In Vivo Imaging of Thyroid Cancer with 99mTc-TR1401 and 99mTc-TR1402: A Comparison Study in Dogs.

Differentiated thyroid cancer (DTC) cells may lose NIS expression and iodine uptake, but usually express TSH receptors (TSHR). Therefore, the aim of our study was to compare two radiolabeled superagonist TSH analogues for DTC imaging. These analogues (namely TR1401 and TR1402) have a higher TSHR binding affinity than recombinant human TSH (Thyrogen®). Radiolabeling was performed with technetium-99m using an indirect method via HYNIC conjugation and was followed by in vitro quality controls and binding assay on TSHR-positive cell lines (ML-1). An in vitro binding assay was also performed and compared with radiolabeled human recombinant TSH. In vivo imaging was performed in four dogs with spontaneous follicular thyroid carcinoma with solid poorly differentiated areas with 99mTc-TR1401 SPECT/CT, 99mTc-TR1402 SPECT/CT, and [18F]FDG PET/CT on different days within 2 weeks. TR1401 and TR1402 were labeled with high specific activity (8.3 ± 1.2 MBq/µg) and retention of their biological activity and structural integrity. Both agonists were able to efficiently bind TSHR receptors expressed by cell lines with dissociation constants (Kd) of 2.7 nM for 99mTc-TR1401 and 0.5 nM for 99mTc-TR1402 compared with 99mTc-Thyrogen (Kd = 8.4 nM). In tumor-targeting experiments, a focal uptake was observed in dogs with spontaneous intraglandular thyroid carcinoma, in which TSHR expression was confirmed by immunohistochemistry. 99mTc-TR1402 provided higher T/B than 99mTc-TR1401 and [18F]FDG (12.9 ± 1.3, 10.2 ± 0.7, and 3.8 ± 0.6, respectively; all p < 0.001). Given these results, 99mTc-TR1402 appears to be a useful tool for in vivo imaging of thyroid cancer.

Radiolabeling of VEGF165 with 99mTc to evaluate VEGFR expression in tumor angiogenesis.

Angiogenesis is the main process responsible for tumor growth and metastatization. The principal effector of such mechanism is the vascular endothelial growth factor (VEGF) secreted by cancer cells and other components of tumor microenvironment. Radiolabeled VEGF analogues may provide a useful tool to noninvasively image tumor lesions and evaluate the efficacy of anti-angiogenic drugs that block the VEGFR pathway. Aim of the present study was to radiolabel the human VEGF165 analogue with 99mTechnetium (99mTc) and to evaluate the expression of VEGFR in both cancer and endothelial cells in the tumor microenvironment. 99mTc-VEGF showed in vitro binding to HUVEC cells and in vivo to xenograft tumors in mice (ARO, K1 and HT29). By comparing in vivo data with immunohistochemical analysis of excised tumors we found an inverse correlation between 99mTc-VEGF165 uptake and VEGF histologically detected, but a positive correlation with VEGF receptor expression (VEGFR1). Results of our studies indicate that endogenous VEGF production by cancer cells and other cells of tumor microenvironment should be taken in consideration when performing scintigraphy with radiolabeled VEGF, because of possible false negative results due to saturation of VEGFRs.

(99m)Tc-labeled-rhTSH analogue (TR1401) for imaging poorly differentiated metastatic thyroid cancer.

BACKGROUND: Differentiated thyroid carcinomas originating from thyroid follicular cells are frequent tumors of the thyroid with relatively good prognosis due to improved surgical techniques and follow-up procedures. Poorly differentiated thyroid cancers, which lose iodine uptake ability, in most cases still express thyrotropin (TSH) receptors (TSHR). Therefore, the aim of this study was to radiolabel a superagonist recombinant human TSH (rhTSH) analogue for imaging poorly differentiated thyroid cancer. METHODS: The TSHR superagonist, TR1401, was labeled with (99m)Tc using an indirect method via succinimidyl-6-hydrazinonicotinate hydrochloride conjugation. In vitro quality controls included SDS-PAGE, cysteine challenge, and cell-binding assay on TSHR positive cell lines (JP09 and ML-1). In vivo studies included tumor targeting experiments in athymic nude CD-1 mice xenografted with several different TSHR positive cells (JP09, K1, and ML-1) and TSHR negative cells (JP02) as control. RESULTS: The superagonist rhTSH analogue TR1401 was labeled with high labeling efficiency (>95%) and high specific activity (9250 MBq/mg). The labeled molecule retained its biologic activity and structural integrity. In tumor targeting experiments, a focal uptake of radiolabeled TR1401 was observed in TSHR positive cells but not in TSHR negative cells. The same observation was made in a dog with spontaneous intraglandular thyroid cancer. CONCLUSIONS: We were able to radiolabel the rhTSH superagonist analogue TR1401 with (99m)Tc efficiently with retention of in vitro and in vivo binding capacity to TSHR. The relative role of such novel radiopharmaceutical versus (131)I scanning of thyroid cancer will require future histopathologic and clinical studies, but it may open new perspectives for presurgical staging of thyroid cancer, and diagnosis of radioiodine negative local relapses and/or distant metastases.

