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Epitope Heterogeneity of Thyroid-Stimulating Antibodies Predicts Long-Term Outcome in Graves’ Patients Treated with Antithyroid Drugs

Department of Internal Medicine, Seoul National University College of Medicine and Clinical Research Institute, Seoul National University Hospital (T.Y.K., Y.J.P., D.J.P., B.Y.C.), Seoul 110-744, Korea; Department of Internal Medicine, Dankook University College of Medicine (H.C.), Cheonan 330-715, Korea; Department of Internal Medicine, Asan Medical Center, University of Ulsan, College of Medicine (W.B.K.), Seoul 110-744, Korea; and Edison Biotechnology Institute and Ohio University College of Osteopathic Medicine (L.D.K.), Athens, Ohio 45701

Address all correspondence and requests for reprints to: Bo Youn Cho, M.D., Ph.D., Department of Internal Medicine, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea. E-mail: bycho{at}plaza.snu.ac.kr.

	Abstract

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Differences in the epitopes of thyroid-stimulating antibodies (TSAbs) from patients with untreated Graves’ disease were compared with long-term response to antithyroid drugs. Epitopes were measured using Chinese hamster ovary cells transfected with wild-type human TSH receptor (TSHR) and two receptor chimeras, wherein TSHR residues 9–165 or 90–165 had been substituted with comparable residues of the LH/chorionic gonadotropin receptor. Of 159 patients studied, 52 (32.7%) exhibited positive TSAb activity with one or both chimera lines (heterogeneous group), and 107 (67.3%) had no activity with either (homogeneous group). Independent of all other parameters, patients with heterogeneous epitopes responded more favorably to oral antithyroid drugs than patients with homogeneous epitopes (65.4% vs. 41.9%, P = 0.011: estimated odds ratio by logistic regression, 2.17). Although most clinical parameters were not different at presentation, significant differences in the size of goiters, total T₃ concentrations, and titers of TSH-binding inhibitory Igs were evident in the successfully treated group (n = 80) by comparison to the group of patients whose treatment failed (n = 79). Alone, these three parameters did not predict outcome; however, when either of these parameters were considered together with epitope heterogeneity, predictability of a positive therapeutic response was increased to nearly 80%. Thus, the presence of TSAbs with a heterogeneous epitope in a patient with Graves’ disease is not only associated with a favorable response to antithyroid drug treatment, it may help predict the response to treatment when the patient is initially seen.

	Introduction

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THE MEASUREMENT AND characterization of autoantibodies to the TSH receptor (TSHR) in Graves’ disease (GD) has the potential to be clinically useful because the autoantibodies are pathognomonic (1, 2). Consistent with this potential, when thyroid-stimulating antibody (TSAb) levels fall to normal after antithyroid drug treatment, relapse is much less likely to occur (3). However, TSAb levels before treatment are not significantly different between groups of patients who achieve permanent remission or who relapse with active disease; TSAb levels are, therefore, of no prognostic value.

TSAbs are not a single population of antibodies. Their epitopes are biased more toward the N terminus of the TSHR ectodomain in 95% of patients but can be heterogeneous (4, 5, 6, 7, 8, 9, 10, 11). Thus, approximately 30% of patients with untreated GD have minor epitopes outside of the N-terminal region of the TSHR extracellular domain in addition to the critical N-terminal epitopes of TSAbs (9, 10, 12). In a study of 66 patients using human TSHR-rat LH/chorionic gonadotropin (CG) receptor chimeric cells, patients with TSAbs having a heterogeneous epitope were more likely to become euthyroid during antithyroid drug therapy than those having TSAbs with a homogeneous epitope (9, 10). However, the population enrolled in that previous study was small and followed only for 6 months.

