Incidence and Distribution

The annual incidence of thyroid cancer varies considerably in different registries, ranging from 1.2-2.6 per 100,000 individuals in men and from 2.0-3.8 per 100,000 in women (106,107). It is particularly elevated in Iceland and Hawaii, being nearly two times higher than in North European countries, Canada and the USA. In Hawaii, the incidence rate of thyroid cancer in each ethnic group is higher than that registered in their country of origin (108), and it is particularly common among Chinese males and Filipino females. Most of the differences are probably due to ethnic or environ¬mental factors (such as spontaneous background radiation) or dietary habits (109), but different standards of medical expertise and health care may also play a role in the efficiency of cancer detection. The American Cancer Society indicated incidence in the USA of nearly 10/100,000 population in 2003. The reported incidence has been increasing at more than 5%/yr for a decade.

In sharp contrast with these data concerning the incidence of clinical thyroid cancer, is the prevalence found in autopsy series or screening programs. Autopsy studies indicate a surprising frequency ranging from 0.01 to over 2.0% (110,111).A survey of consecutive autopsies at Grace-New Haven Hospital found 2.7% of thyroids to harbour unsuspected thyroid cancer (111). Another 2.7% had discrete benign adenomas, and nearly half showed nodularity. The high prevalence may be attributed to careful examination of the gland, but probably also reflects a highly selected group of older patients dying in a hospital. Up to 6% of thyroid glands in autopsied adults in the United States, and over 20% in Japan, also harbour microscopically detectable foci of thyroid carcinoma, which are believed to be of no biologic significance. Altogether autopsy studies suggest that thyroid cancer is in many instances not diagnosed dur¬ing life or is not the immediate cause of death. Both suggestions are in agreement with the rather leisurely growth of the majority of thyroid tumors, especially the frequent small papillary types.
The annual mortality from thyroid cancer in 2003 was 5 per million for men and 6 per million for women (112). The discrepancy between incidence and mortality reflects the good prognosis for most thyroid cancers. Recent statistics suggest about 6 deaths /million in the USA.

Thyroid tumors are rare in children and increase in frequency in each decade.  Carcinomas are two-three times as frequent in women as in men. In the past, it was generally believed that thyroid tumors were more frequent in areas of endemic goiter, and reports from Colombia and Austria support this association (113) (see Chapter 11). More recent studies suggest that in iodine deficient countries the number of nodules is increased and, as a consequence, also the number of thyroid cancers is increased (114). Surveys conducted in the United States found no relation between usual geographic residence and incidence of thyroid cancer.

ETIOLOGY

Most, if not all, thyroid adenomas are monoclonal, as, presumably, are most carcino¬mas (115). Colloid nodules may be either mono-or poly-clonal. Thus tumors represent the persistent growth of the progeny of one cell which has somehow escaped the mechanisms which maintain normal cell division at about once each 8.5 years (116).

The process of oncogenesis is conceived to be a series of events induced by genetic and environmental factors which alter growth control. At the phenomenologic level these factors may be considered as "initiators" and "promoters". Initiators include such agents as chemicals and irradiation which induce tumors, and promoters are agents such as phenobarbital, which in rats augments TSH secretion and radically increases tumor development. In man x-ray treatment is the sole known initiator, and other than elevated TSH, no promoters are known. Compounds such as phenobarbi¬tal, dilantin and PCBs, which are known thyroid tumor promoters in animals through liver microsomal hormone degrading enzyme induction leading to increased thyroid hormone metabolism, do not appear to have a detectable adverse effect in man in doses usually employed (117).

Oncogenes (Fig. 18-11)

We now begin to understand oncogenesis in more details. More than 30 "oncogenes" have been recognized in the human genome. The most likely genetic events in thyroid cancer are reported in Fig. 18-11. These genes, normally silent, can be¬come activated by chromosomal translocations, deletions, or mutations, and then can "transform" normal cells into a condition of uncontrolled growth. Most onco¬genes appear to be closely related to normal growth factors, genes that control cell division, or to hormone receptors. In general, these genes, when turned on, promote cell growth and cell division and depress differentiation. Typically activation of one such gene may not be enough to produce malignancy, but if accompanied by ex¬pression of another oncogene, or if gene mutation or reduplication occurs, the cell may progress toward a malignant potential. Information on expression of oncogenes in human thyroid tissue is rapidly accumulating. Expression of c-myc is stimulated in normal thyroid cells by TSH, and the proto-oncogene is expressed in adenomas and carcinomas. Activating mutations of h-ras at codons 12, 13, and 61, and over expression of h-ras, are found in adenomas and carcinomas, but h-ras mutations are also found in nodular goiter tissue (118), suggesting that h-ras mutations could be an early event in oncogenesis (119). Other studies, it should be noted, find ras mutations uncommon (120).

Figure 18-11. Possible role of oncogene activation, receptor or G-protein mutation, or tumor repressor gene alteration in the induction of thyroid carcinoma.

Santoro and co-workers (121) cloned an oncogene which is frequently and specifically expressed in papillary thyroid cancers. This oncogene is found on chromosome 10 and involves an intrachromosomal rearrangement of the tyrosine kinase domain of the ret oncogene so that it is attached to one of three different promoters, produc¬ing retPTC-1, retPTC-2, and retPTC-3. As a mean, one of these translocation products is found in about 20% of PTC, although in different series a large variation is observed (20-70%). This rearrangement leads to constitutive expression of the oncogene. It has been shown that intra-thyroidal expression of the ret/PTC1 oncogene can induce thyroid cancer (122). BRAF mutations, in the form of point mutations, are the most frequent alterations in papillary carcinoma, and undifferentiated cancers that have arisen from papillary tumors (123), approaching 40% of all PTCs (124).

Recently a mutational change has been associated with follicular cancers. In 5 of 8 follicular cancers, Kroll et al (125) found translocation of the DNA binding domain of PAX8 to domains A-F of the peroxisome proliferator-activater receptor (PPAR) gamma1 gene. The fusion oncogene is able to transform thyrocytes, so appears to be able to produce malignancies (126). Although initially thought to be exclusively present in follicular cancers, it is now known to be present in follicular adenomas as well (127).
Mutation or deletion of the p53 tumor suppressor gene is found in some differentiated thyroid cancers, and many undifferentiated cancers, suggesting that this genetic deletion may be one of the final steps leading to anaplastic cancer growth. A proliferation of studies in this field has provided many clues to thyroid tumorigenesis. Simian virus 40-like sequences are found in many thyroid cancers, as well as other cancers, and the Tag gene sequence found is known to be oncogenic in animal models (128). Mutated and non-functional thyroid hormone receptors are recognized in up to 90% of PTC by one author, suggesting a role in oncogenesis, but other workers find these mutations to be rare (129,130). The tumor suppressor gene TSG101 is over-expressed in most PTCs (131). Overexpression of many other genes -galectin-3, Thymosin beta-10,hTERT, CD97, CD26, VEGF-has been detected, but of course a question always is whether these changes represent the cause or the result of oncogenesis.

Mutations in the proteins involved in the normal TSH-receptor-G protein-adenyl¬cyclase-kinase signal transduction pathway also play a role in tumor formation. Activating TSH receptor mutations have been found by Parma and co-workers (132) to be the cause of most hyperfunctional nodules, and are now known to be common in "hot" nodules in patients with multi-nodular goiter.. These mutations involve the extracellular loops of the transmembrane domain and the transmembrane segments, and are proven to induce hyperfunction by transfection studies. However these mutations are not associated with cancer formation. Mutations of the stimulatory GTP binding protein subunit are also present in some patients with hyperfunctioning thyroid adenomas (133). TSH-R mutations are, however, unusual in thyroid cancer (134), (excepting hyperfunctional adenomas). TSH-R expression tends to be lost as cancers de-differentiate, and persistence of expression is associated with a better prognosis (135).

In addition to positive genetic factors, oncogenesis frequently involves loss of tumor suppressor genes. This has been proven in hereditary retinoblastoma. These genes are normally present on both sets (maternal and paternal) of chromosomes. In retinoblastoma the inherited lack of one suppressor (RB) gene does not cause disease, but if a genetic event (deletion, recombination, mutation, etc.) causes failure of expression of the second allele, cancer ensues. The presence of tumor-specific suppressor genes is often detected because of lack of heterozygosity of chromosomal markers associated with deletions of segments of genetic material. Evidence for characteristic chromosomal abnormalities within tumor cells may lead to recognition of a tumor suppressor gene. Deletions of the tumor suppressor genes, p53 and the RB gene, have been detected in differentiated and undifferentiated thyroid cancer (136). Many chromosomal rearrangements are found in Hurthle cell tumors, and correlate with tumor recurrence (137).

Ret oncogene and Medullary Thyroid Cancer

Studies on patients with MENI and MEN II indicated linkage to chromosomes 11 (138) and 10, respectively. Subsequent studies demonstrated that the ret oncogene is present at 10q11.2. Germline mutations have been detected in this oncogene in all patients with MEN II and MEN III (or IIB), and familial MTC (139). RET is a cell-membrane receptor of the growth factor family, with tyrosine kinase function. In up to 97% of patients with MenIIA, mutations are found in codons 609,611,618, 620, and 630 in exons 10 and 11. These all involve substitutions of other aminoacids for cysteine, and are thought to cause activation of the gene by aberrant disulphide bonding causing dimerization. Similar changes are seen in Familial MTC. In patients with the MENIIB syndrome, almost all,if not all, mutations involve an amino acid substitution of threonine for methionine at codon (918) in exon 16, and are thought to induce a change in substrate phosphorylation. Somatic mutations in ret are present in up to half of patients with sporadic MTC and are almost always in codon 918 (140,141). Gene mutations in this codon are thought to imply a poor prognosis.

Familial tumors

Apparent familial thyroid cancer development has been reported by several clinicians, including cases which seem to show a dominant pattern of inheritance (142,143). Thyroid carcinomas occur rarely as part of several familial syndromes, which may involve hereditable loss of tumor suppressor genes. Papillary cancer can occur as an independent familial syndrome in 5-10% of patienst in different series. Whether the recurrence of PTC represents a true familial aggregation or rather the fortuitous association of PTC in the same family, is still a matter of discussion. However, recent evidence seem to support the existence of a true familial PTC syndrome based on the demonstration that familial PTC display the features of “genetic anticipation” (the disease recurs at an earlier age and at an higher aggressiveness in the 2nd generation compared to the first one) typical of familial diseases (144). In addition, a germline alteration consisting in short telomeres has been demonstrated in familial cases of PTC (145,146), which may be responsible for genomic instability leading eventually to thyroid cancer.

Other, more rare forms of familial thyroid tumors are those associated with complex hereditable diseases. Cowden’s disease is a familial syndrome which includes a variety of hamartomas, multinodular goiter, and carcinomas of several tissues including breast, colon, lung, and thyroid, especially in women (147). Thyroid carcinoma also co-occurs in patients with familial adenomatous polyposis of the colon (148), and can occur in the absence of bi-allelic inactivation of the APC gene. Differentiated thyroid carcinoma is reported to co-occur with chemodactomas of the carotid body, which can be inherited in a familial autosomal dominant form (149). Thyroid carcinoma is also associated with Gardner’s syndrome (150) and Carney’s Syndrome (151). Papillary thyroid carcinoma has been associated with papillary renal neoplasia in a distinct hereditable tumor syndrome. Some patients in the families also have nodular thyroid disease. The predisposing gene has been chromosome 1q21 (152). These syndromes are listed in table 18.5.

Experimental Thyroid Tumor Formation

Thyroid tumors have been induced experimentally in rodents by several procedures having as their common denominator a prolonged increase in pituitary thyrotropin production and thyroid stimulation. Goitrogenic drugs, if administered to animals for a prolonged period, can induce tumors, as numerous investigators (153) have demonstrated. These tumors are typically papillary adeno-carcinomas, and are associated with a diffuse hyperplasia of the thyroid gland. Old rats of some strains appear to develop thyroid cancers spontaneously

Roentgen irradiation of the thyroid and administration of 131I have both induced carcinomas in the experimental animal (154). A combination of 131I injury to the thyroid cell and prolonged administration of a goitrogen is especially likely to produce carcinomas, as shown by Doniach (155). Cell metabolism is altered by 131I, even when small amounts are administered. In the rat, 5 µCi prevents subsequent response to a goitrogenic drug (156). With larger doses the colloid is sparse, the follicles are variable in size, and large eosinophilic acinar cells appear. Very large doses of 131I (producing several thousand rads) to the rat thyroid radically alter cell metabolism, liberate TG within 1 or 2 weeks, and subsequently reduce the efficiency of hormone synthesis. 131I iodine irradiation in rats in doses so low as not to alter hormone biosynthesis immediately inhibits DNA synthesis and cell replication, as shown by a failure to respond to subsequent goitrogenic challenge. The cells also have a shortened life span. Similar inhibition of hyperplasia follows x-irradiation to the thyroid.

Therapeutic doses of 131I to patients also induce atypical nuclei, which may remain for many years (157). The doses of RAI needed to produce neoplastic change in the thyroid glands of animals closely parallel those given in the treatment of thyrotoxicosis in humans. The morphologic changes are intensified by a goitrogenic stimulus and reduced by thyroid hormone treatment.

The effects of radiation may be twofold. The nuclear morphologic changes may derive from an abnormality in cell division or replication of nucleic acids, which may predispose to carcinomatous change. Also, the damaged cell produces less thyroid hormone, and thereby ultimately comes under intense TSH stimulation, as in experiments with goitrogens. Thus, it seems certain that chronic TSH stimulation in animals is associated with the evolution of a neoplasm, especially if it is combined with radiation damage to the cell nuclei. Experimental thyroid tumors induced by 131I are initially TSH dependent. At first, they can be transplanted successfully only into thyroidectomized animals that are producing much TSH. After serial passages through several generations, the tumors may become autonomous and will then grow in a normal host. Partial or complete dependence on TSH is also observed in some human papillary and follicular tumors.

External Radiation and Thyroid Cancer

Duffy and Fitzgerald (158) first made the important observation that a high proportion of children with thyroid carcinoma had received therapeutic x-irradiation to the upper mediastinum or neck during childhood for control of benign lesions such as enlarged thymus, tonsils, or adenoids. Their finding has been amply confirmed (159-166). Winship and Rosvoll (167) studied 562 children with thyroid carcinoma from all parts of the world. Among those for whom adequate historical data were available, 80% had a history of prior x-ray treatment. This relationship is not so obvious for carcinomas developing after age 35. Significant x-irradiation to the head, neck, and chest in childhood increases the frequency of thyroid cancer by 100-fold (168), and the incidence is proportional to the dose, reaching at least 1.7% at 500 rads, or 5.5 cases per million exposed persons per rad each year (Fig. 18-6). Our own data dislose a 7% incidence by 30 years after irradiation (163). The latent period averages 10 -20 years, but tumors occur even after 20-40 years (Fig. 18-7). There appears to be no true threshold, since even doses as low as 9 rads increase the incidence of cancer (160). It is in fact probable that “natural” background radiation may produce many of the spontaneous tumors (169). There is a direct dose-response relationship through 1,000 rads (168). Higher doses of irradiation also induce tumors, and the true dose-response curve in the range 1,000 -5,000 rads in humans is not known. Benign nodules occur with nearly 10 times the frequency of cancers. Interestingly, the type of tumor induced is not different from those occurring spontaneously, and there is no relation between dose and latent period. For some reason, women are more prone to develop radiation-induced tumors than men, and both ethnic and familial factors may influence tumor development (170).

Figure 18-6. Estimated dose response for thyroid cancer in humans from external irradiation. The incidence of carcinomas each year is plotted against the original thyroid irradiation dose. (From Maxon H, Thomas SR, Saenger EL, Buncher ER, and Kereiakes JG. American J Med, 63:967, 1977)

Figure 18-7. Distribution of patients with a history of irradiation to the head and neck, according to the time after irradiation at which they were examined. The majority of patients were seen 20 - 35 years after irradiation, but the incidence of tumors peaked 5 - 10 years earlier. Tumors continued to occur through 40 years after irradiation, and it is not clear that there is a finite latency period.

Probably any x-ray exposure of the thyroid has some carcinogenic potential, although the risk may decrease with age. Adults were extensively treated by x-irradiation for Graves’disease from 1930 to 1950. An increased incidence of carcinoma has been reported in these patients (171). A significant incidence of thyroid neoplasia was observed in patients who received x-ray therapy for thyroid disease (172). These patients were treated at ages up to 34, received 500-1,500 rads, and developed tumors 10-27 years after treatment. In a study of survivors of the atomic blasts at Nagasaki and Hiroshima, an increased incidence of thyroid cancer was found among persons who had received large amounts of radiation (173). Thus, the thyroid of the adult is sensitive to the carcinogenic action of x-rays, although not so sensitive as that of the child.

Radiation-associated tumors of the thyroid continue to occur, although x-ray treatment of thymic enlargement and tonsillar or adenoid hypertrophy has been discontinued since 1959. A recent analysis of 1787 patients treated with X-ray for Hodgkin’s disease found 1.7% to have thyroid cancer (174). The most dramatic and terrifying data emerged from the area around Chernobyl, where thousands of people of all ages received large doses of radiation from external fallout and ingested isotopes, especially short-lived isotopes of iodine. In this epidemic the risk of thyroid cancer is highest among children who were under 9 years and especially under 5 years old at the time of the Chernobyl explosion, and presumably ingested iodide via milk from cows grazing on contaminated forage. The latent period in these children is amazingly short (6 to 7 years), the tumors tend to be relatively aggressive, and are frequently associated with thyroid autoimmunity (175).

Radiation-associated tumors are generally found among younger patients. They are rarely undifferentiated, but some have been fatal. In a review of x-ray associated thyroid tumors at the University of Chicago Thyroid Clinic (163), the latent period among children treated predominantly in adolescence for tonsillar enlargement or acne averaged 20 years. It appears that the peak incidence of lesions is at 10 -25 years after exposure (Fig. 18-7, above), and it is probable that the occurrence of new cancers decreases over time. Among 100 consecutive patients seen in 1973 and 1974, only because they knew of prior radiation exposure, 15% had lesions suggestive of tumor and 7% had cancer proven at operation (162). Favus et al. (176) found a similar incidence of cancer (60/1056) in irradiated patients called back for evaluation. Although one case-controlled study suggests a lack of effect of radiation, the evidence, reviewed by Maxon et al (168), clearly confirms the importance of this problem.

Fig 18-8. The size of radiation associated and non-radiation associated tumors was statistically non-different.

Based on these facts, it has been accepted by most physicians in the field that patients with a history of thyroid irradiation (over 20 rads, and certainly 50 rads) should be located and advised to have an assessment. This evaluation should consist at least of a physical examination and thyroid ultrasound. If one or more clear-cut nodules is found, then FNAC should be performed followed by surgical intervention in case of malignant result. Benign nodules are also found in these glands, with an incidence much higher than that of cancers. Serum TG levels tend to be elevated in irradiated patients, and antithyroid antibodies are more commonly present, but these tests are not of diagnostic value. When excised these glands often show multiple benign as well as malignant nodules as well as areas of fibrosis and hyperplasia (177).

M.P., 52-Year-Old-Woman: Thyroid Radiation and Multiple Gland Abnormalities

This patient was first seen with a history of irradiation for acne during her teens. She subsequently developed telangiectasia of the skin of her face. The month before the examination, she had observed a lump on the right side of the neck. Examination disclosed a 1-cm nodule in the right lobe of the thyroid and some irregularity of the left lobe. Thyroid scintiscan showed a cold nodule of the lower pole of the right lobe. Ultrasound examination of the right lobe identified a partially cystic nodule and a small cystic structure of the left. The FTI was slightly elevated at 10.9. RAIU was above normal. Thyroid antibodies were not present. Thyroid needle aspiration showed cells indicative of malignancy.
Routine blood tests disclosed alkaline phosphatase of 107 units (normal, 25-100 units), calcium 10.9 and 11.5 mg/dl (normal, 8.5-10.2 mg/dl), and phosphate 2.7 ng/dl. Repeated assay of FTI again demonstrated an elevated value of 15 (normal = 6-10.5). The level of parathyroid hormone was 0.65 ng/ml (with a coincident calcium level of 11.5 mg/dl), values indicative of primary hyperparathyroidism.

The patient was treated with potassium iodide for 1 week and admitted for ex¬ploratory surgery. A right upper para-thyroid adenoma weighing 908 mg was found. The adenoma showed areas of cystic degeneration and fibrosis. The thyroid gland was multinodular and was suspicious on frozen section for follicular carcinoma. There was extensive fibrosis around and adherent to the thyroid gland.A near-total thyroidectomy was performed. The gland weighed 17 g. Multiple nodules in the gland measured 1-18 mm in diameter. An 18-mm nodule in the right lobe was iden¬tified as follicular carcinoma. There were, in addition, multiple follicular adenomas and multiple Hurthle cell tumors, focal hyperplasia, and colloid nodules in the right and left lobe.

Postoperatively the patient received thyroid hormone. When seen 1 month after surgery, her calcium level was 9.4 mg/dl, phosphorus 3.5 mg/dl, and parathyroid hormone 0.28 ng/ml (normal). The FTI was 9.2 while taking 0.15 mg L-T4.

This patient developed a cystic parathyroid adenoma with hyperparathyroidism, multiple adenomas of the thyroid, follicular carcinoma, and multiple functioning adenomas that produced thyrotoxicosis. All of these tumors occurred concurrently in a gland showing changes typical of prior radiation exposure.

D.C., 19-Year-Old-Girl: Development of Thyroid Carcinoma, After X-Ray Therapy, While Receiving Thyroid Hormone

At age 10 the patient had respiratory distress and was found to have a superior-anterior mediastinal mass. There was left cervical lymphadenopathy and bilateral supraclavicular lymphadenopathy. Biopsy revealed Hodgkin’s disease of the nodular sclerosing variety. The result of staging laparotomy was negative. She was treated with 4,000 rads to a neck mantle field.

One year later the results of thyroid function tests were normal, but two years after x-ray treatment the FTI was 4.1 and the TSH level was 24 µU/ml. Thyroid hormone replacement therapy was begun, and the patient was carefully monitored over sub¬sequent years with periodic FTI and TSH determinations. Six years after irradiation a2-cm nodule was noted in the left lobe of the thyroid. This nodule was found to be cold on 123-I scan. Fine needle aspiration revealed cells suggestive of malignancy.

At surgery a papillary adenocarcinoma with capsular and vascular invasion was found, and a near-total thyroidectomy was performed. Postoperatively residual thy¬roid tissue was ablated by administration of 30mCi131I.The skeletal survey findings were negative; chest x-ray films and bone films were normal. She has remained free of evidence of thyroid carcinoma or Hodgkin’s disease in the subsequent three years.

This history demonstrates the occurrence of thyroid carcinoma, in a gland heavily irradiated during therapy for Hodgkin’s disease, while the patient was taking ade¬quate replacement doses of thyroid hormone. Possibly the period of X-ray induced hypothyroidism played a role in tumor induction. The tumor developed within six years of the radiation therapy. Fortunately, the tumor was not metastatic and has presumably been eradicated by surgery and RAI ablation of residual tissue.
In our series, postradiation carcinomas averaged 1.7 cm in size (Fig. 18-8), and 14% were below 0.5 cm.

The size distribution was similar to that of non-X-ray-associated tumors. They were more frequently multicentric than those in non irradiated glands and as aggressive, or more so, in behavior than tumors arising without known irradiation (178). The tumors are mainly papillary or follicular, but an occasional anaplastic cancer is also found.In examining patients, it should be remembered that benign and malignant salivary gland neoplasms, neuromas, parathyroid adenomas (179), laryngeal cancer, skin malignancies, and breast cancer also occur with undue frequency in this group of patients.
Thyroid nodularity and cancer also occur as a sequela of nuclear fallout. In the accident at Rongelap in the Marshall Islands (180), individuals received 200-1,400 rads. The incidence of nodularity was 40%, and nearly 6% proved to have cancer. Children in Utah exposed to small amounts of fallout from atomic bomb testing have been proven to develope nodules and possibly carcinomas.

Lack of Association of 131I Treatment and Thyroid Carcinoma

Iodine-131 treatment induces abnormalities in the thyroid gland that persist for many years (181). Giant nuclei, increased mitotic activity, hyperchromatic nuclei, and other abnormalities appear. It seems reasonable that these nuclear changes could lead to carcinomatous degeneration. Chromosomal damage in circulating lymphocytes has also been reported after 131I administration (182). Patients have developed thyroid nodules or tumors after 131I therapy for Graves’ disease; it has been suggested, but not proved, that the highest incidence has been among those treated during childhood. Some of the lesions found in 131I-treated children may actually have been carcinomas, but there has been debate (183) among the various pathologists who examined the specimens. Several reports of isolated instances of cancer after 131I treat¬ment of adults for Graves’ disease have appeared, but the large United States Public Health Service cooperative study failed to show an increased risk in this group (184-186). Studies by Holm et al (186) also failed to show an increase in cancer incidence among persons given 131-I either for diagnosis or for therapy for thyrotoxicosis. These patients were adults, and usually in the 40-60-year age group. Also, very large radiation doses may be less carcinogenic than small ones, and in half or more of these patients, the thyroid has been totally destroyed. Lastly, the follow-up time averages 8-13 years, which may be too soon to see radiation-induced neoplasia. Thus the evidence is reassuring but the question cannot be considered closed.

Thyroid Hyperplasia and Cancer

Chronic stimulation of the thyroid with TSH probably can lead to carcinogenesis in humans, as it can in animals. There are several reports of intensely hyperplastic congenital goiters, untreated for long periods, in which carcinomas have finally developed (187-191). Fortunately, most patients with congenital goiter are recognized and treated with replacement thyroid hormone at sometime during early childhood, so that chronic TSH stimulation does not occur. Interestingly, activating mutations of the TSH-R, which are metabolically like chronic TSH stimulation, lead to benign and not malignant change, as described above.
Relation of Cancer To Other Thyroid Disease

The relationship of thyroid tumors to other thyroid disease is still debated. In the preceding section we discussed whether carcinomas arise from adenomas, occurring either singly or as a component of a multinodular gland. While this must happen rarely, it is not the ordinary course of events. In support of this view one may note, for example, that, whereas adenomas are rarely if ever papillary, approximately 80% of all thyroid carcinomas are papillary. If carcinomas arise from adenomas, one might expect that the majority would be follicular rather than papillary, and this is not the case. Also, although carcinomas, largely of the papillary type, occur in nontoxic nodular goiters with a reported frequency of 4-17% of cases, the age of diagnosis of papillary carcinomas does not follow that for nontoxic goiter (192). Papillary carcinomas occur in children and adolescents, and reach their highest frequency during the middle decades of life. Multinodular goiter, by contrast, is infrequent in childhood, but increases with each decade. The high frequency of carcinomas detected in nodular goiter appears to reflect the efficiency of selection of patients for operation on the basis of suspicious clinical findings in the gland. Although it remains unproven, it is likely that in many or most thyroid adenomas and carcinomas, one specific mutational event leads directly to the development of the specific neoplasm.

Parathyroid adenomas occur in a small percentage of patients with thyroid cancer. The converse relationship may also exist; 2-11% of patients with parathyroid adenomas also have thyroid cancer (193-195). An important reason for this association is the induction of both tumors by X-ray exposure.

Neoplasia and Autoimmune thyroid diseases

An increased incidence of cancer in Hashimoto’s thyroiditis has been reported, Further, focal thyroiditis may occur as an immunologic response to thyroid cancer. In most series in the past the coexistence of Hashimoto’s thyroiditis among thyroid cancer patients was between 2-4%, but the real association, particularly with microcarcinomas, is difficult to be assessed because Hashimoto’s thyroiditis is rarely operated upon. Recently this issue has gained new attention in the era of FNAC, and several reports have found that when a thyroid nodule is associated with autoimmune thyroiditis, the chance of malignancy is significantly higher (9-40%) than in nodules not associated with thyroid autoimmunity (196,197). One possible explanation for this finding might be that patients with autoimmune thyroiditis tend to have higher levels of serum TSH (potential thyroid carcinogen) compared with non-autoimmune patients.

Many reports on Graves’ disease stress a normal or low coincidence of cancer, but several series have reported a significant association between Graves’ disease and thyroid cancer, ranging from 3 to 10% (198-200). However, most of these series were surgical, and the patients were selected for surgery on the basis of suspicious nodules or large goiters. In Graves’ patients treated by radioiodine, no subsequent increase in the discovery of thyroid cancer has been reported.In our review, 4 of 50 patients with thyroid cancer had coincident Graves’ disease (201). Belfiore et al found the risk of thyroid cancer in Graves’ disease to be increased 2-3 fold (198). TSAb can stimulate thyroid cancers when Graves disease coexists, so the idea that TSAb might induce malignant change is tenable, but not proven. It also is possible, but unproven, that continued stimulation of a tumor may make it behave in a more aggressive manner (198,202). Patients with Graves disease and thyroid cancer who underwent total thyroidectomy and 131I ablation fared as well in follow-up as did patients without Graves’ disease.

Micro-carcinomas

The term micro-carcinoma refers to tiny carcinomas (<1 cm), usually papillary and often sclerotic. Such tumors were frequently found in autopsy studies when the thyroids were sectioned completely at 1-2 mm intervals and every abnormality was studied, or during histology of glands operated for large multinodular goiters (203,204). Nowadays, with the extensive use of thyroid ultrasound, micronodules (<1 cm) are frequently discovered in the general population, and proven malignant in a significant percentage. These tumors are probably synonymous with the "occult" tumors described at autopsy, but since they are discovered during life can no longer be considered occult. The term microcarcinomas is now preferred to occult.

Tiny carcinomas, usually papillary and often sclerotic, have been found in 5.7% of thyroids of adults coming to autopsy in the United States (203). This prevalence is noted only when the thyroid is sectioned completely at 1-2 mm intervals and every abnormality is studied. The tumors have a mean diameter of 2 mm, and almost all are under 5 mm. The prevalence is best known in adults (204) and may be lower in young people. Since, in some glands, collections of psammoma bodies also exist in tiny scarred areas, it is hypothesized that such lesions may spontaneously regress. Because of their small size, they are detectable only at surgical or pathologic examination of the gland. These observations, now widely accepted, have provoked much discussion. Certainly most of these tiny tumors cannot be biologically significant, considering the low incidence of clinically recognized cancer. Most of the lesions are probably missed during routine surgical or autopsy pathologic studies. Their cause is unknown. They may be a variant of the occult sclerosing carcinomas described by Hazard, but the latter tumors are usually larger, have a predominant sclerotic component, and clearly do metastasize. It has been suggested that such lesions are in fact the cancers found after thyroid irradiation, but most likely they can explain only a small proportion of radiation-associated tumors. Most of these lesions would go undetected in a standard surgical pathologic examination, and the great majority of radiation-associated lesions are larger. In our own series, the radiation-associated le¬sions were on average 1.7 cm in diameter and only 14% were under 0.5 cm. Whether such "minimal" cancers are in fact the occasional precursors of clinically evident cancers is a moot point. It is clear that they are at present an important pathologic -but not clinical --entity.

PATHOLOGY (Figures 18-9a-h)

Pathologists are agreed that there are peculiar difficulties in the classification and diagnosis of malignant tumors of the thyroid. The histologic changes required for diagnosis of carcinoma include absence of a true capsule, invasion of surrounding normal tissue, invasion of blood and lymph channels, loss of normal follicular architectural arrangements, and cellular abnormalities such as an increase in the ratio of nucleus to cytoplasm, enlarged vesicular nuclei, nuclear folding, increased mitoses, and hyperchromasia of the nucleus. Recently, aneuploidy of nuclear DNA content has been added to this list. Obviously the presence of distant metastases is the most certain criterion. Most students of the disease agree that the ordinary criteria of malignancy have little prognostic value in thyroid tumors, except perhaps in the wildly growing anaplastic tumors. However, it may be noted that pathologists at the Mayo Clinic believe a histologic typing by their criteria provides significant information on prognosis.

Examples of the histologic patterns of several of these tumors are given in Figure 18-9. The papillary adenocarcinoma typically shows tumor cells around a fibrovascular core and, not infrequently, areas of follicular differentiation. Papillary lesions tend to be infiltrative, and encapsulation is rare. Lymphocytic "reactions" are prominent. The cell nuclei have a ground-glass or "cat’s eye" appearance and intracellular inclu¬sions are common. Vascular invasion is rare. Psammoma bodies are often abundant. Multiple intraglandular foci are frequent, especially in children. Areas of lymphocyte infiltration, and even extensive lymphocytic thyroiditis, are common, especially in papillary tumors. Many tumors look much like follicular cancers, but have the characteristic nuclei of papillary cancers. These constitute the "follicular variant" of papillary cancer, and behave more or less as do other papillary cancers.

Figure 18-9. A) Papillary carcinoma of the thyroid. The structure is made up of complex (branches on branches) fibrovascular core structures covered by crowded, overlapping, vesicular nuclei (artifact of fixation). Little colloid is visible. Such histologic foci may be encapsulated, sclerosing, invasive, or multicentric.

Figure 18-9 B) Follicular variant of papillary carcinoma with more typical vesicular nuclei, and hemorrhage in follicular lumens.

Figure 18-9. C) Follicular variant of papillary carcinoma with crowded nuclei showing nuclear folding and peripherally vacuolated colloid.

Follicular adenocarcinomas vary from those with a definite follicular pattern to those with solid sheets of cells. The lesions are more frequently encapsulated, but capsular and blood vessel invasions are typical. The nuclei are normo-or hyper-chromated, or may be quite vesicular. One variant, the so-called malignant adenoma, appears to be nearly benign and can be identified as malignant only by the demonstration of invasion of vessels or capsule, or because of the presence of distant metastases, which may also be composed of normal-appearing thyroid tissue. Hurthle cell carcinomas usually grow as solid sheets of large eosinophilic granular cells with much cytoplasm, and less often with a follicular pattern. An Hurthle cell appearance can also be observed in some papillary tumors. (Figure 18-9d)

Figure 18-9. D) Poorly differentiated follicular carcinoma with oxyphillic features.

Medullary tumors (Fig. 18.9 f-g) have an ominous histologic pattern, with solid masses of cells with large vesicular nuclei (205). There may be considerable associated fibrosis, and deposits of amyloid are a helpful diagnostic point. At the time of initial histologic examination the pathologist should recognize these tumors as entities distinct from the undifferentiated cancers, for the medullary carcinomas have a much better prognosis. Medullary thyroid carcinoma (MTC) is associated with amyloid deposition in the surrounding tissues. Recent studies demonstrated that full-length calcitonin is the sole constituent of amyloid in MTC (206).

In the undifferentiated group of small-cell tumors, giant-cell tumors, and carcinosarcomas, or in the miscellaneous group, the histologic pattern has little resemblance to the original thyroid structure (Fig 18-9e-h).

Figure 18-9. E) An anaplastic carcinoma of the thyroid with pleomorphic giant tumor cell nuclei.

Figure 18-9. F) Medullary (C-cell) carcinoma of the thyroid with amyloid stroma.

Figure 18-9. G) Immunohistochemical anti-calcitonin antibody stain of a medullary carcinoma showing strong red positivity.

Figure 18-9. H) Large cell lymphoma of the thyroid.

The general experience of pathologists has been that, in the absence of irradiation, the substrate in which thyroid tumor forms is usually normal thyroid tissue or displays the changes of multinodular goiter or adenoma in approximately the proportion found in any sampling of the general population (207). There is a slightly increased frequency of association with benign adenomas and with Hashimoto’s thyroiditis (208). Lymphomas are associated with Hashimoto’s thyroiditis, and there is considerable evidence that lymphoma actually evolves from a gland with thyroiditis (209).

Multicentricity is a common feature of thyroid cancer, especially papillary cancer. Innumerable separate foci are sometimes found. Estimates of multicentricity range from 20 to 80% (210,211). Whether this phenomenon represents truly multicentric sites of origin or intrathyroidal dissemination is not clear. This multifocality is thought to be one cause of recurrences in patients treated by subtotal rather than total thyroidectomy.

Both papillary and follicular tumors may appear as small (less than 1.5-cm) tumors surrounded by a densely fibrotic reaction. Although it is frequently said that these "occult" (because they may be found incidentally at operation) tumors are benign, the original report by Hazard (212) and subsequent studies show that cervical lymph node metastases occur (213).
Occasionally pathologic examination suggests conversion of differentiated papillary or follicular cancers into anaplastic forms or conversion of a follicular adenoma into a follicular carcinoma.
An interesting aspect of thyroid tumor pathology is the frequency of metastatic tumors to the thyroid, 5% in the data of Silverberg and Vidone (214) for unselected autopsies and 24% for patients dying of metastatic malignant disease.

Course of the Disease

Clinical signs

Most frequently the tumor is discovered accidentally by the patient or physician as a lump in the neck or may be a fortuitous finding at ultrasound of the neck. Less and less frequently, nowadays, it may appear as a gradually enlarging, painful mass with associated symptoms of hoarseness, dysphagia or dysphonia, or there may be difficulty in breathing. Occasionally a arrives with metastatic nodules in the neck, pulmonary symptoms from metastases, or a pathologic fracture of the spine or hip. Usually there are no symptoms of hyper-or hypothyroidism, but in very rare instances the tumor, usually metastatic follicular carcinoma, can produce enough hormone to cause hyperthyroidism (215,216). (See also Chapter 13)

Upon examination of the neck, carcinoma of the thyroid characteristically appears as an asymmetrical lump in the gland. If it is still within the confines of the gland, it will move with the gland when the patient swallows and may be moveable within the gland. If it has invaded the trachea or neighboring structures, it may be fixed; this is a useful sign. Lymph nodes containing metastases may be found in the supr-aclavicular triangles, in the carotid chain, along the thyroid isthmus, and rarely in the axillary nodes. A sentinel or "Delphic" node above the isthmus may be present. Although carcinoma of the thyroid is typically firm or hard, rapidly growing lesions may sometimes be soft or even fluctuant. When the tumor is poorly differentiated or anaplastic, the lesions may undergo necrosis and discharge through sinuses that developed in the skin of the neck. Patients with thyroid cancer are also prone to develop other cancers, the risk being about double the average. Among these cancers is an excess incidence of leukemia, perhaps related to 131I therapy (217-219).

Age at diagnosis has an important bearing on the patient’s subsequent course. The adverse effect of age on the prognosis increases gradually with each decade (220). For practical assessment purposes, it is clear that patients diagnosed before age 45 have a much better prognosis than those detected later (221). Age is also directly related to the incidence of undifferentiated tumors and to overall mortality. Pregnancy does not seem to worsen the course of established or previously treated thyroid can¬cer (222). Overall, women have a better prognosis than men with cancer (223). Other characteristics of the tumor, including (as would be expected) extraglandular extension, gross invasion of the tumor capsule, and increasing size also carry a worsened prognosis (223).

Papillary Carcinoma-course of disease

Papillary carcinoma has a peak incidence in the third and fourth decades (224). It occurs three times more frequently in women than in men, and accounts for 60-70% of all thyroid cancers in adults and about 70% of those found in children. The disease tends to remain localized in the thyroid gland and in time metastasizes locally to the cervical or upper mediastinal nodes. The lesions are multicentric in 20% or more of patients, especially in children. Using rigid pathologic criteria, perhaps two-thirds of predominantly papillary thyroid cancers are found to have follicular elements. The natural history of these tumors is similar to that of pure papillary lesions (225). The metastases may conform to either histologic pattern. At present, the mixed tumors are lumped together with all other papillary cancers. This tumor tends to be indolent and may exist for decades without killing the host. In a Mayo Clinic series of papillary tumors that were detected because of lymph node metastasis or found incidentally during surgery of the thyroid gland, all the patients were unaffected by the tumors over several decades (224). However, in a recent series from one single institution in Italy, it is apparent that the way of presentation of papillary thyroid cancer has been changing in the last to decades compared to previous years. In particular the authors report increasing number of small tumors and less frequent lymph node metastases at presentation (226)

The frequent occurrence of occult or “minimal’ incidentally found thyroid cancers, usually papillary and under 0.5 cm in size, is described above. However, the term occult has been used in a variety of ways, including reference to tumors with malignant nodes but no obvious primary, or in reference to any tumor under 1.5 cm in diameter. Mayo Clinic reports of papillary tumors under 1.5 cm in diameter, treated with conservative subtotal thyroidectomy and node dissection, have stressed their non-lethal nature, but a 1980 follow-up report on 820 patients treated by this group notes that 6 patients eventually died after spread of tumor from such "occult" primaries (227). Patients with appropriately treated Clinical Class I or II lesions have 96-100% survival even after 15-30 years. Survival lowers to 87% for Class III and 35% for Class IV lesions at 15 years.

While the disease may be aggressive in children, it is distinctly less aggressive in young adults, as compared to patients over age 40 (223).Young patients tend to have small primary lesions and extensive adenopathy, but even with local invasion survival is good (228). When papillary cancer occurs in persons over the age of 45, it may show, on microscopic examination, areas of undifferentiation, and pursue a highly malignant clinical course. The lesions tend to be larger and more infiltrative, and to have fewer local metastases (229). It is possible that persons dying in older age actually have had their disease since youth, and that it has simply evolved into a more malignant phase (230,231).

Papillary carcinoma tends to metastasize locally to lymph nodes, and occasionally produces cystic structures near the thyroid that are difficult to diagnose because of the paucity of malignant tissue. In this case measurement of thyroglobulin in the fluid aspirate is the clue for the correct diagnosis. The presence of nodal metastasis correlates with recurrence (230-232) but has little effect on mortality in patients under age 45. In old studies, cervical adenopathy even seems to confer a protective effect on young people, but this assumption is not confirmed in present series. Indeed in patients over 45, the presence of nodes is associated with greater recurrence rates and more deaths (233,234). The tumors often metastasize elsewhere, especially to lung or bones.

Papillary tumors may metastasize to the lungs and produce a few nodules, or the lung fields may have a snowflake appearance throughout. These tumors are amazingly well tolerated and may allow relatively normal physical activity for 10-30 years. At times, particularly in the follicular variant of papillary thyroid cancer, the pulmonary metastases are active in forming thyroid hormone, and may even function as the sole source of hormone supply after thyroidectomy. The metastases may progress gradually to obstructive and restrictive pulmonary disease. They also may develop arteriovenous shunts, with hypoxia or cyanosis. Such shunts become more prominent during pregnancy, perhaps as an effect of the increased supply of estrogens.

The tall cell variant of papillary carcinoma comprises about 10% of total cases, and as noted by several authors appears to be more aggressive than other forms of the disease (235,236). .
The usual net extra mortality in papillary cancer is not great when compared to that of a control population, perhaps 10-20% over 20-30 years (231,232,234). Mortality is rare in patients diagnosed before age 40, and is much greater in the patients found to be in clinical stages III and IV at initial diagnosis. About one-half of patients ultimately dying from this lesion do so because of local invasion. Frazell and Duffy (237) have noted that papillary carcinoma is not always so benign; they reported 35 patients with "invasive papillary carcinoma," which had a very malignant course.

We found that risk of death from cancer was increased by extrathyroidal invasion (6 fold) or metastasis (47 fold), age over 45 years (32 fold) and size over 3 cm (6 fold). Thyroiditis, multifocality and the presence of neck nodes had no effect on disease-induced mortality.

P.P. 41-Year-Old Man: Long-Term Survival with Papillary Cancer

This man was first seen at age 41. At age 14, while living in Yugoslavia, he had developed a mass in his neck. Tracheostomy was required because of dyspnea, and a biopsy of the mass was performed. A diagnosis of papillary thyroid carcinoma was made, and he was treated with radiotherapy to the neck. Because of an abnormal chest x-ray film, presumed to be due to tuberculosis, he was given streptomycin and isoniazid for 2 months, but this therapy was discontinued when no improvement occurred. Extracts of calf thymus were injected. He was evaluated for hemoptysis at age 18, and chest x-ray films again showed the infiltrates without evidence of change. The patient was not given thyroid hormone replacement therapy at any time. At age 39 he developed fatigue, substernal chest pain, occasional cough without production of sputum, and occasional hemoptysis. A thyroid scintiscan at another hospital revealed poor visualization of the thyroid and uptake of 4%, and 131I uptake in the mediastinum.

Physical examination disclosed hyperpigmentation of the skin in the area of radiotherapy in the neck and supraclavicular areas. There was no evidence of a mass or lymph-adenopathy. The tracheostomy site was well healed. The lungs were clear, BP was 100/70, pulse 68, and respiration 16/min, and there was no cyanosis. Rou¬tine complete blood count, urinalysis, and blood chemistry test results were normal. Chest x-ray films showed multiple nodular densities throughout both lungs and a prominent left hilum. The results of a radiographic skeletal survey and a technetium pyrophosphate bone scan were normal. The FTI was 8.4 and TSH was 4.1 µU/ml. A whole body 131I scan showed multiple areas of abnormal uptake in the area of the thyroid, and satellite areas of focal uptake around the thyroid bed. There was also focal activity in the mediastinum, in both hila and in the lung fields. A 72-hour chest uptake was 8.9%. The serum TG level was 81 ng/ml.

The patient was treated multiple times with RAI and remained well while taking replacement T4 for ten years. At age 51, the tumor grew more rapidly, failed to accumulate 131I, and caused the patient’s death.

This patient developed thyroid carcinoma at age 14 and probably had lymph node and lung metastases at that time. He lived a normal life during the ensuing 27 years, without suppressive thyroxine treatment, and with only intermittent episodes of hemoptysis. The tumor responded at first to 131I but later was uncontrolled. The common benign course of metastatic papillary thyroid carcinoma over many years is clearly shown, as is the equally typical later exacerbation and death.

Follicular Carcinoma

Follicular carcinoma has a peak incidence in the fifth decade of life in the United States and accounts for about one-quarter of all thyroid carcinomas (223,238,239). In past decades, follicular carcinomas comprised up to 50% of thyroid malignancies in Europe. The high incidence may partly be explained by iodine deficiency, but, more likely, was due to histological miss-classification at a time when the follicular variant of papillary thyroid carcinoma was not recognized as papillary but rather was classified as follicular. It is a slowly growing tumor and frequently is recognized as a nodule in the thyroid gland before metastases appear. Variation in the cellular pattern ranges from an almost normal-appearing structure to anaplastic tissue that forms no follicles or colloid. The insular variant of follicular tumor tends to be more aggressive (240). The tumor is three times as common in women as in men. At operation one-half to two-thirds of these tumors are resectable. Tumors that are small and well circumscribed (not surprisingly) tend to be less lethal than those actively infiltrating local structures at the initial operation. Local adenopathy, which is uncommon, probably carries a greater risk, and extensive invasion of the tumor capsule and thyroid tissue increases mortality (241). Local direct invasion of strap muscles and trachea is characteristic of the more aggressive tumors (242). Resectability depends on this feature, and death may be caused by local invasion and airway obstruction. The "minimally invasive" variant has a far better prognosis than the highly invasive variant.
Follicular carcinomas tend to invade locally and metastasize distantly, rather than to local nodes, and are especially prone to metastasize to bone or lung. In a Massachusetts General Hospital series (231), one-half had metastasized at the time the diagnosis was originally established. Bony metastases are usually osteolytic, rarely osteoblastic, and the alkaline phosphatase level is rarely elevated. The tumor and metastases often retain an ability to accumulate and hold iodide, and are therefore sometimes susceptible to treatment with RAI. Indeed, some metastatic tumors synthesize thyroid hormone in normal or even excessive amounts. RAI therapy, as discussed below, improves survival in these patients (241).

Occasionally the primary lesion of a follicular tumor appears to be entirely benign, but distant metastases are found. Invasion of vessels or the capsule, apart from the metastasis, is the only reliable criterion of malignancy. This variant has been called the “benign metastasizing struma” or malignant adenoma. It has a more prolonged course than do other varieties of follicular tumor, and is the type that has offered the best opportunity for the therapeutic use of 131I.
The net extra mortality attributable to follicular cancer in the 10 -15 years after diagnosis is 30 -50% (231,233,235). Of the patients dying from the lesion, three-fourths do so from the effect of distant metastases and the remainder from locally invasive disease.

Hurthle CellTumors

Hurthle cell tumors are histologically distinct from other follicular tumors, but they pursue a similar course. They tend to invade and metastasize locally and have a strong propensity to recur after surgery. The course tends to be prolonged. These carcinomas often do not accumulate 131I. However, in a large survey, Caplan et al (243) found that 4.4% of Hurthle cell neoplasms were hot on scan and 8.9% were warm. Serum TG levels may be normal or elevated. Cheung et al recently studied the presence of ret/PTC gene rearrangements in Hurthle cell tumors and found that many expressed ret/PTC, and also had other evidence of a papillary cancer origin, including focal nuclear hypochromasia, grooves, and nuclear inclusions. Tumors with the ret/PTC gene rearrangement tended to have lymph node metastases, rather than hematogenous spread. Thus Hurthle cell tumors can be classified into Hurthle cell adenomas, Hurthle cell carcinomas, and Hurthle cell papillary thyroid carcinoma (244).

Insular Tumors

A subset of thyroid carcinomas which give a histologic picture of islands of cells -thus "insular" --has been identified (243). These tumors often look like anaplastic cancers, but sometimes are able to concentrate 131I and thus are amenable to this excellent treatment. Whether these are properly considered a variety of follicular cancer is uncertain. The important message is that the histology in this instance does not reliably predict the utility of 131I treatment, suggesting that all patients with thyroid cancer should at some point be studied to determine whether 131I treatment is possible.

Undifferentiated Tumors

Undifferentiated tumors occur predominantly in persons over 50 years of age and constitute an increasing proportion of lesions in each subsequent age decade. Of great interest is the pathologic evidence that such tumors arise, in perhaps half of the cases, in a long-standing benign lesion or in differentiated carcinoma (245). Although 131I therapy for differentiated cancers has been blamed for this dedifferentiation, current evidence is against this hypothesis. Spindle cell, and most giant-cell carcinomas of the thyroid grow rapidly and are very invasive. Local invasion may cause difficulty in breathing or swallowing, and tracheotomy is frequently required. These tumors metastasize to lymph nodes both locally and widely, but not characteristically to bone. Pulmonary metastases are frequent. Some patients present with a tender mass suggesting thyroiditis, and occasionally thyroid destruction induces hyperthyroidism (246). The outlook in this particular group of tumors is poor. By the time the diagnosis is made, the disease has spread in most patients beyond the area that can be attacked surgically, and they die within 6 months to 1 year. A few, perhaps 10%, of these tumors are entirely resectable when first discovered. There is nothing characteristic about the growth pattern of these tumors; their behavior is similar to that of any highly malignant tumor elsewhere in the body. The course of the epidermoid carcinoma and sarcoma of the thyroid is essentially the same. Small-cell anaplastic carcinomas are also found, but probably most tumors so classified in the past are actually lymphomas or lymphosarcomas.

Malignant Lymphomas

Lymphomas of the thyroid gland represent less than 5% of primary thyroid neo¬plasms (247-250). Unlike most other thyroid neoplasms, lymphomas usually appear as rapidly enlarging masses and local symptoms are common. Many patients note pain, hoarseness, dysphagia, and dyspnea or stridor. Hoarseness is often present in the absence of vocal cord paralysis. Rarely, patients may have the superior vena cava syndrome. The mean age at occurrence is 62 years. Primary lymphomas of the thy-roid are two to three times more common in women than in men.

The incidence of hypothyroidism at the time of appearance is variable, ranging from 0 to 60% (251,252). The co-occurrence of pathologic lymphocytic thyroiditis has ranged from 30 to 87%. These figures may underestimate the true incidence, as some patients have only had a biopsy examination and in others the entire gland has been replaced by the lymphoma. The frequent presence of thyroiditis has naturally led to the suggestion that the lymphoma might derive from preexisting thyroiditis.

The co-occurrence of thyroiditis may create difficulties in the proper interpretation of fine needle aspiration cytology. The clinical appearance must be carefully considered in accepting a diagnosis by fine needle aspiration of thyroiditis only or thyroiditis with lymphoma. An excisional or large needle biopsy may be necessary to make the correct diagnosis.

