Prognostic factors for thyroid-stimulating immunoglobulin normalization in moderate-to-severe Graves' orbitopathy: a 36-month longitudinal study.

PURPOSE: The primary objective of this study was to identify predictive factors linked to the normalization of thyroid-stimulating immunoglobulin (TSI) levels in patients diagnosed with active, moderate-to-severe Graves' orbitopathy (GO). The study also tracked the longitudinal changes in TSI levels over a 36-month period following treatment. METHODS: The study population consisted of individuals who were recently diagnosed with active, moderate-to-severe GO and received a 12-week course of intravenous methylprednisolone (IVMP) treatment. A subgroup of patients who did not respond to the initial treatment received an additional 20 Gy of radiation therapy (RTx). TSI levels were monitored at the time of diagnosis, after treatment, and subsequently every 6 months for 36 months. Normalization was defined as a TSI level below 140%. Patients were divdied into two groups with success and failure group depending on whether TSI became normal or not. RESULTS: Out of 83 patients, 36 (43.4%) achieved normalized TSI levels within two years post-IVMP treatment. Lower initial TSI levels (< 425%), absence of additional RTx, and early treatment initiation were associated with a higher likelihood of TSI normalization (P = 0.035, P = 0.028, P < 0.001, respectively). Notably, significant differences in TSI level reduction were observed from 18 months post-treatment between the two groups (P = 0.031). A TSI cutoff value of 413% was identified as predictive for normalization at 24 months (P = 0.002). CONCLUSION: This study is the first to identify key factors that influence normalization of TSI levels in moderate-to-severe Graves' Orbitopathy. It highlights the importance of early treatment decisions, particularly for patients with initial TSI levels above 425%. Despite the treatment, less than half of the patients achieved TSI normalization within 24 months, underscoring the need for additional research to explore the relationship between TSI levels and the clinical manifestations of chronic GO.

Identification and functionalization of thyrotropin receptor antibodies with different antigenic epitopes.

One of the sensitive markers for autoimmune thyroid disease (AITD) clinical identification is thyroid-stimulating hormone receptor antibodies (TRAbs). To quickly distinguish TRAb with distinct antigenic epitopes, a straightforward and uncomplicated technique has not yet been created. The objective of this study is to search for molecular diagnostic targets for different types of AITD {Graves' disease (GD), Graves' orbitopathy (GO), GD with third-degree goiter [GD(3)], hypothyroidism combined with positive TRAb [HT(TRAb+)]} as molecular diagnostic targets. Following action on thyroid cells, differential genes (DEGs) generated by TRAb with distinct antigenic epitopes were detected and identified by RNA sequencing (RNA-Seq), bioinformatics analysis, and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) in the serum of patients with AITD. Using the 5-ethynyl-2'-deoxyuridine (EdU) assay, the effect of coculturing thyroid cells with different antigenic TRAb epitopes on the cells' capacity to proliferate was investigated. Bioinformatics analysis and RT-qPCR validation identified one GD key gene alpha 2-HS glycoprotein (AHSG), two GO key genes [adrenoceptor alpha 1D (ADRA1D) and H2B clustered histone 18 (H2BC18)], two GD(3) key genes [suppressor of cytokine signaling 1 (SOCS1) and cytochrome b-245 beta (CYBB)], and one HT(TRAb+) key gene (MASP2). Correlation analysis and ROC curves showed that the abovementioned genes could be used as molecular diagnostic targets for different types of AITD. Finally, EdU results showed that TRAb inhibited thyroid cell proliferation in the HT(TRAb+) group compared with the normal control group, whereas the remaining three groups promoted thyroid cell proliferation, with a statistically significant difference (P < 0.05). We identified six key genes for different types of AITD, which have diagnostic value for different types of AITD. Meanwhile, we found that TRAbs with different antigenic epitopes in AITD have different biological functions.NEW & NOTEWORTHY We identified six molecular targets of different types of AITD [GD, GO, GD(3), and HT(TRAb+)], which have diagnostic value for different types of AITD. Meanwhile, we found that TRAb with different antigenic epitopes extracted from the sera of patients with AITD had different biological functions, which also provided a new idea for further research on the mechanism of action of TRAb with different antigenic epitopes in AITD.

Epidemiology of thyroid-stimulating immunoglobulin in recent-onset symptomatic thyroid eye disease.