Effects of recombinant human thyroid-stimulating hormone superagonists on thyroidal uptake of 18F-fluorodeoxyglucose and radioiodide.

BACKGROUND: Superagonist analogs of human thyroid-stimulating hormone (hTSH) may stimulate the uptake of (131)I-iodide and (18)F-fluorodeoxyglucose ((18)F-FDG) in thyroid carcinomas to a greater degree than hTSH. We herein report the potency and efficacy of two hTSH analogs, TR1401 and TR1402, to stimulate radioiodide and (18)F-FDG uptake in FRTL-5 cells and compared the effects of hTSH and TR1401 on radioiodide uptake in the thyroid in vivo in mice. METHODS: The effects of hTSH analogs on intracellular levels of cAMP, uptake of (131)I-iodide, and (18)F-FDG were studied in FRTL-5 cells to determine the stimulatory potency and efficacy of the compounds by calculating half-maximum effective concentration (EC(50)) values and maximal stimulatory effects (E(max)). Biodistribution studies (n = 96) and positron emission tomography/computed tomography imaging studies (single animals) on thyroid (125)I/(124)I-iodide uptake were performed with T3-suppressed CD-1 mice in a dose-dependent manner (3, 10, and 30 µg/animal). RESULTS: The EC(50) values of TR1401 and TR1402 demonstrated a 90-fold or 800-fold higher potency for their capacity to increase intracellular cAMP levels in comparison with hTSH (p < 0.05). Similar results were demonstrated for the stimulation of (18)F-FDG uptake. Bovine TSH, TR1401, and TR1402 were 85%-490% more potent to increase iodide uptake than hTSH (p < 0.05). TR1402 was 30% more efficacious to stimulate iodide uptake than hTSH. The agonist-induced increase in radiotracer uptake was paralleled by increases in NIS and GLUT-1 expression. Ex vivo biodistribution studies showed an increased iodide uptake in the thyroid of TR1401-treated mice at the low dose of 3 µg/animal in comparison with hTSH-treated mice (n = 16, p < 0.05). Positron emission tomography/computed tomography imaging studies confirmed the increased thyroidal iodide uptake in TR1401-treated mice in vivo. CONCLUSIONS: TR1401 and TR1402 have considerably higher potency than hTSH to stimulate thyroidal iodide and (18)F-FDG uptake in vitro. Moreover, in vivo studies indicated that at low but not higher doses, TR1401 induced an enhanced ability for the thyroid to concentrate iodide compared with hTSH. These properties makes TR1401 and TR1402 interesting candidates for use in humans to enhance uptake of radioiodine and (18)F-FDG by metastases and recurrences of thyroid carcinoma.

Regulation of uptake of 18F-FDG by a follicular human thyroid cancer cell line with mutation-activated K-ras.

UNLABELLED: Dedifferentiation of thyroid carcinoma is accompanied by increased accumulation of the PET tracer (18)F-FDG. The molecular mechanisms responsible for this phenomenon are poorly understood. Therefore, we studied the regulation of (18)F-FDG uptake by the human follicular thyroid carcinoma cell line ML-1 and the as-yet-unknown oncogene expression of that cell line. The data obtained in ML-1 were compared with those of a well-differentiated thyroid cell line of rat origin (FRTL-5). METHODS: The expression of the thyroid-stimulating hormone (TSH) receptor was investigated by immunocytochemistry, and the expression of the glucose transporters (GLUTs) was determined by Western blotting. Mutation analysis of ML-1 was performed for K-ras codons 12 and 13. The effect of TSH on intracellular cAMP levels was determined by a competitive enzyme immunoassay. Cells were incubated with (18)F-FDG (0.5-1.0 MBq/mL) for 1 h, and tracer uptake was related to protein concentration. The effects of bovine TSH, the cAMP analog (Bu)(2)cAMP, and the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor LY294002 on (18)F-FDG uptake were investigated. RESULTS: The TSH receptor was present in both cell lines. FRTL-5 clearly expressed GLUT-1 and also GLUT-4. In ML-1 only, the expression of GLUT-3 was detected. TSH and (Bu)(2)cAMP had a significant effect on (18)F-FDG uptake or GLUT-1 expression in FRTL-5, but not in ML-1 cells. PI3-kinase inhibition by LY294002 downregulated (18)F-FDG uptake in FRTL-5 by 58% +/- 9% (n = 6) and in ML-1 by 26% +/- 5% (n = 42, both P < 0.05). Mutation analysis of ML-1 cells revealed a Gly12Ser point mutation at codon 12 of the K-ras gene. CONCLUSION: (18)F-FDG uptake in the thyroid carcinoma cell line ML-1 is no longer regulated by TSH or cAMP or mediated by GLUT-1. However, in this cell line, this variable is still governed to some extent by PI3-kinase located downstream to the constitutively active K-ras in the Ras-PI3-kinase-Akt pathway. These data suggest that increases in (18)F-FDG uptake in thyroid carcinomas observed in vivo by PET may reflect activation of intracellular signal transduction cascades by oncogenes.

