Clinical and Experimental Rheumatology 2021; 39: 879-882. BRIEF PAPER Rheumatoid arthritis ABSTRACT Early studies showed that patients with Objective. To examine the association RF-positive RA are characterised by autoantibodies and between individual rheumatoid arthri- lower age of diagnosis as compared to their association with tis (RA) autoantibodies, sex and age at RF-negative patients (2, 3). These stud- age and sex RA onset. ies used isotype-unspecific methods Methods. Anti-CCP2, IgA-, IgG- and based on agglutination or nephelom- E. Pertsinidou1, V.A. Manivel1, IgM-RF were analysed centrally in etry, which primarily, but not exclu- H. Westerlind2, L. Klareskog3,4, baseline sera from 1600 RA patients sively, detect IgM RF. Similarly, the L. Alfredsson5,6, L. Mathsson-Alm1,7, diagnosed within one year of RA symp- occurrence of ACPA has repeatedly 3 3,8 tom onset. Cut-offs for RF isotypes were been shown to associate with younger M. Hansson , S. Saevarsdottir , th J. Askling2, J. Rönnelid1 determined at the 98 percentile based age at RA diagnosis (4, 5). Although on RA-free controls, close to the 98.4% there is a strong co-occurrence of RF 1Department of Immunology, Genetics anti-CCP2 specificity. and ACPA in RA, no studies so far have and Pathology, Uppsala University, Results. Anti-CCP2 was found in 1020 investigated the association between Uppsala, Sweden; 2Clinical Epidemiology patients (64%), IgA RF in 692 (43%), individual RA autoantibodies and age Division, 3Rheumatology Unit, Department IgG RF in 529 (33%) and IgM RF in at RA diagnosis, nor the corresponding of Medicine Solna, Karolinska University 916 (57%) of the patients. When as- association with sex. Hospital, Karolinska Institute, Stockholm, sessed one by one, anti-CCP2 and IgM Although IgM RF is the most common Sweden; 4Section of Rheumatology, RF were both associated with lower age Gastro-enterology, Dermatology, Theme RF isotype among RA patients of Cau- Inflammation, Karolinska University at RA diagnosis. When assessed in one casian ancestry, other RF isotypes ap- Hospital, Stockholm, Sweden; 5Center joint model, the association to IgM RF pear to be more relevant in other geo- for Occupational and Environmental weakened and a strong association be- graphical regions. In Asian populations, Medicine, Region Stockholm, Stockholm, tween IgA RF and higher age at RA di- IgG RF is dominating, or as common 6 Sweden; Institute of Environmental agnosis appeared. IgA RF and IgG RF as IgM (reviewed in (6)), whereas IgA Medicine, Karolinska Institute, Stockholm, associated with male sex, and IgM RF RF showed the highest sensitivity in a Sweden; 7Thermo Fisher Scientific, with female sex, with no difference for Uppsala, Sweden; 8Faculty of Medicine, cross-sectional RA cohort from Khar- School of Health Sciences, University anti-CCP2. toum, Sudan (7). of Iceland, Reykjavik, Iceland. When the model was adjusted for sex, The 1987 ACR classification criteria Eleftheria Pertsinidou, MSc the association between IgM RF and state that RF should be measured “by Vivek Anand Manivel, PhD age disappeared, whereas the strong any method for which the result has Helga Westerlind, PhD associations between IgA RF and high been positive in <5% of normal con- Lars Klareskog, MD, PhD age and between anti-CCP2 and low trol subjects”, and no upper limit of Lars Alfredsson, PhD age at diagnosis remained. Further ad- specificity is set (1). No correspond- Linda Mathsson-Alm, PhD justments for smoking, shared epitope ing recommendations exist for ACPA, Monika Hansson, PhD Saedis Saevarsdottir, MD, PhD and inclusion year did not change the and the 2010 EULAR/ACR classifica- Johan Askling, MD, PhD outcome. Univariate analyses stratified tion criteria do not include any recom- Johan Rönnelid, MD, PhD on anti-CCP2 and IgA RF status con- mendations concerning autoantibody Please address correspondence to: firmed the findings. cut-offs. In practice, most laboratories Johan Rönnelid, Conclusion. Anti-CCP associate with use the cut-offs independently recom- Department of Immunology, low, and IgA RF with high age at RA mended by different manufacturers. Genetics and Pathology, onset. RFs and anti-CCP2 display op- These recommendations do, however, Uppsala University, posing association with sex. These differ considerably. In clinical practice, 57185 Uppsala, Sweden. results underscore that studies on RA diagnostic laboratories often use higher E-mail: [email protected] phenotypes in relation to autoantibod- diagnostic specificity for anti-CCP than Received on December 18, 2020; accepted ies should accommodate age and sex. for RF, although this has never been of- in revised form on February 22, 2021. ficially recommended. © Copyright Clinical and Introduction Therefore, and to comprehensively ad- Experimental Rheumatology 2021. In the 2010 European League against dress the question of the occurrence Key words: rheumatoid arthritis, Rheumatism (EULAR)/American Col- of RA autoantibodies in relation to age of onset, rheumatoid factor, lege of Rheumatology (ACR) classifi- age at onset and to sex, we examined anti-citrullinated protein antibodies cation criteria for rheumatoid arthritis a large cohort of patients with newly (RA), the serology domain includes diagnosed RA, in which we centrally rheumatoid factor (RF) as well as measured anti-CCP2 and all three RF anti-citrullinated peptide antibodies isotypes in sera drawn at the time of Funding: this study received funding from (ACPA), often represented as antibod- RA diagnosis, with cut-offs adjusted to Nordforsk with grant agreement no. 90825. ies against cyclic citrullinated peptide the same level in relation to a matched Competing interests: see page 882. 2 (anti-CCP2). population-based control group.

