0021-972X/03/$15.00/0 Printed in U.S.A. The Journal of Clinical Endocrinology & Metabolism 88(10):4818 – 4822 Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2003-030789 Recombinant Human Thyrotropin Reduces Serum Vascular Endothelial Growth Factor Levels in Patients Monitored for Thyroid Carcinoma Even in the Absence of Thyroid Tissue Chair of Endocrinology, Department of Clinical and Experimental Medicine “F. Magrassi and A. Lanzara” (F.S., G.M., A.C., M.P., M.R., P.M., S.I., G.A., C.C.), Department of General Pathology (M.C.), Second University of Naples; and Department of Molecular and Clinical Endocrinology and Oncology (B.B.), University “Federico II” Naples, 80121 Naples, Italy In this study, we have investigated in vivo the time-dependent effects of TSH on vascular endothelial growth factor (VEGF) production in patients monitored for thyroid carcinoma. Serum VEGF, thyroglobulin (Tg), and TSH levels were assayed at baseline and 6, 24, 30, 48, 72, and 96 h and 1 wk after administration of recombinant human TSH (rhTSH) in 45 thyroidectomized patients affected by differentiated thyroid carcinoma. At baseline, the patients with metastasis (18 cases) showed serum Tg and VEGF values significantly higher than those seen in the cured patients (27 cases). During rhTSH stimulation, the mean VEGF levels decreased significantly in both patient groups. In 60% of patients with metastasis, VEGF nadir occurred at the same time as serum TSH reached the highest values, whereas in 85.7% of the cured patients VEGF decreased after the TSH peak (P ⴝ 0.003). In conclusion, we demonstrate for the first time that shortterm administration of rhTSH in patients monitored for differentiated thyroid carcinoma induces a significant reduction in serum VEGF values even in the absence of thyroid tissue. This result would suggest that TSH may be able in vivo to regulate VEGF production from tissues other than the thyroid gland. (J Clin Endocrinol Metab 88: 4818 – 4822, 2003) T from thyroid cells may be variable in relation to different experimental conditions (24). In in vitro conditions, the duration of TSH exposure is critical in determining the kind of response in terms of VEGF secretion from follicular cells (23, 24). Whether or not similar time-dependent effects of TSH occur in vivo remains to be clarified (22). In this study, we have evaluated the sequential profile of serum VEGF levels during acute stimulation with recombinant human TSH (rhTSH) in patients monitored for thyroid carcinoma. By such a model we have aimed to measure in vivo the time-dependent effects of TSH on VEGF production in relation to the presence or not of thyroid tissue. HE ANGIOGENESIS IS an important process involved in the growth of normal and neoplastic tissues (1–3). Vascular endothelial growth factor (VEGF) is a 34- to 46-kDa glycoprotein playing a central role in the endogenous regulation of angiogenesis by promoting growth and migration of endothelial cells (4). Increased tissue expression and high serum VEGF levels have been reported in patients with cancers of different origins (5–10), especially in the presence of disseminated disease (11–19). VEGF is synthesized and secreted by thyroid follicular cells (18). In patients with goiter, autoimmune thyroid disease, or thyroid carcinoma, thyroid VEGF expression is increased in relation to the extension of the vascular area in thyroid parenchyma (18, 20, 21). In thyroid carcinoma, the degree of expression and the serum levels of VEGF seem to be correlated with the aggressive behavior of disease (15–17, 19, 22). There is evidence to suggest that the VEGF production from thyroid cells is controlled by factors signaling through the cAMP and protein kinase C pathways (23). In humans with thyroid autoimmune disease, serum VEGF levels correlate positively with serum TSH and anti-TSH receptor antibodies (20). In patients with thyroid carcinoma, however, preliminary data would suggest that TSH does not control VEGF production (22). Indeed, experimental evidence suggests that the effects of TSH on VEGF production Abbreviations: CI, Confidence intervals; rhTSH, recombinant human TSH; Tg, thyroglobulin; VEGF, vascular endothelial growth factor; WBS, whole-body scan. Materials and Methods The study group included 45 thyroidectomized patients affected by differentiated thyroid carcinoma (13 with follicular, 20 with papillary, and 12 with follicular