruitment of infiltrating cells in RP lesions.5 Unlike biological treatments that target one cytokine pathway in the inflammatory network, tofacitinib inhibits the intracellular effects of several inflammatory media-tors, resulting in downregulation of the immune and inflamma-tory response.6
Moulis et al showed at least partial efficacy of biological therapy in a significant proportion of patients with refractory RP, despite low complete response rate and modest steroid-sparing effect. There was no single drug having apparent advan-tages in efficacy and safety over other biologics. However, infliximab and adalimumab should be preferred among the TNF antagonists. Tocilizumab was highly effective for almost all features of RP, but with a relative high rate of withdrawal due to adverse drug reactions. Our case report suggests that tofacitinib may constitute an additional therapeutic option for selected patients with RP. However, its efficacy and safety in RP versus non-biological or biological disease-modifying antirheumatic drugs should be confirmed in well-controlled prospective head-to-head studies.
Alexey D Meshkov,1 Pavel I Novikov,1 Evgeny V Zhilyaev,2 Ilia David J Ilevsky,3 Sergey V Moiseev1
1Tareev Clinic of Internal Diseases, Sechenov First Moscow State Medical University, Moscow, Russia2European Medical Center, Moscow, Russia3Municipal Polycinic #64, Moscow, Russia
Correspondence to Professor Sergey V Moiseev, Tareev Clinic of Internal Diseases, Sechenov First Moscow State Medical University Moscow Russia ; clinpharm@ mtu- net. ru
Handling editor Josef S Smolen
Contributors All authors participated in the preparation of the manuscript.
Competing interests None declared.
Patient consent Not required.
Correspondence
Figure 1 Uptake of tracer in laryngeal and tracheal cartilages on positron emission tomography/CT in patient with relapsing polychondritis.
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Provenance and peer review Not commissioned; internally peer reviewed.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
To cite Meshkov AD, Novikov PI, Zhilyaev EV, et al. Ann Rheum Dis 2019;78:e72.
Received 10 April 2018Revised 23 April 2018Accepted 24 April 2018Published Online First 3 May 2018
Ann Rheum Dis 2019;78:e72. doi:10.1136/annrheumdis-2018-213554
RefeRences 1 McAdam LP, O’Hanlan MA, Bluestone R, et al. Relapsing polychondritis: prospective
study of 23 patients and a review of the literature. Medicine 1976;55:193–215. 2 Leroux G, Costedoat-Chalumeau N, Brihaye B, et al. Treatment of relapsing
polychondritis with rituximab: a retrospective study of nine patients. Arthritis Rheum 2009;61:577–82.
3 Moulis G, Sailler L, Pugnet G, et al. Biologics in relapsing polychondritis: a case series. Clin Exp Rheumatol 2013;31:937–9.
4 Moulis G, Pugnet G, Costedoat-Chalumeau N, et al. Efficacy and safety of biologics in relapsing polychondritis: a French national multicentre study. Ann Rheum Dis 2018;77:1172–8.
5 Arnaud L, Mathian A, Haroche J, et al. Pathogenesis of relapsing polychondritis: a 2013 update. Autoimmun Rev 2014;13:90–5.
6 Hodge JA, Kawabata TT, Krishnaswami S, et al. The mechanism of action of tofacitinib - an oral Janus kinase inhibitor for the treatment of rheumatoid arthritis. Clin Exp Rheumatol 2016;34:318–28.
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Miscellaneous
Correction: ADA2 deficiency (DADA2) as an unrecognised cause of early onset polyarteritis nodosa and stroke: a multicentre national study
Caorsi R, Penco F, Grossi A, et al. ADA2 deficiency (DADA2) as an unrecognised cause of early onset polyarteritis nodosa and stroke: a multicentre national study. Ann of Rheum Dis 2017;76:1648–56. doi:10.1136/annrheumdis-2016–210802
The author’s name Romina Gallizi is incorrect and should be Romina Gallizzi.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
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