The majority of thyroid lymphomas are diffuse, large-cell lymphomas (formerly classified as diffuse histiocytic or reticulum cell lymphomas), diffuse mixed small and large cell lymphomas (formerly called diffuse mixed lymphocytic-histiocytic), or diffuse small cleaved-cell lymphomas (formerly classified as diffuse poorly differen¬tiated lymphocytic). Although older series include reports of Burkitt lymphoma, none were reported in larger, more recent series. Areas of diffuse large-cell lymphomas may have features similar to those characteristic of Burkitt’s lymphoma or the Reed-Sternberg cells of Hodgkin’s lymphoma.

Metastatic Carcinomas to the Thyroid

Melanomas, breast tumors, pulmonary tumors, gastric, pancreatic, and intestinal carcinomas, renal carcinomas, lymphomas, carcinomas of the cervix, and tumors of the head and neck may metastasize to the thyroid. Sometimes the first indication of one of these tumors may be the appearance of a lump in the neck. Unless there is evi¬dence for a primary site elsewhere, these tumors are easily mistaken for expanding tumors that have their origin in the thyroid gland. Usually by the time metastases appear in the thyroid, other metastases have occurred and the primary lesion may be discerned.

Cancer in Aberrant Thyroid Tissue

Thyroid tumors occasionally arise in lingual thyroids, along the thyroglossal duct (253), in substernal goiters, and even in struma ovarii. Whenever discovered, the therapeutic approach should the removal of the tumor assoiated with total thyroidectomy to allow subsequent treatment with radioiodine and follow-up based on serum Tg mreasurement.

MEDULLARY CARCINOMA

Hazard et al. (205) first described a unique tumor in the thyroid characterized by sheets of cells with large nuclei, amyloid deposits, fibrosis, multicentricity, and an unexpectedly benign course in view of the solid tumor appearance. Over 50% may have local or distant metastases at diagnosis. The tumors may metastasize locally, or to bones and soft tissues. The thyroid primary tumor and the metastases may show dense calcification on x-ray film. The course tends to be progressive, and 10-year survivorship varies from 50 to 70%. These tumors, which constitute 2-8% of all thyroid cancers, are derived from the "light," or "C," or "parafollicular" cells. These are calcitonin (CT)-secreting cells, distinct from thyroid acinar cells, and are of ultimo-branchial origin.

The tumors may occur sporadically (about 70% of the total) or as part of the MENII syndromes, which constitute about 10-20% of the cases, and are transmitted in families as dominant traits due to activating germline point mutations of the RET proto-oncogene (254,255). In contrast with thyroid epithelial cell tumors, the female to male ratio is near unity. MEN-II (or IIA) includes patients with medullary thyroid cancers, pheochromocytomas, and parathyroid hyperplasia or adenomas. MEN-III (or MEN-IIB) includes medullary thyroid carcinoma, mucosal neuromas, pheochromocytomas, which are usually bilateral and often malignant, occasionally cafe-au-lait spots, and possibly Gardner’s syndrome (mucocutaneous pigmented nevi and small intestinal polyps) (133,256-259). An occasional variant of medullary thyroid cancer appears to contain both CT and TG, suggesting the cells, surprisingly, have features of both medullary thyroid cancer and follicular cancer (260).

Gastrointestinal symptoms including diarrhea, constipation, and rarely megacolon occur in these patients and may occur before the thyroid tumor is detected. Hyperplasia of C cells often precedes the development of familial cancers (261). Medullary tumors derived from the C cells not only secrete CT, but in addition may produce serotonin (with a carcinoid syndrome), prostaglandins, corticotropin releasing factor, and adrenocorticotropic hormone (causing Cushing’s syndrome), histaminase, and somatostatin. Some of these substances, rather than calcitonin, may be the cause of the severe diarrea syndrome associated with metastatic medullary thyroid cancer. Interestingly, expression of somatostatin appears to correlate with improved prognosis (262). Medullary thyroid carcinoma cells in tissue culture have been found to produce Ghrelin, which is an endogenous ligand for the GH secretagogue receptor. MTC is known to produce several gastrointestinal hormones and neuroendocrine peptides, in addition to calcitonin, and including CGRP, ACTH, serotonin, chromogranin A, and vasoactive intestinal peptide, and to this we now add Ghrelin (263). Alcohol ingestion is reported to induce attacks of flushing and diarrhea and to stimulate CT secretion by the tumors (264). The CT secreted by these tumors rarely causes hypocalcemia (265).

Medullary tumors have an ominous histologic pattern, with solid masses of cells with large vesicular nuclei (212). There may be considerable associated fibrosis, and deposits of amyloid are a helpful diagnostic point. At the time of initial histologic examination the pathologist should recognize these tumors as entities distinct from the undifferentiated cancers, for the medullary carcinomas have a much better progno¬sis.

Ret oncogene and MedullaryThyroid Cancer

Studies on patients with MENI and MEN II indicated linkage to chromosomes 11 and 10, respectively (138). Subsequent studies demonstrated that the ret oncogene is present at 10q11.2. Germline mutations have been detected in this oncogene in all patients with MEN II A and MEN IIB, and familial MTC (139). RET is a cell-membrane receptor of the growth factor family, with tyrosine kinase function. In up to 97% of patients with MenII A, mutations are found mostly in codons 609, 611, 618, 620, and 630 in exons 10 and 11. These all involve substitutions of other aminoacids for cysteine, and are thought to cause activation of the gene by aberrant disulphide bonding causing dimerization. Similar changes are seen in Familial MTC. In patients with the MENIIB syndrome, almost all, if not all, mutations involve an amino acid substitution of threonine for methionine at codon 918 in exon 16, and are thought to induce a change in substrate phosphorylation. Somatic mutations in ret are present in up to half of patients with sporadic MTC and are almost always in codon 918 (140,141). Mutations in this codon are thought to imply a poor prognosis (266).

Calcitonin and CEA

The calcitonin assay provides a convenient screening procedure in families with this genetic trait (267). Family members at risk should firstly BE screened for ret oncogene mutation in their blood, and when when recognized as a carrier of the mutation, should be screened by neck ultrasound and calcitonin measurement to assess the presence of the disease. Every member of one of these families with either a thyroid mass or elevated calcitonin levels should have a thyroidectomy. If no thyroid nodule is detected and the serum calcitonin is normal according to the reference range for that specific age group, prophylactic thyroidectomy should be considered in order to take out the thyroid gland before the disease is initiated.

There is controversy regarding the more appropriate age for prophylactic thyroidectomy. Recent ATA guidelines for MTC have suggested different time for surgery according the the age and the mutation found (268) In MEN-IIA, the tumors follow a rather benign course somewhat akin to that of follicular cancer, and usually can be controlled by surgery. MEN-IIB tumors are much more aggressive and often cause death in the second or third decade. TSH suppressive treatment is not efficacious in MTC and has no logical basis.

Secretion of calcitonin by medullary cancer is remarkably increased by calcium or pentagastrin infusion (269). This procedure can be helpful in establishing a diagnosis if available. At present the infusion of pentagastrin (0.5 µg/kg over 5 seconds), with determination of calcitonin levels at 0, 1, 2, 5, 10, and 15 minutes, appears to be the best test. Alternatively stimulation with calcium gluconate, 20 mg/kg, can be done with infusion IV over 1 minute and testing as tor TRH. (This test can induce adverse cardiac effects, and close monitoring is reasonable.) Basal CT values are normally under (depending on the laboratory) 30 pg/ml (269). Values of 30-100 after pentagastrin indicate hyperplasia, and values over 100 typically indicate the presence of cancer. Calcitonin should drop to undetectable levels if the tumor is completely removed surgically. It should be noted that excess production of CT is not unique for medullary cancer, but can occur with granulomatous diseases and other cancers. An alternative is to monitor Calcitonin after Calcium infusion. Patients with the syndrome should also be studied with parathyroid hormone and catecholamine assays in order to determine the presence of other components of the syndrome.

The neoplastic C cells also produce carcinoembryonic antigen (CEA) in large amounts. Serum CEA levels are elevated in medullary cancer with the same frequency as are CT levels (270). Although CEA determination provides another parameter to follow, it does not offer any obvious advantage, and lacks the specificity of CT determinations. Tumor dedifferentiation is associated with a fall of CT and increasing CEA. This is an ominous sign.

Several specialized scanning procedures have been used in MTC. Total body imaging with Tl-201 chloride andTc-99m(V)DMSA have been successful in localizing metastases. 131I MIBG and 131I anti-CEA have been used both for localization and in attempts at therapy (271). Most recently, radiolabelled somatostatin has been used as a whole body scanning agent.
Since patients with MENIIA and IIB, and familial MTC, probably all have a germline mutation of the ret oncogene near the centromere of chromosome 10, and many patients with sporadic MTC have somatic ret mutations, molecular techniques can be used to detect at-risk subjects in recognized families (269). PCR amplification of exons 10, 11, and 16, followed by single strand conformational electrophoretic analysis, or use of specifically designed restriction enzyme sites, allows recognition of most mutations (272). This information is crucial in defining potential risk in young children and identifying need for operation or frequent pentagastrin testing (255), and presumably will also screen out members of families who will not need to be repetitively screened by pentagastrin stimulation tests. Unlike occasional false positives with CT assays, the results of genetic screening are unambiguous (273).
For readers with an interest in a somewhat broader view of oncogenesis, it may be noted that a syndrome entirely analogous to metastatic medullary thyroid carcinoma appears frequently in aged bulls (274). Histologically similar adenomas are also frequently found.The lesion may be due to excessive dietary calcium; whether a similar stimulus could operate in human disease is unknown.

Treatment of MedullaryThyroid Cancer (MTC)

MTC is unique since it can be detected in MEN-IIA or MEN-IIB families by genetic analysis and screening tests measuring pentagastrin-stimulated CT secretion before the disease reaches clinical detectability. In families with familial MTC, MENIIa, and IIB, repeatedly observed elevations to well above the normal range (e.g., peak values of 30-10pg/ml) constitute a basis for operation. In these patients focal tumors or C-cell hyperplasia may be found after near-total or total thyroidectomy is performed. Since C-cell hyperplasia precedes the development of malignancy, it is currently believed logical to operate at this stage of the disease even in young children with MENIIB. Patients with MENIIA or familial MTC are similarly followed, tend to develop tumor later, and 20-40% may never develop cancer (275). International Workshops on MTC have recommended prophylactic thyroidectomy before the age of 5 years in persons harboring mutations of codons 611, 618, 620, and 634 because of their high efficiency in transformation. Consensus on best treatment for patients with other mutations (codons 609,630,768,790,791,804,891) is not clear, with thyroidectomy recommended from ages 5 to 10 (268,276).

Patients with clinical tumor of any size, sporadic or familial, are also treated by near-total or total thyroidectomy. Invasive disease is resected if possible. Since cervical nodal metastasis occur in up to 90% of patients with palpable tumors, a careful mod¬ified radical neck dissection is performed with removal of nodes in the central and ipsilateral compartment. The exploration for nodes should include the upper mediastinum. Bilateral neck dissection may be appropriate in patients with hereditary tumors (277).

Management of Post-operative MTC patients who have positive CT assays

When post-surgical serum CT is undetectable in basal condition and after pentagastrin stimulation, the patient has an almost 100% chance of being in complete and stable remission. In this case follow up should be based on periodic measurement of serum CT, without any instrumental evaluation. After operation, patients with still-detectable serum CT levels should be screened for persistent disease. Imaging should consists of CT of the neck and chest, MRI of the liver and bone scintigraphy. PET scanning may also be informative in selected cases. Some centers used to ablate residual functioning thyroid tissue with 131I, as in papillary or follicular tumors, since this procedure may ablate tiny foci of residual cancer (278,279). However, the value of this procedure is not proven, and this procedure is almost universally abandoned. In patients with local invasive disease, who are above age 45-50, we believe that radiotherapy (5,500-6,000 rads) is useful, although there is disagreement on this point.(v.i.). Occasionally CT levels fall gradually over a year. It is also possible to follow the serum level of CEA as a tumor marker, or CT-related peptide (280). A discordant elevation of CEA in relation to CT may be an indicator of an aggressive tumor. However, in general, CT is the most informative marker.

What to do about the frequently-found elevated CT level without an obvious tumor source is less certain. Residual or recurrent resectable disease should be approached surgically. Neck dissection, if not done previously with a unilateral tumor, should be considered. In the absence of an identifiable source of the CT, careful, extensive, microdissection of the neck and upper mediastinum have been proposed in the hope to eliminate the source of CT (281), but the poor results of such extensive operation have dissuaded many from this approach. Catheterization of the superior vena cava and internal jugulars, with an attempt to localize the tumor by means of multiple venous sampling for CT (242) during pentagastrin stimulation, is possible. The technical success of this approach has been demonstrated, but often the CT level is not reduced to normal, even if operation is subsequently performed on the identified area (282). Radiotherapy may be given to identified non-resectable lesions.

Radiation therapy for MTC

In many cases, no source for the CT is found. Watchful waiting is the approach preferred by some experienced clinicians. Alternatively, mantle irradiation can be recommended. As shown by Simpson (283), medullary tumors are radiosensitive, although a full response may not be seen for many months. This irradiation has been shown to prolong local relapse-free time, but not to clearly improve ultimate prognosis (284). The effectiveness of radiotherapy was evaluated in 24 of 139 patients with medullary thyroid cancer who were given radiotherapy because of advanced local disease at presentation. Only one had normalization of calcitonin, but ten remained free of clinical recurrence. Local relapse was significantly reduced after radiotherapy, but there was no difference in ten year survival between those with and without treatment. These authors believe that radiotherapy does reduce the incidence of loco-regional relapse, but do not advise routine use of radiotherapy because of the relatively favourable long term survival of patients even with elevated calcitonin levels after operation (285). Other investigators question the value of X-ray therapy, contending that patients with MEN-II have multicentric foci from the start, and that radiotherapy may actually worsen prognosis (286). Systemic therapy is reserved for patients with proven symptomatic metastases, and the program is described below.

MTC is associated in MEN-II with parathyroid adenomas and hyperplasia, and in both MEN-IIA and MEN-IIB with pheochromocytomas that are often bilateral and malignant. Parathyroid hormone, VMA, and catecholamine assays should be done to evaluate these problems, and pheochromocytoma, if present, should be treated before the thyroid cancer. Occasionally neuromas cause problems requiring surgery of the intestine or other organs, including the larynx.

BIOLOGICAL FUNCTION OF THYROID TUMORS

Interesting abnormalities in iodide metabolism are typical of thyroid adenomas (described above) and in thyroid carcinomas. In general, the tumors are cold at 131I scintiscan, because they tend to lose the ability to accumulate iodide from the plasma, to bind iodide to TG, or to synthesize thyroid hormone (287). The pattern is one of dedifferentiation, the loss of those functions peculiar to the thyroid gland. Follicular carcinomas tend to retain iodide metabolism more completely than other tumors, and therefore may be susceptible to treatment with RAI. Localization of 131I in tumors, if present at all, is spotty rather than homogeneous.

Occasionally follicular thyroid cancers express high levels of Type2 iodothyronine deiodinase, and cause "consumptive hypothyroidism" (288). This surprising phenomenon has also been observed in patients who have highly vascular tumors which express the deiodinase (289).

In rare cases follicular tumors or their metastases produce significant amounts of thyroid hormone. In many instances the thyroid gland has been completely removed or destroyed, but the metastases have been sufficiently active to maintain the patient in a thyrotoxic state. More frequently, the metastases produce enough hormone to maintain the patient in a euthyroid condition, but not enough to produce thyrotoxicosis. This action indicates that the tumor may be responsive to thyrotrophic hormone, like the observation that thyrotropic hormone can induce growth of the tumors.
Carcinomas usually are associated with elevated serum TG levels. Growing differentiated tumors may cause levels of TG of > 2000 ng/ml. In Hurthle cell tumors and anaplastic cancers, TG levels are usually minimally elevated.

Differentiated thyroid cancers tend to retain a normal affinity and capacity for binding of TSH to their membrane receptors (290), and TSH stimulates cAMP production normally in tumor tissue. Perhaps in keeping with the clinically recognized unresponsiveness to TSH, undifferentiated tumors lack high-affinity TSH receptors. Genetic studies show that in these tumors the expression of the TSH receptor gene, and other thyroid differentiation genes is usually lost (291). In autonomous thyroid nodules, TSH stimulates cAMP accumulation normally; hypersensitivity to the hormone has sometimes been found (292,293).

Immunologic Studies on Tumors

Differentiated thyroid cancers contain TG and microsomal (TPO) antigens cross-reacting with antibody to normal thyroid antigens; this fact can be very useful in identifying metastatic deposits (294). Some tumors tend to lose the microsomal (TPO) antigen (295,296). There is strong evidence that the immune system mounts a defense against the tumors, although an imperfect one. Tumors commonly are infiltrated by lymphocytes, and there is less tendency for nodal metastasis, and a better prognosis, with tumors so involved (207). Antibodies to TG and TPO antigen are more common in cancer patients than in control subjects (254). We have shown, using in vitro assays, that 50% of patients develop immunity to TG, and some react to apparently specific thyroid tumor antigens (297). The tumor antigen is recognized in both autologous and heterologous tumors, and is not present in Graves’ disease tissue. Immunization with tumor preparation has been performed, and in a few cases has led to higher in vivo and in vitro reactivity and apparent partial tumor regression (298). Non-specific immune complexes are found in 10% of patients with differentiated cancers, and specific TG containing complexes are present at low levels in 30% of patients (299). With progress of thyroid cancer, the immune system gradually loses responsivity, as with other tumors (296,298,299).

In vivo, circulating antibodies to Tg and/or TPO are found in nearly one fourth of the patients, and the anti-TG antibodies cause interference in assay of this substance thus making follow-up of patients difficult. Their presence seems not to influence the tumor outcome (300,301). Interestingly, the disappearance of antithyroid autoantibod¬ies during follow-up, correlates with achievement of complete remission, suggesting that elimination of the antigen with successful therapy causes the disappearance of the corresponding autoantigen. Thus, the conversion from antibody positive to antibody negative may be interpreted as a marker of remission, while the persistence of the antigen may be regarded as indirect evidence of persistent disease (302).

The fibrosis and amyloid deposits in medullary cancers might suggest a host antitumor response, and indeed antitumor immunereactivity is detectable in vitro in most of these patients (303).
Sometimes autoimmunity may play an adverse role. Graves’ disease co-occurs with 4-8% of thyroid cancers, whether by chance or through a true association is uncertain. In some patients with the association of thyroid cancer and Graves disease, the thyroid-stimulatory autoantibodies seem clearly to stimulate the growth of the cancer (198,199,200), but overall, it is not clear that the co-occurrence of Graves’ disease causes thyroid tumors to behave more aggressively.

DIAGNOSIS

Most patients with thyroid carcinoma are recognized because of a positive or suspicious FNA done for the clinical or sonographical discovery of a thyroid nodule, either single or in "multinodular" goiter (304). Anaplastic cancers and lymphomas commonly present as a smooth goiter. Stony hardness, fixation to the trachea, and damage to recurrent laryngeal or cervical sympathetic nerves are other important clinical signs. The problems of differentiating carcinoma from other lesions of the thyroid have been reviewed in the preceding discussion of thyroid nodules. A few additional comments are given here. FNA and US studies, constitute the basic and most useful laboratory studies. TSH and fT4 are usually measured to verify metabolic status, and anti-TPO and TG antibodies may be useful in helping differentiate thyroiditis. Isotope scans have a limited role in initial diagnosis.

Thyroglobulin assay-

Although TG assay has been suggested as an important marker for thyroid cancer (305), practice shows that elevated TG levels can be caused by adenoma, multinodular goiter, and other diseases; thus the determination is of little value before operation. Chest xray may be informative but is often omitted. In lesions which extend outside the thyroid, or have metastasis, ultrasound of the neck, CAT scanning of the lungs, and MRI of the neck can provide useful information prior to surgery, and especially when following disease progress.

Fine Needle Aspiration cytology-

Currently, as described previously in the section on diagnosis of thyroid nodules, most reliance is placed on needle aspiration cytology. Fine needle aspiration cytology of cervical nodes under ultrasound guidance also must be remembered as a very useful technique (306).

Ultrasound

Typical features of ultrasonic scanning can suggest malignancy with rather good sensitivity. Microcalcifications, irregular margins, hypoechogeneicity, intranodular blood vessels and round shape are all in support of malignancy and may dictate the need for FNA. The US procedure has replaced other scanning procedures such as angiography, thermography, iodopaque "thyrography", and fluorescent scanning. US is described in the chapter on thyroid nodules and 6c. Very recently thyroid elastosonography has been proposed as a promising new tool to distinguish benign and malignant nodules.

Isotope scanning

Scintiscanning using 131-I is currently usually omitted in the initial evaluation of a possible malignancy. Most of the anatomic information needed is provided by US. Occasionally isotope scanning is useful in demonstration of hyperfunction in a nodule, lack of a lobe, extension below the sternum, or other factors. Demonstrate of failure of the involved area to concentrate RAI used to be important, since malignant lesion are invariably cold at scan. However it in no way proves the presence of cancer, nor does its absence rule out this possibility. Whole body scintiscanning is useful to determine whether lesions in lung or bone are thyroid tumor metastases. Significant accumulation of RAI by the metastasis definitely proves thyroid origin. Occasionally uptake of RAI can be demonstrated in metastases in the neck before surgery. This finding is almost certain evidence for cancer. Usually normal thyroid tissue must be destroyed and TSH elevated before a metastasis will accumulate 131I. 131I whole body scanning in management of previously diagnosed thyroid cancer is discussed later in this chapter. Some tumors accumulate iodide but do not organify it. They are delineated by radiotechnetium scans done during the early "iodide phase" of isotope distribution, but not by scans using131-I or 125-I done at 24 -72 hours, when the storage of organified isotope is primarily recorded. Conversely, some metastases accumulate small amounts of 131-I but are not shown on short-term technetium scans (307).
Recently, PET scan has been introduced and found informative for the imaging of metastatic disease devoid of 131-I uptake. 18-F Fluorodeoxyglucose Positron Emission tomography can localize tumor and determine tumor volume. Large deposits (>125ml) have a very adverse prognostic implication (308). It appears that stimulation of metastatic deposits by elevated TSH makes PET scanning more sensitive (309,310).

CHOICE OF OPERATIVE PROCEDURE

Which operative procedure is indicated when FNA is suspicious or indicative of cancer? (Table 18-6)

Suspicious FNA lesions have a nearly 70-80% chance to be malignant, while an FNA indicative of papillary thyroid cancer is almost always true positive at final histology. Thus, we recommend total (or near-total) thyroidectomy as the initial surgical procedure in these categories, regardless of the size of the nodule. "Near-total" thyroidectomy refers to a procedure which intentionally leaves small portions of thyroid tissue near parathyroid glands or at the entry of the recurrent nerve into the larynx, and is associated with a marked reduction in possibility of hypoparathyroidism and nerve damage. It is frequently used with intended 131I ablation of residual thyroid tissue.

Some authors prefer lobectomy with frozen section examination in case of suspicious FNA. .It must be noted that frozen section carries a high rate of false negative diagnosis, compared to final histology. For this reason, some authors prefer to do total (or near-total) thyroidectomy without performing frozen section. In addition, in many follicular lesions the diagnosis of malignancy can be made only from paraffin sections.

Table 18-6. Suggested Surgical Procedures in Thyroid Cancer

* STT = Subtotal thyroidectomy ** NTT = Near-total thyroidectomy *** MND = Modified neck dissection

Among patients with papillary cancer within the gland, some will have cervical lymph node involvement and others will have no obvious spread. The utility of prophylactic neck dissection is controversial. Sone authoritative centers are in favour but other, including the authors of this chapter, prefer to perform central neck dissection only when there is a preoperative evidence of lymph node metastases at US or intraoperative evidence, The same attitude seems indicated for lymph node dissection of other node chains. Whenever a patient treated with lobectomy is found to have cancer at final histology, the question arise whether to perform completion thyroidectomy. The indication of several guidelines (39) are in favour of completion thyroidectomy, with the exception of patients with unifocal, small, intrathyroidal, papillary thyroid cancers without evidence of lymph node metastases and favourable histology.

The approach proposed here, is based on several observations. Multicentric involvement is reported to range from 25 to 90%. The wide variation of multicentricity (or intraglandular dissemination) can be explained in part by the finding that the incidence of multicentricity is doubled if one does whole gland histologic sections. There is little or no relationship between the size of a solitary nodule and the incidence of intraglandular dissemination, but an increasing degree of histologic malignancy is associated with the frequency of dissemination.

Mazzaferri et al., in their review of 576 cases of papillary carcinoma, found that total thyroidectomy significantly reduced the incidence of recurrences, and recurrences will presumably be correlated with deaths from disease (234). Samaan et al (311) also supported this procedure. Hay et al. evaluated the efficacy of different surgical approaches to treatment of patients with low risk papillary carcinoma at the Mayo Clinic and concluded that more extensive surgery was not associated with lower case specific mortality rates, but was associated with a lower risk of local regional recurrence. Their data supports the use of bilateral resection as the preferable initial surgical approach (312).
Total thyroidectomy carries an increased risk of hypoparathyroidism, recurrent nerve damage, and the necessity for tracheostomy (313). Accidental unilateral nerve damage may reach 5%, but fortunately bilateral injury is rare (314). All surgeons attempt to preserve those parathyroid glands that can be observed and spared, and an attempt is often made to transplant resected glands into the sternocleidomastoid muscles. Reports range from a 1 to a 25% incidence of hypoparathyroidism after total thyroidectomy (234,315).

TUMOR STAGING AFTER SURGERY

Tumor staging is intended to identify the risk of death or recurrence after initial treatment. The most used staging system is the TNM Staging system which combines simplicity with rather good predictive power.

Several other staging systems have been developed. One is the Clinical Class system developed at the University of Chicago and is based simply on the extent of disease (316). Other systems are designed to predict outcome. The EORTC classification pro¬posed by the European Thyroid Association is based on age, sex, histology, invasion, and metastases (317). The Dames classification includes data on age, extent and size of primary, distant metastases, and DNA ploidy (318). MACIS includes data on age, invasion, metastases, size, and completeness of surgery (319). All of the systems appear to be effective in categorizing patients into largely similar low and high risk groups.

Several groups have recently established new criteria for risk assessment based on pathological features combined with clinical features and with the response to initial therapy. The idea is to delay the risk assignement to a time when the response to initial therapy may be evaluated. The first proposal (called “Ongoing Risk Stratification”) came from the Memorial Center in New York (320), where patients were assigned to low or high risk category based on the results of follow-up after initial treatment. Patients in apparent complete remission at that time were defined as low risk, regardless of the initial risk stratification obtained soon after surgery. A second proposal came from an Italian study (321). These authors assigned patients to low or high risk group at the moment of the first evaluation done 8-12 months after surgery and radioiodine ablation (if performed). Patients free of disease (negative neck US, undetectable basal and stimulated serum Tg and no other evidence of disease) were classified at low risk. Patients with any evidence of ersistent disease (including detectable TG) were considered at high risk of recurrence. The authors demonstrated that nearly half of the patients could be shifted from the high risk category (at the time of surgery) to the low risk category. The system was named Delayed Risk Stratification (DRS). One advantage of these delayed risk stratification systems is that gives an idea of the risk of recurrence which is not considered in the TNM classification.

TSH SUPPRESSIVE/REPLACEMENT THERAPY

After operation all patients are kept on TSH-suppressive thyroid hormone therapy with l-thyroxine . At the time or the first post-surgical evaluation, individuals with current active cancer (other than medullary or lymphoma) should continue with TSH-suppressive therapy aimed to a TSH around 0.1 µU/ml. Pushing TSH below this level has not been associated with better outcome, while has been associated with more frequent side effects from clinical or subclinical hyperthyroidism. Patients who are considered free of disease, should have their replacement lowered to provide a TSH in the low-normal range, and ultimately as safety is assured, in the normal range.

CHILDHOOD THYROID CANCER

Some special features of thyroid cancer occurring in children deserve comment. It is, of course, an uncommon disease. The tumors are usually papillary or mixed histologically, and tend to grow slowly, with a high frequency (50 -80%) of neck metastases, but with a relatively favorable prognosis. Very young patients (under age 12) often have relatively aggressive disease. The association with x-ray exposure has already been discussed. As in adults, the incidence in girls is double that in boys. Multicen¬tricity of tumors is found in 30 -80%. Metastases to lungs, usually microscopic, are common (perhaps 20%), but tumor is rarely found in the bones. Lung metastases usually accumulate 131-I and can often be eradicated with this isotope, particularly those not visible with X-rays.

As with adult tumors there is no universally accepted surgical approach, but it is certain that sentiment has swung away from prophylactic and radical neck dissections to a more conservative position (322,323). The operations employed are as described above, and near-total thyroidectomy, done by an experienced surgeon, is favored. Thyroid remnants are destroyed with 131-I in patients with multicentric lesions and in all Clinical Stage II, III, and IV. Detection of metastases is attempted by131-I scanning, as described elsewhere in this chapter. Most childhood metastatic thyroid cancers are found to accumulate sufficient 131-I to allow useful and sometimes curative therapy, often with doses of 75-150 mCi. Presumably children are more likely to suffer side-effects of 131-I therapy, as described below.

Reproductive potential is diminished by large doses of 131-I (324), but an increased incidence of birth defects has to date not been encountered among the relatively few progeny studied (325-327). Thyroid hormone is given to suppress TSH to the 0.1uU/ml area in patients who have known residual or probable residual disease, even though this is known to cause some loss of bone mineral. Although the 10-year survival is from 90 to 95%, long term follow-up demonstrates an eight fold greater than normal mortality (230) and emphasizes the need for comprehensive therapy and long term follow-up.

RAI 131-I ABLATION

Most patients who have had a "total" thyroidectomy, and all patients who have had a subtotal resection, will have some functioning thyroid tissue remaining in the normal position after surgery, and will thus be candidates for 131I ablation. This is done to remove any possible residual tumor in the thyroid bed (thyroid ablation), to make subsequent scans and TG assays more interpretable, and (hopefully) to kill tumor cells elsewhere (adjuvant therapy). There is no unanimity regarding the use of postoperative 131I ablation in Stage I tumors, since absolutely convincing evidence of its value is lacking (234,328). But for all patients with papillary and follicular cancers as a group, 131I ablation correlates with improved survival (232). Our data demonstrate that postoperative 131I ablation correlated with decreased recurrences for all patients with papillary cancers over 1 cm in size. Samaan et al (311), in a review of 1599 patients, observed that 131I treat¬ment was the most powerful indicator for disease-free survival.

Ablation can be accomplished in most instances by one dose of 30 mCi 131I, giving the patients about 10 whole body rads (329). In our practice 80% of patients are ablated successfully with one dose of 30mCi, and the remainder require repeat therapy at the time of their second scan. Other clinicians find this dose insufficient, and give 50-150 mCi as an inpatient treatment. In part this difference may depend upon the surgeon, since small remnants of residual thyroid are more easily ablated than large amounts of residual tissue. Low dose (30 mCi) ablation of thyroid tissue after near-total thyroidectomy was recently reviewed by Roos et al. Surveying many studies, they concluded that 30 mCi was as effective as larger doses in inducing ablation, and since it could be administered without hospitalizing the patient, was an appropriate treatment (330). Doses of 100 mCi may provide more certain ablation with one dose (although at the expense of greater patient radiation) but there is little difference between ablation rates with does of 30-75 mCi. There is no data proving that one method or the other provides superior results in terms of survival. We do not routinely use ablation in patients under age 21 with tumors under 1 cm. Patients with tumors above this size, older patients, or those with multicentricity or a history of neck irradiation are advised to take 131I. This practice is followed in most clinics.

The indications for thyroid ablation, based on levels of evidence have been detailed in recent ATA guidelines (39). Three groups of patients are identified, one (at very low risk of recurrence) in which thyroid ablation is not indicated due to the lack of evidence of any benefit; a second group where the benefit, if any, are not evidence based. In this group, ablation should be offered in selected cases according to the judgement of the treating physician. Finally, a third group, including high risk patients, in which ablation has a strong indication based on good evidence that it may reduce cancer recurrence and possibly deaths.

Irrespective of the protocol and the dose used for ablation, there is always a subgroup of about 20% of patients that will not be successfully ablated with the first RAI course. The factors associated with ablation failure are not fully understood. Ablation failure does not correlate precisely with the dose, with the levels of TSH stimulation, the amount of thyroid residue or the level of urinary iodine excretion (331). In particular, it is not certain whether the use of doses higher than 3.70 GBq would result in any addi¬tional benefit, or whether there is a ’stunning’ effect of the diagnostic dose of 131I on the subsequent ablation rate, although likely to occur. A retrospective analysis was performed of all patients (n=389) with well-differentiated thyroid cancer treated at our institution between 1992 and 2001. The therapeutic dose was the only variable found to be associated with success (odds ratio, 1.96 per 1.85 GBq increment). Our results confirm the presence of a significant percentage of ablation failures (24.4%) despite the use of high ablative doses (3.70-7.40 GBq). Higher therapeutic doses are associated with higher rates of successful ablation, even when administered to patients with more advanced stages. Higher diagnostic doses were not associated with higher rates of ablation failure. (332).
The utility of radioactive iodide treatment of patients with papillary and follicular cancer was recently reviewed in a series of articles by Wartofsky, Sherman, and Schlumberger and their associates. Schlumberger concludes that routine radioactive iodide ablation is not indicated in patients with differentiated thyroid carcinomas of less than 1.5 cm in diameter, and advocates restricting RAI ablation to patients with poor prognostic indicators for relapse or death (333). Wartofsky points out a secondary benefit of postoperative low dose 131I ablation in that, for many patients, it provides a high degree of certainty and peace of mind when subsequent scans are negative and TG is undetectable. Another argument for radioactive iodide ablation and early detection of any recurrence is the data presented by several groups, including Schlumberger and colleagues, that there is a reciprocal relationship between the success of cancer therapy and the size and duration of the lesions.
In patients with Stage II to IV disease, we proceed to destroy all residual thyroid and to treat demonstrable metastases if they can be induced to take up enough 131I. Use of 131I therapy is investigated in these patients, regardless of the histologic characteristics of the resected lesion, although significant uptake rarely is found in Hurthle tumors (243,334) or in patients with anaplastic lesions.

Preparation for 131-I ablation

The "traditional" approach has been to induce hypothyroidism prior to the ablative dose in order to raise TSH and stimulate uptake of RAI in residual thyroid or tumor. This may be done by simply leaving the patient without T4 therapy for 3 weeks post op. Alternatively patients can be given thyroid hormone suppressive therapy for 6 weeks or so after operation, so that any malignant cells disseminated at the time of thyroidectomy will not be stimulated by TSH. The value of this measure is admittedly unknown. Patients then receive 25 µgL-T3 bid for 3 weeks, and therapy is then stopped for at 2-3 weeks to allow endogenous TSH (which may reach 20-60 µU/ml) to stimulate uptake of the 131I by the remaining fragments of thyroid tissue or metastatic lesions in the neck or elsewhere before proceeding with 131I therapy. Diagnostic scans are no longer indicated by several groups and ATA guidelines (39), based on the evidence that they do not offer additional information compared to the post-therapy scan and based on the possibility of stunning. However, if one wants to do it, the usual scanning dose should be no higher than 1 mCi 131-I, and scans are read at 48 or 72 hours, when body background has diminished. If TSH is sufficiently elevated the initial scan can reveal distant metastases as well as residual thyroid gland. If large thyroid tissue remnants are present, TSH may not become very elevated, but will do so after the first ablation dose. Patients with Class I and Class II disease under age 45 are given 30 mCi as an out-patient treatment. Older patients with Class II disease and patients with Class III or IV disease are given doses of 75-100 mCi as an inpatient treatment. A post-therapy whole body scans should be mandatory 3-5 days after the ablative dose of 131I (or after therapeutic doses), since occasionally unsuspected metastasis may be visualized on scans at this time. Serum Tg is always measured at the time of 131-I therapy. At 24 hours after initial ablation, we replace hormone therapy at suppressive doses

Some physicians proceed without prior scanning directly to 131-I ablation 2-4 weeks after surgery and perform a post-therapy scan 5-7 days later. Presumed benefits of this approach are patient convenience, less expense, and avoidance of possible thyroid "stunning" by the scan dose. In fact, stunning has not been demonstrated with the 2mCi 131-I dose, although it may occur. Arguments for doing a pre-ablation scan include finding out the actual percent uptake of the treatment dose in the neck and elsewhere, establishing if in fact there is uptake, and recognizing disease that may dictate a larger initial dose. The final word on these different approaches is not in. Variations on this approach were studied by Pacini et al (335), who compared induced hypothyroidism with rhTSH stimulation. The Pisa group found that either thyroid hormone withdrawal, or hormone withdrawal plus 2 doses of rhTSH, produced higher percentage uptakes and more frequent ablation with 30 mCi doses (in about 80% of cases), compared to rhTSH alone. These results have been confirmed in other series (336), including a randomized, international study (337) which brought the approval of Thyrogen in the preparation of thyroid ablation.

Half-Dose Protocol and Thyroid Hormone Withdrawal

An alternative to rhTSH stimulation for the initial follow-up is the "half-dose" protocol (338). Half the usual dose of thyroxine is given for six weeks. TSH is tested in the fifth week, and if over 20 uU/ml, scanning is done in the sixth week, or preparation is prolonged if needed. On this protocol patients usually feel quasi-normal and conduct normal activities, in contrast to their function during withdrawal. On the half-dose protocol, FT4 falls to just below normal, and TSH on average reaches about 60uU/ml in the sixth week. Patients who start with TSH below 0.1 U/ml may take longer to reach a satisfactory level for Tg testing, which is generally considered to be with TSH at least 30 U/ml.

Recombinate human TSH (Thyrogen)

During induced hypothyroidism, patients may experience a wide range of hypothyroid signs and symptoms which may be severe and may result in a substantial impairment of the patients’ lives and ability to work, and occasional tumor growth. Recombinant human TSH (Thyrogen) has been developed to meet the need for safe, adequate exogenous TSH stimulation in patients with papillary and follicular thyroid carcinoma. In vitro studies have shown that rhTSH can effectively stimulate cAMP production and the growth of human fetal thyroid cells. The in vivo biological efficacy of rhTSH was demonstrated in normal subjects, in whom it is able to increase serum T4 and T3 concentrations and stimulate thyroidal radioiodine uptake. A single dose of 0.1 mg rhTSH is a potent stimulator of thyroid function in normal subjects (339).

A first clinical trial of recombinant human TSH (rhTSH-THYROGEN) (phase I/II) was completed in 1994 in 19 patients after a recent thyroidectomy for differentiated thyroid cancer (340).The encouraging results of this limited study were confirmed in a larger multicenter phase III study conducted between 1992 and 1995 in the USA in 127 patients (341) and in a second phase III multicentric trial, which included USA and European centers (342). The results of this trial can be summarized as follows: scans were similar after rhTSH and thyroid hormone withdrawal in 92% of the patients, with no difference between the two dose regimens investigated. When the results of 131I WBS and post-rhTSH Tg levels were combined, the detection rate increased to 94%. Among the patients with persistent or recurrent disease, 80% had concordant scans, 4% had superior rhTSH scans and 16% had superior withdrawal scans. Interestingly, serum TG levels were detectable in 80% during thyroid hormone therapy and were detectable in all following either rhTSH stimulation or withdrawal of thyroid hormone treatment. However, the TG level reached after rhTSH stimulation was in general lower than that obtained after thyroid hormone withdrawal. Tissue RAI uptakes obtained in the patients undergoing hormone withdrawal were twice the values found after rhTSH, indicating that withdrawal provided a much greater stimulus to thyroid or tumor tissue. However, as noted, the diagnostic results were nearly equal. Quality of life was much better during rhTSH than during hypothyroidism induced by thyroid hormone withdrawal, and side effects were minimal, mainly consisting in mild and transient nausea or headache in less than 10% of patients. No patient has developed detectable anti-rhTSH antibodies, even after receiving repeated courses of rhTSH in successive clinical trials .All together these clinical trials have clearly shown that rhTSH is an effective and safe alternative to thyroid hormone withdrawal during the post-surgical follow-up of papillary and follicular thyroid cancer, although not as sensitive as scanning after hormone withdrawal in some patients. Another factor to consider is the cost, wehich is rougly $ 2000 per treatment, although for the majority of patients in USA this is covered by their insurance. A few patients have been reported with metastases demonstrated on withdrawal scans that were not evident on rhTSH scans (343). It has been found that Thyrogen administration induces a reduction of serum vascular endothelial growth factor, even in the absence of thyroid tissue (344). The clinical significance of this observation, if any, is unknown, but it does imply possible action of rhTSH on receptors other than in thryoid tissue. Use of rhTSH in managing thyroid cancer has recently been extensively reviewed (345). Thanks to many studies confirming the properties of rhtsh in stimulating iodine uptake and Tg production, rhTSH has now considered a standard method of preparation for both thyroid ablation and post-surgical follow-up in patients with any form of differentiated thyroid cancer.

Options in Follow-up scans and treatment-including recently described variations

After surgery and thyroid ablation, the next step is follow-up. The first important time for follow-up is between 8 and 12 months after initial treatment. At this time we want to understand whether the patients have evidence of complete remission or some evidence of persistent or recurrent disease. In the past, the conventional preparation for follow-up was to obtain a diagnostic total body scan with 131I after induction of hypothyroidism, with the same methodology as described for ablation, in order to stimulate uptake of 131-I by residual thyroid tissue or tumor cells and production of TG. In recent years it has become common to omit the diagnostic scans after initial ablation, at least in patients deemed to be at low risk, and relying entirely on measurement of stimulated (after rhTSH administration) serum TG when anti-Tg antibodies are negative (39). In patients known to have residual disease because of elevated baseline TG or ultrasound evidence of metastatic lymph nodes, is to give therapeutic 131-I without preliminary scanning. In several large series, it was demonstrated that at this time of the follow-up, more than 80% of the patients will have evidence of complete remission (negative neck US and undetectable stimulated serum Tg levels). These patients do not require additional tests or imaging and their suppressive hormone therapy should be shifted to replacement targeting serum TSH in the low-normal range. In subsequent years, the chance of these patients to have a recurrence is extremely low (<1%) and thus their follow uo should be solely based on basal Tg measurement and neck US once a year. On the contrary, when neck US is positive for local disease, or the basal or stimulated Tg is elevated the patient should be screened for the localization of the disease and treated accordingly.

FOLLOW -UP TREATMENT BASED ON TG ASSAYS

As assays for thyroglobulin (TG) have become more sensitive and reliable, measurement of TG assumes more and more importance in determining the management of patients followed after thyroidectomy and radioactive iodide ablation treatment for thyroid cancer. Serum TG levels, in the absence of antibodies interfering in the assay, correlate well with tumor burden, although detectable tumor may well be present even in the presence of negative TG assays in individuals who are on replacement thyroid hormone (346). Pacini et al (347) reported that in a retrospective study of 315 patients who had undetectable serum TG in the hypothyroid state at the time of the first control body scan after thyroid ablation, no useful information was provided by the body scan. Thus they propose that diagnostic 131I whole body scans can be avoided in patients with undetectable levels of stimulated TG after initial ablation, and that the patients can be monitored with clinical examination, ultrasound, and serial TG measurements on thyroxin treatment during the subsequent follow-up.
However, some concerns may be noted. The TG assay used in their study recognized a value of <3ng/ml as "undetectable". Also, of the 90 patients in the study with thyroid bed RAIU, 54 received a second course of treatment, and seven received two additional courses. This seems to question the recommended approach (347). In a second study (348) they found that an undetectable TG, when hypothyroid at the time of the first control scan after ablation, predicted complete and permanent remission. They propose that subsequent 131-I scanning is unneeded, and follow-up clinically and by TG assays while on thyroxin, would be sufficient. Mazzaferri and Kloos (349) studied retrospectively 107 patients who were “clinically free of disease” and had undetectable or very low serum TG levels during thyroid hormone therapy. The Tg levels on treatment were all 1 ng/ml or less, and 95% were under 0.5 ng/ml in their assay, which was a commercial (Nichols Institute) chemoluminescent antibody assay. In response to the administration of two doses of recombinant TSH and assay of TG on samples taken on the fifth day, 20% were found to have a value above 2, with values ranging from above 2 up to 18 ng/ml. Twenty percent of the patients who had low or undetectable Tg had elevation above 2 ng/ml after rhTSH stimulation, and many of these patients ended up with therapy. However, the authors found that radioactive iodide whole body scans often failed to localize the source of the elevated TG, which was found after post-therapy scans or by other imaging methods. This study suggests that even with a TG level below 1 while on replacement therapy, persistent disease may sometimes be present and be detected by stimulation using recombinant TSH or thyroid hormone withdrawal.

Wartofsky (350,351) comments on these studies and supports the idea that TG testing, both on suppression and after TSH stimulation, can help in determining therapy. He suggests that, in patients with a serum TG<0.5ng/ml on suppression, and in a low risk category, that stimulation by recombinant TSH and measurement of TG, rather than scanning, is satisfactory. If the TG remains <1, the patients can be evaluated annually with such a stimulation test. In patients with slightly higher TGs, up to 2, he suggests measuring a recombinant TSH stimulated TG, and scanning. In patients with higher TGs, he suggests that thyroid hormone withdrawal and radioactive iodide treatment, without initial scanning, may be appropriate. In a study done by the rhTSH-Stimulated Thyroglobulin Study Group (352) and published in 2002, a cut off level of 1 ng/ml for stimulated TG was taken as the safe level for patients with low risk. This group would presumably be monitored by repeat rTSH stimlated TG assays rather than scans. It is of interest that in this study 14 of the patients with stimulated TG <2ng/ml underwent isotope scanning and 9 were positive. These "could be interpreted as false negative" tests. Five had uptake outside the thyroid bed. This group suggests that patients with stimulated TG above 2 would have subsequent thyroid hormone withdrawal and possible 131-I therapy without scanning. A recent “consensus” statement by a group of thyroidologists also supports the categorization of patients into high and low risk groups, and use of TG as described above for following low risk patients (353).

Whether this approach, with omission of whole body scans, has any adverse effect on long term outcome is not yet known.

STUNNING FROM "TRACER" RAI-

The amount of 131I used for scanning varies from 2-10 mCi, in various clinics, or even larger amounts. Clearly the larger doses detect more lesions, but this rarely alters treatment plans. More importantly, doses of 5-10 mCi have been shown to decrease tumor uptake of the subsequent treatment dose due to "stuning" of the tumor (354). The exact importance of this phenomena is uncertain, but use of a 2mCi dose has seemed to be a reasonable compromise. A study by Lassmann et al (355) call this assumption into question, since they found that even a 2mCi scan dose reduced RAIU by up to 50% in a second procedure done 6 weeks later.This study was carefully performed on a few patients, but it is possible that prolonged stimulation of the thyroid by elevated TSH may have adversely altered iodide kinetics in the remnants during the second RAIU procedure.
Although a reduction of uptake after prior low dose scans has been reported, it is uncertain that this limits the effectiveness of treatment. It was found that ablation rates were equal in individuals who had, or had not, received scans prior to their treatment dose of 131-I (356).

Ingestion of large amounts of iodide, or exposure to contrast dye within 6 (or more) weeks, can prevent RAIU. Uptake can be enhanced by prescribing a low iodine diet for at least two weeks before scanning and therapy, although also this assumption has been questioned by a recent study (331). It is useful to give magnesium citrate to induce bowel emptying prior to the scan. As discussed above, the value of routine follow-up diagnostic scans has been challenged by two European study, who suggest that such scans rarely give information of value in patients who have TG <1 after thyroid ablation (357,358). They suggest testing TG after thyroid hormone withdrawal or rhTSH, and directly treating those with TG above 10 by administration of 100mCi 131-I. This concept is of interest and awaits further examination, but has obvious problems as noted.

HIGH DOSE RAI THERAPY FOR INVASIVE OR METASTATIC DISEASE

Efficacy and morbidity of high activity of 131-I therapy was assessed in 38 patients with locally advanced or metastatic differentiated thyroid cancer (16 follicular, 20 papillary, one Hurthle cell, one insular) who were treated with high activity radioiodine therapy (9 GBq) as the cancers had previously not responded to standard activities of 5.5 GBq.

After high activity treatment, 9.7% of patients suffered grade 3 and 3.2% suffered grade 4 WHO haematological toxicity. Significant salivary gland morbidity was observed (30% dry mouth, 27% salivary swelling). In this study repeated treatment with high activity (9 GBq) in patients with advanced differentiated thyroid carcinoma appeared to be of no apparent benefit and lead to late morbidity (359). However, other investigators have differing results. A retrospective analysis was conducted on 124 differentiated thyroid cancer patients who underwent dosimetric evaluation using MIRD methodology over a period of 15y. One hundred four RAI treatments were performed. A complete response at metastatic deposits was attained with absorbed doses of >100Gy. No permanent BM suppression was observed in patients who received absorbed doses of<3Gy to BM. The maximum administered dose was 38.5 GBq (1,040 mCi) with the BM dose limitation. Dosimetry-guided RAI treatment allowed administration of the maximum possible RAI dose to achieve the maximum therapeutic benefit. Estimation of tumor dose rates helped to determine the curative versus the palliative intent of the therapy (360).

131-I Treatment using empirically determined doses of 100-250mCi

Patients who have significant uptake of 131I in metastases (usually above 0.5% of the tracer) are given 150-250 mCi 131I. This dose can be tolerated without acute radiation sickness, and is below the level that would promote pulmonary fibrosis if diffuse pulmonary metastases are present, unless uptake in the lungs exceeds 50% (see below). Although use of these empirically derived doses is the most common practice, some centers do careful dosimetry with a tracer dose of 131-I prior to therapy, in order to judge the appropriate, or maximal safe, dose. This requires 2-5 days of observation. The methodology and results have been recently discussed (361). Whether administration of maximally large individual doses is more effective than use of somewhat smaller doses of 131-I has not been established. In perhaps four-fifths of patients accumulating 131I, it is possible to administer a dose of RAI that should be useful in destroying tumor. For normal thyroid tissue 10,000-15,000 rads is destructive, and a dose of 20,000 rads or more is probably needed for therapy of cancer. Assuming, for example, a standard 150 mCi 131I dose, and delivery to tumor of about 100 rads per micro Curie retained per gram, a 1% tumor uptake distributed through 10 g of metastatic tissue could provide an effective treatment.

Some groups have attempted to measure tumor volume by use of quantitative PET scanning (362). The effective half-life can be determined from serial counts of the tracer over the metastasis. If 10 g of tumor in the neck accumulated 1% of a 150 mCi dose, and isotope turnover in the tumor was extremely slow, the radiation dose might be as follows: Rads =74X0.19X 150,000X0.01X6 / 10= 12,654 rads.

The question of whether a sub-cancericidal dose should be delivered in patients with low levels of tumor isotope accumulation needs further investigation, since radiobiologic studies suggest that radiation could preferentially spare the more radioresistant cells, ultimately leaving a more lethal tumor. It may be possible to give conventional x-ray therapy after 131I in those instances in which 131I uptake is present but the total dose delivered to the metastasis is less than adequate. This procedure may provide another therapeutic approach to the thyroid cancer patient, but it has not yet been given adequate trial. Maxon et al (363) report that radiation doses of at least 30,000 rads for thyroid ablation, and 8,000 for therapy to metastasis, improve the rate of response.

Lithium was found to be a potent adjuvant in 131I therapy of metastatic well differentiated thyroid cancer. Koong et al administered 600 mg of lithium carbonate orally followed by 300 mg three times daily and adjusted the dose to maintain a lithium concentration of 0.6 – 1.2 mEq/L, which is effective in blocking 131I release from the thyroid. The estimated 131I radiation dose to metastatic tumor tissue was increased on average 2.29-fold. It is possible that lithium would be of value in managing a large fraction of patients treated with radioactive iodide. It must be noted that lithium has a narrow therapeutic index and that serum concentrations must be monitored. Care must be taken in patients with reduced renal function. It would be imperative in patients who are to receive "maximal 131I therapy" that the measurement of 131I retention in blood and body be performed while the patient is receiving lithium prior to therapy. The use of lithium might also reduce the required 131I dosage for ablation of thyroid remnants (364).

It is useful to do a scintiscan on patients who have received therapeutic doses of 131I at 5 -7days following the treatment, thus using the treatment dose as a more pow¬erful scanning dose. While often offering no new information, this may reveal unsuspected metastasis, especially in younger patients who have previously had 131I treatment. Fatourechi et al found that 13% of follow-up scans demonstrated abnormal foci of uptake not seen on diagnostic scans, and changed management in 9% of their patients (334,365).

