We extracted the study population from participants in the Korean Initiative of primary Sjögren’s Syndrome (KISS) study. KISS is a nationwide prospective cohort database established to provide overall clinical data and samples of patients with pSS in Korea. Informed consent was obtained from all participants according to the principles of the Declaration of Helsinki. This study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital of the Catholic University of Korea (KC13ONMI0646). Recruitment began at Seoul St. Mary’s Hospital, a tertiary care university-affiliated hospital and referral center in Seoul, Korea, in October 2013. By July 2017, the database included 501 patients with pSS from 11 other university hospitals across Korea, as well as Seoul St. Mary’s Hospital. At the time of enrollment, all candidates had to fulfill the 2002 American-European Consensus Group (AECG) classification criteria [12] and/or the 2012 ACR criteria [13]. Any secondary cases possibly combined with other systemic autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis (SSc), were excluded from inclusion for the cohort by experienced rheumatologists’ inspecting whether candidates fulfilled classification criteria for other connective tissue diseases. The additional exclusion criteria were radiation history to the head and neck area, chronic hepatitis C or human immunodeficiency virus infections, previous lymphoproliferative disease, sarcoidosis, graft-versus-host disease, amyloidosis, and immunoglobulin G (IgG) 4-related disease. We selected 318 patients who fulfilled the 2016 ACR/EULAR classification criteria [3] for pSS and had data on ACA positivity among all the participants of KISS study. Among them, 53 subjects were positive for ACA, while the remaining 265 were not.

We obtained all the clinical variables from the KISS database and used only baseline data acquired at the time of cohort enrollment in this study. According to the first and second items of the 2002 AECG classification criteria, positivity for subjective sicca symptoms was indicated [12]. As an objective test and a patient-reported symptom severity index for oral dryness, the unstimulated salivary flow (USF) rate and the xerostomia inventory (range, 11 to 55), self-checking questionnaire about severity for dry mouth, were evaluated [14]. The Schirmer I test, tear film break-up time, meibomian gland dysfunction, ocular staining scores (OSS) by both the SICCA method [15] and van Bijsterveld’s system [16], and ocular surface disease index (range, 0 to 100) [17] were measured by ophthalmologists as objective tests related to ocular dryness. All ocular tests were performed for both eyes and worse results were selected for analysis. Minor salivary gland biopsies were obtained and evaluation of the degree of lymphocytic infiltration into the glands was conducted according to the SICCA protocol. The severity of the infiltration was presented as focus scores [18].

Disease activity and long-term damage were evaluated by the ESSDAI [19] and the Sjögren’s syndrome disease damage index (SSDDI) [20], respectively. These indices were calculated by two experienced rheumatologists. A visual analogue scale (VAS) was used for the patient and physician global assessment (range, 0 to 100 mm) of disease activity. The EULAR Sjögren’s syndrome patient-reported index (ESSPRI) was also assessed [21]. EuroQol (EQ)-5 dimensions (5D) time trade-off (TTO) values were derived from the South Korean reference data and EQ VAS scores were used to measure the patients’ disease-related quality of life [22]. Information on EGMs presented earlier or at the time of enrollment for the subjects in each cohort was gathered. The definition of each type of EGM was based on a case report form from the KISS study and included (1) articular involvement, including a history or presence of morning stiffness over 30 minutes or any swollen or tender joints with the exclusion of degenerative arthritis; (2) RP, indicated by the presence of at least two color changes in distal phalanges including pallor, cyanosis, and redness combined with sensory changes, such as pain and tingling; (3) lymphadenopathy, defined as the presence of palpable lymph nodes over 1 cm; (4) pulmonary involvement, including a history or presence of autoimmune-mediated or interstitial lung disease (ILD); (5) cutaneous vasculitis, indicated by the presence of palpable purpura suggesting the extravasation of red blood cells into the dermis; (6) kidney involvement, indicated by the presence of interstitial nephritis combined with renal tubular acidosis or glomerulonephritis combined with proteinuria over 0.5 g/day; (7) liver involvement, including the presence of autoimmune hepatitis (AIH) or autoimmune cholangitis or primary biliary cholangitis (PBC) diagnosed clinically or pathologically with the exclusion of toxic hepatitis and transient elevation of liver enzymes; (8) lymphoma, indicated by a history of lymphoma confirmed by pathology; (9) splenomegaly, defined as a palpable spleen below the left lower costal margin under full inspiration; (10) peripheral neuropathy, indicated by abnormal findings suggesting peripheral neuropathy in electroneurography; (11) myositis, the presence of myositis diagnosed by elevated serum muscle enzymes, such as creatinine kinase and abnormal findings suggesting myositis in electromyography or biopsy; (12) central nervous system involvement, demonstrated by the presence of cranial or optic neuritis or cerebrovascular accident or epilepsy; and (13) autoimmune thyroid disease, indicated by a history or presence of Graves’ disease or Hashimoto’s thyroiditis.

