THE AIM OF postsurgical follow-up in patients with differentiated

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1 /01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism 86(12): Printed in U.S.A. Copyright 2001 by The Endocrine Society Prediction of Disease Status by Recombinant Human TSH-Stimulated Serum Tg in the Postsurgical Follow-Up of Differentiated Thyroid Carcinoma FURIO PACINI, ELEONORA MOLINARO, FRANCESCO LIPPI, MARIA GRAZIA CASTAGNA*, LAURA AGATE, CLAUDIA CECCARELLI, DONATELLA TADDEI, ROSSELLA ELISEI, MARCO CAPEZZONE, AND ALDO PINCHERA Department of Endocrinology and Metabolism, University of Pisa, Pisa, Italy Stimulation with recombinant human TSH (rhtsh) has been introduced in clinical practice as an effective alternative to thyroid hormone withdrawal for the diagnostic follow-up (Tg measurement and 131-iodine whole-body scan) of patients with differentiated thyroid cancer. The present study was specifically aimed to evaluate the utility of rhtsh-stimulated serum Tg measurements in patients with undetectable serum Tg values, on L-T 4 therapy, as the only test to differentiate patients with persistent disease from patients who are disease-free. We studied 72 consecutive patients with differentiated thyroid cancer, previously treated with near-total thyroidectomy and 131-I thyroid ablation. Admission criteria were: an undetectable (<1 ng/ml) serum Tg, on L-T 4 therapy, and negative anti-tg antibodies. The study design consisted of a Tg-stimulation test after rhtsh, during L-T 4, followed by diagnostic WBS and serum Tg measurement off L-T 4. After rhtsh, serum Tg remained undetectable in 41 of 72 patients (56.9%). A negative rhtsh Tg test agreed with an undetectable hypo-tg in 36 of 41 cases (87.8%), all without Abbreviations: DTC, Differentiated thyroid cancer; rhtsh, recombinant human TSH. evidence of metastatic disease at hypo-wbs. In 5 of 41 cases (12.2%), hypo-tg was detectable ( ng/ml), in association with negative hypo-wbs or faint uptake in the thyroid bed. Serum Tg converted from undetectable to detectable after rhtsh in 31 of 72 patients (43.1%), with a peak Tg ranging between 1.2 and 23.0 ng/ml. Hypo-Tg was always detectable in these patients (100% concordance), and it was significantly higher than rhtsh-stimulated Tg (P < ). Hypo-WBS was positive in 23 of 31 patients (74.2%), showing thyroid residues in 12, cervical lymph nodes in 7, and lung metastases in 4 cases. In 8 of 31 cases, hypo-wbs was negative, despite detectable serum Tg. Thus, rhtsh-stimulated Tg was able to detect all cases of documented local or distant metastases. In conclusion, our data indicate that, in patients with undetectable basal levels of serum Tg, rhtsh-stimulated Tg represents an informative test to distinguish disease-free patients (not requiring WBS) from diseased patients (requiring further diagnostic and/or therapeutic procedures). (J Clin Endocrinol Metab 86: , 2001) THE AIM OF postsurgical follow-up in patients with differentiated thyroid cancer (DTC) is the early discovery of persistent or recurrent disease, a condition occurring in nearly 20% of the patients in several series (1 5). After total thyroidectomy and 131-I thyroid ablation, serum Tg measurement and 131-I whole-body scan (WBS) are the most sensitive markers for the presence or absence of disease activity (6, 7). These two methodologies have high levels of concordance, but serum Tg is more sensitive, being positive also in patients with local or distant metastases deprived of iodine uptake and thus not visible at the WBS (8 10). However, a major limitation of serum Tg test is its decreased sensitivity when measured on l-t 4 -suppressive therapy. In this condition, serum Tg may be suppressed to undetectable levels (false negative) in patients with thyroid residues or lymph node metastases and, less frequently, even in patients with distant metastases (6, 11, 12). To increase the sensitivity of serum Tg, thyroid hormone therapy is withdrawn to raise endogenous serum TSH concentrations, which, in turn, will stimulate the release of serum Tg. The consequent state of hypothyroidism is unpleasant for most patients and sometimes results in marked morbidity and impairment of the quality of life (13). The development of a rapid and simple Tg-stimulation test not requiring thyroid hormone withdrawal would certainly facilitate the diagnostic follow-up of DTC patients. Recombinant human TSH (rhtsh), an exogenous source of human TSH, may represent this tool. Recently, stimulation with rhtsh, under l-t 4 suppressive therapy, has been introduced in the clinical practice, as an effective alternative to thyroid hormone withdrawal, for the diagnostic follow-up of DTC. rhtsh has been proven to be effective in promoting both Tg release and 131-I uptake in thyroid cancer cells, without inducing the undesired effects of hypothyroidism, as shown in a preliminary phase I/II study and in two larger phase III clinical trials (14 17). In the second phase III study (16), rhtsh-stimulated Tg predicted the presence of local or distant metastases in 100% of the patients when a stimulated Tg cut-off of 2 ng/ml or more was chosen. Based on this observation, the present study was aimed to specifically evaluate the utility of rhtsh-stimulated serum Tg measurements in patients with undetectable serum Tg values, on l-t 4 therapy, as the only test to differentiate patients with persistent disease (requiring diagnostic imaging and/or therapeutic procedures) from disease-free patients (not requiring further procedures). 5686