This study questioned whether TSAb epitope heterogeneity measured before treatment, rather than TSAb levels, could predict the long-term response to antithyroid drug in GD patients. Before starting treatment, we measured the TSAb activity and epitopes of IgG preparations from GD patients; then followed the patients to determine their therapeutic outcome after antithyroid drug therapy. Persons following the patients had no knowledge of the assay values. We show that patients with a heterogeneous TSAb epitope measured at the start of treatment have a favorable long-term response to antithyroid drug treatment. We, therefore, suggest that the measurement of epitope subtypes, rather than levels of TSAbs, may be a useful pretreatment means of predicting long-term outcome in patients with GD treated with antithyroid drugs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

The cAMP RIA kits were obtained from INCSTAR Corp. (Minnesota, MN); protein A-Sepharose CL-4B columns were from Pharmacia Fine Chemicals (Uppsala, Sweden). The sources of all other materials were the same as reported previously (9).

Patients and sera

We recruited 223 new patients with GD presenting consecutively to the Thyroid Clinic at Seoul National University Hospital, Korea, between August 1993 and November 1997. Patients who had previous histories of relapse after antithyroid drug therapy or who preferred therapy with radioactive iodine or surgery were not included in this study. Pregnancy, postsurgical or postradioiodine relapse, treatment within 12 months before study entry, taking steroids or immunosuppressive drugs, and significant adverse drug reactions to antithyroid drug were exclusion criteria.

Data were retrieved from our thyroid clinic database, which has been used in other studies (9, 12), as well as from case notes. The diagnosis of GD was based on conventional clinical and laboratory criteria, including elevated serum thyroid hormone levels, undetectable TSH by a sensitive RIA, and diffuse goiter with increased ^99mTcO₄^- uptake at scintiscan. Thyroid function and thyroid autoantibodies were measured as described previously (9).

The sizes of goiters were categorized at diagnosis by physical examination by a single physician (B.Y.C.) as absent, small, medium, or large. Eye disease was defined according to the presence of eye signs in categories 2–6 of the NOSPECS classification (13). The following factors were measured at diagnosis (before initiation of treatment) and recorded in the database: gender, age at diagnosis, family history of thyroid disease, presence of eye disease, presence and size of diffuse goiters, autoantibody status and titers, and serum concentrations of total T₃ and free T₄.

This study protocol was approved by the Institutional Review Board of Seoul National University Hospital Clinical Research Institute, and signed informed consent was obtained from each subject before the start of the study.

Protocol of antithyroid drug treatment and definition of therapeutic outcome

The protocol for methimazole treatment was 30 mg/d for one month, 10–20 mg/d from the second month to the time of normalization of the serum free T₄ levels, and a maintenance dose (2.5–5.0 mg/d) thereafter. Propylthiouracil treatment was similar, except that the dose was 10-fold higher. We discontinued medication when both serum TSH and TSH-binding inhibitory Ig (TBII) values normalized during a minimal maintenance dose of antithyroid drugs (14). The clinical outcome after a course of antithyroid drug treatment was classified as either success or failure. The success group included patients who maintained their euthyroid state for at least 12 months after withdrawal of antithyroid drugs. The failure group included patients who relapsed after discontinuation of antithyroid drugs or who could not discontinue their antithyroid drug treatment within 24 months. All patients were followed up by a single physician (B.Y.C.) who had no information on the results of TSAb activities measured in the wild-type or chimeric receptor assays throughout the study.

TSAb assays

Sera were obtained from 223 patients enrolled in this study, before the start of treatment, and from 33 normal individuals who had no clinical history of thyroid disease as well as normal thyroid hormone and TSH levels. The sera were stored at -70 C until IgG preparation. IgGs were extracted by affinity chromatography using protein A-Sepharose CL-4B columns, then lyophilized and stored at -20 C until assay (9).