The 131-I treatment cycle is repeated at 24-52 weeks, as long as there is no evidence of systemic radiation damage, and as long as the metastases continue to accumulate iodide. The total 131I dosage may vary from 150 to (rarely) 2,000 mCi. Both papillary and follicular cancers respond to 131-I therapy. Small metastases from papillary cancer, especially if functional in the lungs but not large enough to be visualized on X-ray, are typically cured. Follicular tumors often have relatively few metastases and high uptake, thus seem ideal targets for therapy. However portions of the metastases, especially in bone, appear to be resistant and finally continue growth despite 131-I treatment. Nevertheless 131-I therapy is beneficial even in advanced and aggressive tumors. Pelikan et al report their experience on the use of radioactive iodide in treating advanced differentiated thyroid carcinoma and report that up to 50% of patients who have distant metastases can be cured by 131I therapy (366). Aggressive high dose radioiodine therapy has been advocated for treatment of advanced differentiated thyroid cancer by Men-zel and colleagues. These physicians gave repeated doses of 300 mCi (11.1 GBq 131I) with mean accumulated total activities of, on average, 55 GBq per patient. Repetitive high dose therapy appeared beneficial in the majority of patients with papillary carcinoma, but the majority of follicular thyroid cancer patients had progressive disease despite treatment (367). The National Thyroid Cancer Treatment Cooperative Study Registry Group recently evaluated the therapy of high risk papillary and non-Hurthle cell follicular thyroid carcinoma. The study confirmed the utility and benefit of radioactive iodide therapy to reduce recurrence and cancer-specific mortality among patients in the high risk group (368). Pittas et al. (369) reviewed an extensive series of 146 patients with documented bone metastasis from thyroid carcinoma seen at Memorial Sloan Kettering in New York City. Bone metastases were most common in vertebrae, pelvis, ribs, and femur, and multiple lesions were present in more than half the cases. Overall ten year survival rate was 35%, and from diagnosis of initial bone metastasis, was 13%. Favorable prognostic signs for survival included radioiodine uptake by the metastases and absence of non osseous metastases. Hurthle cells had a favorable response to treatment, rather surprisingly, whereas un-differentiated thyroid tumors fared the worst.

Arterial embolization has been combined with radioactive iodide treatment for management of large bone metastasis from differentiated thyroid carcinoma with apparent improvement in effect over the use of radioactive iodide alone. In the study by VanTol et al, (370) embolization was not accompanied by any severe complications.

rhTSH is now available for routine use, and allows 131I therapy without induction of hypothyroidism (340). This will probably increase acceptance of scanning and therefor increase the frequency of the procedures. Iodide depletion by dietary control and diuresis, including furosemide or mannitol administration, can also double the fractional uptake of 131I in metastases (371,372). Finally, when the diagnostic scan shows no 131I uptake, even with TSH, the potential benefits from this mode of therapy have been probably exhausted. However, before giving up to 131-I therapy, some authors suggest using high doses of 131-I and obtaining a post-therapy scan, which in some cases may show areas of uptake not seen in the diagnostic scan (see below).

131-I Therapy with "Negative" scans

In some patients tracer studies fail to show uptake, and serum TG is elevated. Some investigators recommend treating these individuals with large doses of 131I (100¬-150 mCi) and report that tumor uptake can be visualized after treatment, and that serum TG may fall (373,374) (Fig. 18-18). The clinical efficacy of this approach is not known. In a few cases reported by Schlumberger et al. (375) and Pineda et al. (376). TG became undetectable, which clearly is a striking and hopeful result. As of this date, there is no data proving that this treatment improves prognosis (377).
The utility of radioactive iodide treatment of patients with papillary and follicular cancer was recently reviewed in a series of articles by Wartofsky, Sherman, and Schlumberger and their associates (333). Sherman and Gopal analyzed the use of 100 mCi doses of 131I for treatment of scan negative TG-positive patients and conclude that this must, at this point, be considered an experimental procedure of uncertain benefit. They argue against its use in young patients with elevated although apparently stable TG values and without radiographic evidence of disease. Fatourechi et al. (378) analyzed results of this treatment in a series of patients treated at the Mayo Clinic and concluded that it rarely produced significant effect, although it possibly helped stabilze disease in patients with micro metastases in the lung. It is clearly ineffective in patients who have metastases large enough to be detected on chest X-ray of CAT . Wartofsky et al. (333) sug¬gests that, rather than initial treatment with 131I of patients who are scan negative and TG-positive, thorough imaging studies are appropriate. These might include a CAT scan of the chest, an MRI of the neck, 99mTc-MIBI, or 18-fluorine fluorodeoxyglucose PET scanning, or even 99mTc-tetrafosmin, or 201TI thallium. Localization of malignant tissue by any of these means may allow surgical excision or external radiotherapy. This series of articles provides many very useful thoughts on management of diffi¬cult patients with recurrent thyroid carcinoma.

Figure 18-18. Changes in serum TG after therapeutic doses of radioiodine in patients with negative basal whole-body scans. The arrows indicate the administration of therapeutic doses of 131-I. The numbers represent individual patients. In most patients, thyroglobulin decreased, although only in a few to a level that would indicate absence of residual disease. (From Pacini et al, J Nuc Med 28:18888-1891, 1987)

Maximal dose protocols

The therapeutic protocol used at Memorial Hospital in New York, by Maxon (363), and as well at some other centers, has for years been designed to give maximal-tolerable radiation doses to cancer patients (231). The dose is calculated on the basis of prior isotope tracer kinetics. The aim is to give a blood dose of under 200 rads, or less than 120 mCi retained at 48 hours, or 80 mCi retained at 48 hours if diffuse lung metastases are present. This method has theoretical advantages since it potentially provides the most cancericidal dose, but the difficulties of calculating the dose and the occasional adverse reactions have so far prevented this method from being generally employed. The dosimetric approach has been carefully reviewed by Van Nostrand et al (361).

Figure 18-19a. Chest radiographs of a patient with extensive follicular thyroid carcinoma prior to (upper) and after 131-I therapy (lower). With 131-I therapy there was resolution of the large pulmonary metastases, but a tumor in the cervical vertebrae progressed and caused instability of the spine.

Figure 18-19b. Chest radiographs of a patient with extensive follicular thyroid carcinoma prior to (upper) and after 131-I therapy (lower). With 131-I therapy there was resolution of the large pulmonary metastases, but a tumor in the cervical vertebrae progressed and caused instability of the spine.

RADIATION PRECAUTIONS

Before radiation therapy, female patients should be carefully screened for pregnancy and lactation. Confirmed or possible pregnancy constitutes a firm contraindication to therapy because of the risk of damage to the fetus.

A patient who has ingested many milliCuries of 131I can cause serious radiation contamination, and appropriate precautions must be followed. If less than 30 mCi 131I is given, it is permissible to have the patient dispose of urine and feces into general sewage. If amounts of 131I greater than 30 mCi are given, the patient should be kept in a private room in the hospital until less than 30 mCi is retained in the body. Urine can be directly disposed in sewage, or can be collected by the patient and stored in bottles behind protective lead shielding. After physical decay, usually after about 6 weeks, it may be discarded in the sewage. Contaminated bedding and utensils should be stored for 10 half-lives (80 days), thoroughly washed, and monitored for residual contamination before being used again. Alternatively, disposable bedding and utensils may be used.

Table 18-9. Radiation Exposure to Personnel During Care of a Patient Who Has Received 100 mCi 131I

Personnel caring for a patient who has received 131I therapy are often concerned about exposure to excessive radiation. This is almost never a real problem. Isotope can, at a practical level, only be passed from the patient to another person via saliva or urine. Monitoring by means of a portable counter is important in making certain that no person receives more than an allowable radiation dose from the isotope in the patient’s body. Table 18-9 gives a rough estimate of the amount of radiation received while performing ordinary hospital tasks at various distances from a patient who has received 131I. In general, all ordinary patient care can be performed without hazard. It is best to avoid close contact between hospital personnel and patient during the first 48 hours after therapy because of undue apprehension that may be induced. However, even after doses of up to 100 mCi, normal personal actvities such as eating at the same table, or driving in the same car, carry no risk to others.
The US Nuclear Regulatory Commission has published new guidelines which allow release of patients treated with isotopes from the hospital if the total effective radiation exposure from the treated person to any other individual is not likely to exceed 5mSv (0.5 rads). Grigsby et al (379) found that when using precautions such as those described above in a group of patients given on average about 100mCi 131-I, the exposure to other individuals in their household and to pets did not exceed this level. Recently, guidelines for the optimal radiation protection after treatment with 131-I have been proposed by the American Thyroid Association (380).

Radiation damage from 131-I Therapy

The use of RAI in large doses is not without hazard. The radiation dose delivered to the whole body, the gonads, or bone marrow is usually assumed to be the same as that of the blood. The blood dose depends on the amount of isotope administered; its distribution space and turnover; the degree of heterogeneity of distribution in the tumor; the uptake, synthesis, and secretion of labeled compound by the tumor; and perhaps other variables. The radiation is usually largely due to inorganic iodide, since little protein bound 131I ordinarily appears in the blood. Sometimes tumor destruction is such that much PB131I appears in the blood and can yield a major fraction of the total whole body radiation dose. As a rough estimate, the blood, gonadal, or bone marrow radiation may be assumed to be 0.3 -1.5 rads/mCi 131I administered (381), or 45-150 rads per treatment with 100 mCi. The genetic risks are discussed in Chapter 11 and are not reviewed here. Ordinarily, when 131I therapy is needed for carcinoma, the necessity of treating the patient outweighs the risks of genetic damage.

Various unwanted effects of radiation may occur in patients receiving large doses of 131I. Mild radiation sickness is seen. Metastatic deposits or surrounding tissues may become painful over 2-4 weeks from radiation-induced inflammation. Damage to the salivary glands can cause sialadenitis, and xerostomia, and can lead to loss of teeth (382). Increasing salivary flow following treatment is partially protective. Ovarian function is often temporarily suppressed (383), and if there are pelvic metastases that collect 131I, the gonads may receive a sterilizing dose. Sperm count may be reduced for months (384). Leukemia occurs with increased frequency in patients who have received large doses of 131I (usually>600 mCi) for cancer (217). Transient or permanent alterations in liver function and lymphoma of the parotid gland have been reported as possible sequelae (385). Pulmonary fibrosis has occurred in patients with functioning lung metastases who have received unusually large doses or who have very active metastases (386). Leukopenia, thrombocytopenia, and anemia are encountered with accumulating doses. A mild effect on the bone marrow is seen with each therapeutic dose, and after several hundred milliCuries, aplastic anemia may develop (387). The hemoglobin level, white cell count, differential count, and platelets should be monitored periodically in order to judge recovery of the marrow between treatments and to prevent excess total radiation damage to the marrow. Large radiation doses may cause transient swelling of metastasis in the brain or spinal canal.

Lin et al (388) recently reviewed pregnancies following 131I treatment of well differentiated thyroid carcinoma among a group of 58 pregnant women and found no evidence of demonstrable adverse effects, but suggest that it would be wise to avoid pregnancy during the first six months after the last administration of 131I. With the exception of possibly increased rate of miscarriages, no other adverse effect of radioiodine has been found on the outcome of 2113 pregnancies after radioiodine treatment and on their offspring (318).

Two special complications need be noted. Occasionally withdrawal of hormone suppression, in preparation for isotope therapy, leads to rapid growth of the tumor, and reinstitution may not seem to return the patient to the prior condition. Special care should be taken if metastases are present in areas such as brain or spinal column, where growth could cause serious sequelae. Glucocorticoids are occasionally given prophylactically in an effort to prevent tumor swelling in this situation .
RAI was introduced into the treatment of thyroid carcinoma with the hope that it would be a panacea for this disease. Unfortunately, the results have not been universally beneficial. Most tumors in children appear to be treatable, and among adults 80-90% of metastatic carcinomas accumulate sufficient 131I to warrant a serious therapeutic trial. Patients who harbor this form of the disease are fortunate, since 131I may totally eradicate the metastases. Even multiple pulmonary metastases occasionally disappear after 131Itherapy (Fig. 18-19a, above). The final value of 131I therapy has been difficult to define, largely because of a lack of controlled series and because of other treatment (especially thyroid hormone) given at the same time (387). Mazzaferri (232) found that 131I ablation and therapy significantly improved the prognosis in papillary cancer by decreasing recurrences. Varma et al. (389) found that RAI treatment had no effect on the survival of persons under age 40 but did lower the death rate of patients over age 40. Leeper (390) concluded that 131I treatment appears clearly to benefit patients under 40 years of age with papillary cancer, but the course of this cancer in older patients is rarely affected; follicular cancers in older patients are treatable, and survival is prolonged even if the disease is not eradicated. Soft tissue lesions, especially of the lung and mediastinum, respond best to 131I. Osseous lesions are often highly functional but are infrequently totally destroyed by 131I (Fig. 18-9b) (391). Lesions detected on whole body scans, with negative bone X-rays, are most likely to be cured. In another report, 59 of 400 patients were considered candidates for 131I therapy after using antithyroid drugs or TSH to stimulate uptake. Of these, 61% with metastatic disease were benefited (362). The follicular, papillary, and mixed cancers responded equally well. Numerous reports indicate that ablation of metastasis with 131I is associated with a better prognosis than failure to ablate, but obviously this outcome may relate to the histologic nature and function of the lesion rather than to the therapy per se.

Disseminated pulmonary metastasis can sometimes be eradicated by 131I, but radiation pneumonitis or fibrosis may be produced and may be fatal (321,386). On first observation of pulmonary metastases, this therapy should be considered, but no more than 75 mCi should ever be deposited in the lungs in one treatment. Progress of the lesion and pulmonary function should be carefully evaluated before and be-tween treatments (389,391,392-394). Occasionally patients present with locally advanced papillary thyroid cancer which is not surgically resectable. In some instances preoperative treatment with radioactive iodide sufficiently reduces the extent of the lesion to allow subsequent definitive surgery (395). One of the most informative study regarding the effects and limitation of 131-I therapy is given by the series of the Institut Goustave-Roussy periodically updated by Schlumberger et al (396). In their most recent publication the authors report on 444 patients treated with 131-I for distant metatsases and the results identified three groups of patients with different outcome after therapy: a group very likely to be cured after a few courses of RAI, represented by young patients with micronodular disease, usually in the lung; a second group whose metatsases can be stabilized but not cured after more than 600 mCi as cumulative doses and a third group including older patients with macronodular disease, partuclarly in the bones, who do not responde to RAI and progress rapidly to the exitus. It is apparent from this study that, continuing RAI therapy after 600 mCi usually has no benefit.

FOLLOW-UP OF CANCER PATIENTS: THE SERUM TG ASSAY

After initial ablation patients are given TSH-suppressive doses of l-thyroxine. This therapy as dual aims: to replace the thyroid hormone function and to inhibit the growth of potential residual disease..Two-three months later serum TSH, free thyroid hormones and Tg concentrations are measured during l-thyroxine treatment. The results of these tests will disclose whether the l-thyroxine dose is adequate in suppressing TSH levels without inducing hyperthyroidism, but will give little information on whether the patient is in remission.
To this purpose, the most informative follow-up period is at 6-12 minths after initial treatment, when the ablative dose of radioiodine should have exerted its effect. At this point, the patients have been already attributed a risk estimate based on the results of the post-ablative WBS and serum Tg measurement (397,398). This information is important at the moment of the new evaluation. If uptake was seen outside the thyroid bed on the post-therapy WBS, the patient must be considered in high risk of recurrence or persistent disease, while if no uptake was seen outside the thyroid bed on the 131-I post-therapy scan, the Patient is considered at low-risk. The 6-12 month control, consists of careful neck ultrasound to detect the lymph node status and serum Tg levels (with negative anti-Tg) after stimulation with exogenous (better) or endogenous TSH. These tests are considered by many authors as sufficient to confirm complete remission (negative US and undetectable stimulated Tg). Other advocate the usefulness of a diagnostic WBS with radioiodine, aimed to ensure that thyroid ablation has been successful and to search for foci of 131-I uptake outside the thyroid bed. However, two independent studies (357,399) have shown that this diagnostic WBS are almost always negative (and sometimes false negative) thus adding very little information to that given by neck US and serum Tg measurement. Indeed, the result of serum Tg measurement is the more sensitive predictor of complete remission or persistent disease (provided that anti-Tg autoantibodies are negative) (400-404). Nearly all patients with local or distant disease have detectable or elevated serum Tg levels, while patients in stable remission have undetectable serum Tg concentrations. Compared to serum Tg measurement, the yield of the diagnostic 131-I WBS is lower. A significant proportion of patients may have elevation of serum Tg in the presence of falsely negative diagnostic WBS. A retrospective study by Cailleaux et al. (357) has shown that when serum Tg off therapy is undetectable, routine diagnostic WBS does not add any further in¬formation on the clinical status of the patient. Similar results have been obtained at the Department of Endocrinology, University of Pisa, (399) in a retrospective series of 315 patients who had undetectable serum Tg off l-thyroxine at the time of the first control after thyroid ablation. None of these patients had evidence of disease activity at WBS, and 99.4% were in complete and stable remission after 12 years of follow-up. Only two patients (0.6%) had recurrence of lymph node metastases which were treated with radioiodine therapy. Based on these studies it is possible that in the future the need for 131-I scanning may be dictated by the results of serum TG during hypothyroidism or by rhTSH-stimulation (342).
After this follow up, low-risk patients (those with an undetectable stimulated serum Tg, negative neck US and negative WBS, when performed) are considered as cured and may be followed with periodic serum Tg measurement during l-thyroxine therapy. Thyroxine therapy may be decreased to maintain a low but not suppressed serum TSH concentration (0.1-0.4 µU/ml). The risk of recurrence is in fact so low in these patients (representing more than 80% of the total) that overdosage of l-thyroxine is unjustified.

As noted the problem of antibody interference in the TG assay makes this test unreliable in 10-15% of patients. However another aspect of the antibodies should be remembered. If patients are free of thyroid cancer and the thyroid has been ablated, it appears that the antigenic stimulation necessary to maintain an anti-TG titer is gradually lost, and these antibodies disappear with a 3-6 year half-life (302). Thus, the level of anti.Tg antibodies may be used as a surrogate marker of disease. Other approaches, including the search of Tg mRNA in the blood, have been proposed, but none has entered clinical practice. Fugazzola et al (405) point out that the combination of TG RIA and TG mRNA assay offer better positive and negative predictive value than TG alone. In some studies TGmRNA analysis has proven much less reliable than serum TG assay (406).

In high risk patients, even if considered cured, suppressive doses of l-thyroxine may be continued for some years, because the risk of relapse is greater. Pujol et al evaluated a series of patients over an average of 95 months and compared those who had TSH values constantly under 0.05 mU/l to those who had all TSH values greater than 1mU/l. A lesser degree of TSH suppression was associated with an increased incidence of relapse, with a shorter average relapse-free survival (407). This observation was not sustained in another study (408). The objective of suppressive therapy in these patients should be to attain a serum TSH level of 0.1 µU/ml or less with normal free T3. In this situation, side effects such as osteoporosis, are not observed (409). Clinical and biochemical evaluation is performed annually. If serum Tg becomes detectable during follow-up, the patient should be evaluated for the presence of disease by neck us, first, and by other imaging if neck ultrasound is negative. Some authors prefer to avoid this procedure and give directly a therapeutic dose of 131-I followed by a post-therapy scan. In the absence of uptake after therapeutic doses of 131-I, any further administration of 131-I is not justified, and the site of Tg production should be searched for by other imaging techniques. If 131-I thyroid ablation has not been performed or if the patient has undergone only partial thyroid surgery (subtotal or lobectomy), follow-up should consist of clinical and ultrasound examination and serum Tg measurement. However, in this case, the sensitivity and specificity of serum Tg assay is reduced. In such patients, ant suspicious of persistent or recurrence disease should prompt to complete the initial treatment with completion thyroidectomy and/or radioiodine ablation.

During follow-up, patients may develop isolated metastases that can be approached surgically. Osseous metastases, especially from follicular cancer, may require radiotherapy or operative procedures for stabilization. Progressive growth of soft tissue or osseous metastases that are not amenable to thyroid hormone, 131I therapy, or radiotherapy should lead to consideration of systemic therapies.

UNDIFFERENTIATED CARCINOMAS

Undifferentiated carcinomas, if operable, may be treated by thyroidectomy and resection of all involved tissue in the neck. 5-10% can apparently be completely resected, and the patient can have a discrete survival, although they are never cured. Prophylactic X-radiation (5,000 rads) should be given postoperatively in all cases.. If the lesion is obviously inoperable, X-ray therapy should be the initial treatment. In some instances, the rapidly growing tumors melt almost completely for a time after X-ray therapy, giving the pa¬tient a gratifying respite from the disease.

Some anaplastic tumors have been treated with alkylating agents, and rarely, a rapidly proliferating and highly undifferentiated tumor has responded with a temporary regression. Chemotherapy is discussed below. Kim and Leeper (410) reported partial results combining low dose adriamycin (10 mg/m2) and hyperfractionated radiotherapy (160 rads twice daily) to 5760 rads. Time of survival was prolonged and local recurrences largely prevented. There has been no confirmation of this study, unfortunately.

A prospective protocol combining surgery, chemotherapy (CT),and hyperfractionated accelerated radiotherapy (RT) was employed in anaplastic thyroid carcinoma. The main toxicity was hematologic. High long-term survival was obtained when RT-CT was given after complete surgery. This protocol avoided local tumor progression, and death was mainly caused by distant metastases (411). The clinical behaviour and therapy of anaplastic carcinoma have recently been reviewed by K. Ain (412).
Despite any combination of therapies, anaplastic thyroid cancer remains one of the most aggressive human cancer, with 80% of the patients dying within 2 6 months and the remaining within 2 years. Novel treatment regimens are badly needed for this tumor.

Lymphomas

Once diagnosed, patients should be staged by chest X-ray, chest and abdominal CAT scans, bone marrow biopsy, and gallium scan. In 20-30% of patients, the lymphoma will be confined within the thyroid gland. Lymph nodes are involved in approximately 60% of the patients, and perithyroidal invasion is present in about half. When patients have undergone appropriate staging procedures, 20-30% will have Stage IIIE (lymphoma present in nodes below the diaphragm) or Stage IVE (distant extranodal metastases) disease. The selection of appropriate therapy requires prior staging of the patient. As these tumors are radiosensitive, external radiation therapy is a satisfactory treatment for Stage I-E (local disease only, no nodal involvement) and has been used for Stage II-E (nodal involvement above the diaphragm only) disease. The dose to the neck is usually 4,000 rads (40 Gy) over 4-5 weeks (248). The radiation port should include the mediastinum even in the absence of clinical involvement. When evaluating the long-term survival of patients with Stage I and Stage II disease, Souhami et al. found that of seven patients treated with radiation to the neck only, five died within 5 years, whereas in five patients receiving radiation to the neck and mediastinum, there were no deaths (249). Patients with unfavorable prognostic fac¬tors such as age >60yr, large mass or tumor necrosis, may be given chemo-therapy after radiotherapy. In radiation-treated patients with local extension or with malignant nodes, the survival after surgery is unaffected by the extent of surgery even when surgery was limited to a biopsy. With obvious extension or nodal involvement, surgery should be limited to obtaining an adequate diagnostic specimen since an attempt at complete excision may damage surrounding structures without improving survival. There is a suggestion that total thyroidectomy may improve the prognosis in patients with intrathyroidal disease only (240).

There is a growing tendency to use combination chemotherapy as the initial definitive treatment for Stage IE and IIE thyroid lymphoma, rather than radiation.. Programs may combine cyclophosphamide, adriamycin, vincristine, and prednisolone ("CHOP"). This approach may increase cure above the 30-50% found with radiotherapy alone (413). Currently most patients with Stage II-E, and patients with Stage III-E or Stage IV-E disease, as well as those who relapse after radiation therapy, are treated with chemotherapy, given every 3-4 weeks for 3-6 cycles, prior to radiation therapy if combined with that treatment. Although previous series report overall 5-year survivals of about 50%, certain subgroups have a more favorable prognosis. With appro¬priate staging to exclude Stage III-E and Stage IV-E patients, the Stanford group has reported a 3-year survival of 83% and at 3 years 75% of their StageI-E and Stage II-E patients had no evidence of disease (414). Matsuzuka et al (415) used both radiation therapy and six courses of "CHOP" chemotherapy, and report 100% survival for 8 years in a group of patients who received this treatment.

RADIATION THERAPY

The general indications for radiation therapy are given in Table 18-11. Other sources should be reviewed for details of the port, dose, and methods of irradiation.
Studies by Tubiana et al (416), Simpson (283), and Riccabona (417) have clearly established the efficacy of radiotherapy in all types of thyroid cancer. Although its therapeutic use is now well accepted, its prophylactic use (e.g., in papillary or follicular lesions with possible residual disease, or in papillary or follicular Stage III lesions after RAI ablation) is controversial. Currently, we would first treat all papillary or follicular invasive or possibly metastatic tumors with RAI. In patients under age 55, it is not clear that x-ray therapy should be added. Over age 55, 131I therapy should be followed by x-ray treatment for Stage III or potentially Stage III lesions, and any recurrence or metastasis not responding to 131I may also be treated (418).

The efficacy of external radiation therapy in the management of thyroid malignancy has been reviewed by Brierley et al, Lin et al, Farahati et al, and Tsang et al. (419-422). Brierley, recommends X-ray therapy in addition to 131I therapy in patients with papillary or follicular cancer who have probable or definitive microscopic residual disease. They gave 30 to 50 Gy over four weeks with the spinal cord dose less than 80% of the total. Radiotherapy was also advised for treatment of anaplastic thyroid carcinoma, in an attempt to achieve local control, using hyperfractionation, possibly with the addition of doxorubicin. Radiotherapy was used for treatment of lymphomas, with a total dose of 35 – 40 Gy given over four weeks, and was typically preceded by the use of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) delivered every three to four weeks in three to six cycles prior to the radiotherapy. Cause-specific survival was 82% inpatients who received combined therapy and 63% in those who got X-ray alone. Combined therapies were given to all patients except those with small bulk disease under 3 cm in size confined to the thyroid, who received radiation alone. Lin et al. (420) evaluated 699 patients with differentiated thyroid cancer who received external radiotherapy after surgery, compared to 172 who did not, and found no evidence that radiotherapy improved survival rate of patients with well differentiated thyroid cancer. These were patients with either neck nodes or local invasion.There were 172 patients who received no radiotherapy versus 32 who did. The patients were in clinical stages 2 and 3 at the time of treatment. Farahati et al (421) looked at patients with parathyroidal tumor invasion and found that adjuvant external radiotherapy improved recurrence-free survival in patients over 40 years of age. Benefit was confined to patients with papillary thyroid carcinoma. Tsang et al (422) also confirmed the benefit of radiotherapy in patients with papillary tumors who have microscopic or macroscopic residual disease.

Note- Spinal cord dose not to exceed *3000 or ** 3500 rads.

In a recent study (423), Hurthle cell carcinoma of the thyroid gland was found to be a radiosensitive tumor. Adjuvant radiation therapy was successful in preventing recurrence in 4 of 5 patients. Salvage radiation therapy was successful in 3 of 5 patients treated with external beam radiation therapy. Palliative radiation therapy provided sustained symptomatic relief at 67% of irradiated sites. Radiation therapy may provide palliative relief from symptomatic metastases, control recurrent tumors, and prevent recurrence of advanced resected tumors.

The indications for MTC have been described above. Stage I-E lymphomas may be treated by chemotherapy or radiotherapy. Anaplastic lesions are currently given radiotherapy after operation. It is possible that chemotherapy should be used instead of or in addition to radiotherapy in these lesions, but studies are needed to establish this point.

The exact dose must be individually determined, but usually the maximal dose is 5,000-6,000 rads, using ortho-or megavoltage, and a fractionated technique over several weeks. Dosage must be planned to assure that the spinal cord receives less than 3500 rads in order to avoid myelopathy.

SYSTEMIC THERAPY FOR METASTATIC DISEASE

Sporadic experience indicates that bleomycin (424,425), adriamycin (426), vinblastine (427), methotrexate (426), cisplatinum and other agents (428) may have value in treating disseminated thyroid tumors. However, none of these drug has given more than palliation. With the advent of new molecules targeting specific metabolic pathways involve in the generation and maintainance of thyroid cancer growth, the so call “tyrosine kinase inhibitor’, chemotherapy has been almost universally abandoned. Recent ATA guidelines (39) states that whenever systemic therapy is needed, the patient should be offered the possibility to be entered in clinical trials using these new molecules. Metastatic papillary or follicular tumors refractory to conventional therapy grow slowly and systemic therapy is not indicated until the tumor is clearly growing progressively despite hormone suppression. The same apply to the treatment of MTC (264).

RE-DIFFERENTIATION’ THERAPY

RETINOIC ACID- Experimental data from in vitro studies suggest that retinoic acid can inducere-differentiation of thyroid cancer cells with regain of iodide concentrating ability. Simon et al (429) studied 28 patients with differentiated thyroid cancer using 1.5 mg/kg retinoic acid per day for five weeks. Iodide uptake increased in eight of the patients and thyroglobulin increased in 63%. Thus retinoid appeared to reinduce iodide uptake in half of the treated patients by redifferentiation. They gave 13-cis-retinoic acid. Side effects occurred in half of the patients but were generally well tolerated. A common side effect was dryness of the skin and mucosal surfaces. The reinduction of 131I uptake allowed radioiodine therapy in several of the patients. The effect on tumor size was uncertain. Retinoic acid redifferentiation therapy has recently been reviewed by Schmutzler and Kohrle (430). They note that, of twenty documented retinoic acid treated patients, at least eight had exhibited a decrease or stabilization in tumor size, and in serum TG levels, in addition to enhanced radioiodide transport. However, this relatively positive review seems enthusiastic, considering the small evidence of improvement observed in most cases. Clearly the ben¬efits of this experimental treatment are as yet uncertain, but it is nevertheless an exciting observation that retinoic acid can have an apparent beneficial redifferentiating effect on some thyroid tumors. Histone deactylase inhibitors also have been found to restore in part the function of NIS, TPO and TG in thyroid tumor cells in vitro (431), and valproic acid also has an action to restore NIS function, probably via the same mechanism (432).
PPARgamma agonists-In vitro studies show that PPAR gamma agonists can slow thyroid tumor cell growth and induce apoptosis (433). Results of clinical trials are awaited.

ONYX-015is an E1B deleted adenovirus that replicates in cells with impaired p53 function. p53 is commonly inactivated in anaplastic thyroid cancers. In vitro studies demonstrate that this virus induced cell death in in vitro trials in anaplastic cancer cell lines, and synergized with treatment with doxorubicin and pacitaxel (434).

Adenoviral vectors producing tk, IL-2, IL-12, and GM-CSF in a cell specific manner are currently under study in animals with encouraging results. Studies in humans are so far very limited, but the methods appear to be safe and effective, especially with the immunomodulator IL-12 (434,435).
A variety of ideas on possible treatments for thyroid cancer have been reviewed by Braga-Basaria and Ringel (436).

TYROSINE KINASE INHIBITORS

Several oncogenes involved in thyroid cancer progression are tyrosine kinase receptor. Molecules that block kinase activity at distal steps in the MAP kinase pathway are logical candidate drugs for thyroid cancer. TKI being tested against differentiated thyroid cancer in clinical trials include motesanib diphosphate, axitinib, gefitinib, sorafenib and sunitinib. None of these is specific for one oncogene protein but they target several TK receptors and pro-angiogenic growth factor receptors. The results of phase II-III clinical trials conducted so far are promising with partial responses ranging 14- 32% and stable disease 50-67% (437). In addition, very recently one of this drug, vandetanib, have been approved for the treatment of progressive, metastatic MTC, based in clear evidence of beneficial effects in clinical trials (438,439). All together, the preliminary results of these trials are promising and indicate that target therapy might become the first line treatment of metastatic refractory thyroid cancer patients in the near future.
Sunitinib is a multitargeted tyrosine kinase inhibitor (TKI) that is is currently approved for the therapy of renal cell carcinoma and gastrointestinal stromal tumor. Some eficacy was found in both differentiated thyoid cancer and medullary cancer in Phase II studies, with partial response rates of 13-30%.

Sorafenib is a similar agent, also achieving significant levels of partial responses and stabilized disease. Several Phase II stuies have been reported, and a Phase III trial has been completed.

Vandetanib has been studies in Phase II trials and in a randomized, placebo-controlled phase III study in patients with both hereditary and sporadic MTC, using vandetanib 300 mg or placebo. A 54% reduction in the risk of progression was observed and also objective tumor responses in 45% of patients. The FDA approved it for the treatment of symptomatic or progressive medullary thyroid cancer in patients with unresectable locally advanced or metastatic disease. Motesanib, Axitinib, Pazopanib, Lenvatinib and several other related TKIs that more specifically block VEGF receptors are in Phase II or III trials.

These agents appear to block progression and in some studies decrease tumor bulk, but are not specifically cytotoxic, and tend to loose efficacy over months, presumably by mutational events in the tumor. Future studies will surely investigate serial or combined treatments, and use with other possibly cytotoxic agents.

Progress in the last few years has reinvigerated therapy for these difficult tumors, but comes with a price. Since TRK Iinhibitors affect many sites in the body, side effects are very common and often serious, but generally tolerable The major side-effects include hypertension in 20-50% of cases, diarrhea in 37-77%, fatigue in 40-50%, weight loss in 20-60%, frequent nausea, skin sensitivity, and very frequent rashes, depending on the study, dosage,and drug (439). Some of the agents induce hypothyroidism during therapy.

Most reports available are the result of clinical investigations, often by consortia made up of physicians specializing in therapy of thyroid cancer, and patients are generally advised to enter such a trial if a newer agent is used.. However these agents gradually spread into more generaized usage, by therapists who may not have extensive experience. The MD Anderson group has published suggested institutional guidelines to help provide safeguards for non-investigative use of TKIs(440).

Presentation of this chapter is supported in part by Genzyme, the makers of rhTSH (THYROGEN)

ABSTRACT

This chapter presents an analysis and a summarized  synthesis of  our present  knowledge  of  the  biology  of  the thyroid gland, phylogeny ,ontogeny ,anatomy ,structure ,general metabolism ,regulatory factors  and  hormones , signalling cascades  and  their regulations , ( eg  TSH ), functions including iodine metabolism  and  thyroid  hormones  synthesis , control  of  gene expression ,differentiation  and  growth  and  cell proliferation .Emphasis is ,when  possible , put on  the  human thyroid. The  original primary  literature,as  well as  reviews ,  over  the last  50  years  are  comprehensively  and  critically analyzed with:600 references .
Controversies are  presented . For complete coverage of this and related topics, please visit www.endotext.org.

PHYLOGENY

The primary event in the phylogeny of the thyroid was the development in living forms of the capability of collecting iodide ion and binding it to protein. These activities have been observed widely among plants and in the invertebrate members of the animal kingdom. Brown algal kelps are the most efficient accumulators of iodide identified with enrichment factors for iodine of up to 10 ⁶ (1). In Laminariadigitata , for example, the iodine content can reach up to 5% of the dry weight. However, only a minor fraction of iodine is stored in the form of iodinated amino acid residues including monoiodotyrosine (MIT) and diiodotyrosine (2) (3) (4). The biochemical pathways involved in iodine uptake, accumulation and metabolism in these algae have still not been fully elucidated. Recent studies suggest that iodide is oxidized by a vanadium-dependent iodoperoxidase (5) yielding more lipophilic iodine species which diffuse across cell membranes and are subsequently sequestered as labile iodine species in the apoplastic compartment (6). Thus, the iodide uptake mechanism utilized by these algae appears very different from that of vertebrate thyroid follicular cells and the organification of iodine is still considered a by-product of the reactive environment.

Even though most invertebrates might not be able to endogenously synthesize TH, a wealth of data indicate that organic iodine species are taken up from the environment (e.g., via the food) and might function as signalling molecules with pleiotropic effects on various aspects of invertebrate physiology (21). In analogy to vitamins, Eales coined the term “ vitamones ” to describe this ancient function of iodinated compounds as external morphogenic signals governing larval development in some invertebrates. Recently, this model has been supported by several experimental studies (10;22;23). Moreover, insilico analyses of genome sequences available for several invertebrate species in conjunction with experimental studies in a few model species corroborate a deep ancestry of iodothyronine signalling, most likely at the origin of deuterostomes (24-26). Orthologs of vertebrate TH receptors (TRs) have been identified in several invertebrate species including deuterostomes and protostomes (26-30). However, functional data for invertebrate TRs are still limited to deuterostomes and, thus, the functional role of TR orthologs identified in platyhelminths, mollusks and crustaceans remains elusive.

Interestingly, the functional characterization of the single TR orthologs of the amphioxus Branchiostomafloridae and Branchiostomabelcheri and of the ascidian Cionaintestinalis consistently revealed a lack of effective TR binding by T3 (26;30;31) . Instead, TRIAC (triiodothyroacetic acid), an acetic T3 derivative, was found to bind strongly to amphioxus TRs, to stimulate coactivator recruitment to the TRs and to activate TR-dependent gene expression (26;30). Despite the lack of T3-TR interaction in amphioxus and ascidian species, exogenous T3 and TRIAC are both effective in stimulating chordate metamorphosis (26). One clue to interprete these findings came from the observation that TRIAC is a major metabolite of T4 and T3 in amphioxus suggesting that metabolism of T3 to TRIAC might be involved in the metamorphosis-stimulating activity of T3 in amphioxus (32). A key role for TRIAC in regulating amphioxus metamorphosis is further supported by the recent identification of a nonselenodeiodinase in the amphioxus B.floridae (33). This deiodinase has a cysteine instead of a selenocysteine in its catalytic center and effectively deiodinates TRIAC and TETRAC (tetraiodothyroacetic acid) but not T3 and T4. Together, these experimental data suggest that TRIAC, or a related derivative, but not T3 is the ancestral TH acting in chordates.

Gorbman (34) has hypothesized that during evolution, organisms became accustomed to a supply of iodotyrosines and iodothyronines derived from external sources, and eventually developed a requirement for the iodinated amino acids. The first evidence of an organ capable of providing iodothyronines and thus, related to the vertebrate thyroid, is found in the protochordates, comprising the subphyla Cephalochordata (amphioxus) and Urochordata (ascidians) (Fig. 1-1). In the origin and evolution of the thyroid gland, the protochordates occupy key positions in phylogeny, because cephalochordates are the most basal in the phylum Chordata and urochordates are the closest living relatives of vertebrates (7). In ascidians and amphioxus, an organ known as the endostyle lies on the floor of the pharynx and connects with the pharynx by a duct. Notably, an endostyle is still present in the basalmost vertebrates, the lamprey larvae (ammocoete).

From the present point of view, the significant evolutionary event was the development of iodination centers within the endostyle. The differentiated endostyles in protochordates and lamprey larvae are histologically divided in “ zones ” containing different cell types (35). In the ascidian C.intestinalis , these iodination centers are present in zones 7, 8 and 9 at the tip of the endostyle (36). The amphioxus endostyle contains seven zones and iodide organification has been observed in zones 5a, 5b, and 6 which are also located at the tip of the endostyle (37). In amphioxus, Barrington (38) has shown that an iodinated glycoprotein is formed in these iodination centers, probably on the surface of the cell. The endostyle secretes a mucus that passes down the duct into the pharynx and thence is moved along into the alimentary canal, presumably carrying iodinated protein along with it. Although early accounts were sometimes conflicting (39), accumulation of radioiodide, peroxidase activity as well as endogenous synthesis of T4 and T3 has been demonstrated in the endostyle of several protochordate species (40-42) (43;44) .

In the ascidian C.intestinales , the proposed organ homology between the endostyle and the vertebrate thyroid was strengthened in recent molecular studies. For Ciona orthologs of vertebrate thyroid-specific marker genes, including the transcription factors Pax2/5/8 , ciTTF 1 and Ci-FoxE , expression was demonstrated in several zones of the endostyle in adult Ciona (45-47) . In addition, insitu hybridization revealed expression of Ciona orthologs of thyroid peroxidase ( ciTPO ) and dual oxidase ( ci-Duox ) in the iodide-concentrating zone 7 of the endostyle (48). An interesting observation was that Pax2/5/8 and Ci-FoxE expression domains overlapped with those of ciTPO and ci-Duox whereas ciTTF 1 expression was not detectable in zone 7. In amphioxus, however, all three transcription factors, Pax2/5/8 , TTF 1 and FoxE4 , were expressed together with TPO in the iodide-concentrating zones (49).

The recent releases of genome sequences of C.intestinales and B.floridae greatly contributed to our understanding of the gene repertoire encoding for components of the thyroid system in protochordates (25;50) . Genome analyses identified orthologs encoding for sodium-iodide symporter-like proteins, thyroid peroxidase, dual oxidase, several deiodinases and a single TR. However, no sequence homologous to thyroglobulin (TG) were identified in protochordate genomes suggesting that other scaffold proteins might be used for iodotyrosine synthesis (51). In amphioxus, a TG-like protein has been described by Monaco et al. (52), but a molecular characterization of this protein is not yet available. Further, no clear homologs of thyrotropin-releasing hormone (TRH) or the two subunits of thyroid-stimulating hormone (TSH) have been detected in protochordate genomes, which is in accordance with the current view that protochordates do not have a pituitary gland (53). Based on a comparison of Ciona and vertebrate genomes, Campbell (51) concluded that some features of the vertebrate thyroid system appear well represented in urochordates but that critical genes involved in the neuroendocrine control of thyroid function are lacking.

Interestingly, a recent analysis of the spatial expression profile of TR mRNA in adult amphioxus demonstrated abundant expression in those endostyle zones associated with iodide organification and TH synthesis (30). This finding raises the possibility of a direct role of TH in the regulation of TH synthesis.

Despite the observation of developmental effects of T3 and T4 in echinoderms (e.g., sea urchin, sand dollar, sea star, sea biscuit) and the identification of several genes encoding for components of a functional TH signalling system (24;25), solid experimental data to corroborate TH synthesis, metabolism and TR-mediated biological activities are not yet available. Although data by Heyland et al. (54;55) suggest endogenous TH production in in sea urchin and sea biscits, the tissues and organs involved have not yet been identified (echinoderms do not have an endostyle).

Figure 1. Phylogeny of the development of the thyroid gland.

The most primitive vertebrates in which a follicular thyroid gland can be definitely demonstrated are the jawless fishes (agnathans). Concerning the origin and the evolution of the thyroid gland, lampreys are of particular interest because they are the only known vertebrates that possess a larval endostyle that directly transforms into a follicular thyroid during metamorphosis (56) (57). Although the endostyle of the lamprey larvae (ammocoete) has a different structure and organization compared to the endostyle of protochordates, physiological and molecular characteristics are very similar. The lamprey endostyle shows iodide uptake and organification, the latter involving a protein that is apparently related to TG (58) (59;60) (61). The TG-like protein undergoes proteolytic digestion in the intestinal tract to liberate T4 and T3 which are probably taken up directly from the gut lumen to enter blood circulation (62;63). Given this pathway of TH synthesis and release, it is of particular interest that high 5 ’ -deiodinase activities were determined in the larval intestine (64). Comparative analyses of thyroid-related genes confirmed the expression of Pax2/5/8 (65) and TTF 1 orthologs (56) (66) in the lamprey endostyle. Interestingly, similar to ascidians, the expression domains of TTF 1 were not clearly overlapping with domains of iodide concentration, TG and T4 synthesis (56;67).

Very high plasma concentrations of TH have been determined in lamprey larvae (68). A unique feature of lamprey developmental endocrinology is a dramatic decrease of circulating TH levels concomitantly with the onset of metamorphosis. This developmental TH profile is in sharp contrast to metamorphosis in amphibians and various fishes where high TH titers are associated with metamorphosis. Moreover, metamorphosis in lamprey larvae can be induced by anti-thyroidal compounds such as perchlorate or methimazole suggesting that the abrupt decline of plasma TH levels might trigger the onset of metamorphosis (69).

During metamorphosis of the ammocoete into the adult lamprey, the endostyle loses its connection with the pharynx and becomes a thyroid composed of scattered follicles (70) These follicles are not encapsulated, but they have the typical biosynthetic functions associated with hormone formation in the adult vertebrates. In the lamprey, the biosynthesis of TG in larval forms has the same characteristics as that formed in thyroid follicles of the adult form, with a 12S as the precursor of the 18-19S protein. Total iodine content of TG is very low (0.002%) and about 5% is present in the form of T3 and T4 (71). Curiously, thyroid activity appears to play no role in the metamorphosis of the ammocoete, while the gland itself undergoes a remarkable morphological changes.

Some relationships of the thyroid to the gastrointestinal tract is apparent in phylogenetic studies. Dunn (72) has actually found ciliated thyroid cells in the mouse and shark, a reminder of the origin of the gland from endoderm. In mammals the gastric mucosa and the salivary glands retain a functional relationship to the thyroid in that they too can concentrate iodide (73), and the salivary gland contains a peroxidase.

Thus, a thyroid capable of forming iodotyrosines and iodothyronines is present in all vertebrates. Its level of function varies widely from species to species and season to season. With the exceptions noted below, thyroid activity in the poikilotherms is very low. Seasonal changes in thyroid activity have been found in both warm- and cold-blooded animals. Certain morphologic changes occur after the biochemical evolution of the thyroid has ceased. In the adult lamprey and in most bony fishes, the gland is not encapsulated. The follicles may be widely scattered, either singly or in small clusters, especially along the course of the ventral aorta and in the kidneys (74). In cartilaginous fish, the thyroid is encapsulated. In the higher vertebrate forms, the thyroid is a one- or two-lobed encapsulated structure.

Function of the Thyroid in Non-Mammalian Species

A functioning thyroid is evident in forms as primitive as lampreys and hagfishes. TH are critically important for the regulation of diverse biological processes associated with development, growth and metabolism in non-mammalian vertebrates (75) (76;77) (78). In particular, the vital importance of TH for the regulation of early developmental processes is not limited to human or mammalian species (79), but is well conserved throughout the vertebrate kingdom (80) (81) (82). In avian species, for example, TH are required for nervous system and skeleton development (83) and TH action has also been demonstrated to regulate both direct larval and metamorphic development in fish (84;85). In amphibians, TH are the primary morphogen regulating postembryonic development (metamorphosis) (81).

Many fish species undergo similar developmental phases as described for amphibians including a larval stage, juveniles and adults (82) with a larvae-to-juvenile transition often associated with metamorphic changes (86). Experimental manipulation of the thyroid status as well as the recent cloning of fish TRs and the characterization of TR developmental expression profiles clearly demonstrated the important role of THs for early fish development (87). At later life stages, TH have been shown to assist in the control of various physiological functions in fish including osmoregulation, metabolism, somatic growth, and behaviour (88) (82). In salmonids, for example, TH are important for migration from fresh water to salt water (smoltification), and the high T4 plasma levels during smoltification represent some of the highest circulating T4 levels in fish (89;90).

Initially, it was believed that TH had little or no stimulatory effect on the oxidative metabolism of cold-blooded species. Now it is known that the effect of the thyroid on metabolic activity in cold-blooded species is strongly dependent on environmental temperature. For example, T4 causes stimulation of metabolism in lizards at 32°C, but not when they are acclimated to low temperatures (91). The thyroid gland is also more active at higher temperatures (23°-32°C) than at 10°-15°C in snakes, fish, amphibians, turtles, and lizards (92). TH levels are also influenced by the nutritional status in both endothermic (birds, mammals) and ectothermic (fish) vertebrates with a decreased T3 and T4 to T3 conversion in caloric deficient states (93).

A most striking effect of TH is the induction of metamorphosis in anuran amphibians, first reported by Gundernatsch in 1912 (94). The physiological role of TH during anuran metamorphosis is best exemplified by the fact that surgical removal of the thyroid gland or chemical blockage of TH synthesis leads to complete cessation of metamorphic development (95). On the other hand, addition of minute amounts of T4 (1 nM) to the rearing water of tadpoles during premetamorphosis leads to a precocious induction of metamorphosis (81). Particularly the metamorphosis of the South African clawed frog Xenopuslaevis has been used for years as a highly successful animal model to understand TH function in a developmental context (81) (96) (97;98) (99). The relationship between the functional state of the thyroid system and the progress of postembryonic development is well documented in this species (81) (100). The premetamorphic period is characterized by rapid growth of tadpoles with only minor morphological changes (101) and TH levels are very low (100). Growth and differentiation of the hind limbs are the earliest TH induced modifications marking the onset of the prometamorphic period during which levels of circulating TH steadily increase (102) stimulating further morphogenesis and differentiation of the limbs. The emergence of fore limbs marks the beginning of metamorphic climax characterized by rapid and dramatic changes in morphology (e.g., intestinal remodelling, complete resorption of gill and tail tissue) under the influence of peaking TH levels (81) (103). Towards the end of metamorphosis, plasma TH decline and low levels are present in juvenile and adult frogs (100).

A fascinating aspect of anuran metamorphosis is that a single type of hormone, TH, induces different tissues and organs to undergo remodelling in a highly coordinated spatio-temporal fashion (81) (104) (105). Similar to mammals, TH synthesis is regulated by thyroid-stimulating hormone (TSH) (98). T4 is considered the major hormone secreted by the thyroid gland while the secretion of T3 is low in X.laevis tadpoles (106). Expression of glycoprotein TSH alpha-and beta subunits mRNAs in the pituitary of metamorphosing X.laevis tadpoles increase from low premetamorphic levels to maximum levels at early climax stages and decrease towards the end of metamorphosis (107) (108). Thus, there is a concurrent increase of TSH  expression, thyroid activity and circulating T4 levels from premetamorphosis to early climax stages. Different hypotheses have been put forward to explain this condition. Some authors stressed the importance of climax stage induction of type II iodothyronine deiodinase (D2) expression in pituitary thyrotrophs as a molecular switch to establish a negative feedback control of TSH synthesis (109). In contrast, other studies could demonstrate negative feedback control of TSH expression by T4 at much earlier stages suggesting that the developmental TSH expression profile is the net result of negative feedback action of circulating TH and a concomitant increase in pituitary stimulation by hypothalamic factors (110) (111). Of relevance for the increased hypothalamic stimulation of pituitary thyrotrophs might be the TH-dependent maturation of the median eminence (112) as well as increased synthesis and release of hypothalamic peptide hormones (113).

The extent to which different plasma proteins such as thyroxine-binding globulin (TBG), transthyretin (TTR) and albumin account for TH binding in the blood varies among different groups of vertebrates (114). In humans and rodents, TBG and TTR are the main THBP, respectively, showing a higher affinity for T4 than T3 (115). TTR is assumed to be the main TH-binding plasma protein in metamorphosing tadpoles and many teleost fish. One characteristic property of amphibian and teleost TTRs is that they display a several-fold higher affinity for T3 than for T4 (116).

In their target cells, the biological action of TH is mediated by activation of nuclear TH receptors (TRs). Two TR subtypes (TR  , TR  ) which are encoded by separate genes have been described in X.laevis (82). Due to pseudo-tetraploidy, there are two TR  (A and B) and two TR  (A and B) in X.laevis (117). Similar to mammals, TRs can bind to TH response elements (TREs) weakly as homodimers whereas heterodimers of TR and 9 cis retinoic acid receptors (RXR) strongly bind with TREs (118). Extensive investigations on the gene expression profiles induced by T3 made X.laevis one of the leading resources for understanding TH action during vertebrate development (119) (120). Gene transcription in X. laevis tadpoles can be either up- or down-regulated by TH (121;122). For the category of genes that are up-regulated by TH, it has been shown that unliganded heterodimeric TR-RXR complexes can bind to TRE but repress transcription through recruitment of corepressor (123).

In X.laevis , low mRNA expression has been demonstrated for TR  and TR  in oocytes and embryos but the putative functions are still poorly defined (124) (125). During postembryonic development, the expression profiles of TR  and TR  show striking differences (81) (126) (127;128) . TR  is expressed at high levels during early premetamorphic stages and expression is maintained at an elevated level throughout metamorphic development (129) (130). TR  shows a more complex developmental expression profile, characterized by low expression during premetamorphosis and a dramatic up-regulation in parallel with the increasing TH plasma levels during prometamorphosis (131). When analyzed in individual tissues, TR  was found to be up-regulated particularly during periods of active tissue remodelling (132) (133). Another important aspect of TH action in X.laevis tadpoles is that exogenous T3 regulates the expression of its cognate receptors (134). Among the most rapid changes in gene expression induced by T3, a dramatic up-regulation of TR  gene expression has been observed in all organs and tissues analyzed (135) (136). In addition, recently developed transgenic models carrying dominant negative and constitutively activated TR mutants could clearly demonstrate the important role of TR in mediating the developmental effects of TH during X.laevis metamorphosis (137).