2 Pacini et al. Serum Tg Measurement after rhtsh J Clin Endocrinol Metab, December 2001, 86(12): Subjects and Methods We studied 72 consecutive patients (51 females) with DTC (66 papillary and 6 follicular), previously treated with near-total thyroidectomy and 131-I thyroid ablation, scheduled for routine diagnostic WBS, regardless of any clinical suspicion of metastatic disease. Mean age at diagnosis was yr (range, yr). Admission criteria were: an undetectable ( 1 ng/ml) serum Tg, on l-t 4 suppressive therapy, and negative anti-tg antibodies at the time of inclusion. The reasons for scheduling the WBS were: to control for thyroid ablation after surgery and radioiodine in 38 patients; a second control after 1 negative test in 3 patients; and to control for disease remission in the remaining 31 patients, who, at the last hypo-wbs before the study, had evidence of disease treated with radioiodine (lymph nodes and/or lung metastases in 13, persistence of thyroid bed uptake after more than 1 course of radioiodine in 13, and positive Tg with negative diagnostic WBS in 5). Study design The study design consisted of a Tg-stimulation test with rhtsh during suppressive therapy (phase 1) followed by withdrawal of l-t 4 to perform a diagnostic 131-I WBS and serum Tg measurement off l-t 4 therapy (phase 2). During phase 1, patients received one injection of rhtsh (0.9 mg im, Thyrogen; Genzyme Transgenics Corp., Cambridge, MA) for 2 consecutive days. Serum samples for TSH and Tg measurements were collected before the first rhtsh injection and during the following days, up to d 5. During phase 2, the patients were rendered hypothyroid by withdrawing l-t 4 for 6 wk. l-t 3 (10 g, four times a day) was given for 3 wk, starting 1 wk after withdrawal of l-t 4. Serum Tg measurement and WBS were performed 2 wk after withdrawal of l-t 3. Written informed consent was signed by all patients. Methods For each patient, all serum Tg and TSH determinations were run in the same assay. Serum Tg was measured using a commercial immunometric assay (DPC, Los Angeles, CA) with a lower detection limit of 0.2 ng/ml and a functional sensitivity of 0.9 ng/ml. The assay is standardized against the certified reference material for human Tg (CRM 457) of the Community Bureau of Reference of the European Commission (18). In our laboratory, the intra- and interassay coefficients of variation of the method are 4.3% and 7.0%, respectively. Based on the functional sensitivity of the assay (0.9 ng/ml), we selected 1 ng/ml as the cut-off value discriminating undetectable from detectable Tg levels. This cut-off was sufficiently validated in the clinical practice in the past years; hundreds of disease-free patients, as assessed by clinical and instrumental examination, had serum Tg less than1 ng/ml. Anti-Tg autoantibodies were measured in all sera by an immunoradiometric assay method (ICN Pharmaceuticals, Inc., Asse Relegem, Belgium). Patients with circulating anti-tg antibodies were not enrolled in the study. No patient converted from antibody-negative to antibodypositive during the period of observation; thus, no patient had to be excluded from the study after enrollment. Serum TSH was measured using an ultrasensitive commercial immunometric assay (DPC). WBS was performed using a one-head -camera (Apex SPX 4000; Elscint Italia, Milano, Italy) with a high-energy collimator, having a sensitivity of 160 cpm/ Ci. Scan speed was 10 cm/min,with a total count of at least 100,000 cpm. Imaging was performed 72 h after the administration of a 4-mCi tracer dose of radioiodine. Results Serum TSH and Tg after rhtsh As shown in Fig. 1, all patients had very low concentrations of basal serum TSH ( mu/liter), confirming a good compliance to l-t 4 -suppressive therapy. rhtsh administration elicited a sharp and prompt increase in serum TSH that peaked at d 3 (24 h after the second rhtsh injection), with mean values of mu/liter (range, mu/liter). As expected, basal serum Tg on l-t 4 was undetectable ( 1.0 ng/ml) in all patients (inclusion criteria). After rhtsh stimulation, serum Tg remained undetectable in 41 of 72 patients (56.9%). In the other 31 patients (43.1%), rhtshstimulated serum Tg rose to detectable levels, with a peak ranging between 1.2 and 23.0 ng/ml (Fig. 2). The rhtshstimulated Tg peak was observed at d 3 (24 h after the second rhtsh injection) in 2 patients (6.4%), at d 4 (48 h after the second rhtsh injection) in 8 patients (25.8%), and at d 5(72 h after rhtsh) in 21 patients (67.8%). Comparison between serum Tg after rhtsh or endogenous TSH stimulation Patients with a negative Tg response to rhtsh. As shown in Fig. 3, in this group, of 41 patients, a negative rhtsh Tg test corresponded with a negative hypo-tg in 36 of 41 cases FIG. 1. Mean values of basal and rhtsh-stimulated serum TSH (n 72). The vertical lines indicate the SD. FIG. 2. Basal Tg (undetectable), peak Tg after rhtsh, and Tg after thyroid hormone withdrawal in 31 patients with positive Tg response after rhtsh.

