Production of rhTSH
TSH is a pituitary glycoprotein composed of an α-subunit common to gonadotropins and a hormone-specific β-subunit. Once the β-subunit of the human TSH gene has been cloned, the encoded protein could be over expressed in a cell system (Chinese hamster ovary cell), by transfection with human α- and β-subunits of complementary or genomic DNA (1,2). With this technique, high quantities of highly recombinant human TSH (rhTSH; Thyrogen ®) can be obtained with the same biological properties as native TSH (3).
In vitro model systems were initially used to test the effects of rhTSH on thyroid function. In a human fetal thyroid cell system, rhTSH is able to activate TSH receptor and to induce Tg secretion and thyroid epithelial cell proliferation (4). Several preclinical studies were conducted in animals to evaluate the pharmacological and toxicological potential of rhTSH. The trials included single-dose and repeat-dose studies in primates and rodents (5, 6). Subsequent studies examined the effects of rhTSH on normal human thyroid function. A single injection  of rhTSH was a potent stimulator of  the release of T4, T3 and thyroglobulin (Tg) and was able to increase thyroid radioactive iodine uptake in normal subjects (7-9).

Clinical use of rhTSH
After its development, rhTSH has been extensively tested in several clinical indications. The main applications are reviewed below.

1. Follow-up of differentiated thyroid cancer
2. rhTSH-aided post-surgical thyroid ablation with  radioiodine
3. rhTSH-aided radioiodine therapy for the treatment of metastatic disease
4. Use of rhTSH for improving radioiodine therapy of nontoxic multinodular goiter
5. Other potential use



1. Follow-up  of differentiated thyroid cancer (DTC)
The availability of large quantities of rhTSH prompted clinical trials in patients with differentiated thyroid carcinoma. Phase I-II dose-finding and pharmacokinetic studies were conducted (10) and followed by two phase III studies (11,12). A first study (phase I/II) was completed in 1994 in 19 patients after a recent thyroidectomy for differentiated thyroid cancer. The study protocol compared the stimulation of ¹³¹I uptake and the release of serum Tg after rhTSH administration (0.9-3.6 mg for 1-3 days) and after T3 withdrawal. The quality of thyroid scan and the number of sites of abnormal uptake coincided in rhTSH and in hypothyroidism scans in 12 (63%) patients. Serum Tg levels increased more than 2-fold in response to rhTSH in 73%  but was significantly lower than that observed after thyroid hormone withdrawal (93%). The encouraging results of this study were confirmed in a larger multicenter phase III study conducted between 1992 and 1995 in the USA in thyroid cancer patients (11). The primary objective of this multicentric study was to compare ¹³¹I whole body scan (WBS) performed after rhTSH injection to that obtained after withdrawal hormone therapy. The study showed that rhTSH stimulated  radioiodine uptake and resulted in significantly fewer symptoms of hypothyroidism. However, in 23% of patients the scan performed after rhTSH was less sensitive for detecting residual or recurrent disease than the scan performed in hypothyroidism. A second phase III multicentric trials including US and European centres (12) was designed to compare two different dose regiments of rhTSH (2 injection of 0.9 rhTSH for 2 consecutive days or 3 injections of 0.9 mg rhTSH, 3 days apart). The trial enrolled 229 patients and the results of the study demonstrated that when using rhTSH as an alternative to hypothyroidism, the combination of WBS and Tg testing had 100% sensitivity for detection of thyroid cancer metastases. No significant difference was seen between the two and the three dose arms of the study.
All together, these trials have clearly shown that rhTSH is an effective and safe alternative to thyroid hormone withdrawal during the post-surgical follow-up of differentiated thyroid cancer. As a result, rhTSH obtained  regulatory approval in late 1998 as a diagnostic methodology in North America by the Food and Drug Administration and in 2001 in Europe by Evaluation of Medicinal Products. The recommendations are to administer rhTSH 0.9 mg for two consecutive days, followed by a tracer dose (4 mCi of ¹³¹I) 24 hours after the last injection of rhTSH and a diagnostic WBS and Tg measurement 48 hours after the tracer dose of ¹³¹I. After the approval, a number of investigators have published their clinical experience with rhTSH for diagnostic monitoring of patients with differentiated thyroid cancer (13-18) confirming the safety and efficacy of rhTSH.
Although serum Tg and diagnostic WBS have been routinely performed in the follow-up of DTC patients, some authors advocate monitoring the stimulated Tg alone for the detection of recurrent or persistent disease in low-risk patients, based on evidence that serum Tg levels is more sensitive for disease detection than diagnostic WBS (19-20). Pacini et al (17) reported similar results when comparing rhTSH-stimulated Tg levels with hypothyroid Tg levels and diagnostic WBS in DTC patients. Similar results were reported in subsequent studies (21,22). However, few patients with evidence of lymph node metastases have falsely negative undetectable rhTSH stimulated Tg (<1.0 ng/ml) but are identified by neck ultrasound. Thus, the combination of rhTSH-Tg plus neck ultrasound provides the best sensitivity (96.3%) and negative predictive value (99.5%) for the detection of persistent/recurrent cancer (18).
Recently, both American Guidelines and European Consensus for the management of DTC patients recommend that rhTSH stimulated Tg levels (in patients with negative Tg antibodies) plus neck ultrasound represent an adequate tool for the follow-up of low-risk patients (23,24).