The TSAbs were measured using Chinese hamster ovary (CHO) cells stably transfected with wild-type human TSHR (WT cells) and transfected with two TSHR-LH/CG receptor chimeras (Mc1 + 2 cells and Mc2 cells) previously described (9). The Mc1 + 2 and Mc2 chimeras have TSHR residues 9–165 and 90–165, respectively, substituted with equivalent residues of the LH/CG receptor. CHO cells were plated in 24-well plates (3–4 x 10⁴ cells/well), fed fresh medium 48 h later, and used 12–24 h later at 100% confluency (5–6 x 10⁵ cells/well). TSAb assays in CHO cells were performed in NaCl-free-Hanks’ balanced salt solution containing 20 mmol/liter HEPES (pH 7.4), 1% BSA, 0.5 mmol/liter 3-isobutyl-1-methylxanthine, and 222 mmol/liter sucrose to make it isotonic (9, 10). Bovine TSH (100 mIU/liter) or the purified IgGs (10 g/liter), dissolved in 300-µl incubation medium, was incubated with cells for 2 h at 37 C; supernatants were aspirated and stored at -20 C. The cAMP released into the medium was measured by RIA (9, 10). TSAb activity, measured as the percentage increase in cAMP production by comparison to assays with equivalent amounts of pooled normal IgG, is defined as positive when the value is greater than 2 SD above the mean value of normal IgG. The positive cutoff values for TSAb activities measured by WT cells, Mc1 + 2 cells, and Mc2 cells were 145%, 145%, and 110%, respectively. All samples were run in duplicate or triplicate. The intraassay and interassay variances in TSAb activity were 9.0–13.9% and 12.7–16.6%, respectively.

Measurements of thyroid hormones and autoantibodies

All assays used commercially available kits as previously described (9). Normal ranges for serum free T₄, and total T₃ concentrations were 12.0–27.5 pmol/liter and 1.31–2.74 nmol/liter, respectively; the normal TSH range was 0.38–4.1 mIU/liter. A titer of greater than 300 U/liter was considered positive for thyroglobulin and thyroid peroxidase antibodies. TBII activity was measured with a radioreceptor assay kit (RSR Limited, Cardiff, UK) as previously described (9, 12). TBII activity was expressed as the percentage inhibition of [¹²⁵I] TSH binding: a TBII value exceeding 15%, which is 2 SD above the mean value of 64 normal samples, was considered positive. The intraassay and interassay variances of TBII activity were 1.7–8.0% and 3.7–10.5%, respectively.

Statistics

All data are presented as mean ± SD. ² tests were used to test for association between two categorical factors, and unpaired t tests were used to assess the relationship between continuous and dichotomous categorical factors. Binary logistic regression was used to determine which factors contributed to the prediction of outcome of treatment. These analyses were performed using SPSS, Inc. for Windows, release 10 (SPSS, Inc., Chicago, IL). P < 0.05 was considered statistically significant.

	Results

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The measurement of GD IgG epitopes and heterogeneity using TSHR-LH/CG receptor chimeric cell lines

Sixty-four of 223 patients (28.7%) were excluded during follow-up for various reasons. Ten patients became pregnant and were excluded. In eight cases, adverse drug reactions were the reasons for withdrawal. Forty-six patients did not appear for follow-up, or did not take their prescribed medication. Finally, 159 patients were evaluated for long-term clinical response to antithyroid drug therapy. We compared the long-term clinical response of these 159 patients with untreated GD to the activities of their IgG, which were measured in the TSHR-LH/CG receptor chimeric cell lines.

Among the 159 patients, 107 (67.3%) had antibodies whose activity completely depended on the N-terminal region of the extracellular domain. Twenty-seven (17.0%) patients were positive when tested using Mcl+2 cells, which have TSHR residues 9–165 substituted by the homologous LH/CG receptor residues. Twenty-two (13.8%) patients were positive when tested using Mc2 cells, which have TSHR residues 90–165 substituted. Three (1.9%) patients were positive in both Mc1 + 2 and Mc2 cells (Table 1). Thus, approximately 30% of patients enrolled in this study had a heterogeneous epitope distribution, with residual activity in Mc1 + 2 cells, Mc2 cells, or both.