879 RA age of onset in relation to autoantibodies / E. Pertsinidou et al.

Patients and methods Fig. 1. Autoantibody Patients distribution in rela- tion to and age tertiles RA patients (n=1600) aged between 18 among 1600 newly and 70 years old from the Epidemio- diagnosed RA patients. logical Investigations in Rheumatoid a: shows the data for Arthritis (EIRA) incident case-control all patients. study were included. All cases (pa- b: the data for females. tients with new-onset RA) were di- c: the data for males. agnosed according to the 1987 ACR classification criteria within one year of first symptoms between 1996 and 2010. Patients included in this study had less than 40 days between the clinical RA diagnosis and inclusion in the EIRA study; blood sampling was performed at inclusion. EIRA controls from the general population were ran- domly selected from the Swedish pop- ulation registry, and were individually matched with RA cases for age, sex and residence. The study was approved by the ethics committee at Karolinska Institutet.

Autoantibody analyses Anti-CCP2 was measured with the Im- munoscan RA ELISA (Eurodiagnos- tica, Malmö, Sweden), using the 25 U/ ml cut-off as suggested by the manufac- turer, corresponding to a 98.4% diag- nostic specificity among 551 RA-free EIRA controls (9). IgG, IgA and IgM rheumatoid factor (RF) were measured with the EliA immunoassay on a Phadia 2500 instrument (Phadia AB, Uppsala, Sweden) and the cut-offs 17.99 IU/mL, 34.32 µg/mL and 9.96 IU/mL were de- termined at the 98th percentile among 624 EIRA controls. In alternative cal- culations, antibody cut-offs were set at the 95.5th percentile.

Statistics T-tests were used to compare the mean age, and χ2 tests to compare the propor- tions of males and females, in patients with and without individual autoanti- bodies. Linear regression was used to evaluate the association between mul- tiple RA autoantibodies and age at RA diagnosis. We performed models fitted with all RA autoantibodies, and models further adjusted for sex, smoking sta- tus (ever/never), year of inclusion and the presence (yes/no) of shared epitope (SE) alleles. p-values <0.05 were con- sidered statistically significant.

880 Clinical and Experimental Rheumatology 2021 RA age of onset in relation to autoantibodies / E. Pertsinidou et al.

Table I. Mean age and gender of 1600 RA patients in relation to the occurrence* of RF isotypes and anti-CCP2. Significant associations are depicted in bold.