Purpose: This study aims to report correlations between thyroid-stimulating immunoglobulin (TSI) and both clinical and radiological parameters in recent-onset symptomatic thyroid eye disease (TED) patients. Methods: A prospective cohort study of TED patients managed at the Chinese University of Hong Kong from January 2014 to May 2022. Serum TSI levels were determined with the functional assay. Outcomes included the Clinical Activity Score (CAS), marginal reflex distance1 (MRD1), extraocular muscle motility restriction (EOMy), exophthalmos, and diplopia. The radiological assessment included cross-sectional areas and signal of extraocular muscles on STIR-sequence MRI. Results: A total of 255 (197 female) treatment-naive patients, with an average onset age of 50 ± 14 years (mean ± s.d.), were included. Elevated pre-treatment TSI level was observed in 223 (88%) patients. There was a weak positive correlation between TSI and CAS (r = 0.28, P = 0.000031), MRD1 (r = 0.17, P = 0.0080), and the size of the levator palpebrae superioris/superior rectus complex (r = 0.25, P = 0.018). No significant correlation existed between TSI and STIR signals. The AUC and optimal cut-off value for clinical active TED were 0.67 (95% CI: 0.60-0.75) and 284% (specificity: 50%, sensitivity: 85%). In total, 64 patients received intravenous methylprednisolone (IVMP) during the study interval, and they had a higher baseline TSI level than those who did not have IVMP (P = 0.000044). Serial post-IVMP TSI among the 62 patients showed a significant reduction compared to the baseline level (P < 0.001). Both the baseline and post-IVMP TSI levels, and percentages of TSI changes were comparable between patients who responded and did not respond to the first course of IVMP. Conclusion: TSI can be a serum biomarker for the diagnosis, prognosis, and treatment response of TED. Further validation should be warranted.

Minor hyperthyroidism with normal levels of thyroid-stimulating hormone receptor antibodies: a case report.

The present report describes for the first time a case of diffuse hyperthyroidism in a 30-year-old female patient who had normal levels of thyroid-stimulating hormone receptor antibodies (TSHR-Ab), slightly elevated plasma levels of thyroid hormones, and slightly increased thyroid blood flow. Seven years before, after severe stress, she had Graves' disease with elevated plasma levels of TSHR-Ab. The patient's recent medical history included mental stress and autonomic dysfunction. This report describes a mild form of hyperthyroidism in terms of elevated plasma levels of thyroid hormones and Doppler ultrasonography data; this condition was first defined as 'minor hyperthyroidism'. The examination data suggest a probable secondary role of the immune system and primary role of the autonomic nervous system in the pathogenesis of Graves' disease.

Dynamic Risk Model for the Medical Treatment of Graves' Hyperthyroidism according to Treatment Duration.

BACKGRUOUND: Changes in thyrotropin receptor antibody (TRAb) levels are associated with the clinical outcomes of Graves' hyperthyroidism. However, the effects of the patterns of TRAb changes on patient prognosis according to the treatment duration of antithyroid drugs (ATDs) are not well established. METHODS: In this retrospective cohort study, 1,235 patients with Graves' hyperthyroidism who were treated with ATDs for more than 12 months were included. Patients were divided into two groups according to treatment duration: group 1 (12-24 months) and group 2 (>24 months). Risk prediction models comprising age, sex, and either TRAb levels at ATD withdrawal (model A) or patterns of TRAb changes (model B) were compared. RESULTS: The median treatment duration in groups 1 (n=667, 54%) and 2 (n=568, 46%) was 17.3 and 37.1 months, respectively. The recurrence rate was significantly higher in group 2 (47.9%) than in group 1 (41.4%, P=0.025). Group 2 had significantly more goiter, thyroid eye disease, and fluctuating and smoldering type of TRAb pattern compared with group 1 (all P<0.001). The patterns of TRAb changes were an independent risk factor for recurrence after adjusting for other confounding factors in all patients, except in group 1. Integrated discrimination improvement and net reclassification improvement analyses showed that model B performed better than model A in all patients, except in group 1. CONCLUSION: The dynamic risk model, including the patterns of TRAb changes, was more suitable for predicting prognosis in patients with Graves' hyperthyroidism who underwent longer ATD treatment duration.

Correlation between extraocular muscle enlargement and thyroid autoantibodies in thyroid eye disease.