Three types of iodothyronine deiodinases (D1, D2, D3) have been identified in vertebrates which differ in tissue distribution, substrate specificity and sensitivity to inhibiting compounds (138). D1 and D2 catalyze primarily the removal of one iodide from the outer tyrosine ring of T4 to produce T3. D3 catalyzes the cleaving of one iodide from the inner tyrosine rings of T4 and T3 generating inactive iodothyronine derivatives (e.g. reverse T3 and diiodothyronines), respectively. In amphibian tadpoles, the coordinated progression of metamorphic development requires a high degree of local control of T3 production which apparently dominates over the general supply of T3, at least during metamorphosis (139). Only recently, a putative X.laevis homologue of mammalian D1 has been identified (140) but neither the expression profile nor the putative regulatory role of D1 during metamorphic development have been characterized so far. However, several studies have investigated the role of D2 and D3 in controlling TH action during metamorphosis of anuran tadpoles (141). The data derived from these studies support the view that both D2 and D3 play a central role in modulating the tissue responsiveness to TH by either increasing intracellular concentrations of biologically active T3 (e.g., D2 in hind limbs) or by preventing TH action via rapid inactivation of T4 and T3 (e.g., D3 in tadpole tail).

Central to the understanding of TH function in lower vertebrates are the manyfold interactions of TH with other hormones (e.g., corticosterone, prolactin, and growth hormone) contributing to a fine-tuning of developmental TH action. For example, several studies have shown that high concentrations of corticosterone can provoke both inhibitory or accelerating effects on amphibian metamorphic development, depending on whether treatment is initiated at early (inhibition) or at late developmental stages (acceleration) (142). Concerning the accelerating effects of corticosterone on the development of tadpoles at late stages, two molecular mechanisms have been proposed including corticosterone -induced increases in T3 receptor binding capacities and corticosterone-induced increases in peripheral deiodination of T4 to T3 (81). Similarly, corticosterone is known to affect peripheral deiodination of T4 to T3 in avian species, including inhibitory effects on D3 and D1 activities in chicken liver (143).

Another hormone that has received a great deal of attention with regard to modulation of TH dependent metamorphic development is prolactin. Early studies using mammalian prolactin preparations could demonstrate antagonistic effects of prolactin on TH action in various peripheral tissues (144). Inhibitory effects on TR autoinduction by TH have been suggested as a primary mechanism of prolactin action to antagonize TH action in peripheral tissues. It should be noted, however, that in a transgenic frog model of prolactin overexpression, no retardation of tadpole development was detectable, with the exception of blocked tail resorption in a limited number of transgene animals (145).

In chicken embryos, GH appears to be a potent inhibitor of hepatic D3 activity resulting in increased T3 availability (146). This GH action on TH metabolism probably represents a physiologically relevant hormonal response linking the nutritional state with the activity of the thyroid system. Given the great diversity and heterogeneity in fish physiology and ecology, it is not surprising that a multitude of hormonal interferences have been described in various model species. The reader is referred to several comprehensive reviews about this hormonal interplay in fish (147-149).

Hypothalamic and Pituitary Control

Hypothalamic-pituitary control of thyroid function in the most primitive vertebrates represented by hagfishes and lampreys is still poorly understood (150) (151). Although the pituitary of these primitive fishes might contain thyrotrophic factors (152), their nature and their hypothalamic-releasing factors are unknown. TRH and TSH were largely ineffective in stimulating thyroid activity as assessed by different experimental protocols (153). Instead, numerous studies support the concept that peripheral deiodination of TH might provide the major regulatory mechanism for the control of thyroid status (154). The intestinal tract seems to be a major site of this TH metabolism in larval lamprey (155).

Neuroendocrine control of thyroid activity has been established for teleost fishes, amphibians, birds and reptiles (156). TRH is regarded as the main regulator of TSH secretion in mammals, and TSH-releasing activity of this peptide hormone has also been observed in various non-mammalian vertebrates. In avian species, injection of TRH has been shown to preferentially increase circulating T3 instead of T4, an effect that was later related to a GH-dependent inhibition of hepatic D3 activity subsequent to a stimulation of somatotrophs by TRH (157). In fact, preferential binding of TRH was demonstrated for somatotrophs in chicken. In recent years, it became clear that another hypothalamic factor, corticotropin-releasing hormone (CRH), is a more potent stimulator of TSH release in non-mammalian vertebrates. In chicken, CRH was found to increase both T3 and T4 plasma levels via stimulation of TSH release (158). Studies by de Groef (159) could show a preferential expression of CRH receptors type 1 (CRH-R1) and type 2 (CRH-R2) in chicken corticotrophs and thyrotrophs, respectively. The role of CRH R2 in mediating the CRH effect on TSH release in chicken was corroborated in further studies using CRH R2-specific agonists and antagonists.

In amphibians, the regulation of TSH release by TRH and CRF appears to be dependent on the life stage. While TRH was able to increase TSH secretion in adult frogs but not in tadpoles (160;161), the finding that TRH stimulates TSH release at least in adult amphibians argues against the hypothesis that the TSH-stimulating activity of TRH has only recently been coopted in association with the development of endothermy. Similar to the studies with chicken, CRF proved to be the most potent releasing factor for TSH in the tadpole pituitary invitro (162;163). In vivo, injection of CRH increased plasma T4 levels in Xenopus tadpoles and accelerated metamorphic development in ranids. In addition, TSH synthesis and release by the amphibian pituitary is under the control of a negative TH feedback (164;165). Few studies have addressed CRH effects on thyrotrophs in fish but the available data indicate that CRH but not TRH stimulates TSH release in salmonids (166).

ONTOGENY

The main anlage of the thyroid gland develops as a median endodermal downgrowth from the tongue. It can be seen in the human embryo at the end of the third week (167). It is located near the primordium of the heart, and as the heart is pulled caudally, the thyroid anlage follows. At the 5 ^th week the thyroglossal duct starts to breakdown. At about the 30th day it has developed into a hollow bilobed structure, and by the 40th day, the original hollow stalk connecting it to the pharyngeal floor atrophies and then breaks. Shortly thereafter the lateral extensions of the median anlage make contact with the ultimobranchial bodies developing from the 4th pharyngeal pouches, the so-called lateral anlage of the thyroid. The ultimobranchial cells or neural cells accompanying them are the origin of calcitonin-secreting C-cells in the thyroid gland and may contribute to the formation of follicular cells as well (168). By the 8th week the cells have a tubular arrangement, and cell clusters are apparent. Two weeks later, when the embryo is approximately 80 mm long, follicles are present. Shortly after this time the follicles contain colloid, and the thyroid accumulates and binds iodide by the 11th-12th week (Fig. 2). Secondary follicles arise by budding from the primary follicles; they increase in number until the embryo reaches a length of about 160 mm. After this time the follicles increase in size, but the number remains the same. Under intense stimulation, the adult thyroid can form new follicles.

Fig. 1-2. Photograph of thyroid tissue from a fetus with a 50-mm crown-rump length, estimated gestational age 64 days. The arrows indicate two intracellular canaliculi. During incubation of the tissue in organ culture in vitro, there was no uptake or fixation of iodide. The figure shows the earliest stage of formation of colloid spaces. The tissue was fixed in ozmium, embedded in Epon epoxy resin, and sectioned at 1-mm thickness (x2,400). (From T.H. Shephard, J. Clin. Endocrinol. Metab., 27:945,1967, with permission).

Fujita and Machino (169) have studied the origins of the follicular lumen in the chick embryo. They found that colloid droplets, 1-5µm in diameter and enclosed by a limiting membrane, first appear within the cytoplasm of parenchymal cells. As the droplets enlarge, they approach the cell membrane and come in contact with the droplets of an adjoining cell. The limiting cell membrane disappears, and the droplets fuse. By an extension of this process to cells close to the original droplet, an acinar structure containing colloid and enclosed by a ring of parenchymal cells is formed. A similar process can be demonstrated in aggregates of isolated thyroid cells in vitro (170).

GROSS ANATOMY

Physical Appearance and Anatomic Location

The Germans call the thyroid the "shield gland" (Schilddrüse), and the English name, derived from the Greek, means the same thing. Such a term gives a most erroneous impression of its shape. It is interesting, however, that in the Minoan culture, a shield was used that had a shape somewhat like that of the mammalian thyroid gland. The gland as seen from the front is more nearly the shape of a butterfly. It wraps itself about and becomes firmly fixed by fibrous tissue to the anterior and lateral parts of the larynx and trachea. Anteriorly, its surface is convex; posteriorly, it is concave. The isthmus lies across the trachea anteriorly just below the level of the cricoid cartilage. The lateral lobes extend along either side of the larynx as roughly conical projections reaching the level of the middle of the thyroid cartilage. Their upper extremities are known as the upper poles of the gland. Similarly, the lower extremities of the lateral lobes are spoken of as the lower poles, although they make no such prominent projections as do the upper (Fig. 1-3).

Fig. 1-3. Gross Anatomy of the thyroid and surroundings (from: Netter FH, The Ciba Collection of Medical Illustrations, vol. 4, Endocrine system and selected metabolic diseases, Ciba, 1965, with permission).

The weight of the thyroid of the normal nongoitrous adult is 6-20 g depending on body size and iodine supply. The width and length of the isthmus average 20 mm, and its thickness is 2-6 mm. The lateral lobes from superior to inferior poles usually measure 4 cm. Their breadth is 15-20 mm, and their thickness is 20-39 mm.

The thyroid is enveloped by a thin, fibrous, nonstripping capsule that sends septa into the gland substance to produce an irregular, incomplete lobulation. No true lobulation or lobation exists. In fact, the gland is throughout a uniform agglomeration of follicles packed like berries into a bag. It has no true subdivisions. The lateral lobes lie in a bed between the trachea and the larynx medially and between the carotid sheath and the sternomastoid muscles laterally. The deep cervical fascia, dividing into an anterior and a posterior plane, lines this bed and makes a loosely applied false or surgical capsule for the lateral portions of the gland. In front are the thin, ribbon-like infrahyoid muscles. The thyroid is molded perfectly to fit the space available between the neighboring structures, and is superficially placed. It can usually be outlined by careful palpation in normal humans, but if the neck is thick and short or the sternomastoid muscles heavily developed, it may be impossible to feel the gland.

The shape and attachments of the organ are important in examination and diagnosis. The relation of the thyroid gland to the parathyroids, which are usually situated on the posterior surface of the lateral lobes of the gland within the surgical capsule, and to the recurrent laryngeal nerves, which run in the cleft between the trachea and esophagus just medial to the lateral lobes, are most important to the surgeon. The relationship to the trachea is important from the point of view of pressure symptoms.

The pyramidal lobe is a narrow projection of thyroid tissue extending upward from the isthmus and lying on the surface of the thyroid cartilage, to the right or left of the prominence of that structure. It is a vestige of the embryonic thyroglossal tract. The importance of the pyramidal lobe is in its relation to developmental anomalies and also in its propensity to undergo hypertrophy when the rest of the thyroid has been removed. Any pathologic process that is diffuse may involve the pyramidal lobe, for example, Graves' disease or Hashimoto's thyroiditis. It becomes thus an item of some importance diagnostically and in thyroid surgery. A pyramidal lobe is found by the surgeon in about 80% of patients.

Blood Supply

The thyroid gland has an abundant blood supply. It has been estimated that the normal flow rate is about 5 ml/g of thyroid tissue each minute. The blood volume of normal humans is about 5 liters and total blood flow 5 liters/min. This mass moves through the lungs about once a minute, through the kidneys once in five minutes, and through the thyroid approximately once an hour. Although the thyroid represents about 0.4‰ of body weight it accounts for 2% of total blood flow. In disease the flow through the gland may be increased up to 100-fold.

This abundant blood supply is provided from the four major thyroid arteries. The superior pair arise from the external carotid and descend several centimeters through the neck to reach the upper poles of the thyroid, where they break into a number of branches and enter the substance of the gland. The inferior pair spring from the thyrocervical trunk of the subclavian arteries and enter the lower poles from behind. Frequently, a fifth artery, the thyreoidea ima, from the arch of the aorta, enters the thyroid in the midline. There are free anastomoses between all of these vessels. In addition, a large number of smaller arteriolar vessels derived from collaterals of the esophagus and larynx supply the posterior aspect of the thyroid. The branching of the large arteries takes place on the surface of the gland, where they form a network. Only after much branching are small arteries sent deep into the gland. These penetrating vessels arborize among the follicles, finally sending a follicular artery to each follicle. This, in turn, breaks up into the rich capillary basket like network surrounding the follicle.

The veins emerge from the interior of the gland and form a plexus of vessels under the capsule. These drain into the internal jugular, the brachiocephalic, and occasionally the anterior jugular veins.

Lymphatics

A rich plexus of lymph vessels is in close approximation to the individual follicles, but no unique role in thyroid function has been assigned to this system. The major normal, if not only, secretory pathway for thyroid hormone is through the venous drainage of the thyroid rather than through the lymphatics, but thyroglobulin is mainly secreted in the lymph.

Innervation

The gland receives fibers from both sympathetic and parasympathetic divisions of the autonomic nervous system. The sympathetic fibers are derived from the cervical ganglia and enter the gland along the blood vessels. The parasympathetic fibers are derived from the vagus and reach the gland by branches of the laryngeal nerves. Both myelinated and nonmyelinated fibers are found in the thyroid, and occasionally in the ganglion cells as well. The nerve supply does not appear to be simply a secretory system. The major neurogenic modifications of thyroid physiology have to do with blood flow and are reviewed in Chapter 4. However neurotransmitters have direct effects on thyroid follicular cells, which vary from one species to another. The physiological relevance of these effects remains to be proved.

The Secretory Unit - The Follicle

The adult thyroid is composed of follicles, or acini. These have lost all luminal connection with other parts of the body and may be considered, from both the structural an functional points of view, as the primary, or secretory, units of the organ. The cells of the follicles are the makers of hormone; the lumina are the storage depots. In the normal adult gland the follicles are roughly spherical and vary considerably in size. The average diameter is 300 microns. The walls consist of a continuous epithelium one cell deep, the parenchyma of the thyroid. The epithelium of the normal gland is usually described as cuboidal, the cell height being of the order of 15 µm. In the resting gland the cells may become flatter. Under chronic TSH stimulation such as occurs with iodide deficiency, the height increases, and the term columnar is applied. Such stimulation, which increases colloid resorption, also leads to a reduction in size the follicular lumen. As a result, the height of the epithelium is often inversely proportional to the diameter of the lumen of the follicle.

Within the follicle and filling its lumen is the homogeneous colloid. This is a mixture of proteins, principally thyroglobulin, but there are other lightweight iodoproteins and serum proteins and albumin, originating from the serum, as well.

In addition to the acinar cells, there are individual cells or small groups of cells that are seen not to extend to the follicular lumen and which may appear as clusters between follicles (Fig. 1-4a,b). These light cells, or C-cells, are a distinct category probably derived from the neural crest via the ultimobranchial body, as shown by studies in quail chicks by Le Douarin and Le Lièvre (171). The C-cells secrete calcitonin (or "thyrocalcitonin") in response to an increase in serum calcium (172). This hormone is important in the regulation of bone resorption and to a lesser extent influences the concentration of serum calcium. Calcitonin acts primarily by suppressing resorption of calcium from bone and therefore lowers plasma free Ca ⁺⁺ levels. C-cells also contain somatostatin, calcitonin gene related peptide, gastrin-releasing peptide, katacalcin and helodermin that could have either a stimulatory or inhibitory activity on thyroid hormone secretion. Their physiological relevance is doubtful (see "Other Regulatory Factors" in this chapter). The C-cells are also the origin of the "medullary" thyroid cancers. In adult human they represent 1% of the cell population.

Outside the follicles two other types of cells populate the thyroid the endothelial cells and fibroblasts. In normal dog thyroids the relative proportions of follicular, endothelial cells and fibroblasts are 70%, 20% and 10% (173).

Fig. 1-4. (A) Light microscopy of a parafollicular cluster (arrow) in relationship to thyroid follicle (TF) (x900). (B) Parafollicular cell in characteristic position between follicular cells and follicular basement membrane, not abutting on colloid (TF) (x4,200). Tissue was obtained from normal thyroid tissue of a 26-year-old woman operated on because of a solitary thyroid adenomatous nodule. Specimens were fixed in glutaraldehyde and embedded in Araldite-502. (From Teitelbaum et al. Nature, 230,1971, with permission).

THE FINE STRUCTURE OF THE THYROID CELLS

The follicular organization and the polarity of the thyrocytes are essential to the specialized metabolism of the organ : with the vectorial transport of thyroglobulin and iodide at the apex, the synthesis of thyroid hormones at the apical membrane, the storage of iodine and thyroid hormone within thyroglobulin in the lumen and endocytosis of thyroglobulin also at the apex. The onset of thyroid function in embryo coincides with the appearance of this structure.

The acinar surface of thyroid parenchymal cells appears to be smooth in the light microscope, but the electron microscope shows that it is covered with tiny villi and some pseudopods. Each cell displays a cilium in the follicular lumen. The base of the cell abuts on a capillary and is separated from it by a two-layer basement membrane visible under the electron microscope. In the usual hematoxylin and eosin stain, the cell cytoplasm is neutrophilic, and colloid droplets may be present. The nucleus is at the base of the cell.

The colloid is variable in tinctorial response but tends to be strongly eosinophilic in resting follicles and pale-staining or even slightly basophilic when the gland is stimulated. In hyperactive follicles the margin of the colloid is scalloped by resorption vacuoles. These vacuoles may represent the "negatives" of the resorption process.

The villi are extensions of cytoplasm which increase cell secretory surface. In acutely stimulated thyroids, pseudopods extend out into the colloid and surround and ingest it by macropinocytosis. Over the course of several hours, the ingested droplets move toward the base of the cell (174). These droplets of resorbed colloid are processed for secretion as hormone by the gland (175).

The resolving power of the electron microscope has been turned upon the thyroid acinar cell by several investigators, among them Wissig (176), Dempsey and Peterson (177), Ekholm and Sjöstrand (178) and Herman (179). Wissig's and Ekholm's findings are presented here in detail and can be taken as typical of the cytologic picture of most species. The entire follicular cell is covered by an uninterrupted plasma membrane (Fig. 1-5 and Fig. 1-6). The apical surface of the cell is dome-shaped and is provided with numerous microvilli that are approximately 0.35 mm tall and 0.07 mm broad. This membrane is composed of two dark layers separated by a single pale layer and is 70 Å thick. Terminal bars join opposing cells at the apical margin, and desmosomes often occur on contacting cell surfaces. Vesicular structures, approximately 60 mm broad, appear in the microvilli and contain material that has the same density as colloid. Beneath the apical border there is a band of cytoplasm that is approximately 0.5 mm wide and devoid of organelles, although microtubular and microfilamentous structures are seen in this area. Beneath this band, a few apical vesicles of 400-15,000 Å are seen, and beneath this area and extending to the base of the cell are the channels of the endoplasmic reticulum, also known as ergoplasmic vesicles. These vesicles, or channels, are limited by a single membrane (the a cytomembrane) approximately 60-70 Å thick, and their outer surface is studded with ribosomes approximately 130-150 Å in diameter. In some areas the membrane covering the cytomembrane is devoid of ribonucleoprotein particles, and in between the vesicles the ribonucleoprotein granules may be seen to lie free. The endoplasmic vesicles are very pleiomorphic. Small vesicles are seen near the apical surface, 50 nm to several microns in diameter, and closed by a single-layer membrane 5 nm in thickness. These droplets appear especially in the apex of the cell and are thought to be secretion droplets. The material within them is frequently quite dense. Large vesicles of up to 1 micron appear especially in stimulated thyroids. These are called colloid droplets because the material within the vesicles is homogeneous and has the density of colloid and results from colloid endocytosis.

Fig. 1-5. A thyroid follicular cell, including: (a) apical vessel of cell; (e) endoplasmic reticulum; (d) colloid droplets; (v) microvilli; (r) ribosomes on endoplasmic reticulum; (g) Golgi apparatus; (m) mitochondrion; (p) plasma membrane; (c) capillary cells; (n) nucleus; (b) basement membrane; (o) open "pore" endothelial cells ; (c) cilium. (Reproduced by permission of the Journal of Ultrastructural Research).

Fig. 1-6. Electron micrographs of rat thyroid. (a) Appearance after inactivation of the gland by two daily doses of T4. The micrograph shows two cell nuclei (N), well-developed rough-surfaced endoplasmic reticulum (RER), Golgi apparatus (G), mitochondria (M), lyosomes (L), and numerous dense, apical (exocytocic) vesicles (V). Because of the T4-induced TSH suppression, no colloid droplets are present (no hormone release); TG synthesis, however, is still going on, as indicated by the dense apical vesicle. (b) Appearance 20 minutes after intravenous administration of TSH (100 mU) to a rat treated with T4 for two days. The most characteristic features of these cells are the large number of colloid droplets (CD) and the almost complete disappearance of dense apical vesicles; TSH has induced an emptying of these vesicles into the follicle lumen. Other organelles are similar to those in Fig. 1-2. Note the close relation relation between colloid droplets and lysosomes (L). At the base of the follicle cells part of a blood capillary (C) is seen. (Micrographs kindly supplied by Professor Ragnar Ekholm, Goteborg, Sweden). The Golgi apparatus is located near the nucleus and consists of small vacuoles and vesicles 400-800 Å in diameter. No nucleoprotein granules are found on the surface of these vesicles. The content of the Golgi vesicles has a density similar to that of secretion droplets.

Numerous rod-shaped or irregular mitochondria are present. Their average diameter is 0.2 mm. They are bordered by a triple-layered membrane 160 Å in width consisting of two opaque layers and a less opaque interposed layer. The inner opaque layer is thrown up into folds, or cristae, which run irregularly, either in the long or the short axis of the mitochondrion.

The nucleus is enclosed within a double-walled envelope whose layers are separated by a less dense area approximately 200 Å thick. The outer nuclear membrane is continuous with the membranes forming the endoplasmic reticulum. The nuclear envelope has characteristic pores 400 Å in diameter.

The abutting plasma membranes of adjacent cells parallel one another and are about 70 Å thick. They are separated by a space 150 Å wide, which contains a material of the same density as the basement membrane. The membrane at the base of the cell is covered on the outer surface by a basement membrane that is approximately 400 Å in width. A thin layer of fibers about 400 Å in diameter may occur at the outer surface of the basement membrane. The basement membrane of the follicular cell is separated by a clear area from the basement membrane of the opposing capillary endothelium. At frequent intervals, the wall of the endothelial cell is interrupted by a pore approximately 450 Å in diameter. Here the lumen of the capillary appears to be in direct contact with the basement membrane of the endothelial cell. The thyroid follicle cells are separated by two layers of basement membrane from the capillaries, but the pores in the endothelial lining of the capillaries may allow, some plasma to come in direct contact with basement membrane. This arrangement should allow free diffusion of materials into and out of the acinar cell.

Ribosomes of the ergastoplasm synthesize thyroglobulin which is processed in the smooth reticulum and Golgi apparatus.

The colloid lumen is sealed by various cell-cell junctions: 1) the tight junctions of the zonula occludens, close to the apical border and which separate the basal from the apical membrane (main protein constituents : occludins); 2) further from the apex are the tight junctions (main protein components : cadherins) and 3) further the desmosome junctions (main protein components : desmogleins and desmocollins). All these junctions are linked to the cytoskeleton. Gap junctions (main protein components : connexins) provide joint channels allowing the passage of small molecules between the cells.

GENERAL METABOLISM OF THE FOLLICULAR THYROID CELL

The metabolism of the thyroid as related to hormone synthesis and secretion is discussed in Chapter 2. In this section, a review of some general aspects of metabolism of the thyroid acinar cell is provided. The metabolism of the thyroid has been studied by all the usual techniques - in vivo in men and mice, in situ, in in vitro perfusion, in slices, cells, homogenates, or subcellular fractions. Several species, including humans, have been investigated, often with obvious and consistent species-related differences. Conditions of tissue preparation and assays have varied widely.

Energy metabolism

Energy supply in the human thyroid cell is necessary for many activities like synthesis of nucleotides, proteins, nuclear acids, lipids, transport functions and other activities like phagocytosis, lysosome movement etc. It is mainly produced by mitochondrial oxidative phosphorylation (about 85%) and to a minor extent by cytosolic aerobic glycolysis (180). Energy metabolism in the human thyroid cell resembles in many aspects that in the dog thyroid. However, the absolute values of oxygen uptake, glucose uptake and lactate formation are significantly less in the human thyroid slices. Adenosine triphosphate (ATP) concentration in the thyroid cell is in the millimolar range and about 90% of ribonucleotides are in the form of triphosphates (181). Free fatty acids are probably the main source of energy in thyroid cells as respiration is maintained for long periods in vitro in the absence of exogenous substrate. As there is hardly any glycogen present in these conditions, most probably free fatty acids are the endogenous substrates. Compartmentation may make glycolytic ATP the main energy source for some membrane functions such as endocytosis of colloid. Indeed inhibition of glycolysis inhibits colloid endocytosis much more than total energy metabolism and addition of glucose counteracts this effect (181).

Mitochondrial inhibitors abolish the stimulatory effect of TSH on thyroid cell respiration (182). Cyclic AMP does not influence mitochondrial respiration in a direct manner. It is therefore assumed that the stimulatory effect of TSH on respiration is secondary to its enhancing effect on energy (i.e. ATP) consuming cellular processes.

Carbohydrate metabolism

The main source of energy delivery for metabolic processes in the thyroid cell are free fatty acids. However, glucose metabolism has an important function in the thyroid for several reasons. About 70% of the glucose taken up by dog or human thyroid slices is transformed to lactate, a further 5% is catabolized through the Embden-Meyerhof pathway and the Krebs cycle (183). Another 6% of glucose carbon is incorporated into protein and less than 1% into lipids and glycogen. The remaining part (about 10%) is oxidized through the hexose monophosphate pathway (HMP). Most of the enzymes participating in the Embden-Meyerhof pathway, HMP and Krebs cycle have been demonstrated in the human thyroid (184) (185) (186). As hexokinase instead of glucokinase is present in the thyroid, the rate of phosphorylation of glucose is probably independent of its concentration because of the low Km of hexokinase for glucose.

Glucose metabolism serves several purposes. The incorporation of glucose carbon into proteins is related to the function of the thyroid cell in protein synthesis i.e. synthesis of thyrogobulin which contains 10% carbohydrate. The metabolism of glucose along the HMP is related to the generation of NADPH and pentoses in this pathway. The production of pentoses is obviously necessary for generation of nucleotides. NADPH production is necessary in several respects (Fig. 7). It is needed for generation of H2O2 for oxidation, organification of iodide and thyroid hormone synthesis. NADPH is also an important cofactor in iodotyrosines deiodination. It is also needed to reduce oxidized glutathione after its generation by GSH peroxidase in the detoxification of the H2O2 leaking in the cell (187).

Fig. 1-7. Postulated NADP oxidation-reduction cycle in thyroid. Four mechanisms of NADPH oxidation are outlined: the reduction of any intermediate X by an NADPH-linked dehydrogenase, the deiodination of iodotyrosines released by thyroglobulinolysis, the generation of H2O2 by THOX (thyroid H2O2 generating enzyme), and the reduction of H2O2 through GSH peroxidase. TG and TGI: uniodinated and iodinated thyroglobulin. +, activation. (From: Dumont JE (187)).

H2O2 generation is stimulated by TSH through cAMP in dog thyroid and by Ca ⁺⁺ and diacylglycerol in all investigated species, including humans and dogs.

TSH enhances carbohydrate metabolism in the dog thyroid (183). During the stimulation there is selective increase in the activity of the HMP whereas incorporation of glucose into proteins and lipids decreases (183). The activity of TSH in this metabolism is probably mediated by cAMP, since this nucleotide can reproduce the TSH effects on glucose uptake, catabolism, incorporation in protein and lipids and on the HMP pathway. TSH also causes an increase in NADPH and NADP+ concentration through increased NAD+ kinase activity (187).

The increased metabolic activity induced by TSH mainly reflects increased consumption by NADPH dependent processes stimulated via cAMP. For instance, the activity of the HMP pathway is predominantly dependent on the availability of the substrate NADP+ generated during oxidation of NADPH (187).

Mitochondrial Respiration

The mitochondrion has appropriately been termed the "powerhouse" of the cell. It provides about 85% of generated ATP in the thyroid cell, only 15% coming from glycolysis. The thyroids of different animal species contain mitochondria with their typical morphology, having electron transport chain, Krebs cycle enzymes, coupled oxidative phosphorylation and good respiratory control (182). The activity of mitochondria is controlled by adenosine diphosphate (ADP) levels. Also respiration linked Ca++ accumulation plays a general and fundamental role in vertebrate cell physiology (188). Free fatty acids are the preferred substrate of oxidation in the unstimulated thyroid, presumably through mitochondrial pathways (189). In thyroids of patients operated for hyperthyroid Graves' disease all enzyme activities studied were increased suggesting an increase in the mitochondrial population in chronically stimulated thyroid cells (181). TSH increases oxygen consumption in thyroid slices by 20 - 30% within a few minutes, independently of exogenous substrates. The increased respiration is oligomycin and antimycin sensitive. Thus, respiration is of largely mitochondrial origin and probably represents the effect of TSH in increasing metabolic activities and consequently ATP consumption (see section on Energy Metabolism) (187). TSH augments oxidation of pyruvate and acetate by thyroid slices. Compounds such as perchlorate, methimazole, iodide, thiocyanate and T4 have no significant direct action on thyroid mitochondria (182). In isolated thyroid mitochodria protein synthesis is dependent on intact electron transport and oxidative phosphorylation. It is inhibited by chloramphenicol but not by cycloheximide (190).

RNA and DNA Metabolism

Chronic TSH stimulation produces cell hypertrophy, and proliferation with a greater increase of RNA than of DNA. Since RNA and DNA synthesis are required for cell growth and division, it is not surprising that TSH stimulation causes rapid and continued increases in synthetic activities. When given in vivo, TSH stimulates uptake and incorporation of RNA precursors within one hour and net RNA increases in about 12 hours (191) (192). TSH stimulates cell uptake and synthesis of purine and pyrimidine precursors (193) (194) and purine and pyrimidine synthesis. Synthesis of both messenger RNA (mRNA) and ribosomal RNA (rRNA) is stimulated by TSH (195). The population of mRNA preferentially synthesized in response to TSH and cyclic AMP is important and includes specific thyroid gene expression such as thyroperoxidase (TPO), Na+/I- cotransporters (NIS) (196), thyroglobulin etc.. RNA degradation is not known to be influenced by TSH.

Formation of polyamines is closely linked to cell growth, although the mechanism is not known. TSH and cAMP enhance ornithine decarboxylase activity, the rate-limiting enzyme in polyamine synthesis (197).

Protein Metabolism

Thyroid tissue is composed of cells and a storage protein, and the kinetic behavior of each compartment varies enormously with the conditions. Thus, in the colloid especially, protein storage and degradation go on concurrently, and content at any time reflects a balance between these activities.

TSH enhances uptake of amino acids by isolated thyroid cells, and stimulates protein synthesis within 30 minutes to 4 hours in some preparations. Because of effects on thyroglobulin (TG) degradation, and dilution of amino acid precursor pools, stimulation of synthesis is more difficult to demonstrate in whole tissues (198) (199) (200). However, if thyroid slices are incubated in high concentration of leucine to obliterate any separate effect of TSH on cell uptake of amino acid, a clear stimulation of protein synthesis by TSH can be demonstrated in vitro in dog thyroid slices (200), and also in isolated thyroid cells but not in primary cultures of dog thyroid cells. Within 12-24 hours of chronic TSH stimulation in vivo, net protein content may be decreased by active TG hydrolysis, but after this, protein content is increased (201). This response remains nearly linear over four to five weeks as thyroid size in animals quintuples. The response is primarily due to production of new cells, since DNA and protein change in parallel.

Huge polysomes (40 to 80 ribosomal units) connected by mRNA have been demonstrated in the thyroid (202) (203) and were shown to incorporate precursors into TG-related peptides. (Fig.1-8). In dog thyroid slices, TSH also shifts thyroid monosomes to polysomes, and this is stimulated by cAMP. This action suggests a direct effect on translation (204).

Fig. 1-8. Electron microscopic photograph of an enormous polysome containing 60 or 70 monosomes, the presumed source of TG synthesis. The arrows point to a thread possibly representing mRNA holding the polysome together. (From Keyhani et al., J. Microscop., 10:269,1971, with permission).

Lipid metabolism

Free fatty acids are the main fuel of the thyroid cell and they may be completely oxidized. Sufficient endogenous substrate is present to sustain respiration for several hours during in vitro incubation of thyroid slices (205) (206) (207). Studies on localization of lipids in human thyroids have shown that small amounts are only present in goitres from thyrotoxic patients, but that appreciable amounts are present in the normal human thyroid i.e. phospholipids, cholesterol and gangliosides: 5.2, 4.3 and 0.12 mmol/kg fresh tissue. C-cells contain most abundantly phospholipids. The human thyroid contains phospholipids in the proportion: phosphatidylcholine (41.8%), phosphathidylethanolamine (26.9%), phosphathidylserine (10.4%), phosphathidylinositol (4.4%), cardiolipin (3.4%), sphyngomyelin (12.4%) (208) (209) (210). TSH enhances the incorporation of precursors into most phospholipids. The effect is believed to reflect a direct stimulation of synthesis of phospholipids. However, as TSH also stimulates phospholipid degradation, increased phospholipids synthesis under the influence of TSH could correspond in part to this accelerated turnover rather than to an accumulation  123  . TSH also stimulates incorporation of inositol into phosphoinositides in a glucose free system. TSH specifically enhances synthesis of phosphatidic acid from glycerophosphate after in vivo administration (211) (212).

Electrolyte Transport and metabolism

The mean resting transmembrane potential as studied in rat, rabbit and guinea-pigs thyroid cells varies between -60 and -70 mV. The magnitude of the membrane potential was found te be dependent mainly upon the gradient for K+ across the membrane. A high intracellular K+ and low Na+ concentration is maintained by ouabain sensitive Na+ - K+ ATPase. The activity of this ATPase varies in direct relation to chronic TSH stimulation, probably corresponding to cell hypertrophy and hyperplasia. There is no evidence for direct action of TSH on this enzyme. Acute stimulation of thyroid cells induces a depolarization of the cell, which is accompanied by a decrease in membrane resistance. The depolarization may correspond to increased permeability to predominantly extracellular cations, such as Na+ or to a decreased permeability to predominantly intracellular cations, such as K+. Administration of TSH or veratridine, a sodium channel agonist, depolarized cultured thyroid cells and increased the secretion of radioiodine from the organically bound pool. Depolarization of the cells by increasing the potassium concentration in the medium failed to promote secretion of radioactive iodine indicating that the sodium influx rather than the depolarization itself, may mediate the secretory response (187).

THYROID REGULATORY FACTORS

In Physiology

Four major biologic variables are regulated in the thyrocyte as in any other cell type: function, cell size, cell number, and differentiation. The first three variables are quantitative and the latter is qualitative. In this chapter we consider the factors involved in these controls in physiology and in pathology, the main regulatory cascades through which these factors exert their effects, and the regulated processes, which are function, proliferation and cell death, gene expression, and differentiation. Whenever possible, we describe what is known in humans.

The two main factors that control the physiology of the thyroid after embryogenesis are the requirement for thyroid hormones and the supply of its main and specialized substrate iodide (Table 1-1). Thyroid hormone plasma levels and action are monitored by the hypothalamic supraoptic nuclei and by the thyrotrophs of the anterior lobe of the pituitary, where they exert a negative feedback. The corresponding homeostatic control is expressed by thyroid-stimulating hormone (TSH, thyrotropin). The hypophysis adjusts its secretion of TSH to the sensitivity of the thyroid, increasing TSH levels when thyrocyte sensitivity decreases (e.g. because of reduced TSH receptor expression) (213) . The TSH receptor is also stimulated by a new different natural hormone cloned by homology, thyrostimuline. The physiological role of this protein is unknown but its level is not controlled by a thyroid hormone feedback and it does not participate in the homeostatic control of the thyroid (214) . Iodide supply is monitored in part through its effects on the plasma level of thyroid hormones, but mainly in the thyroid itself, where it depresses various aspects of thyroid function and the response of the thyrocyte to TSH. These two major physiologic regulators control the function and size of the thyroid - TSH positively, iodide negatively (187;215-217) . These are the specific controls exerted at the level of the thyrocyte itself. The follicular cells themselves probably regulate the other thyroid cells, fibroblasts and endothelial cells through local extracellular signals such as NO, prostaglandins, growth factors etc.

In mice embryo, other unknown factors control differentiation and organ growth which takes place normally in the absence of TSH receptor (218;219) . However, homozygous inactivating mutations of the TSH receptor in familial congenital hypothyroidism were found to be associated with a very hypoplastic thyroid gland (220). Although the thyroid contains receptors for thyroid hormones and a direct effect of these hormones on thyrocytes would make sense (221), as yet little evidence has indicated that such control plays a role in physiol ogy (222). However expression of dominant negative thyroid hormone receptors in mice represses PPARγ expression and induces thyroid tumors in thyroid (223) . Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) at high levels directly stimulate the thyroid, and this effect accounts for the depression of TSH levels and sometimes elevated thyroid activity at the beginning of pregnancy (224-226) .

The thyroid gland is also influenced by various other nonspecific hormones (227). Hydrocortisone exerts a differentiating action in vitro (228) . Estrogens affect the thyroid by unknown mechanisms, directly or indirectly, as exemplified clinically in the menstrual cycle and in pregnancy and by higher prevalence of thyroid disease in females. Growth hormone induces thyroid growth, but its effects are thought to be mediated by locally produced somatomedins (IGF-I). Nevertheless the presence of basal TSH levels might be a prerequisite for the growth promoting action of IGF-I, because a GH replacement therapy did not increase thyroid size in patients deficient for both GH and TSH (229) . The anomalously low endemic goiter prevalence among pygmies living in iodine deficient areas (230), who are genetically resistant to IGF-I, is also compatible with an in vivo permissive effect of IGF-1 and IGF-1 receptor on TSH mitogenic action. Indeed thyroids of transgenic mice overexpressing IGF1 and IGF1 receptor develop hyperplasia and a degree of autonomy vs TSH: their serum TSH is lower and thyroid hormones level normal which shows that they require less TSH to maintain normal thyroid hormone levels (231;232) . In dog and human thyroid primary cultures, the presence of insulin receptors strictly depends on TSH, suggesting that thyroid might be a more specific target of insulin than generally considered (233;234) . It is permissive for TSH mitogenic action in vitro.

Effects of locally secreted neurotransmitters and growth factors on thyrocytes have been demonstrated in vitro and sometimes in vivo, and the presence of some of these agents in the thyroid has been ascertained. The set of neurotransmitters acting on the thyrocyte and their effects vary from species to species (215;235) . In human cells, well-defined direct, but short-lived responses to norepinephrine, ATP, adenosine, bradykinin, and thyrotropin-releasing hormone (TRH) have been observed (215;236;237) . In rat, as evidenced by superior cervical ganglion nervation, sympathetic activity positively modulates function and size of the thyroid (238).

Growth factor signaling cascades demonstrated in vitro can exert similar effects in vivo . In nude mice, the injection of EGF promotes DNA synthesis in thyroid and inhibits iodide uptake in xenotransplanted rat (239) and human thyroid tissues (240). By contrast, the injection of FGF induces a colloid goiter in mice with no inhibition of iodide metabolism or thyroglobulin and thyroperoxidase mRNA accumulation (241) . These effects are the exact replica of initial observations in the dog and other thyroid primary culture system (242),(243-246) . EGF and FGF have since been reported to be locally synthesized in the thyroid gland, as a possible response to thyroxine and TSH (247) respectively. Their exact role as autocrine and/or paracrine agents in the development, function and pathology of the thyroid gland of different species has yet to be clarified (248;249) . HGF does not activate mitogenesis in normal human thyrocyte. The Transforming Growth Factors  (TGF)β constitute another category of cytokines that are produced locally by thyrocytes and influence their proliferation and the action of mitogenic factors (248;250) . TGFβ inhibits proliferation and prevents most of the effects of TSH and cAMP in human thyrocytes in vitro (251;252) . TGFβ is synthesized as an inactive precursor which can be activated by different proteases produced by thyrocytes. TGFβ expression is upregulated during TSH-induced thyroid hyperplasia in rats, suggesting an autocrine mechanism limiting goiter size (253). Activin A and the bone morphogenetic peptide (BMP), which are related to TGFβ,  are also present in thyroid (  MP7 and  MP8A, unpublished) and inhibit thyrocyte proliferation in vitro (254). Unlike TGFβ, they are directly synthesized as an active form. Elements of a Wnt/β catenin signaling pathway (Wnt factors, Frizzled receptors and disheveled isoforms) have been identified in human thyroid and thyroid cancer cell lines (255) . The eventual role in vivo in humans of most of these factors remains to be proved and clarified.

Thyroglobulin has been reported as a negative feedback inhibitor repressing the expression of specific thyroid transcription factors TTF1, TTF2, Pax8 and acting through a putative receptor at the apical membrane (256). However, as previous claims by the same group (the exophtalmic producing factor, ganglioside as the TSH receptor, etc) this one is neither substantiated nor supported by others.

Human thyroid cells contain androgen and estrogen receptors (257). Estrogens promote the growth of these cells (258) which may explain the higher prevalence of thyroid tumors and diseases in women, particularly between puberty and menopause. In mice, thyroid estrogen by downregulating CDKn1B (p27) facilitates the growth effects of the PI3K cascade (259).

In Pathology Mutated constitutively active TSH receptors and Gs proteins cause thyroid autonomous adenomas (260;261) . Mutations conferring higher sensitivity of the TSH receptor to LH/HCG cause hyperthyroidism in pregnancy (262) (263). Pathologic extracellular signals play an important role in autoimmune thyroid disease. Thyroid-stimulating antibodies (TSAbs), which bind to the TSH receptor and activate it, reproduce the stimulatory effects of TSH on the function and growth of the tissue. Their abnormal generation is responsible for the hyperthyroidism and goiter of Graves' disease. The kinetic characteristics of TSH and TSAbs differ: TSH effects on camp accumulation are rapid and disappear rapidly in the absence of the hormone (minutes) while TSAbs effects are slow and persistent (hours) (264) .

Thyroid-blocking antibodies (TBAbs) also bind to the TSH receptor but do not activate it and hence behave as competitive inhibitors of the hormone. Such antibodies are responsible for some cases of hypothyroidism in thyroiditis. Both stimulating and inhibitory antibodies induce transient hyperthyroidism or hypothyroidism in newborns of mothers with positive sera (216). The existence of thyroid growth immunoglobulins has been hypothesized to explain the existence of Graves' disease with weak hyperthyroidism and prominent goiter (265). The thyroid specificity of such immunoglobulins would imply that they recognize thyroid-specific targets. This hypothesis is now abandoned (266-268) . Discrepancies between growth and functional stimulation may instead reflect cell intrinsic factors. Local cytokines have been shown to influence, mostly negatively, the function, growth, and differentiation of thyrocytes in vitro and thyroid function in vivo. Because they are presumably secreted in loco in autoimmune thyroid diseases, these effects might play a role in the pathology of these diseases, but this notion has not yet been proved (215) (269). Moreover in selenium and iodine deficiency plus dietary supplementation of thiocyanate, secretion of TGFβ by macrophages has been implicated in the generation of thyroid fibrosis (270) (271) and the pathogenesis of thyroid failure in endemic cretinism. The overexpression of both FGF and FGF receptor 1 in thyrocytes from human multinodular goiter might explain their relative TSH-independence (272) . On the other hand, the subversion of tyrosine kinase pathways similar to those normally operated by local growth factors (i.e. the activation of Ret/PTC (273) and TRK (274), the overexpression of Met/HGF receptor sometimes in association with HGF (275), or erbB/EGF receptor in association with its ligand TGFα (276) have been causally associated with TSH-independent thyroid papillary carcinomas. An autocrine loop involving IGF-II and the insulin receptor isoform-A is also proposed to stimulate growth of some thyroid cancers (277) . Thyroid cancer cells often escape growth inhibition by TGFβ (278).

REGULATORY CASCADES

The great number of extracellular signals acting through specific receptors on cells in fact control a very limited number of regulatory cascades. We first outline these cascades, along with the signals that control them, and then describe in more detail the specific thyroid cell features: controls by iodide and the TSH receptor.

The Cyclic Adenosine Monophosphate Cascade

The cyclic adenosine monophosphate (cAMP) cascade in the thyroid corresponds, as far as it has been studied, to the canonic model of the β-adrenergic receptor cascade (216) (Fig. 1-9). It is activated in the human thyrocyte by the TSH and the β-adrenergic and prostaglandin E receptors. These receptors are classic seven-transmembrane receptors controlling transducing guanosine triphosphate (GTP)-binding proteins. Activated G proteins belong to the G _s class and activate adenylyl cyclase; they are composed of a distinct α _s subunit and nonspecific β and γ monomers. Activation of a G protein corresponds to its release of guanosine diphosphate (GDP) and binding of GTP and to its dissociation into α _GTP and βγ dimers. α _sGTP directly binds to and activates adenylyl cyclase. Inactivation of the G protein follows the spontaneous, more or less rapid hydrolysis of GTP to GDP by α _s GTPase activity and the reassociation of α _GDP with βγ. The effect of stimulation of the receptor by agonist binding is to increase the rate of GDP release and GTP binding, thus shifting the equilibrium of the cycle toward the α _GTP active form. One receptor can consecutively activate several G proteins (hit-and-run model). The thyroid contains mainly three isoforms of adenylyl cyclase : III, VI and IX (279) . A similar system negatively controls adenylyl cyclase through G _i . It is stimulated in the human thyroid by norepinephrine through α ₂ -receptors. Adenosine at high concentrations directly inhibits adenylyl cyclase. The cAMP generated by adenylyl cyclase binds to the regulatory subunit of protein kinase A (PKA) that is blocking the catalytic subunit and releases this now-active unit. The activated, released catalytic unit of protein kinase phosphorylates serines and threonines in the set of proteins containing accessible specific peptides that it recognizes. These phosphorylations, through more or less complicated cascades, lead to the observed effects of the cascade. cAMP-dependent kinases have two isoenzymes (I, II), the first of which is more sensitive to cAMP, but as yet no clear specificity of action of these kinases has been demonstrated. In the case of the thyroid, this cascade is activated through specific receptors, by TSH in all species, and by norepinephrine receptors and prostaglandin E in humans, with widely different kinetics: prolonged for TSH and short lived (minutes) for norepinephrine and prostaglandins (280). Other neurotransmitters have been reported to activate the cascade in thyroid tissue, but not necessarily in the thyrocytes of the tissue (237). In the thyroid cAMP, besides PKA, activates EPAC (Exchange Proteins directly Activated by cAMP) or Rap guanosine nucleotide exchange factor-1 (GEF-1) and the less abundant GEF-2, which activate the small G protein Rap (281). However, despite high expression of EPAC1 in thyrocytes and its further increase in response to TSH, all the physiologically relevant cAMP-dependent functions of TSH studied in dog thyroid cells, including acute regulation of cell functions (including thyroid hormone secretion) and delayed stimulation of differentiation expression and mitogenesis, are mediated only by PKA activation (282) . The role of the cAMP/EPAC/Rap cascade in thyroid thus remains largely unknown. Activation of PKA inactivates small G proteins of the Rho family (RhoA, Rac1 and Cdc42), which reorganizes the actin cytoskeleton and could play an important role in stimulation of thyroid hormone secretion and induction of thyroid differentiation genes (283) . Of the other known possible effectors of cAMP, cyclic nucleotide-activated channels have not been looked for. For several effects of cyclic AMP (eg NIS and thyroglobulin induction, DNA synthesis) protein kinase A is required.

The cAMP cascade is also controlled by several negative feedbacks. The most important is the activation and induction by PKA of PDE4 D3 and other phosphodiesterases (284) (285)

The thyrocyte is very sensitive to internal c AMP : a mere doubling of its concentration is sufficient to elicit near maximal thyroglobulin phagocytosis (286).

Fig .1.9. Regulatory cascades activated by thyroid-stimulating hormone (TSH) in human thyrocytes. In the human thyrocyte, H 2 O 2 (H2O2) generation is activated only by the phosphatidylinositol 4,5-bisphosphate (PIP 2 ) cascade, that is, by the Ca 2+ (Ca++) and diacylglycerol (DAG) internal signals. In dog thyrocytes, it is activated also by the cyclic adenosine monophosphate (cAMP) cascade. In dog thyrocytes and FRTL-5 cells, TSH does not activate the PIP 2 cascade at concentrations 100 times higher than those required to elicit its other effects. Ac, adenylate cyclase; cA, 3 ’ -5 ’ -cAMP, cGMP, 3 ’ -5 ’ -cyclic guanosine monophosphate; FK, forskolin; Gi, guanosine triphosphate (GTP) binding transducing protein inhibiting adenylate cyclase; Gq, GTP-binding transducing protein activating PIP 2 phospholipase C; Gs, GTP-binding transducing protein activating adenylate cyclase; I, putative extracellular signal inhibiting adenylate cyclase (e.g., adenosine through A 1 receptors); IP 3 , myoinositol 1,4,5-triphosphate; EPAC: cAMP dependent Rap guanyl nucleotide exchange factor; PKA, cAMP-dependent protein kinases; PKC, protein kinase C; PLC, phospholipase C; PTOX, pertussis toxin; R ATP, ATP purinergic P 2 receptor; R TSH, TSH receptor; Ri, receptor for extracellular inhibitory signal I; TAI, active transport of iodide; TG, thyroglobuline; TPO, thyroperoxidase.

The Ca2 ⁺ – Inositol 1,4,5-Triphosphate Cascade

The Ca ²⁺ – inositol 1,4,5-triphosphate (IP ₃ ) cascade in the thyroid also corresponds, as far as has been studied, to the canonic model of the muscarinic or α ₁ -adrenergic receptor – activated cascades. It is activated in the human thyrocyte by TSH, through the same receptors that stimulate adenylyl cyclase, and by ATP, bradykinin, and TRH — through specific receptors. In this cascade, as in the cAMP pathway, the activated receptor causes the release of GDP and the binding of GTP by the GTP-binding transducing protein (G _q ) and its dissociation into α _q and βγ. α _GTP then stimulates phospholipase C. Gs and Gq compete for the same TSH receptor, with a higher affinity for Gs (287-289) . Phospholipase C hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) into diacylglycerol and IP ₃ . IP ₃ enhances calcium release from its intracellular stores, followed by an influx from the extracellular medium. The rise in free ionized intracellular Ca ²⁺ leads to the activation of several proteins, including calmodulin. The latter protein in turn binds to target proteins and thus stimulates them: cyclic nucleotide phosphodiesterase and, most importantly, calmodulin-dependent protein kinases. These kinases phosphorylate a whole set of proteins exhibiting serines and threonines on their specific peptides and thus modulate them and cause many observable effects of this arm of the cascade. Calmodulin also activates constitutive nitric oxide (NO) synthase in thyrocytes. The generated NO itself enhances soluble guanylyl cyclase activity in thyrocytes and perhaps in other thyroid cells and thus increases cGMP accumulation (290). Nothing is yet known about the role of cGMP in the thyroid cell but NO causes vasodilatation.

Diacylglycerol released from PIP ₂ activates protein kinase C, or rather the family of protein kinases C, which by phosphorylating serines or threonines in specific accessible peptides in target proteins causes the effects of the second arm of the cascade (291). It inhibits phospholipase C or its G _q , thus creating a negative feedback loop. In the human thyroid, the PIP ₂ cascade is stimulated through specific receptors by ATP, bradykinin, TRH and by TSH (237) (292) (293). The effects of bradykinin and TRH are very short lived. Acetylcholine, which is the main activator of this cascade in the dog thyrocyte (294), is inactive on the human cell, although it activates nonfollicular (presumably endothelial) cells in this tissue (237).