When patients were divided into two groups, a heterogeneous group containing individuals whose IgG exhibited residual activity in one or both chimeras and a homogeneous group containing individuals whose TSAb activity depended only on epitopes in the N-terminal portion of the extracellular domain, a significant difference in long-term clinical response to antithyroid drug treatment was evident (Table 1). In 52 patients with heterogeneous TSAb epitopes, 34 patients maintained remission for more than 12 months, and the success rate of antithyroid drug therapy was 65.4%. Within the heterogeneous group, there was no statistically significant difference in the success rate between the subgroup those that reacted with either with Mc1 + 2, Mc2, or both. In contrast, 46 (43.0%) of 107 patients whose TSAbs were homogeneous had a successful response to antithyroid drug therapy. Thus, the success rate of antithyroid drug therapy in the heterogeneous epitope group was significantly higher than in the homogeneous epitope group (P = 0.011). The percentage of patients remaining in remission as a function of time after stopping medication was also significantly different between the heterogeneous and homogeneous epitope groups; this is illustrated using survival curves (P = 0.003, Fig. 1).

View larger version (25K): [in this window] [in a new window]   Figure 1. Kaplan-Meier survival estimates for remission after stopping antithyroid drug treatment and TSAb epitope heterogeneity. This compares the heterogeneous group (who had IgGs with residual TSAb activity in the chimeric cells) to the homogeneous group (with epitopes only in the N-terminal portion of the extracellular domain) in 132 patients with GD who could discontinue medication within 24 months of antithyroid drug treatment.

Among the 159 patients evaluated for long-term clinical response to antithyroid drug therapy, 80 (50.3%) were successfully treated and 79 (49.7%) were considered treatment failures by our criteria (Table 3). The mean duration of medication in the successful treatment group was 13.3 ± 4.8 months and they remained euthyroid for an average of 22.2 ± 10.0 months. In contrast, in the group considered treatment failures, the mean duration of remission was 5.7 ± 2.9 months.

Whereas the overall average rate of success was about 50%, the combination of a large goiter, an initially high total T₃ concentration, and an initially high TBII titer yields a significantly different success rate, 25.9 vs. 81.3% (P < 0.001, Table 4). Nevertheless, the combination of having a heterogeneous TSAb epitope and one these potentially adverse clinical parameters (large goiter, high initial T₃ titer, and or high initial TBII titer) yielded a higher success rate than possessing a homogenous TSAb epitope plus one of these clinical parameters (Table 4). For example, a combination of a heterogeneous TSAb epitope and a larger goiter, an initial high total T₃ concentrations (>5.40 nmol/liter), or a high TBII titer (>40%), conferred a 77.8% chance of remission. In contrast, patients with a homogeneous TSAb epitope and one of these parameters had a only 39.3% chance of remission. Interestingly, there was no statistically significant difference in success rate between heterogeneous and homogeneous groups having no goiter, a low T₃ concentration, and a low TBII titer (Table 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we show that the determination of TSAb epitopes before treatment, not simply the presence or the titer of the TSAbs, can be used as a novel marker for predicting the clinical outcome of GD patients treated with oral antithyroid drugs. Thus, the long-term therapeutic outcome for GD patients who have heterogeneous TSAb epitopes is more favorable than that for patients with homogeneous TSAb epitopes. This result extends and confirms our previous short-term study that GD patients with a heterogeneous epitope distribution are more likely to become euthyroid after antithyroid drug treatment (9).

When we decided to pursue epitope mapping studies, we recognized that sera that lack reactivity against Mc1 + 2 and/or Mc2 chimeras could contain autoantibodies to the TSHR that were directed against diverse epitopes that are not distinguished by these chimeric cell lines. The definition of epitope heterogeneity in this study might, therefore, seem somewhat arbitrary. Nevertheless, among several chimeric receptor expressing cell lines, only Mc1 + 2 and Mc2 were those that showed consistent cAMP responses to bovine TSH that were comparable to WT cells. This point is very important in studies using sera from many patients over long periods of time, because this characteristic can be used as a surrogate marker testing functional expression of adequate amounts of receptors on the cell surface. We thus chose these two chimeras as tools to dissect epitope heterogeneity.