PURPOSE: This study aimed to investigate the factors affecting extraocular muscle enlargement in thyroid eye disease (TED). STUDY DESIGN: Retrospective study. METHODS: The thyroid-stimulating hormone (TSH) receptor antibody (TRAb), thyroid-stimulating antibody (TSAb), antithyroid peroxidase antibody (ATPO), and antithyroglobulin antibody (ATG) levels in patients diagnosed with TED who underwent orbital magnetic resonance imaging were assessed. The control group comprised the contralateral eye of patients who underwent orbital magnetic resonance imaging (MRI) for unilateral eyelid tumors or orbital disease. The thickness of the bilateral rectus muscles and superior oblique muscles was measured on orbital MRI. Muscle enlargement was classified as unilateral/bilateral and symmetric/asymmetric. The effects of age, sex, smoking history, TSH, thyroid hormone, and thyroid autoantibodies on the muscle thickness and number of enlarged muscles were assessed by use of simple and multiple regression analyses. RESULTS: The TED and control groups comprised 41 and 44 cases, respectively. The positivity rate of TSAb in patients with TED was 92.7% higher than that of the other autoantibodies. Muscle enlargement was observed in 29 of the 41 cases (70.7%). Older age and higher TSAb levels were identified as significant factors affecting the total muscle thickness and number of enlarged muscles. Bilateral muscle enlargement and asymmetrical muscle enlargement were observed in 17 (58.6%) and 23 (79.3%) of the 29 cases, respectively. The TSAb levels and age had no significant effect on the type of muscle enlargement. CONCLUSIONS: TSAb showed significant associations with extraocular muscle enlargement. Measurement of TSAb, rather than of TRAb, may be more useful for diagnosing extraocular muscle enlargement in patients with TED.

Stimulating thyrotropin receptor antibodies in early pregnancy.

OBJECTIVES: Thyrotropin-receptor antibodies (TRAb) are used to diagnose Graves' hyperthyroidism in pregnant women. Bioassays provide a measure of thyrotropin-receptor stimulatory antibodies (TSI) specifically. The objective was to measure TSI in pregnant women for establishment of a pregnancy-specific cut-off and comparison with immunoassay measurements of TRAb. METHODS: The retrospective Danish study was performed within the North Denmark Region Pregnancy Cohort (2011-2015) that includes stored biobank samples from early pregnancy (median week 10) with immunoassay measurements of thyroid function parameters and TRAb. TSI were measured in the same samples using the Turbo TSI bioassay (Quidel/Ortho-Clinical Diagnostics) with a recommended cut-off of 0.0241 IU/L in non-pregnant adults. A pregnancy-specific TSI cut-off (95-percentile) was established using Regression on Order Statistics. RESULTS: The established TSI cut-off was 0.0418 IU/L (95 % CI: 0.0417-0.0419). Among women with early pregnancy hyperthyroidism (n=438), 43 women (9.8 %) were TSI positive using the established cut-off, and these women had lower TSH (median 0.008 mIU/L) compared to women with TSI levels below 0.0241 (median TSH 0.040 mIU/L) or in the range from 0.0241 to 0.0418 (median TSH 0.033 mIU/L). Among the 438 women with early pregnancy hyperthyroidism, 22 women were positive for TSI and TRAb, 388 were negative for both, and 28 women were positive for either TSI or TRAb. CONCLUSIONS: This is the first study on TSI measurements in a large cohort of early pregnant women. A pregnancy-specific cut-off for TSI was established and agreement in the classification with immunoassay measurements of TRAb was seen in 94 % of cases.

Clinical value of functional thyrotropin receptor antibodies in Serbian patients with Graves' orbitopathy.

PURPOSE: Thyrotropin receptor autoantibodies (TSH-R-Ab) are heterogeneous in their biological function and play a significant role in the pathophysiology of both Graves' disease and Graves' orbitopathy (GO). The clinical significance and utility of determining functional TSH-R-Ab in a Serbian collective were evaluated. METHODS: 91 consecutive patients with GO were included in this study. Total TSH-R-Ab concentration, referred to as TSH-R binding inhibitory immunoglobulins (TBII) was detected using a competitive-binding immunoassay. Stimulating and blocking TSH-R-Ab (TSAb and TBAb) were measured with cell-based bioassays. RESULTS: Stimulating TSAb activity and TBII positivity were detected in 85 of 91 (93.4%) and 65 of 91 (71.4%) patients with GO (P < 0.001). Blocking TBAb activity was observed in only one patient who expressed dual stimulating and blocking TSH-R-Ab activity. The sensitivity rates for differentiating between clinically active versus inactive and mild versus moderate-to-severe GO were 100% and 100% for TSAb, respectively. In contrast, these were 82% and 87% only for TBII. Seven of eight (87.5%) and one of eight (12.5%) euthyroid patients with GO were TSAb and TBII positive, respectively (P < 0.031). TSAb serum levels significantly predicted GO activity compared to TBII (odds ratio, OR, 95%CI: 3.908, 95%CI 1.615-9.457, P = 0.003; versus 2.133, 0.904-5.032, P = 0.084, univariate analysis; and OR 4.341, 95%CI 1.609-11.707, P = 0.004; versus 2.337, 0.889-6.145, P = 0.085 multivariate analysis). CONCLUSION: Stimulating TSAb are highly prevalent in patients with GO and show superior clinical characteristics and predictive potential compared to the traditionally used TBII.