Other Phospholipid-Linked Cascades

In dog thyroid cells and in a functional rat thyroid cell line (FRTL5), TSH activates PIP ₂ hydrolysis weakly and at concentrations several orders of magnitude higher than those required to enhance cAMP accumulation. Of course, these effects have little biologic significance. However, in dog cells, at lower concentrations TSH increases the incorporation of labeled inositol and phosphate into phosphatidylinositol. Similar effects may exist in human cells, but they would be masked by stimulation of the PIP ₂ cascade. They may reflect increased synthesis perhaps coupled to and necessary for cell growth (295).

Diacylglycerol can be generated by other cascades than the classic Ca ²⁺ -IP ₃ pathway. Activation of phosphatidylcholine phospholipase D takes place in dog thyroid cells stimulated by carbamylcholine. Because it is reproduced by phorbol esters, that is, by stable analogues of diacylglycerol, it has been ascribed to phosphorylation of the enzyme by protein kinase C, which would represent a positive feedback loop (296). Although such mechanisms operate in many types of cells, their existence in human thyroid cells has not been demonstrated (297).

Release of arachidonate from phosphatidylinositol by phospholipase A ₂ and the consequent generation of prostaglandins by a substrate-driven process are enhanced in various cell types through G protein – coupled receptors, by intracellular calcium, or by phosphorylation by protein kinase C. In dog thyroid cells all agents enhancing intracellular calcium concentration, including acetylcholine, also enhance the release of arachidonate and the generation of prostaglandins. In this species, stimulation of the cAMP cascade by TSH inhibits this pathway. In pig thyrocytes, TSH has been reported to enhance arachidonate release. In human thyroid, TSH, by stimulating PIP ₂ hydrolysis and intracellular calcium accumulation, might be expected to enhance arachidonate release and prostaglandin generation, but such effects have not yet been proved.

Regulatory Cascades Controlled by Receptor Tyrosine Kinases

Many growth factors and hormones act on their target cells by receptors that contain one transmembrane segment. They interact with the extracellular domain and activate the intracellular domain, which phosphorylates proteins on their tyrosines. Receptor activation involves in some cases a dimerization and in others a conformational change. The first step in activation is interprotein tyrosine phosphorylation, followed by binding of various protein substrates on tyrosine phosphates containing segments of the receptor. Such binding through src homology domains (SH2) leads to direct activation or to phosphorylation of these proteins on their tyrosines and to membrane localization. In turn, these cause sequential activation of the ras and raf proto-oncogenes, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and so on, on the one hand, and phosphatidylinositol-3-kinase (PI-3-kinase), protein kinase B (PKB), and TOR (target of rapamycin) on the other hand. The set of proteins phosphorylated by a receptor defines the pattern of action of this receptor. In thyroids of various species, insulin, IGF-I, EGF, FGF, HGF, but not platelet-derived growth factor activate such cascades (298;299) . In the human thyroid, effects of insulin, IGF-I, EGF, FGF, but not HGF have b een demonstrated (215;300-304) . Transforming growth factor-β, acting through the serine threonine kinase activity of its receptors and intermediate proteins (Smad), inhibits proliferation and specific gene expression in human thyroid cells (251;252;305) . TSH and cAMP do not activate either the MAPK-ERK nor the JUNK and p38 phosphorylation pathways in dog or human thyroids (306).

Cross-Signaling between the Cascades

Calcium, the intracellular signal generated by the PIP ₂ cascade, activates calmodulin-dependent cyclic nucleotide phosphodiesterases and thus inhibits cAMP accumulation and its cascade (307). This activity represents a negative cross-control between the PIP ₂ and the cAMP cascades. Activation of protein kinase C enhances the cAMP response to TSH and inhibits the prostaglandin E response, which suggests opposite effects on the TSH and prostaglandin receptors (308). No important effect of cAMP on the PIP ₂ cascade has been detected. On the other hand, stimulation of protein kinase C by phorbol esters inhibits EGF action.

Cross-signaling between the cyclic AMP pathway and growth factor activated cascades have been observed in various cell types (309;310). In ovarian granulosa cells, FSH through cAMP activates MAP kinases and the PI3 kinase pathway (311). In FRTL5, but not in WRT cell lines, TSH through cAMP activates MAP kinases. In WRT cells but not in PCCl3 cells, TSH and cAMP activate PKB (312;313). Such cross signallings have not been observed in human or dog thyroid cells. Ras, MAPK, p38, Jun kinase and ERK5, as well as PI3 kinase and PKB, are not modulated by cAMP (314;315) (316) (317).

Other growth activating cascades have been little investigated in the thyroid. In dog and human cells TSH or cAMP have no effect on STAT phosphorylations i.e. on the JAK-STAT pathways. The NF  β pathway has not yet been investigated in thyroid cells.

SPECIFIC CONTROL BY IODIDE

Iodide, the main substrate of the specific metabolism of the thyrocyte, is known to control the thyroid. Its main effects in vivo and in vitro are to decrease the response of the thyroid to TSH, to acutely inhibit its own oxidation (Wolff-Chaikoff effect), to reduce its trapping after a delay (adaptation to the Wolff-Chaikoff effect), and at high concentrations to inhibit thyroid hormone secretion (Fig. 1-10). The first effect is very sensitive in as much as small changes in iodine intake are sufficient to reset the thyroid system at different serum TSH levels without any other changes (e.g., thyroid hormone levels), which suggests that in physiologic conditions, modulation of the thyroid response to TSH by iodide plays a major role in the negative feedback loop (217;318). Iodide in vitro has also been reported to inhibit a number of metabolic steps in thyroid cells (319) (320). These actions might be direct or indirect as a result of an effect on an initial step of a regulatory cascade. Certainly, iodide inhibits the cAMP cascade at the level of G _s or cyclase and the Ca ²⁺ -PIP ₂ cascade at the level of G _q or phospholipase C; such effects can account for the inhibition of many metabolic steps controlled by these cascades (321) (322). In one case in which this process has been studied in detail, the control of H ₂ O ₂ generation, that is, the limiting factor of iodide oxidation and thyroid hormone formation, iodide inhibited both the cAMP and the Ca ²⁺ -PIP ₂ cascades at their first step but also the downstream effects of the generated intracellular signals cAMP, Ca ²⁺ , and diacylglycerol on H ₂ O ₂ generation (323). This effect account for the inhibition by iodide of its oxidation i.e. the Wolff-Chaikoff effects (324).

Until now, the mechanism of action of iodide on all the metabolic steps besides secretion fits the "XI" paradigm of Van Sande (325). These inhibitions are relieved by agents that block the trapping of iodide (e.g., perchlorate) or its oxidation (e.g., methimazole) — the Van Sande criteria. The effects are therefore ascribed to one or several postulated intracellular iodinated inhibitors called XI. The identity of such signals is still unproved. At various times several candidates have been proposed for this role, such as thyroxine, iodinated eicosanoids (iodolactone) (326) , and iodohexadecanal (327). The latter, the predominant iodinated lipid in the thyroid, can certainly account for the inhibition of adenylyl cyclase and of H ₂ O ₂ generation (328) (329) (330) . The stimulation of H ₂ O ₂ generation by iodide in follicles of human thyroid is inhibited by methimazole but not by perchlorate.

This suggests that the generation of Xi takes place at the membrane and that the intermediate Xi may diffuse in the membrane but not in the medium (unpublished). It should be emphasized that iodination of the various enzymes, as well as a catalytic role of iodide in the generation of O ₂ radicals (shown to be involved in the toxic effects of iodide), could account for the Van Sande criteria with no need for the XI paradigm (325) (331) . Besides, an inhibition of thyroid secretion by iodide in antithyroid drugs treated hyperthyroid patients suggests a direct Xi independent effect.

Distinct from its inhibitory effects, iodide also activates H2O2 generation and therefore protein iodination in the thyroid of some species including humans. This effect is also inhibited by inhibitors of thyroperoxidase and NIS. It would link the generation of H2O2 to the availability of its cosubstrate iodide (332).

Iodide in vivo, at moderate doses in dog, decreases cell proliferation and the expression of TPO and NIS mRNA but not the synthesis or secretion of thyroid hormones. The downregulation of NIS explains the well known delayed decrease of iodide transport in response to iodide, i.e. the adaptation to the Wolff-Chaikoff effect (333).

Fig 1-10. Effects of iodide on thyroid metabolism. All inhibitory effects of iodide, except in part the inhibition of secretion, are relieved by drugs that inhibit iodide trapping (e.g., perchlorate) or iodide oxidation (e.g., methimazole). All effects are direct inhibitions except the effect on iodide transport which bears on the transcription of Na/I- symporter gene. Three possible mechanisms corrresponding to this paradigm are outlined: generation of O 2 radicals, iodination of target proteins and synthesis of an XI compound. Any of these mechanisms could account for the various steps ascribed to XI inhibition by I - (indicated by slashes).

THE THYROTROPIN RECEPTOR

The Structure of the Thyrotropin Receptor

The receptors for TSH, FSH, and LH/CG are members of the rhodopsin-like G protein-coupled receptor family. As such, the TSH receptor has a “ serpentine ” domain containing seven transmembrane regions with many (but not all) of the features typical of this receptor family. In addition, and a hallmark of the subfamily of glycoprotein hormone receptors (334-336), it has a large (about 400 amino-acid residues) amino-terminal extracellular domain that contains sites that selectively bind TSH with high affinity (337). The higher sequence identity of the serpentine domains of the glycoprotein hormone receptors (about 70%), as compared with the extracellular domains (about 40%, Fig.11 ) suggests that the former are interchangeable modules capable of activating guanine-nucleotide-binding (G) proteins (mainly G  _s ) after specific binding of the individual hormones to the latter (338). Contrary to other rhodopsin-like G protein-coupled receptors, the glycoprotein hormones bind to their respective extracellular domains with high affinity in the absence of the serpentine domain (339-341). The intramolecular transduction of the signal between these two portions of the receptors involves a still incompletely defined mechanism specific to the glycoprotein hormone receptor family (see below). The relatively high sequence identity between the hormone-binding domains of the TSH and LH/CG receptors opens the possibility of spillover phenomena during normal or, even more so, molar or twin pregnancies, when serum CG concentrations are several orders of magnitude higher than are serum TSH concentrations. This provides an explanation to cases of gestational thyrotoxicosis (see below, Chapter 20 and Chapter 56).

Fig. 1-11. Both the the beta subunits of the glycoprotein hormones and the glycoprotein hormone receptors are encoded by paralogous genes. A: Similarity of the aminoacid sequences of the  -subunits of human chrorionic gonadotropin (hCG), luteinizing hormone (LH), thyrotropin (TSH) and follicle stimulating hormone (FSH). Inset: Diagram of the general structure of the receptors for these hormones, showing the ectodomain (extracellular), serpentine (transmembrane) and endodomain (intracellular domain). B: Similarity of the aminoacid sequences of the LH/CG, FSH and TSH receptors (r). Sequence identities are indicated, separately for the extracellular domains of the three receptors. The pattern of shared similarities suggests co-evolution of the hormones and the extracellular domain of their receptors, resulting in generation of specificity barriers. The high similarity of the serpentine domains of the receptors is compatible with a conserved mechanism of intramolecular signal transduction. (Reproduced from Vassart G, Pardo L, Costagliola S. Molecular dissection of the glycoprotein hormone receptors. Trends Biochem Sci 2004;29:119, with permission of the publisher)

The TSH receptor contains six sites for N-glycosylation, of which four are effectively glycosylated (342). The functional role of the individual carbohydrate chains is still debated. It is likely that they contribute to the routing and stabilization of the receptor as it passes through the membrane system of the cell and is inserted into the cell membrane. Alone among the glycoprotein hormone receptors, the extracellular domain of the TSH receptor is cleaved, severing it from the serpentine domain (343). This phenomenon has been related to the presence in the extracellular domain of the receptor of a 50 amino-acid insertion for which there is no counterpart in the FSH receptor or LH/CG-receptor. The initial cleavage step, due to the action of a metalloprotease, takes place at around position 314 (within the 50-amino-acid insertion) from the amino terminus of the receptor, and is followed by removal of approximately 50 amino acids from the amino-terminal end of the serpentine-containing portion of the receptor (344;345). The amino-terminal end of the receptor remains bound to the extracellular end of the serpentine domain by disulfide bonds. The functional importance of this TSH receptor-specific postranslational modification remains unclear. Whereas all wild type TSH receptors on the surface of thyroid follicular cells seem to be in cleaved form, non-cleavable mutant constructs are functionally undistinguishable from cleaved receptors, when expressed in transfected cells (346). Residues 303-366 can be deleted from the hinge region with minimal effects o the function of the receptor (347). When transiently or permanently transfected in non-thyroid cells, wild type human TSH receptors are present at the cell surface as a mixture of monomers and cleaved dimers. There are indications from immunization experiments in mice that cleavage and possible shedding of the aminoterminal portion of the receptor would play a role in the generation of stimulating autoantibodies in patients with Graves disease (348;349) (see Chapter 17).

The TSH receptor is specifically inserted into the basolateral membrane of thyroid follicular cells. This phenomenon involves a signal (amino acids 731-746) unusually localized in the very C-terminal portion of the receptor, at a marked distance from the membrane (350).

The possibility that TSH receptors are present on the cell surface as dimers of cleaved dimers was raised after demonstration that most rhodopsin-like G-protein coupled receptors do dimerize (351). Functional complementation of TSH receptors with mutations in the extracellular and the serpentine domains has been observed after expression of receptor constructs in transfected cells (352). A definitive demonstration that glycoprotein hormone receptors do dimerize in vivo and that dimers interact functionally has been provided by similar complementation experiments performed in LH/CG receptor knockout mice. Mice co-expressing two inactive mutant receptors are fertile (353). Direct demonstration of dimerization of the TSH receptor has been provided by Bioluminescence Resonance Energy Transfer (BRET) and the dimers have been shown to display negative cooperativity for binding of TSH (352). Whether this allosteric behavior of the receptor has (patho)physiological significance remains to be determined..

The Thyrotropin Receptor Gene

The gene coding for the human TSH receptor is located on the long arm of chromosome 14 (14q31)(354;355). It is is organized into 10 exons. The extracellular domain is encoded by a series of 9 exons, each of which corresponds to one or an integer number of leucine-rich repeat segments (see below). The carboxyl-terminal half of the receptor containing the carboxyl-terminal part of the extracellular domain and the serpentine domain is encoded by a single large exon (356), in keeping with the fact that the genes for many G protein-coupled receptor have no introns. A likely evolutionary scenario derives from this gene organization: the glycoprotein hormone receptor genes would have evolved from the condensation of an intron-less classic G protein-coupled receptor with a mosaic gene encoding a protein with leucine-rich repeat segments (357). Triplication of this ancestral gene and subsequent divergence led to the receptors for TSH, FSH, and LH/CG. The existence of 10 exons in both the TSH and FSH receptor genes (as opposed to the 11-exon LH/CG-receptor gene), suggests the following evolutionary steps: first, duplication of an ancestral glycoprotein hormone receptor gene, yielding the LH/CG-receptor gene and the ancestors of the TSH- and FSH-receptor genes. After losing one intron, the latter duplicated subsequently into the TSH- and FSH- receptor genes. The family of the glycoprotein hormone receptors comprises five additional members presenting a similar pattern made of leucine-rich repeats in the ectodomain, upstream of a rhodopsin-like serpentine domain: two of them (LGR7, LGR8) encode insulin-like 3 and relaxin receptors, respectively (358), the remaining three (LGR4, LGR5, LGR6) are orphan receptors involved in regulation of epithelial stem cell biology (359).

The promoter of the TSH receptor gene has the characteristics of a housekeeping gene promoter, being GC-rich and devoid a TATA box. In rats it stimulates transcription from multiple start sites (360) and it contains a functional thyroid transcription factor (TTF)-1 recognition site (361). Expression of the TSH-receptor gene is largely thyroid-specific. Constructs made of a chloramphenicol acetyltransferase reporter gene under control of the 5 ’ -flanking region of the rat TSH-receptor gene are expressed when transfected into FRTL5 cells and FRT cells but not into non-thyroid HeLa or rat liver (BRL) cells (360). However, TSH receptor mRNA has been clearly demonstrated in fat tissue of guinea pigs (362), in adipocytes (363;364) and in ependymal cells of the mediobasal hypothalamus in the mouse, where it plays a role in adjustment of reproductive physiology to the length of the days (365;366) . TSH receptors may also be present in lymphocytes, extraocular tissue, cartilage, and bone, but their functional importance in these tissues is uncertain (367;368). Expression of the TSH receptor in thyroid cells is extremely robust. It is moderately upregulated by TSH in vitro and downregulated by iodide in vivo (369).

Recognition of the Receptor by Thyrotropin

The three-dimensional structures are available for hCG and FSH FSH (370-372) which allows accurate modelization of TSH on these templates. The crystal structure of the human FSHr-FSH complex (373) has confirmed that the ectodomain of glycoprotein hormone receptors belongs to the family of proteins with leucine-rich repeats (LRRs) (374). The concave inner surface of the receptor (Fig.12) is an untwisted, non-inclined b-sheet formed by ten LRRs. Whereas the N-terminal portion of the beta-sheet (LRR1 – 7) is nearly flat, the C-terminal portion (LRR7 – 10) has the horseshoe-like curvature of LRR proteins. The crystal structure of part of the TSHr ectodomain in complex with thyroid-stimulating or – blocking autoantibodies has recently been obtained (375;376). Notably, both the structure of the ectodomain of TSHr and the receptor binding arrangements of the autoantibodies are very similar to those reported for the FSHr-FSH complex. The ectodomain of glycoprotein hormone receptors also contains, downstream of the LRR region, a cysteine cluster domain of unknown structure (the hinge region), involved in receptor inhibition/activation and containing sites for tyrosine sulfation important for hormone binding (see below).

Fig. 1-12. Schematic representation of the structure of the TSH receptor. (A) Two-dimensional representation with indication of the various domains. The blue boxes correspond to amino-terminal and carboxyl-terminal cysteine-rich portions of the extracellular domain, flanking leucine-rich repeats (LRR, yellow box). (B) General view of the follicle-stimulating hormone receptor (FSHr)-FSH crystal structure as a template to model the interaction between TSH and the TSH receptor (692).The concave inner surface of the receptor, formed by ten leucine rich repeats (LRR2 – 9, shown in blue), contact the middle section of the hormone molecule, both the C-terminal segment of the  subunit and the “ seat-belt ” segment of the  -subunit (shown in red). (C) EachLRR is composed of theX1-X2-L-X3-L-X4-X5 residues (where X is any amino acid, and L usually is Leu, Ile, or Val), forming the central X2-L-X3-L-X4 a typical beta-strand, while X1 and X5 are parts of the adjacent loops. (D) Molecular model of the transmembrane domain of the TSH receptor, constructed from the crystal structure of bovine rhodopsin. The color code of the  -carbon ribbons is: transmembrane helix 1 (crimson), 2 (goldenred), 3 (dark red), 4 (gray), 5 (red), 6 (orange), and 7 (blue), and Helix8 (blue). The structures of available class A rhodopsin-like GPCRs are similar at the transmembrane domain.

Replacement by site-directed mutagenesis of the residues facing the hormone in the leucine-rich repeat portion of the TSH receptor (see figure 12 ) with their counterparts in the LH/CG receptor has been performed to assess the structural bases of binding selectivity (377). Exchanging eight amino acids of the TSH receptor for the corresponding amino acids in the LH/CG receptor resulted in a mutant receptor which bound human CG as well as the wild-type LH/CG receptor. While gaining sensitivity to human CG, the mutant receptor retained normal sensitivity to TSH, making it a dual-specificity receptor. It is necessary to exchange 12 additional amino-acid residues to fully transform this mutant receptor into a bonafide LH/CG receptor (378). From an evolutionary point of view, these observations indicate that the specificity of hormone receptors is based on both attractive and repulsive residues, and that residues at different homologous positions have been selected to this result in the different receptors.

Inspection of electrostatic surface maps of models of the wild-type TSH and LH/CG receptors and some of the mutants is revealing in this respect (379;380). The LH/CG receptor has an acidic groove in the middle of its horseshoe, extending to the lower part of it (corresponding to the carboxyl-terminal ends of the  strands). Generation of a similar distribution of charges in the dual-specificity and reverse-specificity TSH receptor mutants suggests that this is important for recognition of human CG. A detailed modelization of the interactions between TSH and the ectodomain of its receptor has been realized (381).

In addition to the hormone-specific interactions genetically encoded in the primary structure of glycoprotein hormone receptors and their ligands, there are important non-hormone-specific ionic interactions involving sulfated tyrosine residues present in the extracellular domains of all three receptors (382). In the TSH receptor, both tyrosine residues of a conserved Tyr-Asp-Tyr motif located close to the border between the extracellular domain and the first transmembrane helix are sulfated ( Fig.13 ), although only sulfation of the first tyrosine of the motif seems to be functionally important (383), contributing importantly to the affinity of the receptor for TSH, without interfering with specificity. The functional role of this postranslational modification of the TSH receptor has been confirmed by demonstration of profound hypothyroidism due to resistance to TSH in mice with inactivation of Tpst2, one of the enzymes responsible for tyrosine sulfation (384;385).

Fig. 1-13. Linear representation of the TSH receptor. Sequences common to all rhodopsin-like G protein-coupled receptors and sequences specific to the glycoprotein hormone receptor gene family are both implicated in activation of the TSH receptor. Key residues are indicated (red dots) as well as conserved motifs: SO3 -- stands for postranslational sulfation of the indicated tyrosine residues (693). The black boxes stand for transmembrane helices and I1-I3, E1-E3, for intracellular and extracellular loops, respectively; LRR, leucine-rich repeats.

Activation of the Serpentine Portion of the Thyrotropin Receptor

Being a member of the G-protein coupled receptor family, the serpentine domain of the TSH receptor is likely to share with rhodopsin common mechanisms of activation (386;387). However, sequence variations in this domain of the glycoprotein hormone receptors suggest the existence of idiosyncrasies associated with hormone-specific mechanisms of activation (Fig. 13). The crystallographic structure of several GPCRs belonging to family A has now been determined (388-396) giving templates for realistic modeling of the serpentine portion of class A GPCRs, including theTSH receptor. A host of artificial and natural mutants of GPCRs have been studied over the past 20 years. This led to scenarios of GPCR activation which have only very recently been confronted with direct structural data (394;397;398). The first activating mutation identified in the α2adrenergic receptor suggested that activation resulted from the release of a structural lock between transmembrane helices 3 and 6 keeping the wild type receptor inactive (399;400). Structural data obtained from opsin at low pH (expected to mimic the active state) (401), a constitutively active rhodopsin mutant (394), and an active state of the α2adrenergic receptor stabilized by a nanobody (398) point to a mechanism of activation involving subtle changes in the conformations of the ligand-binding pocket associated with a more dramatic movement of transmembrane helix 6 (TM6) secondary to rupture of the same TM3-TM6 “ ionic lock ” . The result is the “ opening ” of a cavity between the cytoplasmic ends of TM3, 5 and 6 allowing interaction with- and activation of the G protein (401;402).

The many gain-of-function somatic and germline mutations that have been found in the serpentine domain of the TSH receptor in autonomous toxic adenomas and hereditary nonautoimmune hyperthyroidism (see Chapters 19 and Chapter 25) are expected to trigger a similar conformational change in the TSH receptor (for a complete list of mutations, see http://gris.ulb.ac.be/ and http://www.ssfa-gphr.de/). Of note, a mutation affecting Asp619, at the basis of TM6, which would rupture the TM3-TM6 ionic lock, was amongst the very first activating mutations identified in toxic adenomas (403;404). As many activating mutations affecting a given residue have been found repeatedly over the past 20 years, it is likely that we are getting close to a saturation map for spontaneous gain of function mutations. Combined analyses of these natural mutants with extensive site-directed mutagenesis have identified key interactions implicated in activation of the serpentine portion of the TSH receptor (352;405).

Interaction between the Extracellular and Serpentine Domains of the Thyrotropin Receptor

The dichotomy between hormone binding (to the ectodomain) and activation of the G protein (by the serpentine domain) poses the question of how the activation signal travels intra-molecularly between the two domains after binding of TSH. Two important clues cast light on this issue. Firstly, experiments with aminoterminally truncated receptors demonstrated that the ectodomain exerts a negative effect on the serpentine domain (406;407). Indeed, constructs devoid of the ectodomain display increase signaling via Gαs leading to the notion that in pharmacological terms, the ectodomain behaves as an inverse agonist of the serpentine domain. When compared to wild type receptors fully stimulated by TSH, the activation state of aminoterminally truncated constructs is however far from maximal. Secondly, and more important, mutations of a single residue (Ser 281) present in a highly conserved motif of the ectodomain of glycoprotein hormone receptors (YP S HCCAF) ( Fig. 14 ), result in strong activation of the receptor (408). The segment containing this motif, sometimes referred to as the “ hinge ” region, plays an important role in activation of all three glycoprotein hormone receptors (409). The functional effect of substitutions of S281 in the TSH receptor likely results in a local “ loss-of-structure ” , because the more de-structuring the substitutions, the stronger the activation (410-412). The most active mutants cause increase in cellular cAMP similar to that achieved by saturating concentration of TSH on the wild type receptor (413).

Fig. 1-14. Model for activation of the thyrotropin (TSH) receptor by various agonists. Interactions between the extracellular domain and the serpentine domain are implicated in the activation mechanism. The TSH receptor is represented with its extracellular domain containing a concave, hormone-binding structure facing rightwards, and a transmembrane serpentine domain. The basal state of the receptor is characterized by an inhibitory interaction between the extracellular domain and the serpentine domain (indicated by the  (-) green sign). In the absence of agonist, the extracellular domain would function as a tethered inverse agonist of the serpentine domain. Binding of physiological agonists (TSH  , B; thyrostimulin  2  5, C), or stimulating autoantibodies (D) switches the ectodomain from inverse agonist to full agonist of the serpentine domain (indicated by the  (+) red sign). Mutation of the Ser in position 281of the ectodomain has the same effect (yellow dot, E). Other mutations may activate directly the serpentine domain by breaking silencing locks between transmembrane helices (the example of Asp619Gly is illustrated; blue dot, F). The serpentine domain may also be activated, by binding of low molecular chemical agonists directly to transmembrane segments (yellow star, G). The serpentine domain in the basal state is shown as a compact blue structure. The fully activated serpentine domain is shown as relaxed red structures with arrows indicating activation of G  s.

These results led to the following model for activation of the TSH receptor (Fig.14) (414;415). In the resting state, the extracellular domain would inhibit the activity of an inherently noisy rhodopsin-like serpentine domain. Upon activation, by binding of TSH, or secondary to mutation of S281, a structural module including the hinge region would switch from inverse agonist to full agonist of the serpentine domain. The ability of the strongest S281 mutants to activate the receptor fully in the absence of TSH suggests that the ultimate agonist of the serpentine domain would be the “ activated ” extracellular domain, with no need for a direct interaction between TSH and the serpentine domain. Several arguments support such a model: (i) mutations in the extracellular loops which strongly activate the intact receptor are unable to activate constructs devoid of ectodomain, while mutations affecting the transmembrane helices or the intracellular loops of the same construct are effective. This suggests that, in the intact receptor, the extracellular loops would be part of a module containing the “ hinge region ” involved in activation. (ii) a monoclonal antibody displaying inverse agonistic activity has been generated and its epitope has been localized within the hinge region (416). (iii) the TSH receptor can be activated by molecules, like autoantibodies (see Chapter 17) and thyrostimulin (417), sharing little if any structural homology with TSH. A parsimonious explanation is that these diverse agonists would bind to the ectodomain, switching it into an agonist of the serpentine domainwith no need for additional specific interaction with the serpentine.

Activation by Chorionic Gonadotropin

The sequence similarity between TSH and hCG, and between their receptors, allows for some degree of promiscuous activation of the TSH receptor by CG during the first trimester of pregnancy, when serum human CG concentrations are highest. The inverse relation between serum TSH and CG concentrations in most pregnant women is clear indication that their thyroid gland is stimulated by CG (418) (see Chapter 20 and Chapter 56). While most pregnant women are euthyroid, thyrotoxicosis may occur if CG production is excessive (as it occurs in twin pregnancies or chorionic tumors (see Chapter 20), or in rare women who have a mutant TSH receptor with increased sensitivity to CG (419).

Activation by Thyrotropin-Receptor Antibodies

The serum antibodies found in most patients with Graves' thyrotoxicosis and some patients with hypothyroidism caused by chronic autoimmune thyroiditis can stimulate or block the TSH receptor, respectively (see Chapter 17, Chapter 34). Epitopes recognized by TSAbs have been identified from precise mapping of binding site of murine or human monoclonal antibodies endowed with TSAb activity (420-422). However, the precise mechanisms implicated in activation of the receptor by TSAbs (and by TSH) are still unknown. Although most TSAbs do compete with TSH for binding to the receptor and despite similarity in interaction surfaces the precise targets of the hormone and autoantibodies are likely to be different, at least in part.. It has indeed be shown that the sulfated tyrosine residues, which are important for TSH binding (see above), are not implicated in recognition of TSH receptor by TSH receptor-stimulating antibodies (423). Also, most TSH receptor-stimulating antibodies stimulate cyclic AMP accumulation in cells transfected with TSH receptors more slowly than does TSH (424).

Activation by low molecular weight drug-like chemicals

High-throughput screening of low molecular weight chemical libraries identified specific TSH receptor agonists which were found to bind to the serpentine portion of the receptor (425). Oral administration of the agonist to mice stimulated thyroid, resulting in increased serum thyroxine and thyroidal radioiodide uptake (426). Apart from their interest to unravel the mechanism of activation of the receptor, these molecules constitute leads for development of drugs to use in place of recombinant human TSH, e.g. in patients with thyroid cancer.

Downregulation of the Thyrotropin Receptor

Desensitization of some G protein-coupled receptors involves phosphorylation of specific residues by G-protein receptor kinases (homologous desensitization) or protein kinase A (heterologous desensitization) (427). Acute desensitization of the receptor in the presence of TSH, presumably by phosphorylation, is weak and delayed(428). When compared with other G protein-coupled receptors, the TSH receptor contains few serine or threonine residues in its intracellular loops and intracellular carboxyl-terminal domain that can be phosphorylated, which probably accounts for the limited but definite desensitization and internalization observed in heterologous transfected cells after stimulation by TSH (429). Weak down-regulation, confounded by the long life of both TSHR mRNA and protein, does occur, but has little functional role (430). Chronic administration to mice invivo of stimulating monoclonal antibodies causes sustained hyperthyroidism, with no sign of desensitization (431). Using transgenic mice expressing a cAMP sensor in thyrocytes (432) showed recently that TSH receptors internalized after stimulation by TSH continue to signal to Gαs. This may provide an explanation to the persistence of thyrotoxicosis in patients with TSH-secreting pituitary adenomas and in patients with Graves ’ disease.

CONTROL OF THYROID FUNCTION

Thyroid Hormone Synthesis

Thyroid hormone synthesis requires the uptake of iodide by active transport, thyroglobulin biosynthesis, oxidation and binding of iodide to thyroglobulin, and within the matrix of this protein, oxidative coupling of two iodotyrosines into iodothyronines. All these steps are regulated by the cascades just described.

Iodide Transport

Iodide is actively transported by the iodide Na ⁺ /I ^– symporter (NIS) against an electrical gradient at the basal membrane of the thyrocyte and diffuses, following the electrical gradient, by a specialized channel (pendrin or another channel) (433;434) from the cell to the lumen at the apical membrane. The opposite fluxes of iodide, from the lumen to the cell and from the cell to the outside, are generally considered to be passive and nonspecific. At least five types of control have been demonstrated (319;320;433) .

1. Rapid and transient stimulation of iodide efflux by TSH in vivo, which might reflect a general increase in membrane permeability. The cascade involved is not known.

2. Rapid activation of iodide apical efflux from the cell to the lumen by TSH. This effect, which contributes to the concentration of iodide at the site of its oxidation, is mediated, depending on the species, by Ca ²⁺ and/or cAMP (294;433) . In human cells it is mainly controlled by Ca ²⁺ and therefore by the TSH effect on phospholipase C.

3. Delayed increase in the capacity (Vmax) of the active iodide transport NIS in response to TSH. This effect is inhibited by inhibitors of RNA and protein synthesis and is due to activation of iodide transporter gene expression. This effect of TSH is reproduced by cAMP analogues in vitro and is therefore mediated by the cAMP cascade (187). mRNA expression is enhanced by TSH and cAMP and decreased by iodide (333;435). TSH enhancement of thyroid blood flow, more or less delayed depending on the species, also contributes to increase the uptake of iodide (187). Iodine levels in the thyroid are also inversely related to blood flow (436).

4. Rapid inhibition by iodide of its own transport in vivo and in vitro. This inhibitory effect requires an intact transport and oxidation function, that is, it fulfills the criteria of an XI effect. After several hours the capacity of the active transport mechanism is greatly impaired (adaptation to the Wolff-Chaikoff effect) (320). The mechanism of the first effect is unknown but probably initially involves direct inhibition of the transport system itself (akin to the desensitization of a receptor), followed later by inhibition of NIS gene expression and NIS synthesis (akin to the downregulation of a receptor) (333).

5. Inhibition by iodide of thyroid blood flow. This effect may be direct as it takes place in patients treated with thyroperoxidase inhibitors and therefore does not fit the XI paradigm. By decreasing the iodide input it decreases the uptake.

Iodide Binding to Protein and Iodotyrosine Coupling

Iodide oxidation and binding to thyroglobulin and iodotyrosine coupling in iodothyronines are catalyzed by the same enzyme, thyroperoxidase, with H ₂ O ₂ used as a substrate (437). The same regulations apply to the two steps. H ₂ O ₂ is generated by a NADPH oxidase system of which two proteins DUOX (dual oxidases) or THOX have been identified by cloning (438;439) . The system is very efficient in the basal state inasmuch as little of the iodide trapped can be chased by perchlorate in vivo. Also, in vitro and in vivo the amount of iodine bound to proteins mainly depends on the iodide supply. Nevertheless, in human thyroid in vitro, stimulation of the iodination process takes place even at low concentrations of the anion, thus indicating that iodination is a secondary limiting step. Such stimulation is caused in all species by intracellular Ca ²⁺ and is therefore a consequence of activation of the Ca ²⁺ -PIP ₂ cascade. In many species, phorbol esters and diacylglycerol, presumably through protein kinase C, also enhance iodination (440). It is striking that in a species such as the human, in which TSH activates the PIP ₂ cascade, cAMP inhibits iodination, whereas in a species (dog) in which TSH activates only the cAMP cascade, cAMP enhances iodination. Obviously in the latter species a supplementary cAMP control was necessary (440-442).

Thyroperoxidase does not contain any obvious phosphorylation site in its intracellular tail. On the other hand, all the agents that activate iodination also activate H ₂ O ₂ generation, and inhibition of H ₂ O ₂ generation decreases iodination, which therefore suggests that iodination is an H ₂ O ₂ substrate – driven process and that it is mainly controlled by H ₂ O ₂ generation and iodide supply (440;443). Congruent with the relatively high K _m of thyroperoxidase for H ₂ O ₂ , H ₂ O ₂ is generated in disproportionate amounts with regard to the quantity of iodide oxidized. Negative control of iodination by iodide (the Wolff-Chaikoff effect) is accompanied and mostly explained by the inhibition of H ₂ O ₂ generation. This effect of I ^– is relieved by perchlorate and methimazole and thus pertains to the XI paradigm (325;440).

Iodotyrosine coupling to iodotyrosines is catalyzed by the same system and is therefore subject to the same regulations as iodination. However, coupling requires that suitable tyrosyl groups in thyroglobulin be iodinated, that is, that the level of iodination of the protein be sufficient. In the case of severe iodine deficiency or when thyroglobulin exceeds the iodine available, insufficient iodination of each thyroglobulin molecule will preclude iodothyronine formation whatever the activity of the H ₂ O ₂ generating system and thyroperoxidase. On the other hand, when the iodotyrosines involved in the coupling are present, coupling is controlled by the H ₂ O ₂ concentration but independent of iodide (437). In this case, H ₂ O ₂ control has a significance even at very low iodide concentrations.

H ₂ O ₂ generation requires the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) as a coenzyme and is thus accompanied by NADPH oxidation. Limitation of the activity of the pentose phosphate pathway by NADP ⁺ insures that NADPH oxidation for H ₂ O ₂ generation causes stimulation of this pathway. Also, excess H ₂ O ₂ leaking back into the thyrocyte is reduced by glutathione (GSH) peroxidase, and the oxidized GSH (GSSG) produced is reduced by NADPH-linked GSH reductase. Thus both the generation of H ₂ O ₂ and the disposal of excess H ₂ O ₂ by pulling NADP oxidation and the pentose pathway lead to activation of this pathway — historically one of the earliest and unexplained effects of TSH (187;440).

On the long-term, in vivo or in vitro, the activity of the whole iodination system obviously also depends on the level of its constitutive enzymes. In human thyrocytes H2O2 generation but not TPO is stimulated by direct activation of DUOX by the Gq-PLC-Ca ⁺⁺ cascade while TPO expression, but not DUOX is upregulated by the TSH-cAMP cascade (444). It is therefore not surprising that activation of thyrocytes by the cAMP cascade increases the corresponding gene expression whereas dedifferentiating treatments with EGF and phorbol esters inhibit this expression and thus reduce the capacity and activity of the system. Apparent discrepancies in the literature about the effects of phorbol esters on iodination are mostly explained by the kinetics of these effects (acute stimulation of the system, delayed inhibition of expression of the involved genes).

Thyroid Hormone Secretion

Secretion of thyroid hormone requires endocytosis of human thyroglobulin, its hydrolysis, and the release of thyroid hormones from the cell. Thyroglobulin can be ingested by the thyrocyte by three mechanisms (187;445-447) .

In macropinocytosis , pseudopods engulf clumps of thyroglobulin. In all species this process is triggered by acute activation of the cAMP/PKA cascade and therefore by TSH. Stimulation of macropinocytosis is preceded and accompanied by an enhancement of thyroglobulin exocytosis and thus of the membrane surface (443;448;449). In dog thyroid slices (450) and even (325) primary cultures, TSH and PKA activation acutely induces phagocytosis (451), which appears as the invitro manifestation of the macropinocytosis of thyroglobulin involved in stimulated thyroid hormone secretion. This process might be mediated by inactivation of the Rho family small G proteins), resulting in microfilament depolymerization and stress fiber disruption accompanied by dephosphorylation of cofilin (452) and myosin light chains (453) .

By micropinocytosis , the second process, small amounts of colloid fluid are ingested. This process does not appear to be greatly influenced by acute modulation of the regulatory cascades. It is enhanced in chronically stimulated thyroids and thyroid cells with induction of vesicle transport proteins Rab 5 and 7 (454) (455;456). It probably accounts for most of basal secretion.

A third (hypothesized) process is receptor-mediatedendocytosis ; it is enhanced in chronically stimulated thyroid cells (457-459). The protein involved could be megalin (460) or and asyaloglycoprotein. This process probably accounts for the transcytosis of low hormonegenic thyroglobulin (461) which is found in the serum .

Contrary to the last named, the first two processes are not specific for the protein. They can be distinguished by the fact that macropinocytosis is inhibited by microfilament and microtubule poisons and by lowering of the temperature (below 23°C) (187;462). Whatever its mechanism, endocytosis is followed by lysosomal digestion with complete hydrolysis of thyroglobulin. The main iodothyronine in thyroglobulin is thyroxine. However, during its secretion a small fraction is deiodinated by type I 5 and in man type II 5 -deiodinase to triiodothyronine (T ₃ ), thus increasing relative T ₃ (the active hormone) secretion (463).

The free thyroid hormones are released by the thyroid hormone transporter MC7 an unknown mechanism, which may be diffusion or transport. The iodotyrosines are deiodinated by specific deiodinases and their iodide recirculated in the thyroid iodide compartments. Under acute stimulation, a release (spillover) of amino acids and iodide from the thyroid is observed. A mechanism for lysosome retention of poorly iodinated thyroglobulin on N -acetylglucosamine receptors and recirculation to the lumen has been proposed. Under normal physiologic conditions, endocytosis is the limiting step of secretion, but after acute stimulation, hydrolysis might become limiting with the accumulation of colloid droplets. Secretion by macropinocytosis is triggered by activation of the cAMP cascade and inhibited by Ca ²⁺ at two levels: cAMP accumulation and cAMP action. It is also inhibited in some thyroids by protein kinase C downstream from cAMP. Thus the PIP ₂ cascade negatively controls macropinocytosis (308).

The thyroid also releases thyroglobulin. Inasmuch as this thyroglobulin was first demonstrated by its iodine, at least part of this thyroglobulin is iodinated; thus it must originate from the colloid lumen. Release is inhibited in vitro by various metabolic inhibitors and therefore corresponds to active secretion (448;464). The most plausible mechanism is transcytosis from the lumen to the thyrocyte lateral membranes (449). As for thyroid hormone, this secretion is enhanced by activation of the cAMP cascade and TSH and inhibited by Ca ²⁺ and protein kinase C activation. Because thyroglobulin secretion does not require its iodination, it reflects the activation state of the gland regardless of the efficiency of thyroid hormone synthesis. Thyroglobulin serum levels and their increase after TSH stimulation constitute a very useful index of the functional state of the gland when this synthesis is impaired, as in iodine deficiency, congenital defects in iodine metabolism, treatment with antithyroid drugs, and the like (465). Regulated thyroglobulin secretion should not be confused with the release of this protein from thyroid tumors, which corresponds in large part to exocytosis of newly synthesized thyroglobulin in the extracellular space rather than in the nonexistent or disrupted follicular lumen. In inflammation or after even mild trauma, opening of the follicles can cause unregulated leakage of lumen thyroglobulin.

Transcytosis or leakage from the lumen yields iodinated thyroglobulin while exocytotic thyroglobulin is not iodinated.

Functional Heterogeneity

It has long been known that at any given time the function of the thyroid follicles is not homogeneous. For instance, after injection of radioiodide, some follicles will incorporate important amounts of radioiodine while others will not incorporate at all. Similarly, after stimulation with TSH in in vivo thyroids or in in vitro incubated slices, some cells will develop pseudopods for macropinocytosis whithin 15 min while others submitted to the same stimulus will only respond after one to two hours (175;466).

In a more recent study, Gérard et al (467) (468) showed in human thyroids that while some follicles exhibit marked expression of pendrin, TPO and THOX, others did not. The expressing follicles were those containing iodinated thyroglobulin. They correspond to larger capillary networks and to the expression in the follicular cells of vascular regulators nitric oxide synthase and endothelin. This shows the existence of active and inactive angiofollicular units. It suggests that over time angiofollicular units cycle from active to inactive states and that this is controlled by the follicular cells. It would be interesting to know if the inactive state corresponds to a lower sensitivity to TSH.

CONTROL OF THYROID-SPECIFIC GENE EXPRESSION

The study of specific gene expression and proliferation at the biochemical and mechanistic level requires long term in vitro incubations, i.e. cell culture. It is therefore easy and tempting to rely on cell lines such as the rat thyroid FRTL-5, WRT and PCCl3 cells. However these cells, sometimes different from one lab to another, are different from each other and are very different from the cells in vivo, especially the human cells. Primary cultures are closer to the in vivo situation but they are difficult to obtain and not as reproducible. However, in monolayers, but not in reorganized follicles, follicular structure is fully and cell polarity and structure are partially lost. As most authors generalize the results of work done on their pet systems to "The Thyroid" the literature is very confusing and full of contradictions (469;470). Among the various possibilities demonstrated in such systems only those validated in vivo in transgenic animals and in human cells interest us.

A positive in vivo effect of TSH on general protein synthesis has been well documented. This effect is mimicked by cAMP agonists and is part of the trophic effect of TSH on the thyrocyte. It involves stimulation of transcription and translation; however, the detailed mechanisms implicated are not known. In cells in culture there is no such effect and the TSH cyclic AMP cascade has rather an inhibitory effect. Stimulation of protein synthesis and hypertrophy of the cell in culture results from IGF1 action on PI3 kinase (471). The in vivo effect of TSH might be mediated by IGF-1.

The so-called thyroid-specific genes encode proteins that are either found in the thyroid exclusively, like thyroglobulin and thyroperoxidase, or that, although being also found in a few additional tissues, are primarily involved in thyroid function, like TSH receptor and sodium/iodide symporter. The transcription of these genes in the thyroid appears to rely on the coordinated action of a master set of transcription factors that includes at least the homeodomain protein TTF-1 (also known as Nkx 2.1 or T/ebp or Titf1), the paired-domain protein Pax 8, and perhaps also the forkhead-domain protein TTF-2 (also known as FoxE1) (472;473). Loss of function mutant mice for TTF-1, Pax 8 or TTF-2 have been generated and allowed to identify a crucial role for these transcription factors in the development of the thyroid also. However, as none of these animals develop a normal mature thyroid, they could not be used to investigate the exact role of these key factors in the control of gene expression in the mature thyroid. A conditional loss of function mutant mouse for TTF-1 has also been generated (368). Only partial inactivation of TTF-1 could be achieved in this animal which precluded the analysis of TTF-1 role in the developed thyroid at the molecular level. Most of the work concerning this last aspect has been conducted either in primary cultures of thyrocytes (474) or in immortalized thyroid cell lines like FRTL-5 and PCCl3 (475). Although the data gathered to date agree on most basic aspects, significant differences have sometimes been observed between primary versus immortalized cell models (476). Part of these discrepancies may result from the existence of occasional species-specific differences (477).

The main regulator of thyroid function, the TSH signal, which is predominantly conveyed inside the cell by cAMP and PKA, upregulates the expression of transcription factor Pax 8, both in primary cells (478) and established cell lines (479). However, mice genetically deprived of TSH or of functional TSH receptor do not show reduced amounts of Pax 8 in their thyroids as compared to wild type animals (480) suggesting that compensatory mechanisms may ensure an adequate production of this factor when thyroid development takes place in the absence of the normal physiological stimulus. Besides this control on the amount of Pax 8 protein, there is no firm evidence that TSH, or cAMP, exerts any other control at the level of the master thyroid transcription factors identified presently (476;481;482). The expression of several other transcription factors was shown to be upregulated, often at least transiently, in response to TSH/cAMP in the thyroid, namely c-myc (483) , c-fos (483), fos B, jun B, jun D (484), CREM (485) , NGFI-B (486) and CHOP(487), for example. An hypothetical role in the control exerted by TSH/cAMP on the expression of the thyroid-specific genes has been proposed for some of these factors (488;489), but no final link has been established yet (490). A recent report proposes that the dopamine and cAMP-regulated neuronal phosphoprotein DARPP-32 could play an essential role in this control (382).

It is noteworthy that in addition to its control on the transcription of the individual thyroid-specific genes, which is detailed below, TSH also regulates gene expression by acting at some post-transcriptional steps, as shown in the case of thyroglobulin (491). Finally, many effects of TSH and cAMP on gene expression (including on thyroid-specific genes such as thyroglobulin) might be rather indirect and depend in part on the profound modifications of cell morphology and cytoskeleton that result from PKA activation (245;492).

TGF-β has been shown to downregulate the expression of thyroid-specific genes (493;494). It seems to involve a reduction in the level of Pax 8 activity that is mediated by Smad proteins (495;496). In human thyroid primary cultures, TGF-β inhibits most effects of cAMP on gene expression (252). As above, this might be related in part to an inhibition of morphological effects of TSH/cAMP. In all the species tested so far, EGF strongly represses thyroglobulin and thyroperoxidase gene expression as well as iodide transport (245;430;497-499). FGF has a similar action in some species including bovine (500). The mechanisms have not been explored. The apparent dedifferentiation induced by EGF in dog thyrocytes is associated with an enhanced vimentin expression and a progressive induction of a fusiform fibroblast-like morphology, which is suggestive of an epithelial-mesenchymal transition (501) (502). This process is reversible after elimination of EGF and re-addition of TSH. As recently quantified by SAGE analysis in the thyroid cell line PCCl3, exposure to a high dose of iodide also decreases the expression of most of the thyroid-specific genes within the thyrocyte (389).

Thyroglobulin

The regulatory DNA elements of the thyroglobulin gene have been characterized in several species (472;477;503). The proximal promoter, as defined in transfection experiments, extends over 200 base-pairs and contains binding sites for transcription factors TTF-1, TTF-2 and Pax 8 (see Fig. 1-15). An upstream enhancer element containing binding sites for TTF-1 has been identified in beef and man (504). In the latter, the enhancer region is longer and harbors additional binding sites for TTF-1 and cAMP responsive element binding (CREB) protein (505). Both TTF-1 and Pax 8 proteins were individually shown to exert a major control on thyroglobulin gene transcription (506;507). By contrast, TTF-2 activity appears to be dispensable as the thyroglobulin gene is expressed in cells devoid of TTF-2 protein (508). Synergism in the transcriptional activation of the gene by TTF-1 and Pax 8 appears to rely on a direct interaction between these two factors (509), and on their coordinated action involving both the enhancer and proximal promoter sequences (510). Transactivation of the thyroglobulin promoter by TTF-1 and Pax 8 has been reported to be enhanced by the coactivator TAZ in vitro (396). But the overexpresion of TAZ observed in papillary thyroid carcinoma is not associated with an increased expression of the thyroglobulin gene (397). The coactivator p300 has also been independently reported to be involved in this transactivation mechanism (398). On the other side, poly(ADP-ribose) polymerase-1 was reported to counteract the transactivation of the thyroglobulin promoter by Pax 8 through a direct interaction with this factor impairing its DNA-binding activity (399). Recently, the osteoblast-specific transcription factor Runx2 (also known as Cbfa1 or AML3) was shown to be expressed in the thyroid and to control thyroglobulin gene expression by direct binding to the thyroglobulin proximal promoter region (see Fig. 1-15). Runx2 deficiency in mice causes a marked reduction in thyroglobulin gene expression leading to hypothyroidism (400). Again however, Runx2 overexpression in papillary thyroid cancers is not associated with an increased expression of the thyroglobulin gene (401).

Fig. 1-15. Schematic of the known regulatory elements of thyroglobulin, thyroperoxidase, sodium/iodide symporter and TSH receptor genes. The organization of the proximal promoter and upstream enhancer elements of the different genes is depicted as determined in the species studied so far. Coordinates of the proximal promoters are in base pairs and refer to the transcription start site as +1. The positions of the upstream enhancer elements relative to the transcription start site are not indicated as they vary extensively among the different species.

The known thyroglobulin gene regulatory elements were shown to be sufficient to drive the thyroid-restricted expression of a linked gene in living mice (511). This thyroid-restricted expression likely results from the requirement for the simultaneous presence of both TTF-1 and Pax 8, which occurs in thyroid only. It is associated with the tissue-specific demethylation of thyroglobulin gene sequences (512). Demethylation of the DNA is supposed to relieve the constitutive silencing of the gene (513).

Thyroglobulin gene transcription has been shown to require the presence of circulating TSH in the adult rat (514) and to be highly dependent on an elevated cAMP level in dog thyroid tissue slices, in primary cultured cells (515), and, to a much lower extent, in immortalized thyroid cell lines like FRTL-5 (516). Although they are devoid of classical cAMP-responsive element (CRE), the proximal promoter sequences are essentially involved in this control, as indicated by the observation of TSH/cAMP-induced changes in their chromatin structure (517) and their TSH/cAMP-dependent activity in transfection experiments (518). It has however been demonstrated recently that the onset of thyroglobulin gene expression during thyroid development takes place normally in mouse strains deprived of either circulating TSH or functional TSH receptors (519;520). This may be consistent with the observation that the thyroglobulin gene displays a low level of cAMP-independent transcription in primary cultured thyrocytes (515), which might depend on insulin, as observed in different culture models (521-523). In primary cultures of dog thyrocytes, the transcriptional activation of the thyroglobulin gene by cAMP after transient TSH withdrawal is also delayed as compared to that of the thyroperoxidase gene (515), and, unlike thyroperoxidase gene expression, it requires an active protein synthesis (515). The increase in Pax 8 concentration consecutive to TSH/cAMP stimulation of the thyrocyte is not sufficient to account for the observed control on thyroglobulin gene transcription, as TSH is still required for transcriptional activation even in cells expressing high levels of Pax 8 protein (524). Thus, besides TTF-1 and Pax 8, at least one additional, still unidentified, factor is likely to play a key role in the control of thyroglobulin gene expression, as also suggested by the observation that, in the course of thyroid development, both TTF-1 and Pax 8 are present well before thyroglobulin gene is expressed.