Anti-CCP2 pos/neg p-value IgA RF pos/neg p-value IgG RF pos/neg p-value IgM RF pos/neg p-value n. 1020/580 692/908 529/1071 916/684 Univariate models Age (mean) 51/54 <0.0001 53/52 0.08 52/52 0.86 51/53 0.0042 n (%) n (%) n (%) n (%) Females (%) 727/400 (71)/(69) 0.3315 462/665 (67)/(73) 0.0050 350/777 (66)/(73) 0.0089 665/462 (73)/(68) 0.0287 Multivariate models Std β Std β Std β Std β Age model a1 0.148 <0.0001 -0.154 <0.0001 -0.011 0.7170 0.073 0.0401 Age model b2 0.145 <0.0001 -0.139 <0.0001 -0.002 0.9378 0.056 0.1128 Age model c3 0.146 <0.0001 -0.119 0.0002 -0.001 0.9688 0.059 0.0918 Age model d4 0.147 <0.0001 -0.117 0.0003 -0.001 0.9720 0.059 0.0950 Age model e5 0.152 <0.0001 -0.118 0.0003 -0.001 0.9612 0.059 0.0924

*Occurrence was determined as above the 98th percentile among population controls for all autoantibodies. 1Multivariate analysis including occurrence of anti-CCP2, IgA RF, IgG RF and IgM RF. 2Model a additionally adjusted for sex. 3Model b additionally adjusted for never/ever smoking. 4Model c additionally adjusted for inclusion year. 5Model d additionally adjusted for shared epitope.

Results Table II. Occurrence of individual RF isotypes in relation to age at RA diagnosis among Among the 1600 patients, 1127 (70%) 1600 RA patients, overall and in subsets of patients defined by the presence or absence of were females. The mean (median) age other RA autoantibodies. Significant differences are depicted in bold. at RA diagnosis was 54 (57) years RA Patient subset Mean age (n) Mean age (n) p-value among males, and 51 (54) years among autoantibody antibody negative antibody positive patients patients women (p<0.0001). Anti-CCP2 was found in 1020 (64%) patients, IgA RF IgA RF All patients 51.5 52.6 0.0781 in 692 (43%), IgG RF in 529 (33%) IgA RF Anti-CCP2 negative 53.9 (516) 54.1 (64) 0.8546 and IgM RF among 916 (57%) of the IgA RF Anti-CCP2 positive 48.4 (392) 52.4 (628) <0.0001 IgG RF All patients 52.0 51.9 0.8641 patients. IgM or IgA RF was found IgG RF Anti-CCP2 negative patients 54.0 (529) 52.5 (51) 0.4901 in 981 (61%), IgM, IgA or IgG RF IgG RF Anti-CCP2 positive patients 50.0 (542) 51.8 (478) 0.0263 in 1023 (64%), and IgM, IgG or IgA IgM RF All patients 53.0 51.2 0.0042 IgM RF Anti-CCP2 negative patients 54.0 (489) 53.1 (91) 0.5313 RF or anti-CCP2 in 1143 (71%) of IgM RF Anti-CCP2 positive patients 50.5 (195) 51.0 (825) 0.6031 the patients. The agreement between Anti-CCP2 All patients 53.9 50.9 <0.0001 the occurrence of the RA autoantibod- Anti-CCP2 IgA RF negative patients 53.9 (516) 48.4 (392) <0.0001 ies varied between poor to good, with Anti-CCP2 IgA RF positive patients 54.1 (64) 52.4 (628) 0.2659 Anti-CCP2 IgG RF negative patients 54.0 (529) 50.0 (542) <0.0001 the highest kappa value for anti-CCP2 Anti-CCP2 IgG RF positive patients 52.5 (51) 51.8 (478) 0.3050 and IgM RF (0.63) and the lowest for Anti-CCP2 IgM RF negative patients 54.0 (489) 50.4 (195) 0.0009 anti-CCP2 and IgG RF (0.32; the dis- Anti-CCP2 IgM RF positive patients 53.1 (91) 51.0 (825) 0.0692 tribution is shown in Supplementary Fig. S1). In Figure 1, the autoantibody diagnosis weakened (Table I). Variance isotypes, or for all autoantibodies in- distribution in relation to sex and age inflation factors (VIF) for autoantibod- cluding anti-CCP2, the same pattern (tertiles) is shown. ies varied between 1.4 and 2.0 and was appeared in multiple regression analy- When investigating one RA autoanti- highest for IgM RF (data not shown). ses, with appearance of IgA RF asso- body at a time, anti-CCP2 (p<0.001) IgA RF and IgG RF associated with ciation to high age, loss of IgM RF as- and IgM RF (p=0.0042) were both asso- male sex and IgM RF with female sex, sociation to low age but with remaining ciated with lower age at RA diagnosis. but no sex-difference was seen for anti-CCP2 association to low age (data No such association, rather the oppo- anti-CCP2 (Table I). When the regres- not shown). site, was observed for IgA RF (p=0.08), sion was further adjusted for sex, the These results were corroborated in and for IgG RF no association with age association between IgM RF and age stratified univariate analyses. Whereas was observed (p=0.86; Table I). disappeared, whereas the strong asso- anti-CCP2 was associated with young In models fitted with all RA autoan- ciations between IgA RF and higher age among RF negative patients after tibodies together, the association be- age, and between anti-CCP2 and lower individual stratification for all -indi tween anti-CCP and young age per- age persisted (Table I). Additional ad- vidual RF isotypes, IgM RF showed sisted whereas a strong association justment for smoking, age at inclusion no age dependency after stratification between IgA RF and higher age at RA in EIRA and occurrence of SE did not for anti-CCP2. IgA RF was associated diagnosis appeared, and the association change this pattern (Table I). Also, us- with high age among anti-CCP2 posi- between IgM RF and lower age at RA ing the 95.5th percentile cut-off for RF tive patients (Table II).