3 5688 J Clin Endocrinol Metab, December 2001, 86(12): Pacini et al. Serum Tg Measurement after rhtsh (87.8%). The hypo-wbs in these 36 patients was negative in 30 of them and positive for faint thyroid bed uptake in 6. In 5 of 41 cases (12.2%), there was a discrepancy between negative rhtsh-tg and positive hypo-tg. These hypo-tg values were always very low, ranging between 1.1 and 7.8 ng/ml, and were associated with negative hypo-wbs in 3 cases and with faint radioiodine uptake in the thyroid bed in 2 cases. Patients with a positive Tg response to rhtsh. A response of serum Tg, from undetectable to detectable levels, after rhtsh was observed in 31 patients. As shown in Fig. 3, hypo-tg in these patients was detectable in all (100% concordance). A positive (r 0.40), statistically significant (P 0.02) correlation was found between rhtsh peak Tg and hypo-tg; but, as shown in Fig. 2, hypo-tg was significantly higher than rhtsh-stimulated Tg (P , by Wilcoxon test for paired data). Hypo-WBS was positive in 23 patients (74.2%), showing the presence of thyroid residues in 12, cervical lymph nodes in 7, and lung metastases in 4 cases. Thus, rhtshstimulated Tg was able to detect all cases of local or distant metastases, but, like hypo-tg, was not able to discriminate between different sources of Tg; as shown in Fig. 4, there was a considerable overlap in patients with lung or node metastases, thyroid bed uptake, and negative WBS. In 8 cases, FIG. 3. Correlation between rhtsh-stimulated Tg and hypothyroid Tg in all patients (with and without Tg response to rhtsh). The notes at the bottom report the results of 131-I WBS in hypothyroidism. FIG. 4. Individual values of peak rhtsh-tg and hypo-tg according to the results of 131-I WBS in 31 patients with positive Tg response after rhtsh. hypo-wbs was negative, despite detectable serum Tg levels. In these patients, clinical and instrumental evaluation failed to detect any possible source of Tg. These patients represent the small cohort of Tg /WBS patients present also in several large series of DTC studied with the conventional hypothyroid strategy. Discussion Our study was designed to assess whether Tg stimulation using rhtsh might represent a simple and rapid test in patients with undetectable serum Tg, on l-t 4, for distinguishing those who are disease-free (not to be submitted to WBS) from those with persistent or recurrent disease requiring further procedures. A prerequisite for such a test is that the users are confident with their Tg assay and that the cut-off used is really the one distinguishing detectable from undetectable Tg. Our assay, although able to measure 0.2 ng/ml Tg, has a functional sensitivity of 0.9 ng/ml. This is the reason why we selected 1 ng/ml as the cut-off value. Other laboratories use 2 ng/ml as the cut-off, although their assays have lower functional sensitivities. With this approach, there is a gray zone (e.g. between 1 and 2 ng/ml) in which disease may be present but not considered. Our strategy reduces (and probably eliminates) this gray zone. The results of our study indicate that, in all patients with detectable local or distant metastases, a response of serum Tg to rhtsh did occur, with no false negative results. Compared with the gold standard (serum Tg and 131-I WBS in hypothyroidism), the positive predictive value in these patients was 100%. On the other hand, when rhtsh stimulation produced no detectable change from baseline Tg, all patients were apparently free of disease or (in a small minority) had persistent thyroid bed uptake of very low significance. Similar positive and negative predictive values have been recently reported by Robbins et al. (19), who found that Tg stimulation and 131-I WBS by the traditional hypothyroid preparation or after rhtsh stimulation on thyroid hormone therapy were equally effective in the detection of residual disease in patients with differentiated thyroid carcinoma. In our study, a few patients with scintigraphic evidence of uptake limited to the thyroid bed (with or without detectable serum Tg during hypothyroidism) were missed by rhtshstimulated Tg. Most likely, these apparently false-negative results represent tiny normal residues rather than local tumor. They should not have an impact on the subsequent outcome of the disease, as recently demonstrated by Cailleux et al. (20), who found that, in patients with undetectable serum Tg at the time of performing the control WBS after initial treatment, the rate of recurrence after a long-term follow-up was as low as 0.6%. As with the serum Tg test during hypothyroidism, rhtshstimulated Tg was not specific for neoplastic disease but was observed also in case of thyroid bed uptake or negative WBS, with a diffuse overlap of the individual peak-tg values after rhtsh, among all categories of patients. The cases associated with negative WBS are not surprising and represent the small fraction of patients with detectable serum Tg and negative diagnostic WBS reported in several studies (10, 21 23). A significant correlation was found between peak Tg after