variant of papillary carcinoma) who underwent routine rhTSH (Thyrogen, Genzyme Transgenics Corp., Cambridge, MA)-assisted whole-body radioactive iodine scanning in the year 2002. The inclusion criteria were: 1) total or near-total thyroidectomy and radioactive iodine ablation before the enrollment; 2) no evidence of remnant tissue in thyroid bed uptake; 3) normal platelet count; and 4) no evidence of other malignancy. At the study entry, all patients were in treatment with l-T4 at suppressive doses. The rhTSH was administered, and the whole-body scan (WBS) was performed according to the standard procedure (25, 26). Two doses of 0.9 mg Thyrogen, administered im, were given once daily for the first 2 d. Twenty-four hours later, at 48th hour of study, 2 mCi of 123I were administered orally, and WBS was performed 24 h later. At enrollment, all patients gave informed consent to the study. For the present study, blood samples were drawn at baseline (before 4818 Downloaded from https://academic.oup.com/jcem/article-abstract/88/10/4818/2845793 by guest on 21 May 2020 FRANCESCA SORVILLO, GHERARDO MAZZIOTTI, ANTONELLA CARBONE, MARCO PISCOPO, MARIO ROTONDI, MICHELE CIOFFI, PASQUALE MUSTO, BERNADETTE BIONDI, SERGIO IORIO, GIOVANNI AMATO, AND CARLO CARELLA Sorvillo et al. • rhTSH and VEGF in Thyroid Carcinoma J Clin Endocrinol Metab, October 2003, 88(10):4818 – 4822 4819 Results Twenty-seven patients (60%) were classified as cured at the time of VEGF determination, whereas the remaining 18 patients showed biochemical and morphological evidence of metastatic/persistent disease (Table 1). At baseline, serum TSH values were suppressed in all patients as effect of l-T4 treatment, without significant difference between the two groups (Table 1). At this time, patients with persistent/metastatic disease showed serum Tg and VEGF values significantly higher than those seen in the cured patients (Table 1). During rhTSH stimulation, no significant difference in mean TSH values was found between cured and metastatic patients (Fig. 1). The mean serum peak of TSH (121 ␮U/ml; 95% CI, 109 –134.0) was reached 30 h after the first dose of rhTSH, without significant difference between cured and metastatic patients (Fig. 1). As expected, after TSH stimulation serum Tg remained lower than 2.0 ng/ml (3 pmol/liter) in the cured patients (27 cases). In the other 18 patients, serum Tg levels increased significantly, the peak occurring between 48 h and 1 wk after the first dose of rhTSH (Fig. 2). Figure 3 shows the sequential change in serum VEGF levels after rhTSH administration. The mean VEGF levels decreased significantly in both patient groups, the reduction being more significant in the metastatic patients (P ⬍ 0.001) than the cured patients (P ⫽ 0.01). The individual analysis demonstrated that 36 of 45 patients (15 with metastasis and 21 cured) had a reduction in serum VEGF levels (from ⫺9.7% to ⫺82.9%; median, ⫺51.2%). In most patients with metastasis (9 of 15), the VEGF nadir occurred at the same time as TSH reached the highest serum values (h 30 of the study). However, in most cured patients (18 of 21), the VEGF nadir was reached after TSH peak (in 12 cases after 18 h and in six cases after 42 h). These temporal differences between metastatic and cured patients were significantly different (␹2 ⫽ 10.3; P ⫽ 0.003). Ninety-two hours after the first dose of rhTSH in both patient groups, serum VEGF values were not significantly different from those found at the baseline. In the patients with metastasis, the VEGF reduction was not significantly correlated with the serum Tg increase after TSH stimulation (␳ ⫽ ⫺0.41; P ⫽ 0.09). Discussion This study has demonstrated that the short-term administration of rhTSH in patients monitored for differentiated thyroid carcinoma induces a significant reduction in serum VEGF values, even in the absence of thyroid tissue. Thyroid follicular cells produce VEGF, which is an important factor promoting tissue angiogenesis (18). There is evidence to suggest that VEGF production from thyroid cells is controlled by factors signaling through the cAMP and protein kinase C pathways (23). However, whether or not TSH stimulates VEGF production remains a controversial matter. In monolayer thyroid cells grown in culture, 6 h of stimulation with TSH induces a suppression of VEGF production (23), whereas in the follicular culture an increase in VEGF