In our previous study, we showed that pooled sera from 30 patients with untreated Graves’ hyperthyroidism lost TSAb activities in Mc1 + 2 and/or in Mc2 chimeras by comparison to WT cells (9). This suggested that the major functional epitope of TSAbs involved TSHR residues 90–165. On the assumption that a serum from a patient with GD might have antibodies to a mix of diverse epitopes on the TSHR, we defined a heterogeneous epitope as an antibody population recognizing not only TSHR residues 90–165, but also epitopes outside of this region on TSHR. The sera from a Graves’ patient that completely loses reactivity to the chimeric receptors, especially Mc2, could be regarded as having its major epitopes focused on TSHR residues 90–165 and as being relatively homogeneous by comparison to sera from a GD patient having residual stimulating activities based on epitopes outside of this region.

In our earlier studies (9, 10, 20), we showed that, although the epitopes of TSAbs in the sera of most GD patients are predominantly located on the N terminus portion of the TSHR, some patients have autoantibodies that also react with other portions of the TSHR. In the present study, the percentage of patients who had IgG reactive against minor epitopes located near the C-terminus of the TSHR ectodomain was about 30%, as previously reported (9). We show that there was no statistically significant difference in clinical parameters between patients whose sera had a heterogeneous TSAb epitope and those with a homogeneous epitope. Additionally, the TSAb titer in WT cells did not correlate with TSAb activity in Mc1 + 2 or Mc2 cells (data not shown). Thus, a heterogeneous TSAb epitope was not simply a secondary phenomenon of other clinical parameters or of TSAb titer measured in WT cells. These data confirm results in our previous paper (9).

In this study, the difference in clinical response to oral antithyroid drugs between patients with a heterogeneous (63.4%) or homogeneous (43.0%) TSAb was statistically significant (P = 0.011). Further, TSAb heterogeneity seems to be an independent factor useful for the prediction of therapeutic outcome after antithyroid drug treatment. However, this value, though significant, does not have sufficient positive predictive values for remission or relapse after completion of antithyroid drug treatment to support, by itself, the treatment decision for most patients. We thus sought to determine if other parameters suggested to be predictors of therapeutic outcome could be used in combination with epitope measurements to predict the response to medical treatment. Our study shows that the combination of a heterogeneous TSAb epitope with one of three favorable clinical predictors (small goiter, low initial T₃ titer, or low initial TBII titer), yielded therapeutic success rates of 81.8%. In contrast, the combination of a homogeneous TSAb epitope and three of the unfavorable clinical predictors (large goiter, high initial T₃ titer, or high initial TBII titer), yielded therapeutic success rate 21.1% (Table 4). We amplify this statistical analysis by presenting individual cases whose therapeutic outcome was depended upon TSAb heterogeneity (Table 5).

Clinical features such as size of goiter, severity of biochemical hyperthyroidism, age, and gender were defined as prognostic indicators of the initial response to antithyroid drug treatment in several past studies (18, 21, 22). In the present study, when we considered a single clinical parameter besides TSAb epitope heterogeneity, we showed that smaller goiters (absent or small on clinical examination) and low serum T₃ concentrations were associated with a higher probability of prolonged remission after antithyroid drug treatment. However, we could not find any difference in gender or age that was relevant to therapeutic outcome.