In addition to the full length thyroglobulin mRNA, a shorter transcript accumulates in the rat thyroid in response to TSH stimulation (525). This transcript results from differential splicing and polyadenylation of the primary transcript, and encodes a protein limited to the very N-terminal part of thyroglobulin. As this truncated protein still contains a major hormonogenic site(526), it could suggest that, in conditions in which the balance of thyroid metabolism would favor hormone synthesis over iodine storage (e.g., shortage of iodine), the rat thyrocyte would manufacture a shorter thyroglobulin with a preserved hormonogenic ability but lacking many of the nonhormonogenic tyrosines.

Thyroperoxidase

In the species studied so far, the architecture of the proximal promoter region of the thyroperoxidase gene strikingly resembles that of the corresponding region of the thyroglobulin gene (527;528) (see Fig. 1-15 ). The upstream enhancer element also encompasses a pair of TTF-1 binding sites and contains an additional binding site for Pax 8, as compared to its counterpart in the thyroglobulin gene (529;530). Here again, the combination of the upstream enhancer and proximal promoter supports the synergistic action of TTF-1 and Pax 8 on gene transcription (531). The transcriptional co-activator p300 has also been reported to be involved in the activation of this promoter (413).

Despite the existence of this high similarity, thyroperoxidase gene transcription is more tightly and more rapidly controled by TSH and cAMP than that of the thyroglobulin gene in primary cultured thyrocytes, and does not require a new protein synthesis (515;532). Contrary to the thyroglobulin gene also, the thyroperoxidase gene is not expressed in the absence of circulating TSH or functional TSH receptors in intact animals (533). On the other hand, the constitutive hyperactivation of the cAMP cascade leads to an increased expression of the gene as compared to the normal situation (534). In spite of their lack of a classical CRE, the proximal promoter sequences have been shown to mediate this TSH/cAMP control on transcription in transfection experiments (535). Exposure to a high dose of iodide reduces thyroperoxidase gene expression as well as that of thyroglobulin, sodium/iodide symporter and thyrotropin receptor genes in PCCl3 thyroid cells (389). Low doses of iodide also decrease thyroperoxidase gene expression in vivo, while the expression of thyroglobulin remains unaffected (333). Thus, apart from their basic dependence on the presence of the transcription factors TTF-1 and Pax 8, which insures their shared thyroid-restricted expression, the thyroperoxidase and thyroglobulin genes distinguish themselves significantly regarding the control of their transcription. It is worth mentioning in this context that a synergistic action of Pax8 and pRb, the retinoblastoma protein, appears to be required for thyroperoxidase promoter activation, whereas this is not the case for thyroglobulin promoter activation (415). It has been postulated recently that the hormone-induced developmental activation of the thyroperoxidase gene involves the concerted action of TTF-2 and NF-1, both of which bind neighbouring sequences in the gene promoter (see figure 1-15) resulting in the initial opening of the chromatin structure of the promoter (416).

The existence of a major thyroperoxidase mRNA isoform has been detected in man (536). It appears to encode a protein devoid of its normal enzymatic activity.

Sodium/iodide Symporter

Although the sodium/iodide symporter plays a key role in thyroid hormonogenesis, the expression of the corresponding gene is not restricted to the thyroid. Accordingly, the proximal promoter sequences identified so far do not exhibit a thyroid-specific activity in vitro (537;538), even if this activity may be marginally increased in the presence of TTF-1 (539). The robust and appropriately controlled expression of this gene in the thyroid seems to be mediated essentially by the upstream enhancer element which contains binding sites for both TTF-1 and Pax 8, and a cAMP responsive element (CRE)-like DNA motif which is involved in the control by TSH/cAMP (540;541) (see Fig. 1-15). The cAMP-response element modulator (CREM) has recently been proposed to be involved in this control (422). The Ras oncoprotein was also shown to reduce sodium/iodide symporter gene expression by targeting Protein Kinase A-dependent Pax 8 transcriptional activity (423). As for the thyroperoxidase gene, TSH signaling is indispensable for sodium/iodide symporter gene transcriptional activation in vivo (542;543), and iodide downregulates the expression of the gene (333). In addition, synergy between Pax8 and pRb appears to be required for the activation of both thyroperoxidase and sodium/iodide symporter promoters (415). A very similar control is thus exerted on the expression of both of these genes in the thyroid in spite of the fact that, overal, the known regulatory regions of the thyroperoxidase gene bear more resemblances to those of the thyroglobulin gene than to those of the sodium/iodide symporter gene. It has been reported recently that the bacterial endotoxin lipopolysaccharide enhances sodium/iodide symporter gene transcription through the direct binding of Nuclear Factor-κB (NF-κB) to the upstream enhancer element and its interaction with adjacently bound Pax 8 factor (423) (see Fig. 1-15). The basic Helix-Loop-Helix transcription factor Hairy/enhancer of split 1 (Hes 1), which is part of the Notch signalling pathway, has also been shown to control sodium/iodide symporter gene expression, as well as that of the thyroperoxidase gene, although no binding sites for this factor have been identified within these promoters as yet. Hes 1 appears to be required in mice for developing a functional thyroid and its decreased expression in thyroid tumors parallels the reduction of thyroid differentiation markers expression observed in these cells (424;425).

Thyropin Receptor

Like the gene described above, the TSH receptor gene is also expressed in tissues other than the thyroid. Again, the promoter elements identified presently, which include binding sites for thyroid hormone receptor (TR)-α1/retinoid-X receptor (RXR) heterodimer (544), GA-binding protein (GABP) (545), cAMP responsive element binding (CREB) protein(546)and TTF-1 (547) (see Fig. 1-15), do not display a clear thyroid-specific activity in transfection experiments, as could be expected. Contrary to the promoters described so far, the promoter of the TSH receptor gene does not contain a TATA-box motif, but encompasses a GC-rich region preceding the multiple neighbouring transcription start sites. Consistent with the presence of TTF-1 binding sites in the promoter region, the TSH receptor genes exhibits a decreased activity in animals expressing reduced level of TTF-1 (548). No other regulatory DNA element specifically involved in the thyroid-specific expression of this gene has been identified as yet. On the other hand, DNA demethylation events in the promoter region have been observed in thyroid cells expressing the TSH receptor gene, as compared to non-expressing cells (549).

The control exerted on the expression of the TSH receptor gene in the thyroid seems to be more complex than the ones described previously. Discordant effect of TSH/cAMP on the expression of this gene have been reported depending on the nature of the experimental system used (550). The presence in the promoter region of a CRE-like DNA motif which appears to be able to bind the CREB protein(551), a transcriptional activator directly activated by cAMP, as well as the CREM isoform ICER(552), a transcriptional repressor induced by cAMP, could explain both reported increase and decrease in gene expression following TSH stimulation, depending on the relative amounts of these factors (and likely of other CRE-binding proteins also) preexisting in the studied cells and the kinetics of the individual observations. Moreover, the binding site of the TRα1/RXR heterodimer identified in this promoter encompasses the CRE-like motif (see figure 1-15), which may add a further level of complexity depending on the availability of thyroid hormone in the experimental system.

On the other hand, the possible existence of species-specific differences could also account for the occurrence of seemingly discrepant reports (430). The accumulation of TSH receptor mRNA requires an active protein synthesis in primary cultured dog thyrocytes (430), which is reminiscent of what was observed for the thyroglobulin gene (see above). Considering the facts that, after the TSH receptor gene, the thyroglobulin gene is the most affected in its expression by a reduced TTF-1 availability as compared to the other known thyroid-specific genes(553), and that, alike the TSH receptor gene itself, the thyroglobulin gene is activated independently of the TSH/TSHR signaling during thyroid development(554;555), it suggests that these two genes may share at least partially similar control mechanisms. Recently, thyrotropin receptor mRNA was also shown to be decreased in PCCl3 cells exposed to a high iodide concentration (556).

Control of TSH receptor gene expression has been studied in the FRTL5 cell line (550;557;558), the canine thyrocyte in primary culture (430), cultured human thyrocytes (559;560), and human thyroid cancer (471;561) . The general conclusion emerging from these studies is the extreme robustness of TSH receptor gene expression as compared with the other markers of thyroid cell differentiation (thyroglobulin and thyroperoxidase). In the dog, levels of TSH receptor mRNA remain virtually unchanged in animals subjected for 28 days to hyperstimulation by TSH secondary to treatment with methimazole or to TSH withdrawal achieved by administration of thyroxine (430). In the same study, the effect of TSH or forskolin has been investigated in dog thyrocytes in primary culture. This experimental system has the advantage that the differentiation state of the cells can be manipulated at will: cAMP agonists maintain expression of the differentiated phenotype, whereas agents such as EGF, tetradecanoyl phorbol acetate (TPA), and serum lead to "dedifferentiation" (498). The results demonstrate that the dedifferentiating agents reduce accumulation of the receptor mRNA. However, contrary to what is observed with thyroglobulin and thyroperoxidase mRNA, the inhibition is never complete. TSH or forskolin is capable of promoting reaccumulation of the receptor mRNA, a maximum being reached after 20 hours. As with thyroglobulin but at variance with the thyroperoxidase gene, this stimulation requires ongoing protein synthesis (430). Chronic stimulation of cultured dog thyrocytes by TSH for several days does not lead to any important downregulation in mRNA. Similar data have been obtained with human thyrocytes in primary culture (430;560) . By contrast, n egative regulation of receptor mRNA accumulation has been observed in immortal FRTL5 cells after treatment with TSH or TSAB (550;558). This difference versus human and canine cells must probably be interpreted in the general framework of the other known differences in phenotype and regulatory behavior of this cell line as compared with primary cultured thyrocytes (see below) (562) .

The effect of malignant transformation on the amounts of TSH receptor mRNA has been studied in spontaneous tumors in humans (471;561), in a murine transgenic model of thyroid tumor promoted by expression of the simian virus-40 large T oncogene(563), and in FRTL5 cells transformed with v- ras (557). In the two last models, expression of the TSH receptor gene was suppressed: the tumor or cell growth became TSH independent. In the transgenic animal model, loss of TSH receptor mRNA seemed to take place gradually, with early tumors still displaying some TSH dependence for growth. In the human tumors a spectrum of phenotypes was observed. As expected, anaplastic tumors had completely lost the receptor mRNA, as well as other markers of thyrocyte differentiation (thyroglobulin and thyroperoxidase). In papillary carcinoma, variable amounts of TSH receptor mRNA were invariably found (561), even in the tumors that had lost the capacity to express the thyroglobulin or thyroperoxidase genes (561). These data agree well with the observations of thyrocytes in primary culture: expression of the TSH receptor gene is robust and it persists in the presence of agents (or after several steps in tumor progression) that promote extinction of the other markers of thyroid cell differentiation. This evidence leads to the conclusion that the basic marker of the thyroid phenotype is probably the TSH receptor itself, which makes sense: the gene encoding the sensor of TSH — the major regulator of thyroid function, growth, and differentiated phenotype — is virtually constitutive in thyrocytes. From a pragmatic viewpoint, these data provide a rationale for the common therapeutic practice of suppressing TSH secretion in patients with a differentiated thyroid tumor (564).

Thyroid oxidases

Two distinct genes, ThOX1 and ThOX2 (also known as DUOX-1 and -2), both significantly related to the gene encoding the phagocyte NADPH oxidase gp91 ^Phox , are expressed in the thyroid essentially but not only (565;566). In the dog, ThOX mRNAs accumulate in response to TSH/cAMP stimulation (567). This effect is much less apparent in man (568), and in the rat conflicting results were obtained in vivo and in the established FRTL-5 cell line respectively (569). Two other genes encoding proteins required for the maturation and function of the thyroid oxidases were identified in the close vicinity of both ThOX/DUOX genes, and named DUOXA-1 and -2 respectively (570). Proper hydrogen peroxide production requires that each DUOXA protein associates with the corresponding DUOX protein (i.e. DUOXA1 with DUOX1 and DUOXA2 with DUOX2) at the cell membrane (571). In both ThOX-DUOXA pairs, the genes encoding the ThOX and DUOXA partners are arranged in a head to head configuration, the tiny DNA region separating the two transcription starts acting as a bidirectional promoter (439). Noteworthy, ThOX1+DUOXA-1 and ThOX2+DUOXA2 promoters exhibit totally unrelated architectures, the former displaying all features of a CpG-rich island, the second containing canonical TATA-box and Initiator elements (see Fig. 1-16 ). This fundamental difference in promoter organization suggests that both genes pairs are subjected to distinct transcriptional controls. In transient transfection experiment, both cloned ThOX-1 and -2 promoters do not show thyroid-cell restricted activity, do not respond to TSH or cAMP (440), and do not appear to depend on transactivation by either TTF-1 or Pax 8 (441), at least in the transcriptional direction investigated in these studies. However, an independent study identified the endogenous ThOX-2 promoter as a target for either Pax8 or TTF-1 (442). A likely explanation for this discrepancy is that this transactivation involves regulatory sequences other than the ones identified and cloned so far (440;441).

Fig. 1-16. Organization of the bidirectional promoters of ThOX and DUOXA genes as determined in the rat (439). The transcription starts are symbolized by arrows and the distances in base pairs separating them are indicated in both cases.

Other thyroid-specific genes

Recently, a few other genes have been found to be highly expressed in the thyroid and/or to play an important role in this tissue, and have been added to the list of the previously known thyroid-specific genes. Notably, the gene encoding Pendrin, a protein at least partly involved in the apical export of iodide ions, has been shown to be under the control of TTF-1 (443). The Tensin 3 gene has been reported to present a particularly high expression level in the thyroid as compared to other tissues, and to exhibit decreased levels of expression in thyroid tumors, but no data are available as yet regarding the molecular mechanisms involved (444). Serial analysis of gene expression (SAGE) aiming at the identification of genes preferentially expressed in the thyroid led to the isolation of C16orf89 encoding a protein of presently unknown function. The expression of C16orf89 is stimulated by TSH and parallels that of the sodium-iodide symporter during thyroid development (445). Genetic analysis of the predisposition to hypothyroidism in mice containing only one intact allele encoding TTF-1 and Pax 8 identified Dnajc17 as a gene highly expressed in the thyroid and playing an essential role in thyroid development. This gene encodes a protein belonging to the type III heat-shock protein-40 family (446).

CONTROL OF GROWTH AND DIFFERENTIATION

Thyroid Cell Turnover The thyroid is composed of thyrocytes (70%), endothelial cells (20%), and fibroblasts (10%) (proportions measured in dog thyroid) (173). Human thyroids: 80% follicular cells for 20% stromal endothelial cells and fibroblasts 80% (572;572). In a normal adult the weight and composition of the tissue remain relatively constant. Because a low but significant proliferation is demonstrated in all types of cells, it must be assumed that the generation of new cells is balanced by a corresponding rate of cell death (215;572;573) . The resulting turnover is on the order of one per 5 to 10 years for human thyrocytes, that is, six to eight renewals in adult life, as in other species. In one child the turnover was 2 per year (573) . Normal cell population can therefore be modulated mainly at the level of proliferation but also secondarily of cell death. In growth situations, that is, either in normal development or after stimulation, the different cell types grow more or less in parallel, which implies coordination between them (248;574-576). Because TSH receptors and iodine metabolism and signaling coexist only in the thyrocyte in thyroid, this cell, sole receiver of the physiologic information, must presumably control the other types of cells by paracrine factors such as FGF, IGF-I, NO, and the like (577). The successful isolation of human thyroid endothelial cells will allow a more detailed study of these interactions (578) . TSH has been demonstrated to upregulate the production of vascular endothelial cell growth factor (VEGF) by human thyrocytes (579) . It is interesting in this regard that the vascular support of the follicles reflects their activity suggesting the concept of angiofollicular units (580;581) .

The Mitogenic Cascades

The study of the control of thyroid cell proliferation has been much confused by the unwarranted extrapolation of data obtained in different model systems, including different rat thyroid cell lines at different stages in their evolution, to the human thyroid. Sentences like “ Agent X stimulates pathway Y in PCCl3 cells, thus human thyroids could ne treated by agent Z which inhibits agent X action ” are not acceptable even if only implied (582).

In the thyroid at least three families of distinct mitogenic pathways have been well defined (Fig. 1-17): (1) the hormone receptor – Gs-adenylyl cyclase – cAMP-dependent protein kinase system, (2) the hormone receptor – tyrosine protein kinase pathways, and (3) the hormone receptor – Gq-phospholipase C cascade (215;583) . The thyroid also autoregulates its size by an unknown mechanism. Thyroxin treated dogs, and humans, compensate the loss of one thyroid lobe independently of TSH (333).

The receptor – tyrosine kinase pathway may be subdivided into two branches; some growth factors, such as EGF, induce proliferation and repress differentiation expression, whereas others, such as FGF in dog cells or IGF-I and insulin, are either mitogenic or are necessary for the proliferation effect of other factors without being mitogenic by themselves, but they do not inhibit differentiation expression (521;584) . In human thyroid cells, IGF-I is required for the mitogenic action of TSH or EGF but by itself it only weakly stimulates proliferation (304). In dog and human thyrocytes in primary cultures, after induction of insulin receptors by TSH, physiological concentrations of insulin permit the proliferative action of TSH (233;585) . In PCCl3 and rat cells, and in mouse thyroid in vivo (586), IGF-I is weakly mitogenic per se (587), whereas in pig thyroid cells it produces a strong effect (588).

Fig 1-17a. Mitogenic pathways in the thyroid. Data from the thyroid cell systems are integrated into the present general scheme of cell proliferation cascades. In the first line, known activators of various cascades in dog and human thyroid cells are shown. Various levels indicate a time sequence and postulated causal relationships from initial interaction of extracellular signal with its receptor to endpoints: proliferation and differentiation expression. In dog but not in human thyroid cells, acetylcholine through muscarinic receptors activates the phospholipase C cascade. PCNA, proliferating cell nuclear antigen; cAPK, cyclic adenosine monophosphate-dependent kinase; CDK, cyclin-dependent kinase; DAG, diacylglycerol,  + , stimulation;  + , inhibition; ;  ♦ + , induction; GFR, growth factor receptor; ODC, ornithine decarboxylase; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PLC, phospholipase C; RSK, ribosomal S6 kinase.

It should be noted that TSH directly stimulates proliferation while maintaining the expression of differentiation. Differentiation expression, as evaluated by NIS or by thyroperoxidase and thyroglobulin mRNA content or nuclear transcription, is induced by TSH, forskolin, cholera toxin, and cAMP analogues (215). These effects are obtained in all the cells of a culture, as shown by in situ hybridization experiments (521) . They are reversible; they can be obtained either after the arrest of proliferation or during the cell division cycle (499;521) . Moreover, the expression of differentiation, as measured by iodide transport, is stimulated by concentrations of TSH lower than those required for proliferation (304).

All the proliferation effects of TSH are mimicked by nonspecific modulators of the cAMP cascade, that is, cholera toxin and forskolin (which stimulate adenylate cyclase), cAMP analogues (which activate the cAMP-dependent protein kinases), and even synergistic pairs of cAMP analogues acting on the different sites of these two kinases (288;304;589) . They are reproduced in vitro and in vivo by expression of the adenosine A ₂ receptor, which is constitutively activated by endogenous adenosine (534), and by constitutively active Gsα (590) and cholera toxin (591) . They are inhibited by antibodies blocking G _s (592) . Inhibition of cAMP-dependent protein kinases (PKA) inhibits the proliferation and differentiation effects of cAMP (593;594). Moreover, stimulation of PKA by selective cAMP analogs that do not activate EPAC proteins is sufficient to fully mimic mitogenic effects of TSH and forskolin in dog thyrocytes (593). There is, therefore, no doubt that the mitogenic and differentiating effects of TSH are mainly and probably entirely mediated by cAMP-dependent protein kinases. A complementary role of the Rap guanyl nucleotide exchange factor EPAC and of Rap has been proposed in rat thyroid cell lines (595;596) but not observed in canine thyroid primary cultures (593).

EGF also induces proliferation of thyroid cells from various species (215;304;597) . However, the action of EGF is accompanied by a general and reversible loss of differentiation expression assessed as described above (498). The effects of EGF on differentiation can be dissociated from its proliferative action. Indeed, they are obtained in cells that do not proliferate in the absence of insulin and in human cells, in which the proliferative effects are weaker, or in pig cells at concentrations lower than the mitogenic concentrations (215).

Finally, the tumor-promoting phorbol esters, the pharmacologic probes of the protein kinase C system, and analogues of diacylglycerol also enhance the proliferation and inhibit the differentiation of thyroid cells. These effects are transient because of desensitization of the system by protein kinase C inactivation.

Activation of the Gq/phospholipase C (PLC)/PKC cascade by a more physiological agent such as carbamylcholine in dog thyroid cells does not reproduce all the effects of phorbol esters. In particular, prolonged stimulation of this cascade by carbamyl choline permits the cAMP-dependent mitogenesis of dog thyrocytes (598), but unlike phorbol esters it does not induce proliferation in the presence of insulin (599) . The Ras protooncogene is strongly activated by phorbol esters but more weakly by carbachol (317). Thus we cannot necessarily equate the effects of phorbol esters and prolonged stimulation of the PLC cascade. The dedifferentiating effects of phorbol esters do not require their mitogenic action either. Thus the effects of TSH, EGF, and phorbol esters on differentiation expression are largely independent of their mitogenic action (215).

In several thyroid cell models, very high insulin concentrations are necessary for growth even in the presence of EGF. We now know that this prerequisite mainly reflects a requirement for IGF-I receptor (215;302;587;600) . It is interesting that in FRTL5 cells, as in cells from thyroid nodules, this requirement may disappear, probably because the cells secrete their own somatomedins and thus become autonomous with regard to these growth factors (302;601) . By contrast, in primary cultures of normal dog and human thyrocytes, very low concentrations of insulin, acting on insulin receptors, are sufficient to support the mitogenic effects of TSH and cAMP when insulin receptors have been induced to high levels by TSH (233;602) . This puzzling regulation, which is reminiscent of the induction of insulin receptors during the differentiation of adipocytes, suggests that thyroid might well be revealed as a more specific target of circulating insulin than hitherto recognized.

In the action of growth factors on receptor protein tyrosine kinase pathways, the effects on differentiation expression vary with the species and the factor involved: from stimulation (e.g., insulin, as well as IGF-II in dog and FRTL5 cells) (603) to an absence of effect (604), to transitory inhibition of differentiation during growth (FGF and HGF in dog cells (605;606) to full but reversible dedifferentiation effects (EGF in dog and human cells) (498;607) . Ret/PTC rearrangements, activating mutations of Ras, as well as oncogenic mutation of B-Raf, which are responsible of most differentiated carcinoma, constitutively activate the signaling cascades of growth factors (608-610) .

The kinetics of the induction of thymidine incorporation into nuclear DNA of dog thyroid cells is similar for TSH, forskolin, EGF, and TPA. Whatever the stimulant, a minimal delay of about 16 to 20 hours takes place before the beginning of labeling, that is, the beginning of DNA synthesis (611). This time is the minimal amount required to prepare the necessary machinery. For the cAMP and EGF pathways, the stimulatory agent has to be present during this whole prereplicative period; any interruption in activation (e.g., by washing out the stimulatory forskolin) greatly delays the start of DNA synthesis (612). This limitation explains why norepinephrine and prostaglandin E, which also activate the cAMP cascade, do not induce growth and differentiation: the rapid desensitization of their receptors does not allow a sustained rise in cAMP levels.

The three main types of mitogenic cascade, specifically, the growth factor – protein tyrosine kinase, phorbol ester – protein kinase C, and TSH-cAMP cascades, are fully distinct at the level of their primary intracellular signal and/or the first signal-activated protein kinase (215).

Iodide actually inhibits the cAMP and the Ca ²⁺ -phosphatidylinositol cascades and in a more delayed and chronic effect decreases the sensitivity of the thyroid to the TSH growth response. These effects are relieved, according to the general paradigm of Van Sande, by perchlorate and methimazole (325;613).

Steps in the Mitogenic Cascades (Fig. 1-17)

The phenomenology of EGF, TPA, and TSH proliferative action cells has been partially elucidated using dog thyroid primary cultures (215;474;614). The mechanisms of TSH/cAMP mitogenic effects have also been investigated using immortal rat thyroid cell lines (FRTL-5, WRT and PC Cl3 cells) (313). Whereas the signaling cascades involved in the action of growth factors and IGF-I are likely to be well conserved in the different thyroid systems, as generally observed in the other cell types, the mechanistic logics of cell cycle regulation by cAMP has disappointingly turned out to strongly diverge in the various thyroid in vitro models (615). These divergences do not only reflect species differences (215). Among the apparently similar rat thyroid cell lines, or even among different subclones of FRTL-5 cells, major differences have been observed (616). For instance, the PI3 kinase/PKB cascade is activated by cAMP in WRT cells (617), but inhibited by cAMP in PC Cl3 cells (618) . The induction of c-jun by TSH/cAMP in FRTL-5 cells and its repression by cAMP in WRT cells (619) as in dog (620) and human thyrocytes likely reflect major differences in upstream signaling cascades, and should result in divergent expression of downstream target genes, such as cyclin D1. Cyclin D1 synthesis, an accepted endpoint of mitogenic cascades, is indeed induced by cAMP in FRTL-5 and PC Cl3 cells, but rather repressed by cAMP in dog and human thyroid primary cultures (621;622) . The reasons for such discrepancies are unclear. Some signaling features, when they lead to selective proliferative advantages, might have been acquired during the establishment and continuous cultures of the cell lines and stabilized by subcloning. Many mechanisms demonstrated in the dog thyroid primary culture system so far apply to normal human thyrocytes (623), but much remains to be defined (624) . In the following lines, we thus rely mostly on these systems.

Three biochemical aspects of the proliferative response occurring at different times of the prereplicative phase have been considered. The pattern of protein phosphorylation induced within minutes by TSH is reproduced by forskolin and cAMP analogues. It totally diverges from the phosphorylations induced by EGF and phorbol esters (625). EGF, HGF and phorbol ester actions rapidly converge on the activation of Ras (317) and the resulting activation of p42/p44 MAP kinases and p90 ^RSK (316;626;627) . PI-3-kinase and its effector enzyme PKB are activated for several hours only by insulin and IGF-I, the effect of EGF being short lived (628) . This activity is therefore the one specific feature of insulin action and presumably the mechanism of the facilitating effect on mitogenesis. In dog thyrocytes, only HGF can trigger cell proliferation in the absence of insulin/IGF-I; this is explained by the fact that only this factor strongly activates both PI3 kinase and MAP kinase cascades (629) . Only insulin, IGF-I and HGF also markedly enhance general protein synthesis and induce cell hypertrophy (630). By contrast, TSH and cAMP are very unique as mitogens, as they do not activate Ras, the PI3kinase/PKB pathway, or any of different classes of MAP kinases in dog thyrocytes (316;317;631;632). TSH and cAMP also do not activate MAPkinases in human thyrocytes (633). The phosphorylation and activation of p70 ^S6K and thus likely of mTOR cascade constitutes the only early convergence point of growth factor and cAMP-dependent mitogenic cascades (634;635). A recent study has demonstrated the crucial role of this cascade for TSH-elicited thyroid follicular hyperplasia invivo in mice (636). Indeed, as found in dog thyroid primary cultures (637) and PCCl3 cells (Blancquaert and Roger, unpublished), TSH stimulates in mice the mTOR/ p70 ^S6K axis without activating PKB, and a rapamycin derivative abrogates the hyperplastic (but, interestingly, not the hypertrophic) responses to TSH (638). The cAMP-dependent mitogenesis and gene expression also appears to require the phosphorylation by PKA and activity of CREB/CREM transcription factors (639;640).

As in other types of cells, EGF and TPA first enhance c-fos and c-myc mRNA and protein concentrations in dog thyrocytes. On the other hand, TSH and forskolin strongly, but for a short period, enhance the c-myc mRNA concentration and with the same kinetics as the enhancement of the c-fos mRNA concentration by EGF/TPA. In fact, cAMP first enhances and then decreases c-myc expression. This second phenomenon is akin to what has been observed in the fibroblast, in which cAMP negatively regulates growth. As in fibroblasts, EGF and TPA enhance c-jun, junB, junD, and egr1 expression. However, as in fibroblasts, activators of the cAMP cascade decrease c-jun and egr1 expression. c-Jun is therefore not, as has been claimed, a gene whose expression is universally necessary for growth (484;620) .

The investigation of the pattern of proteins synthesized in response to the various proliferation stimuli has suggested very early that the proliferation of dog thyroid cells is controlled during G1 phase by at least two largely distinct, cAMP-dependent or cAMP-independent, pathways (641;642). Recent microarray analyses have confirmed and extended this concept in human thyrocytes (643;644). Nevertheless, the different mitogenic cascades are expected to finally modulate the level and activity of proteins that are the primary regulators of the cell cycle machinery.

As generally considered, mitogenic signals regulate mammalian cell cycle by stimulating the accumulation of D-type cyclins and their assembly through a ill-defined mechanism with their partner the cyclin-dependent kinases (cdk) 4 and 6. These complexes operate in mid-to-late G1 phase to promote progression through the restriction point, and thus commit cells to replicate their genome (645). In the current model, this key decision depends on the initiation by cyclin D-cdk complexes of the phosphorylation of the growth/tumor suppressor protein pRb, which triggers the activation of transcription factors, including those of the E2F family, the synthesis of cyclin E and then cyclin A, and cdk2 activation by these cyclins. Activated cdk2 in turn further phosphorylates pRb and other substrates and initiates and organizes the progression through the DNA synthesis phase (646). The down regulation of cdk inhibitors of the CIP/KIP family, including p27 ^kip1 , by mitogenic factors and/or their sequestration by cyclin D-cdk complexes participate to cdk2 activation, but their proposed role of adaptor and/or nuclear anchor for cyclin D-cdk complexes suggests positive influences on cell cycle progression as well (647). These mechanisms have been well studied in dog thyroid cells (Fig. 1-17). As expected, the different mitogenic stimulations (TSH, cAMP, growth factors) require the activity of cdk4 (648), and converge on the inactivating phosphorylation of pRb and related proteins p107 and p130 (649), on the phosphorylation and nuclear translocation of cdk2, and on the induction of cyclin A and cdc2 (650). These effects are dependent on insulin action (651;652). What is strikingly different between the cascades is the mechanism of D-type cyclin-cdk4 activation. TSH, unlike all the other known mitogenic factors, does not induce the accumulation of cyclins D (653), but it paradoxically stimulates the expression of the cdk “ inhibitor ” p27 ^kip1 (654). However the predominant cyclin D3 is required for the proliferation stimulated by TSH, but not in the proliferation of dog thyrocytes stimulated by EGF or HGF that induce cyclins D1 and D2 in addition to increasing cyclin D3 levels (653). The formation and the nuclear translocation of essential cyclin D3-cdk4 complexes depend on the synergistic interaction of TSH and insulin (653;655). These complexes are absent from cells stimulated by TSH or insulin alone. Paradoxically, in the absence of insulin TSH inhibits the basal accumulation of cyclin D3 (656). On the opposite insulin alone stimulates the required cyclin D3 accumulation and it overcomes in large part the inhibition by TSH (657), but it is unable to assemble cyclin D3-cdk4 complexes in the absence of TSH. In the presence of insulin, TSH (cAMP) unmasks some epitopes of cyclin D3 and induces the assembly of cyclin D3-cdk4 complexes and their import into nuclei (653;658) where these complexes are anchored by their association with p27 ^kip1 (659;660). This also sequesters p27 away of cdk2 complexes (661), thus contributing to cdk2 activation. Moreover, cAMP exerts an additional crucial function in very late G1 phase to stimulate the enzymatic activity of cyclin D3-cdk4-p27 complexes, which involves the stimulation of the activating Thr172-phosphorylation of cdk4 (662). TGF  selectively inhibits the cAMP-dependent proliferation of dog thyrocytes by preventing the association of the cyclin D3-cdk4 complex with nuclear p27 ^kip1 and the Thr172-phosphorylation of cdk4 (663;664) (665)

Fig. 1-17b: Targets of cell cycle regulatory effects of TSH, insulin/IGF-1 and TGFβ, as demonstrated in the dog thyroid primary culture system. Diamond/rectangle arrowheads represent inductions/repressions: the other dashed arrows are activations (+) and inhibitions (-). TSH (cAMP) does not induce cyclins D but assembles and then activates the cyclin D3-cdk4-p27 holoenzyme. IGF-1 and insulin allow the accumulation of the required cyclin D3. TGFβ inhibits the nuclear translocation of the cyclin D3-cdk4 complex, its association to p27 and its activation by TSH(cAMP). See text for full explanation.

The investigation of cell cycle regulatory proteins has thus clearly established that both cdk4 activation and pRb phosphorylation result from distinct but complementary actions of TSH and insulin, rather than from their interaction at an earlier step of the signaling cascades (666;667) (Fig. 1-17). Together with the fact that the necessary increase of cell mass before division depends on insulin/IGF-I but not TSH (630), these observations provide a molecular basis for the well established physiological concept that in the regulation of normal thyroid cell proliferation, TSH is the “ decisional ” mitotic trigger, while locally produced IGF-I and/or circulating insulin are supporting “ permissive ” factors (215). Of note, in all these experiments, the facilitative action of insulin can be replaced by activation of the Gq/PLC cascade by carbamylcholine (668).

Studies of protein phosphorylation, proto-oncogene expression, and cell cycle regulatory proteins in dog thyrocytes allow discrimination between two models of cAMP action on proliferation in this system: a direct effect on the thyrocyte or an indirect effect through the secretion and autocrine action of another growth factor. If the effect of TSH through cAMP involved such an autocrine loop, one would expect to find faster kinetics of action of the growth factor and at least some common parts in the patterns of protein phosphorylation and protein synthesis induced by cAMP and the growth factor. The results do not support such an hypothesis, at least for the growth factors tested (215) (Fig. 1-16). Moreover, the data on cAMP action in the dog and human thyrocyte systems do not support a major role for various mechanisms involving cross-signaling of cAMP with growth factor pathways, as claimed in rat thyroid cell line studies (reviewed and discussed in (669) ). Indeed, in primary cultures of normal human thyrocytes, EGF+serum increases cyclin D1 and p21 accumulation, and it stimulates the assembly and activity of cyclin D1-cdk4-p21. By contrast, TSH (cAMP) represses cyclin D1 and p21, but it stimulates the activating phosphorylation of cdk4 and the pRb-kinase activity of preexisting cyclin D3-cdk4 complexes (670). Cyclin D1 or cyclin D3 are thus differentially used in the distinct mitogenic stimulations by growth factors and TSH, and potentially in hyperproliferative diseases generated by the overactivation of their respective signaling pathways.

The validity of these concepts in vivo has been established by using transgenic mice models. The expression in thyroid of oncogene E7 of HPV-16, which sequestrates pRb protein, leads to thyroid growth and euthyroid goiter. Expression in the thyroid of the adenosine A ₂ receptor, which behaves as a constitutive activator of adenylyl cyclase, induces thyroid growth, goitrogenesis, and hyperthyroidism (534). Similar, albeit weaker phenotypes are obtained in mice expressing constitutive Gs (the G protein activating adenylyl cyclase) (671) or cholera toxin (672) . A contrario, the expression in thyroid of a dominant negative CREB provokes a marked thyroid hypotrophy, suggesting the crucial role of CREB and its activating phosphorylation by PKA (673) . By contrast, transgenic mice overexpressing both human IGF-I and IGF-I receptor in their thyroid (TgIGF-I – TgIGF-IR) and the downregulation of PTEN the PIP3 3 ’ phosphatase develop only a mild thyroid hyperplasia and respond to some extent to a goitrogenic effect of antithyroid drugs while maintaining a comparatively low serum TSH level. This indicates some autonomy of these thyroids, as in acromegalic patients, and a much greater sensitivity to endogenous TSH (674) . Very recently, thyrocyte-specific deficiency of Gq/G ₁₁ (the G proteins activating PLC  ) in mice was shown to impair not only the TSH-stimulated iodine-organification and thyroid hormone synthesis, but also TSH-dependent development of goiter (675). It remains to be defined whether this impaired follicular cell hyperplasia could result in part from the lack of induction of VEGF and angiogenesis (676) which normally accompany goitrogenesis. Nevertheless, the phenotype of these mice underscores the role in TSH-dependent goitrogenesis of PLC, which is activated by TSH but even more strongly by neurotransmitters. Noteworthy, section of inferior laryngeal nerve in rats was similarly found to impair both thyroid function and growth stimulated by TSH (677) . Moreover, activation of Gq /PLC by carbamycholine can facilitate cAMP-dependent mitogenesis in dog thyrocytes cultured without insulin or IGF-I (678) . On the other hand, expression of Ret, which is a rearranged constitutive growth factor receptor, in papillary thyroid carcinoma (PTC), leads to growth, cancer, and hypothyroidism (679;680).

Proliferation and Differentiation (Fig 1-18)

Fig 1-18. Main controls of the principal biologic variables of the human thyrocyte. EGF, epidermal growth factor; FGF, fibroblast growth factor; GH, growth hormone; HGF, hepatocyte growth factor; I – , iodide; IGF-I, insulin-like growth factor; IFN, interferon; IL-1, interleukin-1; TGF  , tumor growth factor-  ; TNF, tumor necrosis factor; TSAb, thyroid-stimulating immunoglobulins; positive control (stimulation);  + : negative control (inhibition)  .

Fig 1-18. Main controls of the principal biologic variables of the human thyrocyte. EGF, epidermal growth factor; FGF, fibroblast growth factor; GH, growth hormone; HGF, hepatocyte growth factor; I - , iodide; IGF-I, insulin-like growth factor; IFN, interferon; IL-1, interleukin-1; TGF  , tumor growth factor-  ; TNF, tumor necrosis factor; TSAb, thyroid-stimulating immunoglobulins; positive control (stimulation);  + : negative control (inhibition)  .

The incompatibility at the cell level of a proliferation and differentiation program is commonly accepted in biology. In general, cells with a high proliferative capacity are poorly differentiated, and during development such cells lose this capacity as they progressively differentiate. Some cells even lose all potential to divide when reaching their full differentiation, a phenomenon called terminal differentiation. Conversely, in tumor cells, proliferation and differentiation expression are inversely related. Activation of Ras and p42/p44 MAPkinases, induction of c-jun, sustained expression of c-myc, induction of cyclin D1 and down regulation of p27 ^kip1 , all have been shown to be causatively associated not only with proliferation, but also with loss of differentiation in a large variety of systems, sometimes independently of proliferation effects. It is therefore not surprising that in thyroid cells the general mitogenic agents and pathways, phorbol esters and the protein kinase C pathway, EGF, and in calf and porcine cells, FGF and the protein tyrosine kinase pathway, induce both proliferation and the loss of differentiation expression. The effects of the cAMP cascade are in striking contrast with this general concept. Indeed, TSH and cAMP induce proliferation of dog thyrocytes while maintaining differentiation expression; both proliferation and differentiation programs can be triggered by TSH in the same cells at the same time (521). This situation is by no means unique because neuroblasts in the cell cycle may also simultaneously differentiate. It is tempting to relate this apparent paradox to the unique characteristics of the cAMP-dependent mitogenic pathway, such as the lack of activation (or even the inhibition) of the Ras/MAPkinase/c-jun/cyclin D1 cascade, as demonstrated in dog and human thyrocytes. For instance, if one generalization could be made about proto-oncogenes, it is the dedifferentiating role of c-myc. A rapid and dramatic decrease in c-myc mRNA by antisense myc sequences induces differentiation of a variety of cell types. It is therefore striking that in the case of the thyrocyte, in which activation of the cAMP cascade leads to both proliferation and differentiation, the kinetics of the c-myc gene expression appears to be tightly controlled. After a first phase of 1 hour of higher level of c-myc mRNA, c-myc expression is decreased below control levels. In this second phase, cAMP decreases c-myc mRNA levels, as it does in proliferation-inhibited fibroblasts. It even depresses EGF-induced expression. The first phase could be necessary for proliferation, whereas the second phase could reflect stimulation of differentiation by TSH (483;681). The specific involvement of cyclin D3 in the cAMP-dependent mitogenic stimulation of dog and human thyrocytes, but not for their response to growth factors, is also interesting in this context (653;682) . Indeed, unlike cyclins D1 and D2, cyclin D3 is highly expressed in several quiescent tissues in vivo, and its expression is not only stimulated by mitogenic factors but also induced during several differentiation processes associated with a repression of cyclin D1 (683). We have recently shown that the differential utilization of cyclin D1 or cyclin D3 affects the site specificity of the pRb-kinase of cdk4, including in dog and human thyrocytes (684;685). In addition to inhibiting E2F-dependent gene transcription related to cell cycle progression, pRb plays positive roles in the induction of tissue-specific gene expression by directly interacting with a variety of transcription factors, including Pax8 in thyroid cells (686). Whether, the selective utilization of cyclin D3 in the TSH cascade, associated with a more restricted pRb-kinase activity, could allow the preservation of some differentiation-related functions of pRb thus remains to be examined.

We now consider the distinct cAMP-dependent mitogenic pathway, which appears to be adjuncted to the more general mechanisms used by growth factors, as pertaining to the specialized differentiation program of thyroid cells (309). In dog thyrocytes, the proliferation in response to serum or growth factors specifically extincts their capacity to respond to TSH/cAMP as a mitogenic stimulus (687). Similarly, in less differentiated thyroid cancers generated by the subversion of growth factor mechanisms, the TSH-dependence of growth is generally found to be lost.

Because the cell renewal rate is very low in the thyroid (once every 8 years in adults), the role of apoptosis is unimportant. However, under different circumstances the apoptotic role can greatly increase, such as after the arrest of an important stimulation in vitro (688) and in vivo (689) (690;691).

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294. Raspé E, Dumont JE. Control of the dog thyrocyte plasma membrane iodide permeability by the Ca2+-phosphatidylinositol and adenosine 3',5'-monophosphate cascades. Endocrinology 1994; 135:986-995.

295. Mockel J, Laurent E, Lejeune C, Dumont JE. Thyrotropin does not activate the phosphatidylinositol bisphosphate hydrolyzing phospholipase C in the dog thyroid. Molecular and Cellular Biology 1991; 82(2-3):221-227.

296. Mockel J, Lejeune C, Dumont JE. Relative contribution of phosphoinositides and phosphatidylcholine hydrolysis to the actions of carbamylcholine, thyrotropin, and phorbol esters on dog thyroid slices: regulation of cytidine monophosphate-phosphatidic acid accumulation and phospholipase-D activity. II. Actions of phorbol esters. Endocrinology 1994; 135:2497-2503.

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298. Coulonval K, Vandeput F, Stein RC, Kozma SC, Lamy F, Dumont JE. Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin. Biochemical Journal 2000; 348:351-358.

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300. Van Keymeulen A, Dumont JE, Roger PP. TSH induces insulin receptors that mediate insulin costimulation of growth in normal human thyroid cells. Biochemical and Biophysical Research Communications 2000; 279(1):202-207.

301. Thompson SD, Franklyn JA, Watkinson JC, Verhaeg JM, Sheppard MC, Eggo MC. Fibroblast growth factors 1 and 2 and fibroblast growth factor receptor 1 are elevated in thyroid hyperplasia. Journal of Clinical Endocrinology and Metabolism 1998; 83(4):1336-1341.

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649. Coulonval K, Maenhaut C, Dumont JE, Lamy F. Phosphorylation of the three Rb protein family members is a common step of the cAMP-, the growth factor, and the phorbol ester-mitogenic cascades but is not necessary for the hypertrophy induced by insulin. Experimental Cell Research 1997; 233(2):395-398.

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651. Coulonval K, Maenhaut C, Dumont JE, Lamy F. Phosphorylation of the three Rb protein family members is a common step of the cAMP-, the growth factor, and the phorbol ester-mitogenic cascades but is not necessary for the hypertrophy induced by insulin. Experimental Cell Research 1997; 233(2):395-398.

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654. Depoortere F, Dumont JE, Roger PP. Paradoxical accumulation of the cyclin-dependent kinase inhibitor p27kip1 during the cAMP-dependent mitogenic stimulation of thyroid epithelial cells. Journal of Cell Science 1996; 109(Pt 7):1759-1764.

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659. Depoortere F, Pirson I, Bartek J, Dumont JE, Roger PP. Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1). Molecular Biology of the Cell 2000; 11(3):1061-1076.

660. Coulonval K, Bockstaele L, Paternot S, Dumont JE, Roger PP. The cyclin D3-CDK4-p27(kip1) holoenzyme in thyroid epithelial cells: activation by TSH, inhibition by TGFbeta, and phosphorylations of its subunits demonstrated by two-dimensional gel electrophoresis. Experimental Cell Research 2003; 291(1):135-149.

661. Depoortere F, Pirson I, Bartek J, Dumont JE, Roger PP. Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1). Molecular Biology of the Cell 2000; 11(3):1061-1076.

662. Paternot S, Coulonval K, Dumont JE, Roger PP. Cyclic AMP-dependent phosphorylation of cyclin D3-bound CDK4 determines the passage through the cell cycle restriction point in thyroid epithelial cells. Journal of Biological Chemistry 2003; 278(29):26533-26540.

663. Depoortere F, Pirson I, Bartek J, Dumont JE, Roger PP. Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1). Molecular Biology of the Cell 2000; 11(3):1061-1076.

664. Coulonval K, Bockstaele L, Paternot S, Dumont JE, Roger PP. The cyclin D3-CDK4-p27(kip1) holoenzyme in thyroid epithelial cells: activation by TSH, inhibition by TGFbeta, and phosphorylations of its subunits demonstrated by two-dimensional gel electrophoresis. Experimental Cell Research 2003; 291(1):135-149.

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INTRODUCTION

Thyrotoxicosis is defined as the clinical syndrome of hypermetabolism resulting from increased free thyroxine (T4) and/or free triiodothyronine (T3) serum levels (1). The term thyrotoxicosis is not synonymous with hyperthyroidism, the elevation in thyroid hormone levels caused by an increase in their biosynthesis and secretion by the thyroid gland (Table 1) (2). For example, thyrotoxicosis can result from the destruction of thyroid follicles and thyrocytes in the various forms of thyroiditis, or it can be caused by an excessive intake of exogenous thyroid hormone. It should also be noted that the elevation of free thyroid hormone levels does not always result in thyrotoxicosis in all tissues. In the syndrome of Resistance to Thyroid Hormone (RTH), dominant negative mutations in the thyroid hormone receptor bets ( TRbeta ) result in decreased thyroid hormone action in tissues where TRbeta is the predominant receptor, for example in the liver and the pituitary, whereas other tissues such as the heart, which express mainly TRalpha, show signs of increased thyroid hormone action (See Chapter 16 D). The most common form of thyrotoxicosis is Graves' disease, which is discussed in Chapter 10. This chapter reviews other etiologies of thyrotoxicosis (Table 1). The determination of the etiology of thyrotoxicosis is of importance in order to establish a rational therapy.

TSH: Thyroid-stimulating hormone. Gs: stimulatory G protein subunit. hCG = human chorionic gonadotropin.

TOXIC ADENOMA

Definition and Epidemiology

Clinical presentation

Patients with toxic adenomas present with signs and symptoms of thyrotoxicosis and/or a thyroid nodule. The signs and symptoms of thyrotoxicosis do not differ from other etiologies. Features suggestive for Graves' disease such as endocrine ophthalmopathy, (pretibial) myxedema and acropachy are missing. The onset of thyrotoxicosis is often insidious and more common in older patients, who typically have larger adenomas. However, a toxic adenoma has even been documented as a cause of neonatal hyperthyroidism (8). Mechanical symptoms such as dysphagia or hoarseness are uncommon. Autonomously functioning nodules may remain stable in size, grow, degenerate or become gradually toxic. In one series, 10% of patients followed for 6 years became thyrotoxic (3). Thyrotoxicosis may develop independent of age, but is much more common in nodules ov er 3 cm in diameter (up to 20%). By sonography, the critical volume at which hyperthyroidism occurs is about 16 ml (9). Changes in nodule size were followed in 159 patients during a period of 1 to 15 years (3). An increase in size was seen in only 10%, 4% of nodules decreased, and a loss of function due to degenerative changes was observed in 4 nodules. Eight percent developed overt thyrotoxicosis during a follow-up of 3 to 5 years, and 3% developed subclinical hyperthyroidism (3).

Diagnosis

The measurement of serum TSH with a sensitive third-generation assay represents the best biochemical marker to establish the diagnosis of thyrotoxicosis because TSH and FT4 have an inverse log-linear relationship and a small decrease or increase in FT4 is thus associated with an exponential change in TSH levels (10). If the TSH is suppressed, measurement of serum (free) T4 and T3 permit to ascertain the severity of the thyroid overactivity. A thyroid scan can be performed with 123iodine, 131iodine, or 99technetium-labeled pertechnetate (11). Iodine isotopes, which are not only trapped but also organified in the thyroid, are preferred because 3-8% of nodules that appear functioning on pertechnetate scanning are nonfunctioning on radioiodine scanning. A scan will show uptake of the isotope that is either limited to the nodule, or preferential uptake in the adenoma compared to the surrounding tissue (Figure 1). Scintigraphically, an AFTN may be warm (uptake similar to surrounding tissue), hot (uptake increased without suppression of surrounding tissue), or toxic (uptake increased and suppression of the surrounding tissue). A toxic nodule is associated with overt or subclinical hyperthyroidism. A warm nodule may develop into a hot nodule and ultimately into a toxic adenoma. Toxic adenomas are usually larger in size and often more than 3 cm in size (3, 12). In the case of incomplete suppression of the surrounding tissue, autonomous function of the nodule can be established by a suppression test. After the administration of thyroid hormone (e.g. 75 ï¿&frac12;g of levothyroxine for 2 weeks, followed by 150 ï¿&frac12;g for 2 weeks), a repeat thyroid scan would fail to show remaining uptake in the non-autonomous tissue because of the suppression of serum TSH, thereby unmasking the autonomy of the nodule (13). However, this procedure has no practical consequences and is therefore unnecessary in clinical practice. Ultrasound will confirm the presence of a solitary nodule and may show a small contralateral thyroid lobe. There is no indication to perform fine needle aspiration in patients with toxic adenomas because the risk of a thyroid carcinoma is extremely low and cytological evaluation will not permit distinguishing between a follicular adenoma and a follicular carcinoma (14, 15).