secretion occurs after 72 h of TSH exposure (24). Therefore, the duration of TSH stimulation in vitro is critical in determining the response of follicular cells in terms of VEGF TABLE 1. Baseline characteristics of 45 patients monitored by rhTSH for differentiated thyroid carcinoma Whole population Cases (n) Age [mean ⫾ SEM (yr)] Sex (M/F) Baseline TSH (␮U/ml) [geometric mean (95% CI)] Tg (ng/ml) [geometric mean (95% CI)] VEGF (pg/ml) [geometric mean (95% CI)] 45 37.1 ⫾ 2.2 18/27 0.07 (0.05– 0.9) 0.84 (0.63–1.18) 145.5 (111.0 –188.7) Cured patients 27 34.0 ⫾ 1.7 9/18 0.07 (0.05– 0.1) 0.48 (0.41– 0.56) 112.2 (89.0 –169.0) Metastatic patients 18 41.7 ⫾ 4.5 9/9 0.06 (0.05– 0.07) 2.23 (1.6 –3.0) 221.4 (194.4 –247.8) P 0.09 0.35 0.34 ⬍0.001 0.01 The patients were categorized as cured (27 cases) and metastatic (18 cases) according to the biochemical and morphological data obtained during rhTSH stimulation. Age was expressed as mean ⫾ SEM. Serum Tg (ng/ml ⫽ pmol/1.5), TSH (␮U/ml ⫽ mU/liter), and VEGF (pg/ml ⫽ pmol/0.02) were expressed as geometric mean and 95% CI. The means and the frequencies were compared using Pearson t test and ␹2 test, respectively. Downloaded from https://academic.oup.com/jcem/article-abstract/88/10/4818/2845793 by guest on 21 May 2020 the first rhTSH dose), and 6, 24, 30, 48, 72, and 96 h and 1 wk later. At each time point, serum VEGF, TSH, and thyroglobulin (Tg) levels were assayed. Patients were categorized as cured or metastatic on the basis of serum Tg values and WBS data obtained 96 and 72 h, respectively, after the first administration of rhTSH (27). Cured patients were defined for having serum Tg values below 2.0 ng/ml (3.0 pmol/liter) and negative WBS during stimulation with rhTSH. The patients with serum Tg greater than or equal to 2 ng/ml (3.0 pmol/liter) with or without positive WBS were defined to have metastatic or persistent disease. Serum VEGF measurements were performed in batches using CytElisa Human VEGF sandwich enzyme immunoassay (AMS Biotechnology, Abingdon, Oxon, UK) on frozen (⫺85 C) aliquots. Mouse monoclonal antibodies generated against human isoform 165 of VEGF protein were used as capture antibodies. Sensitivity and intraassay variation coefficient were 18.6 pg/ml (0.37 pmol/liter) and 7.5%, respectively. Twenty-five healthy subjects, with age and sex comparable to those of the study group, were enrolled as controls for the validation of VEGF assay. In these subjects, serum VEGF levels ranged from 20.5 pg/ml (0.41 pmol/liter) to 401.3 pg/ml (8.0 pmol/liter). All assays were performed in duplicate. Serum TSH was assayed by an immunoradiometric method (DiaSorin, Saluggia, Italy). The detection limit of the assay and the intraassay and interassay variation expressed as coefficients of variation were 0.01 ␮U/ml, 3.1%, and 4.2%, respectively. In our laboratory, normal values for TSH were 0.3–3.5 ␮U/ml. Serum Tg levels were assayed by AutoDelfia human Tg assay (PerkinElmer Life Sciences, Wallac Oy, Turku, Finland). The detection limit of the assay and the intraassay and interassay variation expressed as coefficients of variation were 0.2 ng/ml (0.3 pmol/liter), 3.8%, and 4.7%, respectively. Data were analyzed using SPSS (SPSS, Inc., Chicago, IL) statistical package. Normally distributed data were expressed as mean ⫾ sem, unless otherwise stated. For not normally distributed data, a logarithmic (log) transformation was used to give a data skew value of 0 ⫾ 1. After log transformation, VEGF, Tg, and TSH were expressed as geometric mean values with 95% confidence intervals (95% CI). Repeated measures were compared by ANOVA test with Bonferroni’s post hoc test. Unpaired data and frequencies were compared using Pearson t test and ␹2 test, respectively. Relationships among variables were sought using Spearman’s correlation coefficient. Statistical significance was assumed when P was less than or equal to 0.05. 