2. rhTSH-aided post-surgical thyroid ablation with  radioiodine

Initial treatment of DTC patients consists of thyroidectomy followed by ¹³¹I thyroid remnant ablation. The rationale for thyroid remnant ablation is to decrease the risk of clinical tumor recurrence (25) and improve the sensitivity and specificity of follow-up testing with periodic serum thyroglobulin measurement and radioiodine scanning (26). Traditionally, withdrawal of thyroid hormone has been used to optimize the trapping and retention of radioiodine by increasing endogenous TSH levels. Since 1995, rhTSH has been employed in clinical trials for  post-surgical thyroid remnant ablation in DTC patients. Early studies, using a fixed dose of 30 mCi of 131I, suggested that the use of  rhTSH in LT4 substituted subjects was less efficient than in hypothyroid subjects (27,28).  A recent  study, using a different protocol comparing the effect of 131I therapy in hypothyroidism or rhTSH in LT4 substituted subjects found no significant difference in the  rate of successful remnant ablation between rhTSH stimulation (81.2%) and hypothyroidism preparation (75%) (29). An international, randomized, multicenter, controlled study  was designed to investigate whether preparation of patients with rhTSH while on LT4 therapy was equivalent to preparation by LT4 withdrawal. This study demonstrated comparable remnant ablation rate (100%) in patients prepared for ¹³¹I remnant ablation with 100 mCi by either administering rhTSH or withholding thyroid hormone. rhTSH-prepared patients maintained a higher quality of life and received less radiation exposure to the body. In addition, this study demonstrated that the additional iodine content of pills did not produce interference with successful ablation (30). Based on these results, on February 2005, the use of rhTSH as a preparation for post-surgical thyroid ablation has been approved by European Medicine Agency (EMEA) in low-risk DTC patients using 100 mCi of 131-I.

     3.  rhTSH-aided radioiodine therapy for the treatment of metastatic disease
 Although  not approved for the use in the treatment of DTC metastases, rhTSH as been employed in over 100 patients with metastatic disease  as preparation for 131-I therapy. A systematic review (31) of these patients demonstrated that significant radioiodine uptake in post-therapy WBS obtained after rhTSH-aided 131I therapy  was present in 75% of the cases and that 36% of them had positive response from this modality (complete or partial remission stabilization). Although insufficient for final conclusion these preliminary data may be a rational for future prospective studies.

4. Use of rhTSH for improving radioiodine therapy of nontoxic multinodular goiter

Patients with nontoxic multinodular goiter require treatment when compressive symptoms are present. The treatment of choise is usually thyroidectomy but radioactive iodine has been occasionally employed with significant effect in reducing thyroid volume and ameliorating symptoms and signs of  compression (32). In recent years rhTSH has been used in patients with multinodular goiter with the aim of increasing the uptake of radioiodine in the goiter, particularly in the “cold nodules”. Huysmans et al (33-35) demonstrated that administration of a single-low (0.01 or 0.03 mg) dose of rhTSH considerably increased the RAI uptake in patients with multinodular goiter and caused a more homogeneous distribution of radioiodine within the goiter stimulating 131I uptake in relatively “cold areas”. One year after treatment mean thyroid volume was approximately reduced by  40%. Other studies, using high dose of rhTSH demonstrated significant  goiter reduction but also the occurrence of  severe, transient  hyperthyroidism after 131-1 therapy (36-40). Based on these results, pre-treatment with rhTSH seems a promising alternative to thyroid surgery for the treatment of non toxic multinodular goiter. However the optimal dose and timing of both rhTSH and 131-I as well as the criteria for eligible patients remains to be determined in randomized controlled studies.

5. Other potential use

- To increase diagnostic sensitivity of FDG-PET
2-[¹⁸F] Fluoro-2-Deoxy-D-Glucose-Positron Emission Tomography Scanning (FDG-PET) is indicated in selected patients with biochemical evidence (elevated serum Tg levels) of metastatic disease but negative imaging (41). In vitro studies showed that  rhTSH increases FDG uptake by enhancing glucose transport and glicolitic activity in cultured thyrocytes (42, 43). Subsequently Chin et al (44) confirmed these findings in vivo and showed that  rhTSH can increase the sensitivity and specificity of FDG-PET for localization of metastatic disease in DTC patients.

- To potentiate the cytotoxic effect of chemotherapy
Chemotherapy represents the only therapeutic option in most poorly differentiated carcinoma, although its effect is limited and short lasting. The cytotoxic activity of chemotherapic drugs is maximal when tumor cells are proliferating but commonly DTC patients are treated with suppressive doses of LT4 to block the TSH-stimulated cell proliferation. In this condition, the efficacy of chemotherapy may be limited. An experimental trial in patients with poorly differentiated thyroid cancer provided preliminary evidence that delivering cytotoxic drugs in metastatic thyroid cancer patients under elevated levels of either exogenous (rhTSH) or endogenous TSH levels enhanced the rate of positive response to the chemotherapic regime (45).

-  rhTSH in differential diagnosis of congenital hypothyroidism (CH)

Recently rhTSH has been successful tested in the differential diagnosis of different form of CH. Injection of rhTSH produce serum Tg elevation and radioiodine uptake in ectopic thyroid or dyshormonogenetic goiter while no results was observed in patients with thyroid agenesis. (46).