Treatment

In patients with overt thyrotoxicosis, definitive forms of treatment include surgical excision of the nodule, treatment with radioactive iodine, or percutaneous ethanol injection (11, 16). Treatment with antithyroid drugs is used infrequently as it requires long-term therapy and a relapse will almost invariably occur after discontinuation of the medication. Surgical excision permits to achieve a rapid and permanent control of hyperthyroidism with a very low operative complication rate. The disadvantage of a surgical approach includes the risks of general anesthesia and the potential complications of thyroid surgery. Usually the patient is treated preoperatively with antithyroid drugs and beta-blockers. The incidence of hypothyroidism after operation is low, but may occur. In a series of 60 patients operated for AFTNs, 6.6% became hypothyroid after operation (17). Two of these patients had previously received therapeutic doses of 131iodine or long-term treatment with antithyroid drugs. In a series of 35 patients with a solitary toxic adenoma, lobectomy resulted in 30 euthyroid and 5 hypothyroid outcomes, although hypothyroidism was only temporary in 3 patients (4). It remains unclear why some of these patients remained permanently hypothyroid after lobectomy; information about the presence of autoantibodies and the morphology of the contralateral lobe is not provided in this study. Generally, it is believed that long-term suppression of the thyroid gland does not lead to permanent inactivation after suppression is relieved. Administration of 131iodine is a widely used therapeutic modality for patients with toxic adenomas. The main disadvantage consists in the possibility of permanent hypothyroidism in a subset of patients. In a study by Goldstein et al., 23 patients were followed for 4 to 16.5 years and 8/23 (35%) developed hypothyroidism (18). The incidence of hypothyroidism was not related to nodule size, the level of thyroid function, or the administered dose of 131iodine. In a similar study by Mariotti et al. on 126 patients, 5/126 (4%) developed overt hypothyroidism 1 to 10 years after 131iodine therapy (19). There was no relationship between the development of hypothyroidism, nodule size or the administered dose of 131iodine (19). Hypothyroidism occurred in 9.7% of patients with an euthyroid hot nodule treated with 131iodine, and in only 1.5% of patients with a toxic adenoma. When antithyroglobulin and/or antithyroid microsomal antibodies were present, the prevalence of hypothyroidism after 10 years was 18% versus 1.4% in antibody-negative patients. In two studies, evaluating 48 and 45 patients 6 months after radioiodine therapy, hypothyroidism could not be documented in any of the patients (20, 21). In a more recent study by Bolusani et al. on 105 patients with solitary autonomous nodules, the cumulative incidence of hypothyroidism was 11% at 1 year, 33% at 5 years, and 49% at 10 years (22). The development of hypothyroidism was not associated with age, sex, radioiodine dose, radioiodine uptake, or degree of suppression of extranodal tissue on scintiscans. The predictors of occurrence of hypothyroidism were pretreatment with antithyroid medications and a positive thyroid antibody status. Antibody-positive patients showed an earlier progression towards hypothyroidism than did antibody-negative patients (22). In aggregate, these results suggest that longer follow-up periods may uncover hypothyroidism more frequently and that the development of hypothyroidism may often be related to the presence of thyroid autoantibodies, but less to the administered dose of 131iodine and nodule size. In patients treated with antithyroid drugs prior to radioiodine therapy, the increase in TSH may reactivate suppressed thyroid tissue and iodide uptake resulting in damage by 131iodine. Some clinicians administer levothyroxine for two weeks prior to therapy in order to assure that the tissue surrounding the toxic adenoma is suppressed. In some instances, high doses of 131iodine in the nodule may provide enough radiation to the surrounding tissue that its function is seriously damaged. It is noteworthy  that therapy with 131iodine may trigger the development of humoral thyroid autoantibodies (23). For example, about 5% of patients treated with 131iodine for toxic or euthyroid multinodular goiter develop stimulating TSH receptor antibodies and Graves' disease (24). Hence, hypothyroidism may, in part, result from the development of humoral autoantibodies in patients with toxic adenomas treated with 131iodine. An alternative to surgery and 131iodine therapy for toxic adenomas consists in the use of percutaneous ethanol injection into the nodule under ultrasound guidance (11). The injection results in necrosis and thrombosis of small vessels. Side effects include local pain and, in rare cases, recurrent nerve damage. In studies evaluating the outcomes at 12 or 30 months, about 85% of patients were euthyroid (25, 26). Results of ethanol injection in relatively large AFTNs (diameter 3 to 4 cm) are also favorable, particularly in patients with subclinical hyperthyroidism (27, 28). Surpisingly, previous ethanol injection did not hinder histological assessment in 13 patients who ultimately underwent surgical excision of their nodule (29). Percutaneous laser thermal ablation (LTA) is a more recently introduced technique for the debulking of thyroid nodules and has also been used for the debulking of anaplastic thyroid cancer (30). In hyperfunctioning nodules, LTA induced a nearly 50% volume reduction with a variable frequency of normalization of thyroid-stimulating hormone levels (31, 32). Most patients become and remain euthyroid after treatment. However, serum TSH measurement at yearly intervals is necessary in order to detect those patients, especially with circulating thyroid autoantibodies, who will eventually develop hypothyroidism.

Pathogenesis

Chronic stimulation of the cAMP cascade results in enhanced proliferation and function of thyrocytes (33). Hence, any molecular alteration leading to constitutive activation of the cAMP pathway in a thyroid follicular cell is expected to result in clonal autonomous growth and function, and ultimately in a toxic adenoma (33). In line with this concept, somatic mutations were first discovered in the GNAS1 gene encoding the stimulatory Gs alpha  subunit in toxic adenomas (34-36). Stimulatory Gs alph amutations impair the hydrolysis of guanine triphosphate (GTP) to guanine diphosphate (GDP), resulting in persistent activation of adenylyl cyclase. Stimulatory Gs alpha mutations are also found in 35-40 percent of somatotroph tumors in acromegalic patients (37), and mosaicism for Gs alpha mutations with onset during blastocyst development causes the McCune Albright syndrome (38). The observation that site-directed mutagenesis of a residue in the third intracellular loop of the alpha 1b-adrenergic receptor can lead to constitutive activation of this G protein-coupled receptor (GPCR) in the absence of ligand led to the search and detection of naturally occurring activating mutations in numerous GPCRs (39). Somatic mutations in the TSH receptor were first discovered in toxic adenomas (40). The initially characterized mutations were clustered in the third intracellular loop and the sixth transmembrane domain of the receptor, but a wide variety of activating somatic mutations have been found in subsequent studies (see Chapter 16 A) (41-44). Mutations conferring constitutive activity occur in the entire transmembrane domain, as well as in the carboxy-terminal region of the extracellular domain. All mutations increase basal cAMP levels, but only a few amino acid substitutions activate the phospholipase C (PLC) cascade in a constitutive manner. Inositoltriphosphate (IP3) accumulation in response to TSH is usually retained. The reported prevalence of TSH receptor mutations in toxic adenomas varies widely, but is as high as 80% (43, 45). For example, in a study on 33 toxic adenomas from 31 patients from Belgium, 27/33 of adenomas were positive for a somatic mutation in the TSH receptor (46). In contrast, in a Japanese study that analyzed the part of the gene encoding the third cytoplasmic loop and the sixth transmembrane segment, only 1/38 toxic adenomas harbored a functionally silent mutation (45). Differences in sampling technique and methodological approach, as well as variations in iodine intake, may contribute to the reported differences (47). It is now well established that somatic, constitutively activating TSH receptor mutations play a predominant role in the pathogenesis of AFTNs, while Gs aalpha mutations are less common (48). It is likely that other somatic mutations are involved in the pathogenesis of the monoclonal toxic adenomas that are negative for mutations in the TSH receptor and Gs alpha(49). Functionally, some of the mutations may alter the positions of the transmembrane helices, thereby mimicking the conformational changes induced by binding of ligand. Alternatively, some mutations may alter the structure of domains that inhibit coupling of the receptor to G proteins in the absence of TSH (50, 51). Activating mutations in the extracellular domain appear to result in a relief of a negative constraint present in the unliganded carboxy-terminal part of the extracellular domain (8, 43, 52-54). It has been suggested that iodine deficiency may be a predisposing factor for the development of AFTNs (55). Based on the fact that autonomous (multi)nodular goiters develop also in iodine-sufficient regions and that there is often a hereditary predisposition, others propose that hereditary and acquired heterogeneity among the thyrocytes play a fundamental role in the pathogenesis of AFTNs and that iodine deficiency only serves as a modulating factor (56).

Pathology

On macroscopic examination, a solitary toxic nodule is surrounded by normal thyroid tissue that is functionally suppressed. Toxic adenomas are histologically classified as encapsulated follicular neoplasms or adenomatous nodules without a capsule (57). Hemorrhage, calcifications and cystic degeneration are commonly present. In a study on 51 solitary adenomas, functional and pathologic characteristics were determined and compared to normal surrounding tissue (58). The adenomas displayed a higher number of cycling cells in the periphery of the adenomas, a high level of iodide trapping because of a high level of sodium/iodide symporter (NIS) gene expression, a high thyroperoxidase (TPO) mRNA and protein content, and low H2O2 generation. The adenomas secreted higher amounts of thyroid hormone than the quiescent tissue (58). The proliferation index was determined in 20 toxic adenomas using labeling with proliferating cell nuclear antigen (PCNA) and Ki-67 epitope as markers (59). In line with the slow growth of these lesions, cell proliferation was found to be modestly increased compared to the surrounding tissue (59). Malignant AFTNs are uncommon. In a study of 306 patients presenting with AFTNs, Horst et al. did not find any thyroid malignancy (5). Sandler et al. concluded that most reported hyperfunctioning carcinomas resulted from the coexistence of small malignancies in or adjacent to a benign hot lesion (14). Isolated cases of carcinomas in hot nodules have, however, been reported (4, 60-62). Smith et al. reported the occurrence of 3 carcinomas in 30 consecutive patients operated for solitary hot nodules (62). In a series of 164 patients, 3 of 29 patients treated surgically were diagnosed with thyroid cancer (63). It is an open question whether the diagnosis of cancer would be established in all these cases using modern histological criteria and molecular markers (64). In most instances, the presence of an AFTN argues against the presence of a malignant lesion.

TOXIC MULTINODULAR GOITER

Definition

Hyperthyroidism may occur due to AFTNs in a multinodular thyroid gland and this is discussed in detail in Chapter 17.

Clinical presentation

In addition to the signs and symptoms associated with hyperthyroidism, patients with large toxic multinodular goiters may also have dysphagia, shortness of breath, stridor, or hoarseness.

Diagnosis

The diagnostic approach is in general similar to patients with a solitary AFTN, but cross-sectional imaging with computer tomography and pulmonary function tests need to be considered in a subset of patients in whom compression by the goiter is evident or suspected.

Treatment

Therapeutically, surgery and radioiodine therapy are the most commonly used therapeutic modalities.

Pathogenesis

While the mechanisms underlying the development of nodules are of complex nature (65), it has become apparent that hyperfunctioning adenomas within multinodular goiters or autonomous areas within euthyroid goiters may also harbor somatic gain-of-function mutations in the TSH receptor (66-68). It is noteworthy that the mutations may differ among the adenomas within the same multinodular goiter (66). This observation is consistent with studies demonstrating distinct clonal origins of different thyroid adenomas within the same multinodular goiter (69). For example, in two adenomas from the same goiter, one neoplasm harbored a M453T mutation, the second adenoma a T632I substitution (66). In another study, L632I and F631L mutations were found in two distinct lesions within the same goiter, whereas another patient had two distinct toxic nodules with the same I630L mutation (67). These studies reveal that the pathogenesis of hyperfunctioning adenomas does not differ between solitary toxic adenomas and multinodular goiters. In a study analyzing hyperfunctioning and nonfunctioning areas from patients with toxic multinodular goiters, gain-of-function TSH receptor mutations were detected in 14 of 20 hyperfunctioning areas, whereas no mutation was identified in nonfunctioning nodules (70). On microscopic analysis, only two of the hyperfunctioning areas corresponded to classic adenomas surrounded by a capsule, whereas the remainder had the characteristic features of hyperplastic lesions. The development of multinodular goiters had been associated with a D727E germline polymorphism in the TSH receptor (71), but this finding could not be corroborated in other studies (72, 73). Constitutively activating TSH receptor mutations have also been detected in autoradiographically hyperfunctioning areas of goiters from euthyroid patients (74). The observation that TSH receptor mutations are rare in nonfunctioning adenomas, even if of monoclonal origin (68, 75), indicate that distinct mechanisms must be implicated in the abnormal growth leading to nonfunctioning nodules (65).

HYPERTHYROID THYROID CARCINOMA

Definition and Epidemiology

As discussed above, hyperfunctioning nodules are most commonly benign. Rarely, follicular carcinoma is associated with thyrotoxicosis. Ehrenheim compiled 20 such cases in 1986 (76), and Salvatori et al. reviewed 54 similar cases reported in the literature (77). Age and sex distribution in these patients does not differ from that of patients with follicular carcinoma without thyrotoxicosis.

Clinical presentation

Most commonly, thyrotoxicosis and thyroid carcinoma are diagnosed at the same time because of signs of thyrotoxicosis and the finding of a thyroid nodule prompting fine needle aspiration.

Diagnosis

Patients with thyrotoxic thyroid cancer have predominantly T3 thyrotoxicosis (78). Thyroglobulin levels are elevated. Cytology reveals most commonly follicular thyroid cancer (79), but hyperfunctinoning papillary thyroid cancer has also been documented (80). In patients who are on levothyroxine substitution after total thyroidectomy, the presence of hyperfunctioning metastases may not be readily apparent. Gradual reduction or withdrawal of levothyroxine therapy is necessary in order to recognize whether the thyrotoxicosis is caused by excessive exogenous levothyroxine or hyperfunctioning metastases. Whole-body scanning with radioiodine is used for the localization of the hyperfunctioning metastases.

Treatment

Treatment of patients with functioning thyroid carcinomas does not differ from the therapy of thyroid cancer patients without thyrotoxicosis, but appropriate control of the hyperthyroid state with antithyroid drugs and beta-blockers is important before submitting a patient to thyroid surgery or 131iodine therapy. Exacerbation of the hypermetabolic state with precipitation of thyroid storm has been reported in a patient undergoing radioiodine therapy for metastatic thyroid carcinoma without prior control with antithyroid drugs (81).

Pathogenesis

In well-differentiated thyroid cancers, mutations in the Gs alphA subunit and the TSH receptor genes occur only very rarely (36, 80, 82-86). Although constitutive activation of the cAMP pathway results in enhanced growth, it is not thought to be sufficient for malignant transformation of otherwise normal thyrocytes. A few patients with hyperthyroidism due to autonomously functioning thyroid cancers harboring mutations in the TSH receptor have, however, been identified. For example, Russo et al. reported a patient who presented with hyperthyroidism and increased uptake in two nodules, but suppressed uptake in the remainder of the gland (80). After surgical removal of the right thyroid lobe, histological examination revealed the presence of an insular papillary carcinoma with lymph node and lung metastases. Mutational analysis of the TSH receptor gene documented a somatic mutation, D633H, in DNA isolated from the primary tumor and metastatic tissue. Another mutation that activates both the cAMP and the IP3 pathways, I486F, was found in a hyperfunctioning well-differentiated follicular carcinoma in a patient presenting with hyperthyroidism and increased radioiodine uptake within the thyroid mass (86). It is conceivable that concomitant activation of these two signaling casca des may promote transformation. In a patient with an Hurthle cell carcinoma, Russo et al. identified a L677V TSH receptor mutation (85). Basal cAMP levels were increased in transfected Chinese hamster ovary (CHO) cells, but IP3 accumulation has not been determined. A somatic M453T substitution has been identified in a 11-year-old girl with a hyperfunctioning nodule and a papillary carcinoma (87). The same mutation has been found in the germline of two patients with congenital hyperthyroidism, but there was no suggestion that it is oncogenic (88, 89). Interestingly, however, overexpression of the M453T TSH receptor mutation in the FRTL-5 rat cell line was sufficient to induce neoplastic transformation as assessed by growth in semisolid medium and athymic mice (90). Follicular carcinomas have also been reported in patients with Graves' disease (91-93). It has been suggested that long-standing stimulation through TSH receptor-stimulating antibodies may play a role in the pathogenesis of these neoplasias (94). Whether thyroid carcinomas affecting patients with underlying Graves' disease behave more aggressively, as suggested by some authors (95), remains uncertain (96).

FAMILIAL NON-AUTOIMMUNE HYPERTHYROIDISM WITH TSHR MUTATIONS

Definition and Epidemiology

Autosomal dominant familial hyperthyroidism without evidence of an autoimmune etiology has been first described by Thomas et al. in 1982 (Chapter 16 A) (97). Currently 27 families with a total of 152 affected individuals with non-autoimmune familial hyperthyroidism have been reported (For recent review see: (98)). The hyperthyroidism is caused by monoallelic gain-of-function germline mutations in the TSH receptor.

Clinical presentation

The typical signs associated with autoimmune hyperthyroidism, i.e. the presence of stimulatory TSH receptor antibodies, endocrine ophthalmopathy, myxedema, lymphocytic infiltration of the thyroid gland, are absent. The age of onset of hyperthyroidism is variable and depends, in part, on the activity of the mutated allele. The majority of patients has a goiter.

Diagnosis

Affected individuals have a suppressed TSH and elevated peripheral hormones in the absence of TSH receptor-stimulating antibodies and TPO antibodies. The family history is key in order to demonstrate familial clustering suggestive for an autosomal dominant disorder. Ultimately, the diagnosis requires sequence analysis of the TSH receptor gene in order to evaluate it for the presence of a monalllelic mutation. If the mutation is unknown, functional in vitro analyses are needed to demonstrate that the mutated allele confers constitutive activity to the receptor.

Treatment

In order to achieve permanent cure, it is necessary to destroy all thyroid tissue, either by thyroidectomy followed by radioiodine therapy, or radiotherapy alone (98). In younger patients, temporary therapy with thionamides can be considered. Because the condition may not be readily recognized and confused with Graves' disease, patients treated with thionamides or insufficient amounts of radioiodine have frequent relapses (98).

Pathogenesis

The molecular basis of hereditary non-autoimmune hyperthyroidism was elucidated by detecting activating germline mutations in the TSH receptor in the family reported by Thomas et al. (51). Gain-of-function mutations are by definition dominant, and alteration of one allele is thus sufficient for generating the phenotype. Interestingly, the onset of hyperthyroidism may vary in carriers of the same mutation in a given kindred. Hence, other factors, for example genetic background and/or iodine intake, appear to modulate the phenotypic expression (97, 99, 100).

SPORADIC NON-AUTOIMMUNE HYPERTHYROIDISM

Autoimmune neonatal hyperthyroidism is rare and occurs in less than 2% of newborns that are the offspring of a mother with a history of Graves' disease (101), a condition with an estimated incidence of about 2 of every 1000 pregnancies (97). In these infants, the congenital hyperthyroidism is caused by transplacental passage of stimulating TSH receptor autoantibodies (102). Antibody-induced neonatal hyperthyroidism usually resolves within the first few months of life as the maternal antibodies are cleared from the circulation. Occasionally, thyroid hormone may be fluctuating between elevated and decreased levels because of the concomitant presence of stimulating and blocking antibodies (103). Constitutively activating germline neomutations in the TSH receptor have been found in a total of 15 patients with sporadic congenital non-autoimmune hyperthyroidism (Chapter 16 A) (104)(For recent review see:(98)). Congenital hyperthyroidism due to a toxic adenoma harboring a somatic TSH receptor mutation was reported as an unusual variant (8). The patients with non-autoimmune congenital hyperthyroidism must be differentiated from the much more common and transient autoimmune form of neonatal hyperthyroidism, because these patients have pronounced hyperthyroidism requiring a more aggressive therapeutic approach that may necessitate surgery and ablative radiotherapy early in life. Several of the children with severe neonatal hyperthyroidism were reported to have mild mental retardation (104-106), suggesting that high levels of thyroid hormone may have a negative impact on brain development (107). Alternatively, mental development may have been impaired because of premature closure of the cranial sutures. A subset of these children had proptosis (88, 89). Computer tomography of the retroorbital tissue in one of these children did, however, not demonstrate infiltration of the eye muscles (88).

TSH-SECRETING PITUITARY ADENOMA

Definition and Epidemiology

TSH-secreting adenomas (TSHomas) account for less than 2% of all pituitary adenomas and are a rare cause of thyrotoxicosis (Chapter 13) (108, 109). TSHomas and RTH form the two syndromes of  "inappropriate TSH secretion", defined by normal or elevated TSH levels in combination with increased (free) T4 and T3 levels.

Clinical presentation

Patients with TSHomas present with signs and symptoms of hyperthyroidism and an enlarged thyroid. In patients with RTH, the phenotype is more complex as some tissues are resistant to the action of the elevated peripheral hormones and thus hypothyroid, whereas other tissues can be excessively stimulated. The physiological negative feedback normally exerted by thyroid hormones is not operating in both conditions. TSHomas secrete TSH in an autonomous fashion, in RTH the thyrotropes are resistant to the high levels of thyroid hormone. Most patients are older, but TSHomas have also been documented in children (110).

Diagnosis

Magnetic resonance imaging (MRI) of the pituitary will reveal a pituitary adenoma in patients with TSHomas. TSH is formed of a specific  beta subunit and the glycoprotein alpha subunit common to TSH, FSH and LH/CG. Some clinicians measure the glycoprotein alpha subunit as a marker to distinguish between TSHomas and RTH. The alpha subunit and the beta subunit /TSH ratio are often elevated in patients with TSHomas (111). However, in one series of TSHomas, normal alpha subunit levels were observed in more than 60% of the patients, particularly in microadenomas (112). The TSH secreted by TSHomas is normal in terms of amino acid sequence, but has variable biological activity and is secreted in fluctuating amounts (113). Compared to controls, TSH burst frequency and basal secretion are increased, TSH secretion patterns are more irregular, but the diurnal rhythm is preserved at a higher mean in all patients (114). A more sensitive and specific test than measuring the alpha subunit consists in the T3 suppression test (80-100 µg of T3 per day per 8-10 days), which does not result in complete inhibition of T3 secretion in patients with TSHomas (108, 112, 115). An alternative consists in the TRH-stimulation test, but TRH is currently not available in the United States. After injection of TRH (200 µg i.v.) TSH and the alpha subunit do not increase in patients with TSHomas (108).

Treatment

Transsphenoidal surgery is the cornerstone for therapy of TSHomas. Complete resection may not be possible because these tumors can invade the sinus cavernosus and other adjacent structures. Prior to surgery, the hyperthyroidism should be controlled with thionamides and beta-blockers. In patients with residual tumor tissue and persistent secretion of TSH, both -knife radiotherapy (10 ï¿&frac12; 25 Gy) and medical therapies can be considered. The latter include the use of somatostatin analogues, such as octreotide and lanreotide. TSHomas express somatostatin receptors and somatostatin analogues are highly effective in reducing TSH secretion by neoplastic thyrotropes (116). If tolerated, somatostatin analogues are effective in reducing TSH secretion in more than 90% of patients with consequent normalization of thyroid hormone levels and restoration of the euthyroid state. Tumor shrinkage does occur in about 45% of patients (117). Dopamine receptors are also present in TSHomas and dopamine agonists such as bromocriptine or cabergoline have been used in order to control TSH secretion (108). The response is, however, highly heterogeneous and best in tumors secreting both TSH and prolactin. In the case of a surgical cure, the postoperative TSH is undetectable and may remain low for weeks or months, causing central hypothyroidism. Permanent central hypothyroidism may also occur due to the mass effect exerted by the tumor or after radiotherapy. Thus, transient or permanent substitution therapy with levothyroxine may be necessary. Long-term evaluation of all pituitary axes is important, particularly in patients who underwent radiotherapy, in order to recognize and treat anterior pituitary deficiencies in a timely manner.

Pathogenesis

The molecular mechanisms leading to the formation of TSHomas remain unknown. TSHomas have been shown to be monoclonal by X-inactivation analyses suggesting that they arise from a single cell harboring one or several mutations in genes controlling proliferation and perhaps function (108).

RESISTANCE TO THYROID HORMONE

The syndrome of Resistance to Thyroid Hormone (RTH) is described in detail in Chapter 16 D. RTH is defined by elevated circulating levels of free thyroid hormones due to reduced target tissue responsiveness and normal, or elevated, levels of TSH (118, 119). Patients with RTH typically present with goiter. Their metabolic state may appear euthyroid or include signs of hypo- and hyperthyroidism. With the exception of the first studied kindred, a single sibship harboring a deletion of the entire coding sequence of the entire TRbeta gene and a recessive pattern of inheritance, RTH is most commonly caused by monoallelic mutations of the TRbeta gene. The mutation can be inherited in an autosomal dominant manner or occur as de novo mutation. The mutant receptors act in a dominant negative fashion to block the activity of the normal allele, thereby explaining the dominant inheritance. The gene defect remains unknown in about 15% of subjects with a RTH phenotype. It is likely that mutations in cofactors that are required for normal TR function are involved in the pathogenesis of RTH in these patients. The generation of mice with targeted deletion of TRbeta, as well as TR knockin models, have been essential for elucidating the physiology of thyroid hormone action and the pathophysiology of RTH (120).

HCG-INDUCED GESTATIONAL HYPERTHYROIDISM

Thyrotoxicosis and other forms of thyroid dysfunction in the pregnant patient are discussed in detail in Chapter 14.

Definition and Epidemiology

Gestational transient thyrotoxicosis of non-autoimmune origin is caused by stimulation of the TSH receptor through hCG (121, 122). hCG-induced hyperthyroidism occurs in about 1.4 % of pregnant women, mostly when hCG levels are above 70-80,000 IU/l (123, 124) .

Clinical presentation

Many signs and symptoms of hyperthyroidism are not specific and overlap with those of normal pregnancy (125). Hence, the accuracy of clinical diagnosis is limited. Because of the decrease in the levels and bioactivity of hCG later in pregnancy, hCG-induced gestational hyperthyroidism is usually transient and limited to the first 3-4 months of gestation. In a subset of women, the manifestations of hCG-induced hyperthyroidism are more severe and they are often associated with hyperemesis. Goodwin et al. studied the relationship of serum hCG, thyroid function, and severity of vomiting among 57 hyperemesis patients and 57 controls matched for gestational age (126). Hyperemesis patients had significantly greater mean serum levels of hCG, free T4, total T3, and estradiol, and lesser serum TSH concentrations compared to controls. The degree of biochemical hyperthyroidism and hCG concentration correlated directly with the severity of vomiting. The hyperemesis may be caused by a marked hCG-induced increase in estradiol levels (122). However, the relation between hyperemesis and gestational hyperthyroidism varies among patients, and additional, unidentified mechanisms may be involved.

Diagnosis

The diagnosis is established by measuring TSH, free or total T4, and T3. The physiological decrease in TSH levels and the increase in total thyroid hormone concentrations associated with the increase in thyroxine-binding globulin (TBG) have to be considered when interpreting the results. TBG levels increase in response to elevated estradiol levels and plateau by about 20 weeks of gestation (127). Therefore, total T4 and total T3 levels increase by approximately 1.5 fold. If free T4 levels are determined by analogue assays, serum concentrations are usually significantly lower than values in non-pregnant women (128).

Treatment

Treatment with antithyroid medications is often not necessary. Women with hyperemesis need therapy with antiemetics. In patients in whom total T4 levels are higher than 1.5 times the upper reference range, therapy with antithyroid drugs may be indicated. Propylthiouracil (PTU) is the preferred medication during the first trimester and methimazole during the remainder of pregnancy in the United States (129, 130). A review by Mandel and Cooper has specifically addressed the use of thionamides during pregnancy and lactation (131). Overtreatment with antithyroid drugs can result in hypothyroidism in the fetus. Therefore, free T4 should be kept close or slightly above the normal range with the lowest possible dose of antithyroid drugs.

Pathogenesis

The pathophysiology of hCG-induced gestational thyrotoxicosis has been reviewed by Hershman (132). hCG and TSH share the common glycoprotein alpha subunit and the beta subunit is highly homologous. At high doses, hCG cross-reacts with the TSH receptor, and this stimulation can lead to an increase in secretion of T4 and T3, with subsequent suppression of TSH secretion (124, 133). The levels of hCG and TSH are inversely correlated during the first trimester (121). Free T4 levels determined between weeks 6-20 of gestation increase and show a linear relationship with the rising hCG levels (134). The thyroid gland of normal pregnant women may be stimulated by hCG to secrete slightly excessive quantities of T4 and induce a slight suppression of TSH, but it only induces overt hyperthyroidism in a subset of pregnant women. The increased secretion of hCG result only in the physiological decrease in TSH levels that are characteristic for the first trimester of pregnancy, or in overt hyperthyroidism. Of note, elevations of hCG are particularly pronounced in twin pregnancies (135). In a study characterizing the activity of hCG on the human thyroid gland, 1.0 U hCG was found to be roughly equivalent to 0.27 mU of TSH (136). LH also has intrinsic thyroid stimulating activity, but it is lower compared to hCG. TSH-binding and TSH-induced adenylyl cyclase stimulation are more effectively inhibited by desialylated variants of hCG than unmodified hCG (137). Nicked hCG preparations, obtained from patients with trophoblastic disease or by enzymatic digestion of intact hCG, showed approximately 1.5- to 2-fold stimulation of recombinant hTSH receptor compared with intact hCG (122). Deglycosylation and/or desialylation of hCG enhance its thyrotropic potency. Basic hCG isoforms with lower sialic acid content extracted from hydatiform moles were more potent in activating the TSH receptor. From these and other studies it seems that the biological effect of hCG is predominantly confined to hCG containing little or no sialic acid. hCG has also been found to increase iodide uptake in cultured FRTL-5 cells and it also causes a dose related increase of adenylyl cyclase activity and thymidine uptake (138, 139).

FAMILIAL HYPERSENSITIVITY TO HCG

An unusual form of familial gestational hyperthyroidism caused by a mutant TSH receptor displaying hypersensitivity to normal levels of hCG has been identified by Rodien et al. (140). The index patient had a history of two miscarriages that were accompanied by hyperemesis. Subsequently, she had two pregnancies that were complicated by hyperthyroidism, severe nausea and vomiting. She did not have any antibodies against the TSH receptor or TPO. Her hCG levels, determined during the second pregnancy, were in the normal range for the first trimester. The patient' s mother had a history of one miscarriage and two pregnancies that were complicated by hyperemesis gravidarum. Sequence analysis of the TSH receptor gene in the proband and her mother revealed the presence of a monoallelic point mutation resulting in the substitution of K183R. Functional studies in COS-7 cells transfected with the mutated receptor documented no differences in membrane expression, and similar levels of basal and TSH stimulated cAMP accumulation. In contrast to the wild-type TSH receptor, which reacts only minimally to high doses of hCG, the K183R mutant is hypersensitive to hCG, although it still is 1000 times less responsive to hCG than the LH/CG receptor. The K183R TSH receptor mutation is unique because sensitivity is increased for hCG but remains unaltered for the cognate ligand TSH (140). This observation also supports the possibility of an hCG-independent connection between hyperthyroidism and hyperemesis gravidarum.

TROPHOBLAST TUMORS: HYDATIFORM MOLES AND CHORIOCARCINOMA

Definition and Epidemiology

Gestational trophoblastic diseases comprise hydatiform moles, invasive moles, choriocarcinomas and placental site trophoblastic tumors (141). Hydatiform moles and choriocarcinomas that secrete high amounts of hCG can cause hyperthyroidism (142). In 1955 Tisne et al. described a patient with molar pregnancy that had increased thyroidal uptake of radioactive iodine and clinical signs of hyperthyroidism (143). Earlier reports also described molar pregnancies in combination with hyperthyroidism and in all cases a rapid return to normal thyroid function occurred after removal of the mole (143). In men, choriocarcinomas can arise in the testis and cause hyperthyroidism by secreting hCG (144). In a study of 20 patients with gestational trophoblastic neoplasias, 2 patients were overtly thyrotoxic and this was confirmed by elevated serum T4 levels (145). These 2 patients had extremely high serum (3,220,000 IU/l and 6,720,000 IU/l) and urine hCG levels, which correlated closely with TSH-like activity exerted by the serum of these patients in a mouse thyroid bioassay. Patients with moderately increased serum hCG levels (110,000-310,000 IU/l) associated with trophoblastic neoplasia were euthyroid. A similar correlation between serum hCG levels and thyroid stimulating activity in both serum and urine was found in women who had widely metastatic choriocarcinoma and marked hyperthyroidism (145). In another patient with gestational choriocarcinoma serum thyroid stimulating activity correlated precisely with serum T4, with the beta subunit of hCG, and with the quantification of the tumor burden (146). Hyperthyroidism associated with choriocarcinoma in the male is extremely rare, but has been reported repeatedly (122). Orgiazzi et al. compiled four cases from the literature and reported a patient who had choriocarcinoma of the colon associated with gynecomastia and hyperthyroidism (147). Thyroid stimulating activity, measured by a mouse bioassay, was detected in the serum. Serum thyroid stimulating activity was partly inactivated by antibovine-TSH antiserum, but was completely neutralized by anti-hCG antiserum.

Clinical presentation

Most women with hydatiform moles present with uterine bleeding in the first half of pregnancy. The size of the uterus is large for the duration of gestation (141). Many women with molar pregnancies have nausea and vomiting, some have pregnancy-induced hypertension or (pre)-eclampsia. The signs and symptoms of thyrotoxicosis are present in some women, but they may be obscured by toxemic signs. The characteristic features belonging to Graves' disease are missing. The thyrotoxicosis is usually not severe because of a relatively short duration. Women with choriocarcinomas present within one year after conception. The tumor may be confined to the uterus, more frequently it is metastatic to multiple organs such as the liver and lungs, among others. In men, choriocarcinomas of the testes is often widely metastatic at initial presentation. Gynecomastia is a common finding.

Diagnosis

Measurement of serum hCG concentrations is needed for the diagnosis of moles and choriocarcinomas, and hCG serves as a sensitive and specific tumor marker during therapy and surveillance (145). In women, hCG concentrations are significantly higher than those found during normal pregnancies. Ultrasonography of the uterus shows a characteristic "snowstorm" pattern. The diagnosis of thyrotoxicosis relies on the measurement of TSH, (free) T4 and T3. elevated thyroid hormone levels. Thyroidal radioiodine uptake is elevated.

Treatment

Hydatiform moles are treated by suction rather than curettage (148). Serum T4, T3, TSH, and hCG levels normalize rapidly after removal of the mole. Choriocarcinomas can be divided into two groups: 1) a low risk group treated by monotherapy, most often with methotrexate or actinomycine D and a success rate close to 100%, and 2) a high risk group treated with polychemotherapy (etoposide, methotrexate, actinomycine D, cyclophosphamide, vincristine) with a response of about 86%. In patients that are not responding to chemotherapy, the 5-year survival rate is about 43%. Longitudinal measurement of hCG as specific and sensitive tumor marker is key for long-term surveillance (148). Placental-site trophoblastic tumors, a rare form of gestational trophoblastic disease that does not secrete hCG, requires stage-adapted management with surgery, or surgery in combination with chemotherapy (149).

STRUMA OVARII

Definition and Epidemiology

Struma ovarii is a rare tumor consisting primarily of thyroid components occurring in a teratoma or dermoid in the ovary (150). It forms less than 1% of all ovarian tumors and 2 to 4 % of all ovarian teratomas; 5 to 10% are bilateral, and 5 to 10% are malignant (151, 152). Thyrotoxicosis occurs in about 8% of affected patients (153).

Clinical presentation

The clinical presentation may include the finding of an abdominal mass, ascites, pelvic pain, and, rarely, a pseudo-Meigs syndrome with pleural effusions (154). A subset of women present with subclinical or overt thyrotoxicosis. Goiter is only presented in patients with associated thyroid disease. For example, coexistence of Graves' disease and struma ovarii has been reported (155).

Diagnosis

In patients with thyrotoxicosis, TSH is suppressed and T3 and T4 levels are elevated. Thyroglobulin is secreted by benign and malignant ovarian strumae. Radioiodine uptake will reveal uptake in the pelvis, while the uptake in the thyroid is diminished or absent (156). Cross-sectional imaging with computed tomography or magnetic resonance imaging will demonstrate of uni- or bilateral ovarian masses (156). CA125 may be elevated (154). Malignant thyroid tissue shows the characteristic patterns of papillary or follicular thyroid cancer and can be positive for mutations in BRAF (157). Metastasis is uncommon but has been reported repeatedly (158).

Treatment

Unilateral or bilateral open or laparoscopic oophorectomy is the primary therapy (159). Thyrotoxic women should be treated with antithyroid drugs and, if needed, with beta-blockers prior to surgery. In the case of malignant lesions, the patient should undergo thyroidectomy followed by treatment with 131iodine (157). The subsequent surveillance for residual or recurrent thyroid cancer does not differ from primary thyroid carcinomas.

IODINE-INDUCED HYPERTHYROIDISM

I. IODINE-INDUCED THYROTOXICOSIS

Definition and Epidemiology

An excess of iodine through dietary intake, drugs or other iodine-containing compounds can lead to thyrotoxicosis through increased thyroid hormone synthesis in the presence of underlying thyroid disease, particularly multinodular goiters that contain zones of autonomy (160, 161). Iodine-induced thyrotoxicosis (IIT) has been recognized as early as 1821 by Coindet, who reported that goitrous individuals treated with iodine developed hyperthyroidism (162). The condition is now commonly called Jod-Basedow (Jod = iodine in German; Karl von Basedow = German physician describing the signs of thyrotoxicosis associated with exophthalmos and goiter, i.e. Graves' disease) (163). IIT may occur in patients from endemic goiter areas, patients with multinodular goiters in non-endemic areas, individuals with Graves' disease, and in individuals without previously apparent thyroid disease (164). The sources of iodide leading to IIT are manifold (Table 13-2). IIT has been reported after initiating iodine supplementation, but also with the use of iodinated drugs, contrast agents, and food components (165, 166). Use of non-ionic contrast agents does not prevent the development of IIT (167).

For adults, the Dietary Reference Intake for iodine is 150 μg (168). The Tolerable Upper Intake Level for adults has been set to 1,100 μg/day and was assessed by analyzing the effect of supplementation on TSH (169). The thyroid gland needs no more than 70 μg/day to synthesize the required daily amounts of T4 and T3 (170). The higher recommended daily allowance (RDA) levels of iodine are recommended for optimal function of a number of organs such as the lactating breast, gastric mucosa, salivary glands, oral mucosa, thymus, epidermis, and the choroid plexus. The normal thyroid protects itself from acute excessive amounts of iodide by the Wolf-Chaikoff effect, which consists of an immediate reduction in iodide uptake, iodide organification, thyroid hormone biosynthesis and secretion (171). Remarkably, most individuals with a normal thyroid gland also tolerate a chronic excess of 30 mg up to 2 g iodide per day without clinical symptoms (161). Thyroid function tests remain within the reference range although T4 and T3 drop, and TSH rises (161). However, in some individuals, even exposure to modest amounts of excessive iodine can induce IIT or hypothyroidism. Fears that iodine supplementation would lead to IIT led to opposition against iodination programs in Switzerland, but initiation of salt supplementation with very low doses of iodine ( 3.75 parts per million) were shown to be safe (172). This contrasts with the observations from other iodination programs using higher amounts of iodide. For example, a steep rise of IIT has also been documented in 1966 in Tasmania (Australia), an area of iodine deficiency with a high prevalence of goiter (173). This was associated with the addition of potassium iodide to bread in early 1966. The increased incidence occurred predominantly in subjects older than 40 years, in whom a rise in incidence from 50 to a maximum of 130 cases per 100,000 was seen between 1967 and 1968. By 1974, the incidence decreased to the pre-epidemic level. Most thyrotoxic patients had nodular goiters and few patients had underlying Graves' disease. Later it was recognized that there was already a pre-epidemic increase in the incidence of thyrotoxicosis caused by the use of iodofor disinfectants on dairy farms (174, 175). Recent supplementation programs using inadequately high amounts of iodide in endemic goiter regions in Zimbabwe and eastern Zaire also resulted in a significant number of cases of severe and long-lasting IIT (176, 177). Three years after starting supplementation with iodized salt in China, the prevalence of overt hyperthyroidism in three cohorts was 1.6%, 2.0% and 1.2%, irrespective of the nutritional iodide intake, which varied between mildly deficient, adequate or excessive ((178, 179). The incidence of IIT during the first three years of supplementation was not determined. In these three communities, the cumulative 5-year incidences of overt hyperthyroidism for the 4th to 8th year of supplementation were 0.4%, 1.2% and 1.0%. At first glance, this seems to indicate a very low risk of IIT. However, the calculated 1-year incidence rates are 80, 240 and 200 per 100,000 individuals per year, figures that are much higher than the incidence rates published in other countries (180).

In some patients, iodine excess causes overt clinical hypothyroidism. Patients with a history of Graves' disease treated with radioiodine or partial thyroidectomy, partial thyroidectomy for thyroid nodules, or autoimmune thyroiditis appear to be particularly predisposed to iodine-induced hypothyroidism (181-184). Even relatively small excessive doses of 750 ug may be sufficient to induce hypothyroidism (185).

Clinical presentation

The clinical presentation includes the typical signs of thyrotoxicosis and in most patients the finding of a multinodular goiter. Other patients may have underlying autoimmune thyroid disease. A pre-existing thyroid disorder is been present in at least 20% of patients.

Diagnosis

The diagnostic considerations are the same as for toxic nodular goiters.

Treatment

Spontaneous reversal to an euthyroid state may occur after a mean period of 6 months in about 50% of patients. Return to euthyroidism may be preceded by subclinical hypothyroidism (186). In patients with multinodular goiters, the therapeutic considerations are the same as discussed for toxic nodular goiters.

Pathogenesis

In a classical study, four euthyroid patients with a single autonomous nodule from the slightly iodine-deficient Brussels region received a supplement of 500 μg iodide per day (187). This caused a slow but constant increase of thyroid hormone. After four weeks, the patients became hyperthyroid . Later studies confirmed the original interpretation that the nodules were not producing excessive amounts of thyroid hormones because of the low iodine intake, but that they became toxic once presented with high amounts of iodine (188). The autonomy of function was secondary to gain-of-function mutations in the TSH receptor (54). Individuals with multinodular goiters living in iodine-replete regions can also develop hyperthyroidism, albeit at much higher doses of iodine (up to 180 mg) (189). Taken together, these observations confirm that individuals with (multi)nodular goiters are particularly prone to developing IIT (161). In regions with iodine deficiency, nodular goiters disappear slowly after the introduction of iodine supplementation and the incidence of hyperthyroidism then gradually decreases over the years (190, 191). More recent data suggest that autonomous nodules are not the only explanation for the pathogenesis of IIT. Several human and animal studies suggest that chronic excessive iodine intake may modulate thyroid autoimmunity and lead to thyrotoxicosis in genetically susceptible individuals. Mice prone to developing autoimmune disease that are first fed an iodine-deficient diet and then switched to iodine excess develop dose-dependent ultra-structural changes in thyrocytes that are consistent with autoimmune disease (192). A necrotic effect of excessive amounts of iodide has been demonstrated in vivo in various animal species and also in human thyroid follicles in vitro (193). Epidemiologic studies performed in China, Turkey and Denmark suggest that supplementation with iodized salt increases the prevalence of autoimmune thyroid disease, resulting in clinical or subclinical hypothyroidism (178), autoimmune hyperthyroidism (194), or both (195). In a population-based, cross-sectional study with 1085 participants exposed to excessive amounts of iodide (40-100 mg/kg salt) from Brazil, the prevalence of chronic autoimmune thyroiditis was 16.9%, women were more commonly affected (21.5 versus 9.1%), 8% were hypothyroid, and 3.3% were hyperthyroid (196). The authors concluded that the excessive iodine may have increased the prevalence of autoimmune thyroid disease and hypothyroidism in this population (196). In Denmark, a moderately iodine-deficient country, introduction of iodized salt at a dose that was calculated to increase iodine intake by only 50 ug per day, an increased incidence of hyperthyroidism was found mainly in younger patients between the age of 20 and 39 years and was presumably induced by autoimmune thyroid disease (194). In contrast, a study on children in Morocco did not find an effect of iodine supplementation on thyroid autoimmunity (197). It is noteworthy that IIT was also observed in individuals who probably had normal thyroid glands (164, 186, 198). It is well recognized that contrast agents may cause IIT. They contain between 30-50% of iodine and several grams are used freqeuently for radiological studies. Individuals with multinodular goiters or subjects who live in countries where iodine intake is low are at particular risk for developing IIT secondary to exposure to contrast agents (160). Clinicians should be aware that IIT often develops several weeks after their administration. Follow-up of such patients after radiological procedures is therefore advisable and in some cases prophylactic therapy with methimazole prior to the administration of contrast agents may be indicated. Considering the wide use of contrast agents, the probability of inducing IIT by these substances appears to be relatively low. However, the incidence of IIT appears to be inversely related to the nutritional iodine intake.

II. AMIODARONE INDUCED THYROID DISEASE

Definition and Epidemiology

Amiodarone is a widely used anti-arrhythmic agent used for the therapy of ventricular and supraventricular arrhythmias, among them atrial fibrillation. It has a very high iodine content of 37.3% by weight. About 3 mg of iodine are released into the circulation per 100 mg of amiodarone ingested. For comparison, the RDA for iodide is 150 μg per day. Its molecular structure has some similarities with iodothyronines and it may interfere with thyroid hormone transport into cells and with intracellular thyroid hormone metabolism and action (Figure 13-2) (199, 200). Amiodarone interferes with 5'-monodeiodination of thyroid hormones leading to a decrease of both intra- and extracellular T3 concentrations.

Amiodarone-induced hyper- and hypothyroidism play an important role in clinical practice (200, 201). Amiodarone-induced thyrotoxicosis (AIT) is more common in iodine-deficient regions, but also occurs in patients with a normal nutritional iodine intake. Amiodarone-induced hypothyroidism is usually seen in iodine-sufficient areas.

Reported incidences of AIT vary between 0.003% and 11.5%. In a study involving 1448 patients treated with amiodarone, 30 developed AIT (164). AIT is differentiated into two forms. AIT Type I is caused by increased hormone synthesis because of exposure to high amounts of iodine, AIT Type II results from cytotoxic destruction of thyrocytes (200). Hypothyroidism occurs predominantly in patients with preexisting thyroid autoimmune disease and in areas of normal iodine intake (199, 202). AIT is more common in men than in women (203).

Clinical presentation

The signs of thyrotoxicosis are not apparent in all patients with AIT. They may be obscured by the underlying cardiac condition. Some patients have a nodular goiter.

Diagnosis

The total or free T4 levels are elevated in euthyroid, hypothyroid and hyperthyroid patients treated with amiodarone because of its inhibition of 5'-monodeiodination. In hyperthyroid patients, TSH is suppressed and T3 is elevated. The distinction between AIT Type I and II can be difficult on clinical grounds. The radioiodine uptake is typically low to normal in AIT Type I, and low to suppressed in AIT Type II. Serum interleukin 6 levels are normal to high in AIT Type I and markedly elevated in AIT Type II, but there is significant overlap and the test is of insufficient sensitivity. On Doppler ultrasound, AIT Type I is associated with normal to increased vascularity with patchy distribution, while Type II shows absent vascularity (204, 205).

Treatment

The therapy of AIT is a challenge. An algorithm for the management of patients with AIT is shown in Figure 13-3 (200). If possible, amiodarone should be discontinued. Patients with type 1 AIT are preferably treated with methimazole (initially 40-60 mg/day, followed by gradual adjustment of the dose), but the response to thionamides is modest. In selected patients, treatment with potassium perchlorate (1 g/day for 4 to 6 weeks) can be considered. Potassium perchlorate is a drug that can cause aplastic anemia and its use should be limited to patients who cannot be controlled by methimazole, or who are allergic to thionamides. For patients with AIT Type II, prednisone (0.5 - 0.7 mg /kg body weight per day) can be used for several months. Because the distinction between AIT Type I and II is difficult and not always clear, and because some patients have mixed forms of AIT, these therapies are occasionally combined. Patients with a history of AIT type II are at risk for developing hypothyroidism if exposed to high amounts of iodide.

Pathogenesis

Two distinct mechanisms result in AIT. AIT Type I results from the iodine-induced increase in thyroid hormone synthesis. Patients developing AIT Type I usually have a preexisting nodular goiter. AIT Type II is caused by cytotoxic effects of the medication that results in the release of preformed thyroid hormones.

Pathology

On electron microscopy imaging, AIT Type II shows characteristic multilamellar lysosomal inclusions and intramitochondrial glycogen inclusions, and a morphological picture of thyrocyte hyperfunction (206). No inflammatory changes are present.

THYROIDITIS

Any form of thyroiditis can be associated with a thyrotoxic phase because the disruption of thyroid follicles can result in an increased release of stored iodothyronines. The thyrotoxic phase may be followed by transient or permanent hypothyroidism.

I. ACUTE OR SUBACUTE (DE QUERVAIN'S) THYROIDITIS

Definition

This disorder, which is discussed in Chapter 19, leads to temporary thyrotoxicosis in approximately half of the patients due to discharge of stored hormone from the thyroid gland.

Clinical presentation

Patients with subacute thyroiditis often present with a history of a preceding respiratory tract infection (207). They may have fever, malaise, and soreness, and the gland is exquisitely tender on palpation and often displays a substantially increased consistency.

Diagnosis

The laboratory findings will fluctuate with the course of the disease and typically present with initial thyrotoxicosis, followed by a hypothyroid phase. The thyroid function may normalize or result in permanent underfunction. The erythrocyte sedimentation rate is markedly elevated. Thyroid antibodies are usually not detectable. The radioiodine uptake is extremely low or absent. Thyroglobulin levels are elevated because of the destruction of thyroid follicles.

Treatment

Symptomatic treatment with non-steroidal anti-inflammatory drugs (NSAID) or aspirin is often sufficient. A subset of patients needs therapy with prednisone for variable amounts of time. Addition of a beta-blocker should be considered based on the severity of the thyrotoxic signs. Therapy with levothyroxine may be necessary during the hypothyroid phase of the illness. While the majority of patients recover completely, about 10% of cases develop permanent hypothyroidism.

Pathogenesis

The pathogenesis is thought to involve a viral infection.

Pathology

Cytology and histology show characteristic giant cells.

II. SILENT OR PAINLESS THYROIDITIS

Definition and Epidemiology

Silent thyroiditis is characterized by lymphocytic infiltration and can lead to thyrotoxicosis and hypothyroidism (208). Although the terms silent thyroiditis and painless thyroiditis are used most commonly, many other names have been used for this disorder including sporadic thyroiditis (208), destructive thyroiditis (209), hyperthyroiditis (210), spontaneously resolving lymphocytic thyroiditis (211), transient painless thyroiditis (212), painless thyroiditis with transient hyperthyroidism (213), painless subacute thyroiditis (214), occult subacute thyroiditis (215), atypical thyroiditis (216) and transient thyrotoxicosis with lymphocytic thyroiditis (217). Silent thyroiditis has been diagnosed frequently in the 1970s, but its incidence seems to be lower now. A retrospective survey conducted in Wisconsin from 1963 through 1977 showed that silent thyroiditis was not found until 1969 and was uncommon up to 1973 (212). The frequency then increased and silent thyroiditis was thought to be responsible for about 20% of all cases of thyrotoxicosis in this geographical area (211). This high incidence has not been reported in other regions of the United States, Asia and Europe. In a study from Japan, an incidence of 10% was found in the 1980s, but in New York it was only 2.4% (218). Schneeberg reported data obtained from a random poll; the obtained data suggest that silent thyroiditis was uncommon in Argentina, Europe and the East- and the West coast of the United States, but occurred more frequently around the Great Lakes and in Canada (219). The variable incidence rates may be due to an ascertainment bias or to the development of thyrotoxicosis secondary to the ingestion of meat contaminated with bovine thyroid tissue. Two epidemics of thyrotoxicosis thought to reflect silent thyroiditis were found to be explained by meat contamination (220, 221). Affected patients are mostly between 30 and 60 years of age and the female to male ratio is about 1.5: 1 (211). The condition is currently rarely recognized.

Clinical presentation

Patients present with abrupt onset of thyrotoxicosis that can be associated with the development of a goiter or enlargement of a preexisting goiter. Repeated episodes may occur in the same individual (211). In a review on 112 patients, 68 were female and the age at onset was 32.4 +/- 18.5 in females and 24.9 +/- 8.2 years in males (213). None of the patients presented with thyroid pain. The duration of the thyrotoxic phase was variable, but for the most part, it lasted less than one year. The mean duration was 3.6 months (range 1-12.5). Symptoms began 2.5 - 2.2 months preceding the initial evaluation. This period is shorter than is usually seen with Graves' disease and much shorter than in patients with toxic multinodular goiter. Exophthalmos and pretibial myxedema were absent. The thyroid gland is typically firm in consistency. Forty three percent of patients had an enlarged thyroid, which was generally symmetrical and enlargement was in most instances mild. The clinical course of the disease consists usually of an initial hyperthyroid phase, followed by a hypothyroid phase, and subsequent restoration of a euthyroid metabolic state (Figure 13-4). 57 out of 112 patients became euthyroid and did not develop clinical hypothyroidism. After a brief period of euthyroidism, transient biochemical hypothyroidism developed in 17 patients. In 32 patients clinical hypothyroidism was present (213).

Development of Graves' disease, after painless thyroiditis has been documented and TSH receptor antibodies have been found in these patients (222).