Adjuvant Rituximab-Exploratory Trial in Young People With Graves Disease.

CONTEXT: Remission rates in young people with Graves hyperthyroidism are less than 25% after 2 years of thionamide antithyroid drug (ATD). OBJECTIVE: We explored whether rituximab (RTX), a B-lymphocyte-depleting agent, would increase remission rates when administered with a short course of ATD. METHODS: This was an open-label, multicenter, single-arm, phase 2 trial in young people (ages, 12-20 years) with Graves hyperthyroidism. An A'Hern design was used to distinguish an encouraging remission rate (40%) from an unacceptable rate (20%). Participants presenting with Graves hyperthyroidism received 500 mg RTX and 12 months of ATD titrated according to thyroid function. ATDs were stopped after 12 months and primary outcome assessed at 24 months. Participants had relapsed at 24 months if thyrotropin was suppressed and free 3,5,3'-triiodothyronine was raised; they had received ATD between months 12 and 24; or they had thyroid surgery/radioiodine. RESULTS: A total of 27 participants were recruited and completed the trial with no serious side effects linked to treatment. Daily carbimazole dose at 12 months was less than 5 mg in 21 of 27 participants. Thirteen of 27 participants were in remission at 24 months (48%, 90% one-sided CI, 35%-100%); this exceeded the critical value (9) for the A'Hern design and provided evidence of a promising remission rate. B-lymphocyte count at 28 weeks, expressed as a percentage of baseline, was related to likelihood of remission. CONCLUSION: Adjuvant RTX, administered with a 12-month course of ATD, may increase the likelihood of remission in young people with Graves hyperthyroidism. A randomized trial of adjuvant RTX in young people with Graves hyperthyroidism is warranted.

A rapid homogenous bioassay for detection of thyroid-stimulating antibodies based on a luminescent cyclic AMP biosensor.

Graves' disease (GD) is an autoimmune disease caused by antibodies to the thyroid stimulating hormone receptor (TSHR). The FDA-cleared Thyretain™ TSI bioassay is a highly specific method to detect thyroid stimulating antibodies (TSAb/TSI) in the blood of patients with autoimmune thyroid disease (AITD), particularly GD. To simplify the workflow of this bioassay and to support a semi-quantitative result, we have generated a stable CHO-K1 cell line expressing both a chimeric TSH receptor (TSHR-Mc4) and a luciferase-based homogeneous cAMP biosensor (GS luciferase). Here, we describe a rapid, real-time, homogenous bioassay (Turbo™ TSI Bioassay) to directly assess the functional activity of TSI and produce results in International Units of IU/L. The Turbo™ TSI bioassay works by measuring changes in the intracellular cAMP level induced by a G-protein coupled receptor (G-PCR) signaling cascade which is triggered by the binding of TSI to the TSHR. Upon binding to cAMP, the GS luciferase reporter is activated through conformational changes and generates light that can be measured in intact cells with a luminometer. The LoD and LoQ of the assay were determined to be 0.016 IU/L and 0.03 IU/L, respectively and the preliminary assay cutoff was determined to be 0.024 IU/L by ROC analysis using the Thyretain™ TSI bioassay results as reference. The analytical performance of the Turbo™ TSI bioassay is comparable to the Thyretain™ TSI bioassay as evidenced by similar EC50 values for a TSHR stimulating monoclonal antibody (M22). The specificity of the Turbo™ TSI bioassay was demonstrated by showing no response to a high concentration of a human monoclonal TSHR blocking antibody (K1-70). The precision of the assay was excellent with an overall within-laboratory precision <15% CV. When testing 198 clinical samples, the positive and negative percent agreement between the Turbo™ TSI and the Thyretain™ TSI bioassays were 98.7% and 93.5%, respectively. While both bioassays yield equivalent analytical and clinical performances, the Turbo™ TSI bioassay is much simpler to perform. It does not require cell culture, sample dilution, washing or cell lysis steps, resulting in a dramatically reduced turnaround time from about 21 h to 60 min. In addition, the same cell line showed its capability of detecting thyroid blocking antibodies (TBAb/TBI) in a competitive format. The Turbo™ TSI bioassay is user-friendly and is a very promising advancement to aid the diagnosis of autoimmune thyroid disease (AITD).