Clinical and Experimental Rheumatology 2021 881 RA age of onset in relation to autoantibodies / E. Pertsinidou et al.

Discussion all autoantibody cut-offs were set at matoid arthritis. Clin Rheumatol 2004; 23: In our study, we confirm the previously 98% specificity, but the same results 121-2. 4. RÖNNELID J, WICK MC, LAMPA J et al.: described association between anti- were obtained using 95.5% specificity Longitudinal analysis of anti-citrullinated CCP2 and younger age at RA diagnosis for all antibodies, or 95.5% for RF iso- protein/peptide antibodies (anti-CP) during but also demonstrate that the associa- types and 98% for anti-CCP2, which is 5 year follow-up in early rheumatoid arthri- tion between IgM RF and age is lost in close to the common practice in many tis: anti-CP status is a stable phenotype that predicts worse disease activity and greater multivariate analysis. Instead, a strong clinical laboratories. All cut-off set- radiological progression. Ann Rheum Dis opposite association between occur- tings are in agreement with the ACR 2005; 64: 1744-9. rence of IgA RF and higher age at diag- classification criteria (1) and yielded 5. BOETERS DM, MANGNUS L, AJEGANOVA S et al.: The prevalence of ACPA is lower in nosis appears. the same results with opposite age as- rheumatoid arthritis patients with an older These data imply that the previously sociations for anti-CCP2 and IgA RF. age of onset but the composition of the reported association between RF and In conclusion, we demonstrate that the ACPA response appears identical. Arthritis young age (2, 3) is secondary, and that occurrence of individual RA-associated Res Ther 2017; 19: 115. 6. TOO CL, RÖNNELID J, MAT YUSOFF Y et al.: the primary association is between autoantibodies at the time of RA diag- Increased IgG rheumatoid factor-positivity ACPA and young age at diagnosis. The nosis are differently associated with in the Asian rheumatoid arthritis patients strong opposite association between age at diagnosis and sex. Therefore, irrespective of ethnicity. Open J Rheumatol IgA RF and higher age at RA diagno- future studies evaluating the impact of Autoimmune Dis 2014; 4: 43-51. 7. ELSHAFIE AI, ELBAGIR S, ALEDRISSY MIE, sis is a new finding. Whereas univari- individual autoantibodies in RA should ELAGIB EM, NUR MAM, RÖNNELID J: Occur- ate analyses indicated a mere one-year adjust for or otherwise accommodate rence of anti-CCP2 and RF isotypes and their difference in age, the difference in- age and sex. Such adjustments might be relation to age and disease severity among creased to a statistically significant even more important in the future due Sudanese patients with rheumatoid arthritis. Clin Rheumatol 2019; 38: 1545-53. four years difference in multivariate to migration, as different autoantibody 8. BIZZARO N, PREGNOLATO F, van BOEKEL analysis including all autoantibodies, isotypes predominate among RA pa- MA et al.: Preliminary evaluation of the first and in univariate analysis stratified for tients of different ethnicities (6, 7). international reference preparation for an- anti-CCP2. This difference remained ticitrullinated peptide antibodies. Ann Rheum Dis 2012; 71: 1388-92. after adjustment for sex, smoking and Acknowledgments 9. RÖNNELID J, HANSSON M, MATHSSON-ALM shared epitope, when the previously This study was performed within the L et al.: Anticitrullinated protein/peptide anti- described association between IgM Nordic collaborative project NORA