Cytopenia and hypergammaglobulinemia were defined according to the hematological and biological domains of ESSDAI, respectively [19]. Leukopenia was white blood cell counts < 4.00 × 103/mm³, neutropenia was neutrophils < 1.5 × 103/mm³, anemia was defined as a hemoglobin concentration < 12 g/dL, thrombocytopenia was platelet counts < 150 × 103/mm3, and hypergammaglobulinemia was defined as IgG levels > 16 g/L. Patients with cytopenia possibly caused by other medical conditions, such as nutrients deficiencies, drugs, or anemia of chronic disease, were excluded from the analysis. The presence of anti-nuclear antibodies (ANA) and rheumatoid factor (RF) was evaluated using an indirect immunofluorescence assay on HEp2 cells and immunoturbidimetric assay, respectively. A titer of 1:320 was used as a cut-off value to determine positivity for ANA, whereas values over 20 IU/mL were regarded as positive for RF. All autoantibodies, as well as ACA were measured using commercial enzyme-linked immunosorbent assays (ELISA) without subtyping for each possible subset of autoantibodies.

Continuous variables were assessed about normal distribution using the Kolmogorov-Smirnov test. The non-normally distributed variables are expressed as medians and interquartile range (IQR) and were analyzed with the Mann-Whitney U test. The chi-squared test and Fisher’s exact test were used for categorical variables. IBM SPSS Statistics version 24.0 (IBM Corp., Armonk, NY, USA) was used for the statistical analysis. Statistical significance was considered at p < 0.05.

The median age of all selected subjects was 54 years (Table 1). As described in the methods, 53 of the 318 study patients were ACA-positive for a positivity rate of 16%. The ACA-positive pSS patients (median, 56 years [IQR, 48 to 62]) were older than the ACA-negative patients (median, 54 years [IQR, 45 to 60]; p = 0.042). Disease duration was marginally longer in the ACA-negative group. Most patients were female (98.7%) and had ocular and oral symptoms of dryness (98.1% and 97.8%, respectively). Although the frequency of USF positivity was not different between the two groups, the xerostomia inventory scores were higher in the ACA-positive group than in the ACA-negative group (median, 42 vs. 37), which meant that patients in the former group felt more symptoms of oral dryness than patients in the latter group did. Although the absolute values for the Schirmer I test and tear break-up time of the ACA-positive group were higher than those in the ACA-negative group (p = 0.038 and p = 0.028, respectively) among the ocular tests used to measure the severity of dry eyes, there were no differences in the proportion of positivity for Schirmer I test defined according to the 2016 ACR/EULAR criteria (regarded as positive if < 5 mL in 5 minutes). Differences in other ocular tests including the OSS, by both the SICCA method and van Bijsterveld’s method, and the ocular surface damage index, were not significantly different between the two groups. The degree of lymphocytic infiltration into the minor salivary glands represented by focus scores was also similar between the ACA-positive group and the ACA-negative group. The ratio of positive histopathologic findings in the exocrine glands, defined as focal lymphocytic sialadenitis according to the definition in the 2016 criteria, showed the same tendency in both groups.

VAS scores for disease activity evaluated by each physician and patient were not significantly different between the two groups, as shown in Table 1. The VAS scores in the ACA-positive group were lower than those in the ACA-negative group in the ESSPRI pain domain (p = 0.041), whereas the overall scores in the index showed no inter-group differences. EQ-5D TTO and EQ VAS scores, which could reflect the patients’ disease-related quality of life, were not significantly different between the two groups.

The overall scores for both the ESSDAI and SSDDI (Tables 2 and 3, respectively), which represent the degree of disease activity and disease-associated long-term damage, respectively, were not statistically different between the two groups. We also compared the frequencies of positivity for each domain of the ESSDAI and SSDDI. The status of ACA positivity did not make inter-group differences in each domain of both indices.

Among all EGMs, articular involvement, including arthralgia and arthritis, occurred less (32.1% vs. 47.7%, p = 0.037), whereas liver involvement (11.3% vs. 0.8%, p < 0.001) and RP (49.1% vs. 15.2%, p < 0.001) presented more frequently in the ACA-positive group (Fig. 1). Among the six patients with liver involvement in the ACA-positive group, three had PBC, one had AIH, and the other two patients had both PBC and AIH. All the patients with liver involvement described above were diagnosed by pathologic confirmation. Although there was no statistical significance, seven cases of pulmonary involvement were observed in both study groups (two in the ACA-positive group while five in the ACA-negative group). All pulmonary involvements were confirmed as ILD by using computed tomography. A radiological type of ILD in all pulmonary involvements was presented as ‘nonspecific interstitial pneumonia.’

The ACA-positive group presented a lower prevalence of leukopenia compared to the ACA-negative group (17.3% vs. 33.5%, p = 0.021), whereas the absolute white blood cell count was not statistically different between the two groups, as shown in Table 4. The median hemoglobin concentration of the ACA-positive group was higher than that in the other group (p = 0.043), but the proportion of patients with anemia was not different significantly. Nearly all patients in the ACA-positive group showed ANA-positivity, whereas RF was less likely to be present in this group (both p < 0.001). Both the frequency of hypergammaglobulinemia and the median of IgG level were lower in the ACA-positive group (p = 0.006 and p < 0.001, respectively), which could indicate a relatively lower activation of B-cells compared to the ACA-negative group. Among all the extractable nuclear autoantibodies that could have been evaluated in our study, anti-Ro/SSA antibodies, anti-La/SSB antibodies, and anti-topoisomerase antibodies showed statistically significant disparities between the two groups. The presence of anti-Ro/SSA antibodies and anti-La/SSB antibodies was significantly less frequent (p < 0.001 and p = 0.003, respectively), whereas the anti-topoisomerase antibody was present more frequently in the ACA-positive group (p = 0.006).