A comparison of short-term changes in health-related quality of life in thyroid carcinoma patients undergoing diagnostic evaluation with recombinant human thyrotropin compared with thyroid hormone withdrawal.

CONTEXT: Thyroid carcinoma requires lifelong monitoring with serum thyroglobulin, radioactive iodine whole body scanning, and other imaging modalities. Levothyroxine (L-T4) withdrawal for thyroglobulin measurement and whole body scanning increases these tests' sensitivities but causes hypothyroidism. Recombinant human TSH (rhTSH) enables testing without L-T4 withdrawal. OBJECTIVE: Our objective was to examine the impact of short-term hypothyroidism on the health-related quality of life (HRQOL) of patients after rhTSH vs. L-T4 withdrawal. DESIGN, SETTING, AND PATIENTS: In this multicenter study, the SF-36 Health Survey was administered to 228 patients at three time points: on L-T4, after rhTSH, and after L-T4 withdrawal. INTERVENTIONS: INTERVENTIONS included administration of rhTSH on L-T4 and withdrawal from thyroid hormone. MAIN OUTCOME MEASURES: Mean SF-36 scores were compared during the two interventions and with the U.S. general population and patients with heart failure, depression, and migraine headache. RESULTS: Patients had SF-36 scores at or above the norm for the general U.S. population in six of eight domains at baseline on L-T4 and in seven of eight domains after rhTSH. Patients' scores declined significantly in all eight domains after L-T4 withdrawal when compared with the other two periods (P < 0.0001). Patients' HRQOL scores while on L-T4 and after rhTSH were at or above those for patients with heart failure, depression, and migraine in all eight domains. After L-T4 withdrawal, patients' HRQOL scores were significantly below congestive heart failure, depression, and migraine headache norms in six, three, and six of the eight domains, respectively. CONCLUSIONS: Short-term hypothyroidism after L-T4 withdrawal is associated with a significant decline in quality of life that is abrogated by rhTSH use.

Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships.

This review focuses on recent advances in the structure-function relationships of thyroid-stimulating hormone (TSH) and its receptor. TSH is a member of the glycoprotein hormone family constituting a subset of the cystine-knot growth factor superfamily. TSH is produced by the pituitary thyrotrophs and released to the circulation in a pulsatile manner. It stimulates thyroid functions using specific membrane TSH receptor (TSHR) that belongs to the superfamily of G protein-coupled receptors (GPCRs). New insights into the structure-function relationships of TSH permitted better understanding of the role of specific protein and carbohydrate domains in the synthesis, bioactivity, and clearance of this hormone. Recent progress in studies on TSHR as well as studies on the other GPCRs provided new clues regarding the molecular mechanisms of receptor activation. Such advances are a result of extensive site-directed mutagenesis, peptide and antibody approaches, detailed sequence analyses, and molecular modeling as well as studies on naturally occurring gain- and loss-of-function mutations. This review integrates expanding information on TSH and TSHR structure-function relationships and summarizes current concepts on ligand-dependent and -independent TSHR activation. Special emphasis has been placed on TSH domains involved in receptor recognition, constitutive activity of TSHR, new insights into the evolution of TSH bioactivity, and the development of high-affinity TSH analogs. Such structural, physiological, pathophysiological, evolutionary, and therapeutic implications of TSH-TSHR structure-function studies are frequently discussed in relation to concomitant progress made in studies on gonadotropins and their receptors.