4820 J Clin Endocrinol Metab, October 2003, 88(10):4818 – 4822 Sorvillo et al. • rhTSH and VEGF in Thyroid Carcinoma FIG. 2. Serum Tg (ng/ml ⫽ pmol/1.5) profile after administration of recombinant TSH (rTSH)in the cured (27 cases, open circles) and metastatic (18 cases, filled circles) patients affected by differentiated thyroid carcinoma. The values were expressed as geometric mean and 95% CI. *, P ⬍ 0.05 vs. baseline values (ANOVA followed by Bonferroni post hoc tests); ‡, P ⬍ 0.05 cured vs. metastatic (Pearson t test for unpaired data). production. However, to our knowledge there are no data demonstrating similar time-dependent effects of TSH in vivo on VEGF production. In this study, the patients with metastasis from differentiated thyroid carcinoma had serum VEGF levels significantly higher in comparison to the cured patients. This finding is in agreement with previous observations suggesting that VEGF is likely involved in the progression of neoplastic thyroid disease toward the occurrence of metastasis (14 –17). However, the acute TSH administration induced a significant decrease in serum VEGF levels. Such a reduction occurred at the same time that the highest values of serum TSH were reached. Thereafter, VEGF levels increased again, promptly reaching the baseline values, whereas serum TSH levels reduced progressively toward the normal values. Such a dynamic profile could explain the previous observation of Tuttle et al. (22) who did not demonstrate any significant modification in serum VEGF levels 3 d after the second dose of rhTSH. The present study did not allow us to demonstrate the mechanisms responsible for the VEGF reduction after TSH stimulation. There is no evidence to suggest that the low VEGF values may have reflected the existence of circulating protein not recognized by the immunoassay due to eventual structural modifications induced by TSH. Likewise, there are no data in literature about the possible effects of TSH on the clearance of VEGF. However, our results would be in agreement with the experimental evidence demonstrating that TSH down-regulates VEGF synthesis in thyroid cells grown in culture (24). In particular, TSH could have inhibited the VEGF production either directly or by releasing Tg from neoplastic cells (24). In fact, it has been demonstrated that intrafollicular Tg exerts a suppressive effect on the expression of thyroid-specific genes, including that for VEGF (24, 28). Although in the patients with metastasis the degree of VEGF reduction was not significantly correlated with the percentage increase in serum Tg values, a local inhibitory Downloaded from https://academic.oup.com/jcem/article-abstract/88/10/4818/2845793 by guest on 21 May 2020 FIG. 1. Serum TSH (␮U/ml ⫽ mU/liter) profile after administration of recombinant TSH (rTSH) in the cured (27 cases, open circles) and metastatic (18 cases, filled circles) patients affected by differentiated thyroid carcinoma. The values were expressed as geometric mean and 95% CI. *, P ⬍ 0.05 vs. baseline values (ANOVA followed by Bonferroni post hoc tests). Sorvillo et al. • rhTSH and VEGF in Thyroid Carcinoma J Clin Endocrinol Metab, October 2003, 88(10):4818 – 4822 4821 effect of Tg on VEGF synthesis cannot be ruled out. The involvement of Tg in addition to the direct effects of TSH may have been responsible for the earlier and more significant decrease in VEGF levels found in the metastatic patients compared with the cured patients in whom Tg was not produced. It is noteworthy that VEGF reduction occurred even in patients who did not show biochemical and morphological evidence of thyroid tissue. The temporal relationship between the rhTSH administration and VEGF reduction is suggestive for a specific effect of TSH in these cases. The TSH receptor expressed in tissues other than thyroid gland may have mediated the suppressive effects of TSH on VEGF production in the absence of thyroid tissue (29, 30). TSH receptor has been found in many extrathyroidal tissues where it seems to be expressed as a functional protein (29, 30). However, the physiological significance of such an ectopic localization is still unclear. The reduction in VEGF levels after TSH stimulation in our patients without biochemical evidence of thyroid tissue would confirm that TSH is biologically active in tissues other than thyroid gland. In addition to rhTSHmediated effects, TSH at very high levels could have interacted with receptors for other similar hormones as the consequence of a spillover phenomenon (31). According to this hypothesis, TSH may have regulated the VEGF synthesis in extrathyroidal tissues even in the absence of specific receptors (32, 33). In conclusion, our study confirms that serum VEGF levels are higher in patients with metastasis from thyroid carcinoma when compared with the patients without biochemical evidence of disease. 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