4 Pacini et al. Serum Tg Measurement after rhtsh J Clin Endocrinol Metab, December 2001, 86(12): rhtsh and hypo-tg. However, rhtsh-stimulated Tg was usually lower than hypo-tg; and, in some cases, the increment from baseline was very small. Several explanations may be offered for the Tg increments after thyroid hormone withdrawal being higher than those after rhtsh. Methodological problems or interfering substances might account for the discrepancy; but, with the exception of circulating anti-tg autoantibodies, they have not been described. We favor the possibility that, during endogenous TSH stimulation, Tg synthesis and secretion may be more continuous and prolonged, and that its clearance rate may be lower, compared with exogenous stimulation. Whatever the cause, this finding poses some potential problem in the interpretation of the serum Tg results. Even minor changes (such as 4 ng/ml) from undetectable baseline serum Tg might be associated with lung metastases. Thus, any detectable value of rhtshstimulated Tg must be taken into account as potential expression of relevant disease requiring extensive diagnostic evaluation. The same limitation, but to a lesser extent, may occur also in patients studied during hypothyroidism (6). In addition, one must be aware that the basal and stimulated samples for serum Tg measurement should be tested in the same assay, to avoid false-positive elevation caused by the intraassay coefficient of variation; and, even within the same assay, one has to be sure that minor elevations do not fall within the range of the interassay coefficient of variation. In patients with positive response to rhtsh, serum Tg started to rise as soon as d 2 in 45% of thecases, at d 3 in 32%, and afterd3in23%. In the last case, it is too late to administer a tracer dose of radioiodine for diagnostic WBS, taking advantage of the 2 previous injections of rhtsh. For 131-I imaging or therapy, a second course of rhtsh needs to be administered. A possible way to overcome this limitation might be to administer the tracer dose of radioiodine 72 h after the last injection of rhtsh. The feasibility of this approach needs to be addressed in future studies. As an alternative, the diagnostic WBS may be abandoned, and these patients may be directly scheduled for radioiodine therapy and posttherapy WBS in the hypothyroid state. In conclusion, our data indicate that Tg measurement, after rhtsh stimulation, represents a rapid, comfortable, and informative test to distinguish disease-free patients (not requiring a diagnostic 131-I WBS) from diseased patients (requiring further diagnostic and/or therapeutic procedures). On this basis, in patients with basal undetectable levels of serum Tg and negative anti-tg autoantibodies, we propose a diagnostic algorithm consisting of Tg measurements before and after rhtsh stimulation (d 4 and 5). Patients with a Tg response need to undergo diagnostic 131-I WBS (and/or other imaging techniques). Whether this diagnostic WBS should be performed under rhtsh stimulation or after thyroid hormone withdrawal is still debated. The general feeling is that the diagnostic hypothyroid WBS is more sensitive, as reported in the first phase III clinical study by Ladenson et al. (15), who found that the hypothyroid WBS was significantly superior to rhtsh WBS. However, this finding was not confirmed in the second phase III clinical trial by Haugen et al. (16) and, more recently, in the study by Robbins et al. (19). A possible alternative (that we favor) is to avoid the diagnostic WBS and to schedule the patient directly for 131-I therapy (after thyroid hormone withdrawal) and posttherapy WBS. Patients with no Tg response have no measurable disease and are highly likely to be in remission. These patients may safely continue their follow-up program based on periodical clinical evaluation, neck ultrasound, and serum Tg measurement, on l-t 4. Acknowledgments We thank Prof. Lucia Grasso for invaluable assistance in the validation of the Tg measurements. Received April 18, Accepted August 22, Address all correspondence and requests for reprints to: F. Pacini, M.D., Department of Endocrinology, Via Paradisa, 2, Pisa, Italy. fpacini@endoc.med.unipi.it. This work was supported in part by grants from Associazione Italiana Ricerca sul Cancro, European Communities INCO-Copernicus projects IC-15-CT , and Ministero dell Università e della Ricerca Scientifica e Tecnologica * M.G.C. is a Fellow from the Clinical and Experimental Department of Medicine and Pharmacology, University of Messina, Italy. L.A. is a recipient of a fellowship from Federozione Italiana Ricerca sul Cancro. References 1. 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