body multiplexing defines an extended group of ACPApositive rheumatoid arthritis patients RF and younger age was lost. The as- and received funding from Nordforsk with distinct genetic and environmental deter- sociation between IgA RF and higher with grant agreement number 90825. minants. Ann Rheum Dis 2018; 77: 203-11. age at diagnosis seems to be restricted 10. MEEK B, KELDER JC, CLAESSEN AME, van to RA. Whereas IgA RF is specifically Competing interests HOUTE AJ, TER BORG EJ: Rheumatoid factor isotype and Ro epitope distribution in pri- increased in primary Sjögren’s syn- J. Rönnelid has been a member of the mary Sjogren syndrome and rheumatoid ar- drome compared to in RA patients with Scientific Advisory Board of Thermo thritis with keratoconjunctivitis sicca. Rheu- keratoconjunctivitis sicca investigated Fisher Scientific. L. Mathsson-Alm is an matol Int 2018; 38: 1487-93. in parallel (10), occurrence of IgA RF employee of Thermo Fisher Scientific. 11. PEEN E, MELLBYE OJ, HAGA HJ: IgA rheu- matoid factor in primary Sjogren’s syn- is associated with more than ten years J. Askling has received research grants drome. Scand J Rheumatol 2009; 38: 46-9. earlier age at first visit in patients with from Abbvie, Astra Zeneca, BMS, Eli 12. ARNASON JA, JONSSON T, BREKKAN A, primary Sjögren’s syndrome (11). Lilly, MSD, Pfizer, Roche, Samsung SIGURJONSSON K, VALDIMARSSON H: Rela- A number of previous studies have re- Biologics, Sanofi, UCB, Novartis. tion between bone erosions and rheumatoid factor isotypes. Ann Rheum Dis 1987; 46: ported an association between occur- The other authors have declared no 380-4. rence of IgA RF and the degree of ra- competing interests. 13. EGGELMEIJER F, OTTEN HG, DE ROOY HH, diological destruction in RA (12, 13). DAHA MR, BREEDVELD FC: Significance of Other studies have shown that patients References rheumatoid factor isotypes in seronegative rheumatoid arthritis. Rheumatol Int 1990; 10: with RA onset at higher age present with 1. ARNETT FC, EDWORTHY SM, BLOCK DA et al.: The American Rheumatism Association 43-6. more erosions (14, 15), however IgA RF 1987 revised criteria for the classification of 14. CALVO-ALEN J, CORRALES A, SANCHEZ- was not investigated in these studies. It rheumatoid arthritis. Arthritis Rheum 1988; ANDRADA S, FERNANDEZ-ECHEVARRIA is intriguing to hypothesise that these 31: 315-24. MA, PENA JL, RODRIGUEZ-VALVERDE V: 2. DEAL CL, MEENAN RF, GOLDENBERG DL Outcome of late-onset rheumatoid arthritis. elderly patients might show more radio- et al.: The clinical features of elderly-onset Clin Rheumatol 2005; 24: 485-9. logical destructions due to occurrence rheumatoid arthritis. A comparison with 15. KHANNA D, RANGANATH VK, FITZGER- of IgA RF in patients diagnosed at high- younger-onset disease of similar duration. ALD J et al.: Increased radiographic damage er age, as we have shown in this report. Arthritis Rheum 1985; 28: 987-94. scores at the onset of seropositive rheuma- 3. JACOBSEN S: Young age of onset is associ- toid arthritis in older patients are associated The associations observed in this study, ated with increased prevalence of circulating with osteoarthritis of the hands, but not with were independent on how cut-offs were IgM rheumatoid factor and antinuclear anti- more rapid progression of damage. Arthritis determined. In the primary calculations bodies at presentation in women with rheu- Rheum 2005; 52: 2284-92.

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