Patients who opted for radioiodine therapy or surgery were not included in our study and received appropriate ablative treatment. We also excluded women who became pregnant during treatment or the follow-up period because pregnancy itself induces immune tolerance and resolution of hyperthyroidism in GD patients. In contrast, Allahabadia et al. (22) enrolled all their patients in one cycle of antithyroid drug treatment, and allocated patients who failed after one cycle to definitive therapy such as radioiodine or surgery. The exclusion of subjects who became pregnant was not described (18, 22); also, there is a possibility that women of childbearing age—younger than the typical patients—tended not be allocated to definite therapy because of the potential harmful effects of radioiodine therapy on future pregnancies. Because the therapeutic outcome of women who became pregnant during medication or follow-up could be affected more by pregnancy itself than by the antithyroid drug treatment, the difference in inclusion criteria between our study and previous studies may contribute to the conflicting results about age and gender as prognostic factors.

In this study, a substantial portion of patients (28.7%) were excluded during follow-up due to pregnancy, adverse drug reactions, or noncompliance with drug treatment. When we compared the baseline clinical characteristics between the dropout and those who defaulted follow-up, the dropout had significantly larger goiter (data not shown), which was proven to be a poor prognostic factor in the present study. However, the percentage of dropouts in the heterogeneous and homogeneous TSAb groups was similar (23.5% and 31.0%, respectively, P = 0.33). Thus, a selection bias that might be caused by the dropouts did not substantially influence the therapeutic result nor the predictive value of TSAb epitope heterogeneity. Those who defaulted follow-up were likely to have mild disease and went into remission with a short course of treatment.

Initial TBII titers were statistically different when related to therapeutic outcome, but TSAb titers measured by WT cells were not. This disparity between TBII and TSAb results was reported in the previous study. The cause of this disparity may be antibodies with TSH-blocking activity that are detected in the TBII assay but mask TSAb activity in the cell bioassay by inhibiting TSAb-stimulated cAMP production (23, 24, 25).

Another well-known parameter associated with therapeutic outcome is the TBII titer at the end of treatment (3). In our protocol of antithyroid drug therapy, treatment withdrawal was determined by the evolution of the TBII level, i.e. the rate of fall of TBII measurements. The validity of this protocol had been determined in our previous report (14). Thus, all patients who could discontinue medication had negative TBII values at the end of treatment, regardless of long-term therapeutic outcome. We could not, therefore, correlate TBII status at the end of the drug course with therapeutic outcome. Unfortunately, we did not measure TSAb titers with WT cells at the end of treatment, which was also considered of prognostic value in the meta-analysis (3).

The basis for the difference in long-term outcome after antithyroid drug treatment between groups of GD patients with heterogeneous or homogeneous TSAb epitopes is not clear. We considered the possibility that blocking autoantibodies to the C-terminus of TSHR might be more prevalent in patients with heterogeneous TSAb epitopes. This possibility was evaluated in our previous study but no supporting evidence was found (12). Sera from GD patients contain substantial titers of TSH-blocking antibodies that react only with the N-terminal portion of extracellular TSHR. These cannot be measured using chimeric TSHR receptors that substitute the N-terminal portion of TSHR (20, 26). Because these are usually TBIIs and because TBII values in separate assays fall during treatment independent of long-term outcome, as noted above, these are unlikely to explain the prognostic significance of TSAb epitope heterogeneity. Nevertheless, an evaluation of the relationship of the heterogeneity of TSH-blocking antibodies as well as TSAb epitopes is needed. Another consideration needing evaluation is that TSAbs with heterogeneous epitopes represent a population more prevalent in early stages of the disease process and that oral antithyroid drug treatment is more effective early in the development of GD.

In conclusion, patients with heterogeneous TSAb epitopes when first seen have favorable long-term responses to antithyroid drug treatment. Thus, measurement of TSAb epitopes in patients with GD hyperthyroidism before treatment may be useful to predict long-term outcome after antithyroid drug treatment.

	Acknowledgments

 

	Footnotes

 
This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01-PJ1-PG3-20500-0014).

Abbreviations: CG, Chorionic gonadotropin; CHO, Chinese hamster ovary; GD, Graves’ disease; TBII, TSH-binding inhibitory Ig; TSAbs, thyroid-stimulating antibodies; TSHR, TSH receptor.

Received March 12, 2002.

Accepted September 23, 2002.

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