Discussion
Accurate assessment of disease status is important for the management of JSLE.12 Through analysis of autoantibody-based subgroups and longitudinal seroconversion, a deeper understanding of JSLE can be attained. We found differences existed in clinical manifestations and disease activity in the subdivision of JSLE based on autoantibodies. Seroconversion overtime was common in JSLE. Negative seroconversion rates were rather frequent for anti-DNA antibodies, while anti-Ro/La antibodies were relatively persistent. Positive seroconversion existed and may be followed by flare.
IF-ANA has a capital role and serves as an entry criterion in the 2019 SLE classification criteria from the European Alliance of Associations for Rheumatology and ACR,1 but data on switched patterns and titre fluctuation were scarce. The distribution in the present study was dominated by homogeneous (H, AC-1) (n=41, 47.13%) and speckled (S, AC-4) (n=40, 45.98%) at diagnosis, similar to that found in previous SLE cohorts.8 13 The switched pattern from H to S was found and decreasing IF-ANA titres were observed during follow-up. According to the previous study, anti-DNA and anti-nucleosome antibodies produced by plasmablasts lead to the H staining, whereas anti-RNP antibodies generated by long-lived plasma cells, less susceptible to conventional immunosuppressive therapy, lead to the S pattern.14 We also found that negative seroconversion rate was rather frequent for anti-nucleosome and anti-dsDNA. Thus, it seems that the switched pattern from H to S during treatment may be related to the easier elimination of the plasmablasts than the long-lived plasma cells in the bone marrow.14 However, the staining pattern was stable overtime in the majority of our cases (84.21%). Furthermore, both the pattern and titres were not correlated with SLEDAI scores, in agreement with prior studies.8 This finding may be related to the impact of various autoantibodies on ANA staining patterns and titres.14 15 Thus, evaluating IF-ANA staining patterns and titres may not be helpful during follow-up and could not be applied to evaluate the disease activity of JSLE. It seems that the analysis of ANA fine specificities is an essential complement to the IF-ANA study.
Through two-step cluster analysis with autoantibody-defined phenotypes, our study showed that the cases could be categorised based on autoantibody-defined phenotypes and the subgroups bear differential patterns of clinical manifestations in JSLE. Two important organ impairments, LN and NPSLE, differed between the groups at diagnosis. SELENA-SLEDAI scores in subgroup 1 were significantly higher than in subgroup 2. The phenomenon of renal involvement and lower SELENA-SLEDAI in the anti-nucleosome/Sm/DNA/RNP group was also put forward among adults with SLE.6 This may be explained by the results that positive anti-dsDNA, anti-histone and anti-nucleosome were associated with LN. Furthermore, the higher prevalence of LN found in the anti-Sm/RNP group corresponded with previous studies.4 6 On the other hand, positive anti-ribosomal P protein was associated with NPSLE. In addition, more positive seroconversion events were observed in subgroup 1 than in subgroup 2. Therefore, the division of the JSLE into distinct disease subsets may help to predict important organ involvement and to further design treatment strategies.
The cluster of SLE on the basis of antibodies varied from studies.4 6 This may be due to the different onset of disease, population and experimental methods of testing the antibodies. The final model selected in this study was based on adequate goodness of fit of the Silhouette coefficient and clinical interpretability. In our study, aPL antibodies were not included in the final cluster analysis. This may be related to the small sample size. According to previous studies, the prevalence of SLE with positive aPL antibodies varies from 20% to 50% in adult patients with SLE and from 11% to 87% in patients with JSLE (depending on different aPL subtypes and study cohorts), though aPL syndrome is rare.16 17 However, the interplay between aPLs and JSLE remains uncertain. Prior research of aPL analysis in patients with JSLE did not include the other autoantibodies, and thus the results may not be convincing enough because the impact of the other autoantibodies was neglected in the disease course.18–20 Further studies with the increasing sample size and the prospective design will be necessary.
Most measurements of subdivisions based on autoantibodies were performed in a cross-sectional design.6 However, autoantibodies vary over time. To our knowledge, seroconversion study for JSLE was scare. In our study, two cases (2.63%) lost ANA positivity over time. Ever-positive rate (15.3%–61.1%, respectively, depending on autoantibody specificity) and seroconversion rates fell within the range of prior findings detected by ELISA,8 a more sensitive technique than DID. Thus, the fluctuation in this study was not a consequence of the low sensitivity of the DID assay. From a practical point of view, it is worthwhile to validate fluctuation by DID, a widely used assay, during follow-up.
Our study found that negative seroconversion rate was rather frequent for anti-nucleosome (57.1%) and anti-dsDNA (51.3%). The same phenomenon was found among adults with SLE.7 8 It may be related to their origin from newly generated plasmablasts that require proliferation for their differentiation and maintenance,14 and therefore, are more susceptible to immunosuppressive therapy. On the other hand, the relatively persistent expression pattern of anti-Ro/La/RNP/Sm antibodies was considered to be related to their origin from long-lived plasma cells in the bone marrow.14 As a result of the less frequent seroconversion rate of anti-Ro/La/RNP/Sm antibodies, whether it is necessary or cost-effective to repeat testing for these antibodies against extractable nuclear antigens (anti-ENA), remains controversial.21 However, anti-Ro/ SSA and anti-dsDNA co-positivity was recently reported to be associated with progression to end-stage renal disease in juvenile LN,22 suggesting the necessity to retest anti-Ro/SSA regardless of its relatively low seroconversion rate. Furthermore, the higher prevalence of LN found in the anti-Sm/RNP group corresponded with previous studies.4 6 Therefore, analysis of serological conservations of both anti-DNA and anti-ENA may provide some information for assessing and guiding treatment, despite different conservation rates among autoantibodies.23
Although not as frequent as negative seroconversion, we found that the phenomenon of positive seroconversion still existed (negative seroconversion rates: 16.1%~57.1% vs positive seroconversion rates: 9.1%~19.4% of cases, respectively, depending on autoantibody specificity). The median interval to positive seroconversion varied from 2 to 5 years for the nine autoantibodies. In two cases of our study, positive seroconversion of anti-RNP/Sm happened 3 years after the first diagnosis of JSLE, followed by the occurrence of newly onset LN at the fifth year’s follow-up. This finding of the production of IgG autoantibodies several years before organ involvement may be concordant with the prior study by Arbuckle et al24 that ANA fine specificities were reported to be detected several years before the symptoms of SLE onset. The phenomenon was considered to be related to the years auto-reactive B cell needed to mature through episodic exposure to immunogenic autoantigens.25 On the other hand, for patients already diagnosed with JSLE, we found that flare-free survival of patients with positive seroconversion was significantly lower than those without seroconversion and those with negative seroconversion. Therefore, it is worthwhile to retest the array of autoantibodies during follow-up, and positive seroconversion may provide a valuable perspective for assessing flare.
The limitations of our study were as follows. First, the size of our cohort was small. We will increase the sample size in future study. A prospective study will be necessary. Second, quantitative detection through ELISA will be applied, and autoantibody titres will be gathered and analysed. Third, due to the retrospective study design, there was an absence of the British Isles Lupus Assessment group index (BILAG) or the Systemic Lupus International Collaborating Clinics damage index (SDI), genomic variants26 and serological data, such as cytokines.27 Appropriate advanced biomarkers28 may be introduced in further study to provide more insights into the pathogenic mechanisms of JSLE. Nevertheless, our study shed light on better understanding of JSLE subphenotypes and seroconversions.