Diagnosis

During the first phase of the disease, discharge of hormone from the inflamed thyroid results in increases in serum T4, T3 and a decrease in serum TSH. During this phase, there is no uptake of radioactive iodine in the thyroid. If thyrotoxicosis factitia is considered in the differential diagnosis, measurement of serum thyroglobulin levels is useful. During ingestion of levothyroxine, little or no thyroglobulin is present whereas serum thyroglobulin levels are elevated in silent thyroiditis. In 17 out of 71 patients with silent thyroiditis, moderate elevations of antithyroglobulin antibodies were present (213). Antimicrosomal antibodies were examined in 53 patients using the complement fixation test or by microsomal fluorescence. Using the former technique, 22 patients had positive antibodies, and by the latter 4 out of 7 were positive (213). In a small series of 7 patients with silent thyroiditis evaluated with a more sensitive radioimmuno assay (RIA) for human antithyroglobulin antibodies, all were positive (209). The white blood cell count is generally normal. In 53 episodes, 34 had elevated erythrocyte sedimentation rate (ESR), but it was greater than 40 mm/hr in only 8 (213). This contrasts with the typical marked elevation of the ESR in patients with subacute thyroiditis and helps to differentiate the two conditions. T4 and T3 reach subnormal levels in the hypothyroid range in 40% of patients (213). After the hypothyroid phase, patients gradually enter the euthyroid phase, heralded by an increase in thyroid hormone levels and resumption of thyroidal radioactive iodine uptake. The hypothyroid phase may last several months. In 26 episodes, patients became euthyroid after a mean period of 62 months after the onset of the hyperthyroid symptoms. TSH levels may increase during the recovery phase, and can remain elevated for many months. The delayed increase of TSH is due to its suppression during the thyrotoxic phase. Permanent hypothyroidism occurs in about 7% of patients with silent thyroiditis, but a subset of patients may ultimately become permanently hypothyroid. The echogenicity is decreased and a correlation between the decrease in the echo signal at the onset and nadir of the T3 level has been suggested (223) (Figure 13-5).

Treatment

As thyrotoxicosis is usually mild in silent thyroiditis, there is often no need for any treatment. In some patients, therapy with a beta-blocker can be considered during the thyrotoxic phase. In patients with more severe thyrotoxicosis, administration of NSAIDs and prednisone may be of benefit (224). After the thyrotoxic phase, many patients become temporarily hypothyroid and therapy with levothyroxine should be initiated in symptomatic patients. After a few months, levothyroxine therapy should be gradually withdrawn in order to assess whether the hypothyroidism is transient or permanent. Only a small proportion of patients remain permanently hypothyroid. Some patients, who initially recovered, may ultimately develop permanent thyroid failure (225). In a series of 54 patients, Nikolai et al. reported that about half of the patients developed permanent hypothyroidism (225). This is in contrast with subacute thyroiditis where permanent hypothyroidism is less common.

Pathogenesis

Although the disease was earlier considered to be a mild form of subacute (De Quervain's) thyroiditis, there is now convincing evidence that it is a lymphocytic thyroiditis (208, 209, 212-217, 222, 226). Many patients with silent thyroiditis have a personal or a family history of other autoimmune diseases, thereby indirectly supporting the concept that it is an autoimmune thyroiditis (227). There is no significant association with viral infections (211). There is a significant association with HLA genotype DR3. Postpartum thyroiditis (see below) is considered to be a form of silent thyroiditis occurring after delivery (228).

Pathology

On histological examination, follicles are disrupted and infiltrated by lymphocytes and plasma cells (211, 229). The infiltration is diffuse and/or focal, sometimes with the formation of lymphoid follicles. The follicular cells are heterogeneous in appearance. They can be cuboidal or columnar when stimulated by TSH. Some of the hypertrophic follicular cells have an oxyphilic cytoplasm (Hï¿&frac12;rthle or Ashkenazy cells). Thyroid tissue obtained during the hypothyroid or early recovery phase may show regenerating follicles with little colloid. In some patients, persistent mild lymphocytic thyroiditis is seen. Fibrosis is usually minimal, but can be extensive in some cases. Occasionally multinucleated giant cells, which are characteristic of subacute thyroiditis, are observed. The histological picture of postpartum thyroiditis is identical.

III. POSTPARTUM THYROIDITIS

Postpartum thyroiditis is considered to be a subform of silent (painless) thyroiditis (213). This condition is discussed in Chapter 14.

IV. HASHIMOTO'S THYROIDITIS

Occasionally, Hashimoto's thyroiditis is accompanied by mild symptoms of thyrotoxicosis, particularly in the early phases of the disease (230). This condition is discussed in Chapter 8.

THYROTOXICOSIS FACTITIA

Definition

Factitious thyrotoxicosis is due to the voluntary or involuntary intake of supraphysiological amounts of exogenous thyroid hormone (231). Most commonly, it is iatrogenic, either intentionally in order to suppress TSH in thyroid cancer patients or unintentionally in patients treated for primary hypothyroidism. In both instances, subclinical thyrotoxicosis is more common. The risk of atrial fibrilliation is increased in patients with long-standing suppression of TSH (232). Several cardiac parameters can be affected (233), but the severity of these effects is somewhat controversial (234). Suppressive doses of thyroid hormones can also affect bone mineral density (235), but this has not been confirmed in all studies (236). Non-iatrogenic thyrotoxicosis factitia can occur in patients of all ages with psychiatric illnesses (231, 237). In addition, some patients may take excessive amounts of thyroid hormones, sometimes prescribed by physicians, for weight loss, treatment of depression, or infertility (207). These patients often deny the intake of thyroid hormones or an excessive intake. In these instances, a heightened suspicion is needed in order to readily diagnose the disorder. Thyrotoxicosis induced by excessive thyroid hormone intake due to consumption of meat containing bovine thyroid tissue has been reported repeatedly. For example, two events of this so called  "hamburger thyyrotoxicosis" have been documented in the United States (220, 221). Inclusion of the thyroid in neck muscle trimmings is now prohibited by US Department of Agriculture regulations. Accidental dosing with veterinary levothyroxine preparations has also been reported as a cause of thyrotoxicosis factitia (238).

Clinical presentation

Patients are clinically thyrotoxic, however they do not show signs of endocrine ophthalmopathy. In patients with a history of thyroid cancer, the history and the finding of a necklace scar readily provide an explanation. The thyroid may be small because of long-standing suppression of TSH.

Diagnosis

Serum TSH is suppressed, (free) T4 and T3 levels are variably elevated. The T4 and T3 levels depend on the type of ingested thyroid hormone preparation. Both T4 and T3 are high with excessive intake of levothyroxine, while only T3 is elevated with the intake of T3 preparations. With combination therapies, the T4 and T3 increases are variable depending on the relative amounts. Poisoning with T3 may be particularly severe (239), but even very high doses are often well tolerated, especially by children (240). When the diagnosis of thyrotoxicosis factitia is suspected, measurement of serum thyroglobulin levels is useful. During ingestion of levothyroxine, little or no thyroglobulin is present. In contrast, serum thyroglobulin levels are elevated in silent thyroiditis. Mariotti et al. performed thyroglobulin measurements in 6 women with thyrotoxicosis factitia (241). They used a sensitive thyroglobulin assay and excluded the presence of thyroglobulin antibodies that can potentially interfere with the assay. In all 6 women thyroglobulin was undetectable in the serum (241). However, thyroglobulin levels may not be reliable in patients who have anti-thyroglobulin antibodies. In these patients, measurement of fecal T4 can be used to distinguish endogenous and exogenous thyroid hormone excess (242). The thyroidal uptake of radioiodine or technetium are decreased. Doppler sonography shows absent thyroidal vascularity and low-normal peak systolic velocity (243). In contrast, these signs are increased in Graves' disease (243). Thus, factitious thyrotoxicosis is not difficult to differentiate from thyrotoxic Graves' disease, toxic adenoma or toxic multinodular goiter, or subacute thyroiditis. However, it may be difficult to readily distinguish silent thyroiditis from thyrotoxicosis factitia. In both situations, radioiodine uptake is very low or absent. In silent thyroiditis the serum thyroglobulin is, however, elevated. Suppressed radioactive uptake of the thyroid gland in combination with thyrotoxicosis may also exist in patients with hyperfunctioning metastases of well-differentiated thyroid carcinomas (76, 77). However, in these extremely rare patients, the thyroglobulin levels are elevated and radioactive iodine uptake will be detected in metastases by using whole body scanning.

Treatment

In most patients, adjustment or discontinuation of the thyroid hormone preparation is sufficient to normalize thyroid function tests. Patients with surreptitious intake of thyroid hormones for eating disorders or psychiatric illnesses can be difficult to treat and may need psychiatric consultation and assistance. In patients with severe intoxication, beta-blockers can be useful. Gastric lavage, induced emesis, activated charcoal, and, very rarely, plasmapheresis and exchange transfusion, can be considered in patients seen after acute ingestion of large amounts of thyroid hormone (231).

SUMMARY

Thyrotoxicosis can has a broad spectrum of etiologies (Table I). While it is most commonly caused by Graves' disease, it is of importance to recognize other etiologies in order to choose the most appropriate therapeutic option and long-term surveillance. Toxic adenomas are characterized by a single hyperactive nodule in the thyroid leading to clinical and biochemical thyrotoxicosis. Autonomous or toxic adenomas are most commonly caused by somatic gain-of-function mutations in the TSH receptor or the stimulatory Gs alpha subunit. A toxic adenoma is readily recognized on a thyroid scan. Toxic adenomas appear to be more common in countries with a low iodine intake. The possibility of developing thyrotoxicosis in a patient with a hot nodule with a diameter of 3 cm or larger is 20% in 6 years. This risk is substantially less in smaller nodules. Older patients with a hot nodule are more likely to become toxic as compared to younger patients. Definitive treatment consists in the administration of 131iodine, surgical removal of the nodule, or, less commonly used, percutaneous ethanol injection. The likelihood of malignancy in a toxic nodule is very low. In multinodular goiters, several nodules display an autonomous function. The pathogenesis is complex but may also include activating TSH receptor mutations. In addition to hyperthyroidism, some patients present with compressive signs. The diagnostic and therapeutic approach is in general similar to patients with a toxic adenoma, but may need cross-sectional imaging and pulmonary function tests in some patients. Therapeutically, surgery and radioiodine therapy are the most commonly used modalities. Well-differentiated thyroid carcinomas are only rarely associated with thyrotoxicosis. Treatment of patients with functioning thyroid carcinomas does not differ from the therapy of thyroid cancer patients without thyrotoxicosis, but appropriate control of the hyperthyroid state with antithyroid drugs and beta-blockers is important before submitting a patient to thyroid surgery or 131iodine therapy. Familial and sporadic forms of non-autoimmune hyperthyroidism are uncommon. They are caused by inherited or de novo germline gain-of-function mutations in the TSH receptor. Inappropriate TSH secretion by a TSH-secreting pituitary tumor is a rare cause of hyperthyroidism. Transsphenoidal surgery, in combination with radiotherapy and somatostatin analogues in some patients, are the therapies of choice. During pregnancy, transient gestational thyrotoxicosis may be due to stimulation of the TSH receptor by high levels of hCG. In a single instance, a mutation in the TSH receptor conferring hypersensitivity to hCG has been reported. Hydatiform moles or a choriocarcinomas can lead to high hCG levels and thyrotoxicosis. Hydatiform moles are treated by suction. Choriocarcinomas can now be treated successfully in most patients with chemotherapy. Struma ovarii, thyroid tissue in a ovarian teratoma, rarely causes hyperthyroidism. Most patients with struma ovarii are clinically and biochemically euthyroid. Treatment consists of surgical removal of the teratoma. Administration of moderate or high doses of iodine may induce thyrotoxicosis in patients with or without apparent pre-existing thyroid disease. There are numerous sources of iodine, for example drugs, contrast agents, disinfectants, and food components. A notorious iodine-containing agent is the anti-arrhythmic drug amiodarone, which may induce thyrotoxicosis because of its high iodine content and/or a drug-induced thyroiditis. Any form of thyroiditis can be associated with a thyrotoxic phase because the disruption of thyroid follicles can result in an increased release of stored iodothyronines. The thyrotoxic phase may be followed by transient or permanent hypothyroidism. All forms of thyroiditis can be associated with a thyrotoxic phase because the disruption of thyroid follicles can result in an increased release of stored iodothyronines. The thyrotoxic phase may be followed by transient or permanent hypothyroidism. The uptake of radioiodine is very low or absent in the thyrotoxic phase and serum thyroglobulin levels are high. Clinical thyrotoxicosis is often mild and treatment with beta-blocking agents is often sufficient. Although the majority of patients recover, a substantial subset of patients develops hypothyroidism in later years. Therefore, regular assessment of thyroid function is necessary. Thyrotoxicosis factitia, the excessive intake of exogenous thyroid hormones, can be iatrogenic, or due to voluntary or involuntary intake of thyroid hormones. The uptake of radioiodine is low and thyroglobulin levels are also very low or undetectable. The thyroid may be small. The therapy consists in appropriate dose adjustment or discontinuation of exogenous thyroid hormone.

ACKNOWLEDGMENT

This chapter is, in part, based on the previous version written by Dr. Georg Hennemann. These contributions are gratefully acknowledged.

Archived

This chapter has been superceded by  newer chapters (See Homepage). However this Chapter, written originally by Dr Samuel Refetoff and updated by Drs Franklyn and Shephard, remains a treasure trove of information on many now-obscure thyroid tests, and references. For that reason we maintain it as a part of our Archive for use of MDs who may wish to investigate a bit of the history of thyroid testing. L De Groot, MD

The possibility of thyroid disease is considered when signs or symptoms suggest hyper- or hypothyroidism or some physical abnormality of the thyroid gland. Evaluation of the patient should include a thorough history and physical examination. Since most thyroid diseases require prolonged periods of treatment, it is crucial that a firm diagnosis be established before embarking on such a program. Further, a number of medications, in particular those used in the treatment of thyroid disease, may alter the results of thyroid function tests in such a way that reinvestigation after therapy has begun may provide ambiguous results.

EVALUATION BY LABORATORY TESTS

Thyroid tests can be classified into broad categories according to the information they provide at the functional, etiologic, or anatomic levels ( Table 6-1 ).

1. Tests that directly assess the level of the gland activity and integrity of hormone biosynthesis. These tests such as thyroidal radioiodide uptake and perchlorate discharge are carried out in vivo.

2. Tests that measure the concentration of thyroid hormones and their transport in blood. They are performed in vitro and provide indirect assessment of the level of the thyroid hormone dependent metabolic activity.

3. Another category of tests attempts to more directly measure the impact of thyroid hormone on peripheral tissues. Unfortunately, tests available to assess this important parameter are nonspecific, since they are often altered by a variety of nonthyroidal processes.

4. The presence of several substances, such as thyroid autoantibodies, usually absent in healthy individuals, are useful in establishing the etiology of some thyroid illnesses.

5. Invasive procedures, such as biopsy, for histological examination or enzymatic studies are occasionally required to establish a definite diagnosis. Gross abnormalities of the thyroid gland, detected by palpation, can be assessed by scintiscanning and by ultrasonography.

6.The integrity of the hypothalamo-pituitary-thyroid axis can be evaluated by (a) the response of the pituitary gland to thyroid hormone excess or deficiency; (b) the ability of the thyroid gland to respond to thyrotropin (TSH); and (c) the pituitary responsiveness to thyrotropin-releasing hormone (TRH). These tests are intended to identify the primary organ affected by the disease process that manifests as thyroid dysfunction; in other words, primary (thyroid), secondary (pituitary), or tertiary (hypothalamic) malfunction.

7.Lastly, a number of special tests will be briefly described. Some are valuable in the elucidation of the rare inborn errors of hormone biosynthesis, and others are mainly research tools.

Each test has inherent limitations, and no single procedure is diagnostically adequate for the entire spectrum of possible thyroid abnormalities. The choice, execution, application and interpretation of each test requires the understanding of thyroid physiology and biochemistry dealt with in the preceding chapters. Thyroid tests serve not only in the diagnosis and management of thyroid illnesses but also to better understand the pathophysiology underlying a specific disease.

In Vivo Tests of Thyroid Gland Activity and Integrity of Hormone Synthesis and Secretion

Common to these tests is the administration to the patient of radioisotopes that cannot be distinguished by the body from the naturally occurring stable iodine isotope (127I). In contrast to all other tests, these procedures provide a means to directly evaluate thyroid gland function. Formerly these tests were used in the diagnosis of hypothyroidism and thyrotoxicosis, but this application has been supplanted by measurement of serum TSH and thyroid hormone concentrations in blood. Also, alterations of thyroid gland activity and in handling of iodine are not necessarily coupled to the amount of hormone produced and secreted. The tests are time consuming, relatively expensive and expose the patient to irradiation. Nevertheless, they still have some speccific applications including the diagnosis of inborn errors of thyroid hormonogenesis. Administration of isotopes is required for thyroid gland scanning used to demonstrate ectopic thyroid tissue and to establish the etiology of some forms of thyrotoxicosis. Finally, measurement of the thyroidal radioiodide uptake can be used as a means for estimating the dose of radioiodide to be delivered in the therapy of thyrotoxicosis and thyroid carcinoma.

To understand the physiological basis of this category of tests, one should remember the following facts. Iodine is an integral part of the thyroid hormone molecule. Although several other tissues (salivary glands, mammary glands, lacrimal glands, the choroid plexus, and the parietal cells of the stomach) can extract iodide from blood and generate a positive tissue to serum iodide gradient, only the thyroid gland stores iodine for an appreciable period of time. ¹ Since the kidneys continually filter blood iodide, the final fate of most iodine atoms is either to be trapped by the thyroid gland or to be excreted in the urine. When a tracer of iodide is administered to the patient, it rapidly becomes mixed with the stable extrathyroidal iodide pool and is thereafter handled identically as the stable isotope. Thus, the thyroidal content of radioiodine gradually increases and that in the extrathyroidal body pool gradually declines, until virtually no free iodide is left. Normally this end point is reached between 24 and 72 hours.

From data of the radioiodide uptake by the thyroid gland and/or urinary excretion and/or stable iodide concentration in plasma and urine, the following parameters can be derived: (1) the rate of thyroidal iodine uptake (thyroid iodide clearance), (2) the fractional thyroid radioactive iodide uptake (RAIU), (3) the absolute iodide uptake (AIU) by the thyroid gland, and (4) the urinary excretion of radioiodide, or iodide clearance. After the complete removal of the administered radioiodide from the circulation, depletion of the radioisotope from the thyroid gland can be monitored by direct counting over the gland. Reappearance of the radioiodine in the circulation in protein-bound form can be measured and can be used to estimate the intrathyroidal turnover of iodine and the secretory activity of the thyroid gland.

The foregoing tests can be combined with the administration of agents known either to normally stimulate or to inhibit thyroid gland activity thus providing information on the control of thyroid gland activity. Administration of radioiodide followed by scanning allows us to examine the anatomy of functional tissue. The latter two applications of in vivo tests utilizing radioiodide will be discussed under their respective headings.

The potential hazard of irradiation resulting from the administered radioisotopes should always be kept in mind. Children are particularly vulnerable, and doses of X-rays as small as 20 rads to the thyroid gland are associated with increased risk of developing thyroid malignancies. ² However, it must be noted that there is no proven danger from isotopes used for the diagnosis of thyroid diseases. ³ In vivo administration of radioisotopes is absolutely contraindicated during pregnancy and in breast-feeding mothers because of placental transport of isotope and excretion into breast milk.

A number of radioisotopes are now available. Furthermore, provision of more sophisticated and sensitive detection devices has substantially decreased the dose required for the completion of the studies. Table 6-2 ^5-7 lists the most commonly used isotopes for in vivo studies of the thyroid. Isotopes with slower physical decay, such as 125I and 131I, are particularly suitable for long-term studies. Isotopes with faster decay, such as 123I and 132I, usually deliver a lower irradiation dose and are advantageous in short-term and repeated studies. The peak photon energy gamma emission differs among isotopes, allowing the execution of simultaneous studies with two isotopes.

Thyroidal Radioiodide Uptake (RAIU)

This is the most commonly used thyroid test requiring the administration of a radioisotope. It is usually given orally in a capsule or in liquid form and the quantity accumulated by the thyroid gland at various intervals of time is measured using a gamma scintillation counter. Correction for the amount of isotope circulating in the blood of the neck region, by subtracting counts obtained over the thigh, is of particular importance during the early periods following its administration. A dose of the same radioisotope, usually 10%, placed in a neck "phantom" is also counted as a "standard". The percentage of thyroidal radioactive iodide uptake (RAIU) is calculated from the counts cumulated per constant time unit.

The percentage of RAIU 24 hours after the administration of radioiodide is most useful, since in most instances the thyroid gland has reached the plateau of isotope accumulation, and because it has been shown that at this time, the best separation between high, normal, and low uptake is obtained. Normal values for 24-hour RAIU in most parts of North America are 5 to 30 percent. In many other parts of the world, normal values range from 15 to 50 percent. Lower normal values are due to the increase in dietary iodine intake following the enrichment of foods, particularly mass produced bread (150 µg of iodine per slice), with this element. ⁸ The inverse relationship between the daily dietary intake of iodine and the RAIU test is clearly illustrated in Figure 6-1. The intake of large amounts of iodide (>5 mg/day), mainly from the use of iodine-containing radiologic contrast media, antiseptics, vitamins, and drugs such as amiodarone, suppresses the RAIU values to a level hardly detectable using the usual equipment and doses of the isotope. Depending upon the type of iodine preparation and the period of exposure, depression of RAIU can last for weeks, months, or even years. Even external application of iodide may suppress thyroidal radioiodide uptake. The need to inquire about individual dietary habits and sources of excess iodide intake is obvious.

Figure 6-1. Relation of 24 hour thyroidal radioiodide (I131) uptake (RAIU) to dietary content of stable iodine (I12 7 ). The uptake increases with decreasing dietary iodine. With iodine intake below the amount provided from thyroid hormone degradation, the latter contributes a larger proportion of the total iodine taken up by the thyroid. Under current dietary habits in the United States, the average 24-hour thyroidal RAIU is below 20 percent.

The test does not measure hormone production and release but merely the avidity of the thyroid gland for iodide and its rate of clearance relative to the kidney. Disease states resulting in excessive production and release of thyroid hormone are most often associated with increased thyroidal RAIU and those causing hormone underproduction with decreased thyroidal RAIU (Figure 6-2, below). Important exceptions include high uptake values in some hypothyroid patients and low values in some hyperthyroid patients. Increased thyroidal RAIU with hormonal insufficiency co-occur in the presence of severe iodide deficiency and in the majority of inborn errors of hormonogenesis (see Chapter 20 and 16 ). In the former, lack of substrate, and in the latter, a specific enzymatic block of hormone synthesis cause hypothyroidism poorly compensated by TSH-induced thyroid gland overactivity. Decreased thyroidal RAIU with hormonal excess is typically encountered in the syndrome of transient thyrotoxicosis (both de Quervain's and painless thyroiditis), ⁹ ingestion of exogenous hormone (thyrotoxicosis factitia), iodide-induced thyrotoxicosis (Jod-Basedow disease), ¹⁰ and in patients with thyrotoxicosis on moderately high intake of iodide (see Table 6-3 ). High or low thyroidal RAIU as a result of low or high dietary iodine intake, respectively, may not be associated with significant changes in thyroid hormone secretion.

Figure 6-2. Examples of thyroidal RAIU curves under various pathological conditions. Note the prolonged uptake in renal disease due to decreased urinary excretion of the isotope and the early decline in thyroidal radioiodide content in some patients with thyrotoxicosis associated with a small but rapidly turning over intrathyroidal iodine pool.

Various factors including diseases that affect the value of the 24-hour thyroidal RAIU are listed in Table 6-3 . Several variations of the test have been devised which have particular value under special circumstances. Some of these are briefly described.

Early Thyroid RAIU and 99mPertechnetate Uptake Measurements

In some patients with severe thyrotoxicosis and low intrathyroidal iodine concentration, the turnover rate of iodine may be accelerated causing a rapid initial uptake of radioiodide, reaching a plateau before 6 hours, followed by a decline through release of the isotope in hormonal or other forms (Figure 6-2, above). Although this phenomenon is rare, some laboratories choose to routinely measure early RAIU, usually at 2, 4 or 6 hours. Early measurements require the accurate determination of background activity contributed by the circulating isotope. Radioisotopes with a shorter half-life, such as 123I and 132I, are more suitable.

Since thyroidal uptake in the very early period following administration of radioiodide reflects mainly iodide trapping activity, 99mTc as the pertechnetate ion (99mTcO4-) may be used. In euthyroid patients, thyroid trapping is maximal at about 20 minutes and is approximately 1% of the administered dose ¹¹ . This test, when coupled with the administration of T3, can theoretically be used to evaluate thyroid gland suppressibility in thyrotoxic patients treated with antithyroid drugs (see below).

Perchlorate Discharge Test

This test is used to detect defects in intrathyroidal iodide organification. It is based on the following physiological principle. Iodide is "trapped" by the thyroid gland through an energy-requiring active transport mechanism. Once in the gland, it is rapidly bound to thyroglobulin and retention no longer requires active transport. Several ions, such as thiocyanate (SCN-) and perchlorate (ClO4-), inhibit active iodide transport and cause the release of the intrathyroidal iodide not bound to thyroid protein. Thus, measurement of intrathyroidal radioiodine loss following the administration of an inhibitor of iodide trapping would indicate the presence of an iodide-binding defect.

In the standard test, epithyroid counts are obtained at frequent intervals (every 10 or 15 minutes) following the administration of radioiodide. Two hours later, 1g of KClO4 is administered orally and repeated epithyroid counts continue to be obtained for an additional 2 hours. In normal individuals, radioiodide accumulation in the thyroid gland ceases after the administration of the iodide transport inhibitor but there is little loss of the thyroidal radioactivity accumulated prior to induction of the "trapping" block. A loss of 5% percent or more indicates an organification defect (see Chapter 16 ). ¹² The severity of the defect is proportional to the extent of radioiodide discharged from the gland and is complete when virtually all the activity accumulated by the gland is lost (see Fig. 16-2, below). The test is positive in the inborn defect of iodide organification, which can be associated with deafness (Pendred's syndrome), during the administration of iodide organification blocking agents, in many patients with thyroiditis, or following treatment with radioactive iodide.

Figure 6-2. Examples of thyroidal RAIU curves under various pathological conditions. Note the prolonged uptake in renal disease due to decreased urinary excretion of the isotope and the early decline in thyroidal radioiodide content in some patients with thyrotoxicosis associated with a small but rapidly turning over intrathyroidal iodine pool.

Measurement of Hormone Concentration and Other Iodinated Compounds and Their Transport in Blood

Measurements of T4 and T3 in serum and the estimation of their free concentration have become the most commonly used tests for the evaluation of the thyroid hormone-dependent metabolic status. This approach results from the development of simple, sensitive, and specific methods for measuring these iodothyronines and because of the lack of specific tests for the direct measurement of the metabolic effect of these hormones. Other advantages are the requirement of only a small blood sample and the large number of determinations that can be completed by a laboratory during a regular workday.

The thyroid gland is the principal source of all hormonal iodine-containing compounds or their precursors and peripheral tissue are the source of the products of degradation. Their chemical structures, and normal concentrations in serum are given in Figure 6-3. It is important to note that the concentration of each substance is dependent not only upon the amount synthesized and secreted but also upon its affinity for carrier serum proteins, distribution in tissues, rate of degradation, and finally, clearance.

Figure 6-3: Iodine-containing compounds in serum of healthy adults. a. Iodothyronine concentration in the euthyroid population are not normally distributed. Thus, calculation of the normal range on the basis of 95% confidence limits for a Gaussian distribution is not accurate. b. Significant decline with old age. c. Probably an overestimation due to cross-reactivity by related substances.

The main secretory product of the thyroid gland is t4t3 being next in relative abundance. Both compounds are metabolically active when administered vivo. They synthesized and stored as a part larger moleculethyroglobulin.

¹⁸ Under normal circumstances, only minute amounts of Tg escape into the circulation. On a molar basis, it is the least abundant iodine-containing compound in blood. With the exception of T4, Tg, and small amounts of DIT and MIT, all other iodine-containing compounds found in the serum of normal man are produced mainly in extrathyroidal tissues by a stepwise process of deiodination of T4. ¹⁹ An alternative pathway of T4 metabolism that involves deamination and decarboxylation but retention of the iodine residues gives rise to TETRAC and TRIAC. ^20,21 Conjugation to form sulfated iodoproteins also occurs. ²² Circulating iodoalbumin is generated by intrathyroidal iodination of serum albumin. ²³ Small amounts of iodoproteins may be formed in peripheral tissues ²⁴ or in serum ^25,26 by covalent linkage of T4 and T3 to soluble proteins. Although the physiological function of circulating iodine compounds other than T4 and T3 remains unknown, measurement of changes in their concentration is of research interest.

Measurement of Total Thyroid Hormone Concentration in Serum

Iodometry. Iodine constitutes an integral part of the thyroid hormone molecule. It is thus not surprising that determination of iodine content in serum was the first method suggested almost six decades ago for the identification and quantitation of thyroid hormone. ²⁷ Measurement of the Protein-Bound Iodine (PBI) was the earliest method used routinely for the estimation of thyroid hormone concentration in serum. This test measured the total quantity of iodine precipitable with the serum proteins, ²⁸ 90% of which is T4. The normal range was 4 - 8 µg I/dl of serum.

Efforts to measure serum thyroid hormone levels with greater specificity and with lesser interference from nonhormonal iodinated compounds, led to the development of the butanol extractable iodine (BEI) and T4I by column techniques. All such chemical methods for the measurement of thyroid hormone in serum have been replaced by the ligand assays which are devoid of interference by even large quantities of nonhormonal iodine-containing substances.

Radioimmunoassays. Concentrations of thyroid hormones in serum can be measured by radioimmunoassays (RIA). The principle of these assays is the competition of a hormone (H), being measured, with the same isotopically labeled compound (H*) for binding to a specific class of IgG molecules present in the antiserum [antibody (Ab)]. H is the ligand and the Ab is either a polyclonal antiserum to H or a monoclonal IgG. The reaction obeys the law of mass action. Thus, at equilibrium, the amount of H* bound to Ab to form the complex Ab-H* is inversely proportional to the concentration of H, forming the complex Ab-H, provided the amounts of Ab and H* are kept constant.

AbH* + [H] AbH + H*

The radioisotope content in Ab-H* or in the unbound (free) H* is determined after their separation by precipitation of the antibody-ligand complex or adsorption of the free ligand. Some RIAs are carried out with the Ab fixed to a solid support, reacting with H and H* in solution. Increments of known amounts of H are added to a series of reactions to construct a standard curve that describes the curvilinear stoichiometric relationship between Ab-H* and H. It can be converted to a straight line by a number of mathematical transformations, such as the logit-log plot. Blank reactions contain H* but not specific Ab or, a large excess of H in a full reaction. ²⁹ The sensitivity of the assay is dependent upon the affinity of the Ab and specific activity of H*. Under optimal conditions, as little as 1 pg of H can be measured.

In assays for thyroid hormones, the hormone needs to be liberated from serum binding proteins, mainly TBG. Methods to achieve this include extraction, competitive displacement of the hormone being measured, or inactivation of thyroxine-binding globulin (TBG). ^31-34 Rarely, some patients develop circulating antibodies against thyronines that interfere with the RIA carried out on unextracted serum samples. Depending on the method used for the separation of bound from free ligand, values obtained may be either spuriously low or high in the presence of such antibodies. ^38,39

A wide choice of commercial kits is available for most RIA procedures, making these assays accessible to all medical centers. RIAs have been adapted for the measurement of T4 in small samples of dried blood spots on filter paper and are used in screening for neonatal hypothyroidism. ⁴⁰

Non-radioactive Methods. More recently, assays have been developed that are based on the principle of the radioligand assay but do not use radioactive material. These assays, which use ligand conjugated to an enzyme have largely replaced RIAs. The enzyme-linked ligand competes with the ligand being measured for the same binding sites on the antibody. Quantitation is carried out by spectrophotometry of the color reaction developed after the addition of the enzyme substrate. ⁴² Both homogeneous [enzyme-multiplied immunoassay technique (EMIT)] and heterogeneous [enzyme-linked immunosorbent assay (ELISA)] assays for T4 have been developed. ^43-45 In the homogeneous assays, no separation step is required, thus providing easy automation. ⁴³ In one such assay, T4 is linked to malate dehydrogenase, inhibiting the enzyme activity. The enzyme is activated when the T4-enzyme conjugate is bound to T4-specific antibody. Active T4 conjugates to other enzymes, such as peroxidase ⁴⁴ and alkaline phosphatase, ⁴⁵ have also been developed. The assay has been adapted for the measurement of T4 in dried blood samples used in mass screening programs for neonatal hypothyroidism. ⁴⁵ Other non-radioisotope immunoassays use fluorescence excitation for detection of the labeled ligand, a technique which is finding increasing application. Such assay methods utilize a variety of chemiluminescent molecules such as 1,2-dioxetanes, luminol and derivatives, acridinium esters, oxalate esters and firefly luciferins, as well as many sensitizers and fluorescent enhancers. ^45a One such assay which employes T4 conjugated to ß-galactosidase and fluoresence measurements of the hydrolytic product of 4-methyl-umbelliferyl-ßD-galactopyranoside has been adapted for use in a microanalytical system requiring only 10µl of serum. ^45b

Serum Total Thyroxine (TT4). The usual concentration of TT4 in adults ranges from 5 to 12 µg/dl (64 - 154 nmol/L). When concentrations are below or above this range in the absence of thyroid dysfunction, they are usually the result of an abnormal level of serum TBG. The hyperestrogenic state of pregnancy and administration of estrogen-containing compounds are the most common causes of a significant elevation of serum TT4 levels in euthyroid persons. Less commonly, TBG excess is inherited. ⁵⁰ Serum TT4 is virtually undetectable in the fetus until midgestation. Thereafter, it rapidly increases, reaching high normal adult levels during the last trimester. A further acute but transient rise occurs within hours after delivery. ⁵¹ Values remain above the adult range until 6 years of age, but subsequent age related changes are minimal so that in clinical practice, the same normal range of TT4 applies to both sexes and all ages.

Small seasonal variations and changes related to high altitude, cold, and heat have been described. Rhythmic variations in serum TT4 concentration are of two types: variations related to postural changes in serum protein concentration ⁵⁶ and true circadian variation. ³¹ Postural changes in protein concentration do not alter the free T4 (FT4) concentration.

Although levels of serum TT4 below the normal range are usually associated with hypothyroidism, and above this range with thyrotoxicosis, it must be remembered that the TT4 level may not always correspond to the FT4 concentration which represents the metabolically active fraction (see below). The TT4 concentration in serum may be altered by independent mechanisms: (1) an increase or decrease in the supply of T4 , as seen in most cases of thyrotoxicosis and hypothyroidism, respectively; (2) changes due solely to alterations in T4 binding to serum proteins; and (3) compensatory changes in serum TT4 concentration due to high or low serum levels of T3. Conditions associated with changes in serum TT4 and their relationship to the metabolic status of the patient are listed in Table 6-4.

Serum TT4 levels are low in conditions associated with decreased TBG concentration, the presence of abnormal TBG's with reduced binding affinity (see Chapter 16 ) or when the available T4-binding sites on TBG are partially saturated by competing drugs present in blood in high concentrations (see Table 5-2 ). Conversely, TT4 levels are high when the serum TBG concentration is high. The person remains euthyroid provided the feedback regulation of the thyroid gland is intact.

Although changes in transthyretin (TTR) concentration rarely give rise to significant alterations in TT4 concentration, ⁵⁷ the presence of a variant serum albumin with high affinity for T4 ^58,59 or antibodies against T4 ^38,39 produce apparent elevations in the measured TT4 concentration, whereas the metabolic status remain normal. The variant albumin is inherited as an autosomal dominant trait termed familial dysalbuminemic hyperthyroxinemia (FDH) (see Chapter 16 ).

Another possible cause of discrepancy between the observed serum TT4 concentration and the metabolic status of the patient is divergent changes in the serum TT3 and TT4 concentrations with alterations in the serum T3/T4 ratio. The most common situation is that of elevated TT3 concentration. The source of T3 may be endogenous, as in T3 thyrotoxicosis, or exogenous, as during ingestion of T3. In the former situation, contrary to the common variety of thyrotoxicosis, elevation in the serum TT3 concentration is not accompanied by an increase in the TT4 level. In fact, the serum TT4 level is normal and occasionally low. ⁶⁰ This finding indicates that in T3 thyrotoxicosis the hormone is predominantly secreted as such rather than arising from the peripheral conversion of T4 to T3. Ingestion of pharmacologic doses of T3 results in thyrotoxicosis associated with severe depression of the serum TT4 concentration. A moderate hypersecretion of T3 can be associated with euthyroidism and a low serum TT4 concentration. This circumstance, occasionally referred to as T3 euthyroidism, may be more prevalent than T3 thyrotoxicosis. It is believed to constitute a state of compensatory T3 secretion as a physiologic adaptation of the failing thyroid gland, such as after treatment for thyrotoxicosis, in some cases of chronic thyroiditis, or during iodine deprivation. ^61,62 Serum TT4 concentration is also low in normal persons receiving replacement doses of T3. Conversely, serum TT4 levels are above the upper limit of normal in 15-50% of patients treated with exogenous T4. ⁶³ Because of the relatively slow rate of metabolism and large extrathyroidal T4 pool, the serum concentration of the hormone varies little with the time of sampling in relation to ingestion of the daily dose. ⁶⁴

Serum Total Triiodothyronine (TT3). Normal serum TT3 concentrations in the adult are 80-190 ng/dl (1.2 - 2.9 nmol/L). While sex differences are small, those with age are more dramatic. In contrast to serum TT4, TT3 concentration at birth is low, about one-half the normal adult level. It rises within 24 hours to about double the normal adult value followed by a rapid decrease over the subsequent 24 hours to a level in the upper adult range, which persists for the first year of life. ⁵¹ A decline in the mean TT3 level has been observed in old age, although not in healthy subjects. ^52,53 so that a fall in TT3 may refelct the prevalence of nonthyroidal illness rather than to age alone. ⁶⁷ Although a positive correlation between serum TT3 level and body weight has been observed, it may be related to overeating. ⁶⁸ Rapid and profound reductions in serum TT3 level can be produced within 24-48 hours of total calorie or only carbohydrate deprivation. ^69-71

Most conditions causing serum TT4 levels to increase are associated with high TT3 concentrations. Thus, serum TT3 levels are usually elevated in thyrotoxicosis and reduced in hypothyroidism. However, in both conditions the TT3/TT4 ratio is elevated relative to normal euthyroid persons. This elevation is due to the disproportionate increase in serum TT3 concentration in thyrotoxicosis and a lesser diminution in hypothyroidism relative to the TT4 concentration. ⁷² Accordingly, measurement of the serum TT3 level is a more sensitive test for the diagnosis of hyperthyroidism, and that of TT4 more useful in the diagnosis of hypothyroidism.

There are circumstances in which changes in the serum TT3 and TT4 concentrations are either disproportionate or in opposite direction ( Table 6-5 ). These include the syndrome of thyrotoxicosis with normal TT4 and FT4 levels (T3 thyrotoxicosis). In some patients, treatment of thyrotoxicosis with antithyroid drugs may normalize the serum TT4 but not TT3 level, producing a high TT3/TT4 ratio. In areas of limited iodine supply ⁶² and in patients with limited thyroidal ability to process iodide, ⁶¹ euthyroidism can be maintained at low serum TT4 and FT4 levels by increased direct thyroidal secretion of T3. Although these changes have a rational physiologic explanation, the significance of discordant serum TT4 and TT3 levels under other circumstances is less well understood.

The most common cause of discordant serum concentrations of TT3 and TT4 is a selective decrease of serum TT3 due to decreased conversion of T4 to T3 in peripheral tissues. This reduction is an integral part of the pathophysiology of a number of nonthyroidal acute and chronic illnesses and calorie deprivation (see Chapter 5 ). In these conditions, the serum TT3 level is often lower than that commonly found in patients with frank primary hypothyroidism. Yet, these persons do not present clear clinical evidence of hypometabolism. In some individuals, decreased T4 to T3 conversion in the pituitary gland ⁷⁵ or in peripheral tissues ⁷⁶ is thought to be an inherited condition.

A variety of drugs may also produce changes in the serum TT3 concentration without apparent metabolic consequences (see Chapter 6 ). Drugs that compete with hormone binding to serum proteins decrease serum TT3 levels, generally without affecting the free T3 concentration ( Table 5-5 ). Some drugs, such as glucocorticoids, ⁷⁷ depress the serum TT3 concentration by interfering with the peripheral conversion of T4 to T3. Others, such as phenobarbital, ⁷⁸ depress the serum TT3 concentration by stimulating the rate of intracellular hormone degradation. The majority have multiple effects. These effects are combinations of those described above, as well as inhibition of the hypothalamic-pituitary axis or thyroidal hormonogenesis. ⁷⁹

Changes in serum TBG concentration have an effect on the serum TT3 concentration similar to that on TT4 (see Chapter 16 ). The presence of endogenous antibodies to T3 may result in apparent elevation of the serum TT3 but as in the case of high TBG, it does not cause hypermetabolism. ³⁸

Administration of commonly used replacement doses of T3, usually in the order of 75 µg/day or 1 µg/kg body weight per day, ⁸⁰ results in serum TT3 levels in the thyrotoxic range. Furthermore, because of the rapid gastrointestinal absorption and relatively fast degradation rate, the serum level varies considerably according to the time of sampling in relation to hormone ingestion. ⁶⁴

Measurement of Total and Unsaturated Thyroid Hormone-Binding Capacity in Serum

Because the concentration of thyroid hormone in serum is dependent on its supply as well as on the abundance of hormone-binding sites on serum proteins, the estimation of the latter has proved useful in the correct interpretation of values obtained from the measurement of the total hormone concentration. These results have been used to provide an estimate of the free hormone concentration, which is important in differentiating changes in serum total hormone concentration due to alterations of binding proteins in euthyroid patients from those due to abnormalities in thyroid gland activity giving rise to hypermetabolism or hypometabolism.

In Vitro Uptake Tests: In vitro uptake tests measure the unoccupied thyroid hormone-binding sites on TBG. They use labeled T3 or T4 and some form of synthetic absorbent to measure the proportion of radiolabeled hormone that is not tightly bound to serum proteins. Because ion exchange resins are often used as absorbents, the test became known as the resin T3 or T4 uptake test (T3U or T4U), describing the technique rather than the entity measured.

The test is usually carried out by incubating a sample of the patient's serum with a trace amount of labeled T3 or T4. The labeled hormone, not bound to available binding sites on TBG present in the serum sample, is absorbed onto an anion exchange resin and measured as resin-bound radioactivity. Values correlate inversely with the concentration of unsaturated TBG. Various methods use different absorbing materials to remove the hormone not tightly bound to TBG. ⁸³ Labeled T3 is usually used because of its less firm yet preferential binding to TBG. Depending upon the method, typical normal results for T3U are 25-35% or 45-55%. Thus, it is more valuable to express results of the uptake tests as a ratio of the result obtained in a normal control serum run in the same assay as the test samples. Normal values will then range on either side of 1.0, usually 0.85-1.15.

The uptake of the tracer by the absorbent is inversely proportional to the amount of unsaturated binding sites (unoccupied by endogenous thyroid hormone) in serum TBG. Thus, the uptake is increased when the amount of unsaturated TBG is reduced as a result of excess endogenous thyroid hormone or a decrease in the concentration of TBG. In contrast, the uptake is decreased when the amount of unsaturated TBG is increased as a result of a low serum thyroid hormone concentration or an increase in the concentration of TBG. Since the test can be affected by either or both independent variables, serum total thyroid hormone and TBG concentrations, the results cannot be interpreted without knowledge of the hormone concentration. As a rule, parallel increases or decreases in both serum TT4 concentration and the T3U test indicate hyperthyroidism and hypothyroidism, respectively, whereas discrepant changes in serum TT4 and T3U suggest abnormalities in TBG binding. However, abnormalities in hormone and TBG concentrations may coexist in the same patient. For example, a hypothyroid patient with a low TBG level will typically show a low TT4 level and normal T3U result (Figure 6-4). Several nonhormonal compounds, due to structural similarities, compete with thyroid hormone for its binding site on TBG. Some are used as pharmacologic agents and may thus alter the in vitro uptake test as well as the total thyroid hormone concentration in serum. A list is provided in Table 5-2 .

Figure 6-4. Graphic representation of the relationship between the serum total T4 concentration, the RT3U test, and the free T4 (FT4) concentration in various metabolic states and in association with changes in TBG. The principle of communicating vessels is used as an illustration. The height of fluid in the small vessel represents the level of FT4; the total amount of fluid in the large vessel, the total T4 concentration; and the total volume of the large vessel, the TBG capacity. Dots represent resin beads and black dots, those carrying the radioactive T3 tracer (T3*). The RT3U test result (black dots) is inversely proportional to the unoccupied TBG binding sites represented by the unfilled capacity of the large vessel. (From S. Refetoff, Endocrinology, L.J. DeGroot (ed). 1979, Grune & Straton Inc.)

TBG and TTR Measurements.

The concentrations of TBG and TTR in serum can be either estimated by measurement of their total T4-binding capacity at saturation or more usually measured directly by immunologic techniques. ^87,88

TBG concentration in serum can be determined by RIA, ⁸⁸ and both TBG and TTR can be measured by Laurell's rocket immunoelectrophoresis, ^89,90 by radial immunodiffusion, ⁹¹ or by enzyme immunoassay; ⁸⁷ commercial methods are available. The true mean value for TBG is 1.6 mg/dl (260 nmol/L), with a range of 1.1 - 2.2 mg/dl(180 - 350 nmol/L) serum. In adults, the normal range for TTR is 16 - 30 mg/dl (2.7 - 5.0 µmol/L). The concentrations of TBG and TTR in serum vary with age, sex, pregnancy, and posture. Determination of the concentration of these proteins in serum is particularly helpful in evaluation of extreme deviations from normal, as in congenital abnormalities of TBG. In most instances, however, the in vitro uptake test, in conjunction with the serum TT4 level, gives an approximate estimation of the TBG concentration.

Estimation of Free Thyroid Hormone Concentration

A minute amount of thyroid hormone circulates in the blood in a free form, not bound to serum proteins. It is in reversible equilibrium with the bound hormone and represents the diffusible fraction of the hormone capable of traversing cellular membranes to exert its effects on body tissues. ⁹⁴ Although changes in serum hormone-binding proteins affect both the total hormone concentration and the corresponding fraction circulating free, in the euthyroid person the absolute concentration of free hormone remains constant and correlates with the tissue hormone level and its biologic effect. Information concerning this value is probably the most important parameter in the evaluation of thyroid function as it relates to the metabolic status of the patient.

With few exceptions, the free hormone concentration is high in thyrotoxicosis, low in hypothyroidism, and normal in euthyroidism even in the presence of profound changes in TBG concentration, ⁹⁷ provided the patient is in a steady state (see Fig. 5-4). Notably, free T4 (FT4) concentration may be normal or even low in patients with T3 thyrotoxicosis and in those ingesting pharmacologic doses of T3. On occasion, the concentration of FT4 may be outside the normal range in the absence of an apparent abnormality in the thyroid hormone-dependent metabolic status. This is frequently observed in severe nonthyroidal illness during which both high and low values have been reported. ^98-100 As expected, when a euthyroid state is maintained by the administration of T3 or by predominant thyroidal secretion of T3, the FT4 level is also depressed. More consistently, patients with a variety of nonthyroidal illnesses have low FT3 levels. ¹⁰¹ This decrease is characteristic of all conditions associated with depressed serum TT3 concentrations due to a diminished conversion of T4 to T3 in peripheral tissues (see Chapter 5 ). Both FT4 and FT3 values may be out of line in patients receiving a variety of drugs (see below). Marked elevations in both FT4 and FT3 concentrations in the absence of hypermetabolism are typical of patients with resistance to thyroid hormone (see Chapter 16 ). The FT3 concentration is usually normal or even high in hypothyroid persons living in areas of severe endemic iodine deficiency. Their FT4 levels are, however, normal or low. ⁶²

Direct Measurement of Free T4 and Free T3. Direct measurements of the absolute FT4 and FT3 concentrations are technically difficult and have, until recently, been limited to research assays. In order to minimize perturbations of the relationship between the free and bound hormone, these must be separated by ultrafiltration or by dialysis involving minimal dilution and little alteration of the pH or electrolyte composition. The separated free hormone is then measured directly by radioimmunoassay or chromatography. ^97,97a These assays are probably the most accurate available, but small, weakly bound, dialyzable substances or drugs may be removed from the binding proteins and the free hormone concentration measured in their presence may not fully reflect the free concentration in vivo.

Isotopic Equilibrium Dialysis. This method has been the "gold standard" for the estimation of the FT4 or FT3 concentration for almost 30 years. It is based on the determination of proportion of T4 or T3 that is unbound, or free, and is thus able to diffuse through a dialysis membrane, i.e., the dialyzable fraction (DF). To carry out the test, a sample of serum is incubated with a tracer amount of labeled T4 or T3. The labeled tracer rapidly equilibrates with the respective bound and free endogenous hormones. The sample is then dialyzed against buffer at a constant temperature until the concentration of free hormone on either side of the dialysis membrane has reached equilibrium. The DF is calculated from the proportion of labeled hormone in the dialysate. The contribution from radioiodide present as contaminant in the labeled tracer hormone should be eliminated by purification ⁹⁸ and by various techniques of precipitation of the dialyzed hormone.102 FT4 and FT3 levels can be measured simultaneously by addition to the sample of T4 and T3 labeled with two different radioiodine isotopes.103 Ultrafiltration is a modification of the dialysis technique. ⁹⁸ Results are expressed as the fraction (DFT4 or DFT3) or percent (%FT4 or %FT3) of the respective hormones which dialyzed and the absolute concentrations of FT4 and FT3 are calculated from the product of the total concentration of the hormone in serum and its respective DF. Typical normal values for FT4 in the adult range from 1.0 to 3.0 ng/dl (13 - 39 pmol/L) and for FT3 from 0.25 to 0.65 ng/dl (3.8 - 10 nmol/L).

Results by these techniques are generally comparable to those determined with the direct, one step, methods (see below) but are more likely to differ with extremely low or extremely high TBG concentrations or in the presence of circulating inhibitors of protein binding, especially in situations of non-thyroidal illness. ^{104, 104a,104b} The measured DF may be altered by the temperature at which the assay is run, the degree of dilution, the time allowed for equilibrium to be reached and the composition of the diluting fluid. ¹⁰⁵ The calculated value is dependent on an accurate measurement of total T4 or T3 and may be incorrect in patients with T4 or T3 autoantibodies. Some of these problems, particularly those arising from dilution, may be superceded by commercially available dialysis methods or ultrafiltration methods of free from bound hormone which do not necessitate serum dilution.

Index Methods. As the determination of free hormone by equilibrium dialysis is cumbersome and technically demanding, many clinical laboratories have used a method by which a free T4 index (FT4I) or free T3 index (FT3I) is derived from the product of the TT4 or TT3 (determined by immunoassay) and the value of an in vitro uptake test (see below). While not always in agreement with the values obtained by dialysis, these techniques are rapid and simple. They are more likely to fail at extremely low or extremely high TBG concentrations, in the presence of abnormal binding proteins, in the presence of circulating inhibitors of protein binding , and their reliability has been questioned in patients with non-thyroidal illness.

The theoretical contention that the FT4I is an accurate estimate of the absolute FT4 concentration can be confirmed by the linear correlation between these two parameters. This is true provided results of the in vitro uptake test (T3U or T4U) are expressed as the thyroid hormone binding ratio (THBR), determined by dividing the tracer counts bound to the solid matrix by counts bound to serum proteins. ¹⁰⁶ Values are corrected for assay variations using appropriate serum standards and are expressed as the ratio of a normal reference pool. ^106,107 The normal range is slightly narrower than the corresponding TT4 in healthy euthyroid patients with a normal TBG concentration. It is 6.0 - 10.5 µg/dl or 77 - 135 nmol/l when calculated from TT4 values measured by RIA. In thyrotoxicosis, FT4I is high and in hypothyroidism it is low irrespective of the TBG concentration. Euthyroid patients with TT4 values outside the normal range as a result of TBG abnormalities have a normal FT4I. ⁸³ Lack of correlation between the FT4I and the metabolic status of the patient has been observed under the same circumstances as those described for similar discrepancies when the FT4 concentration was measured by dialysis.

Methods for the estimation of the FT3I are also available ¹⁰³ but are rarely used in routine clinical evaluation of thyroid function. Like the FT4I, it correlates well with the absolute FT3 concentration. The test corrects for changes in TT3 concentration resulting from variations in TBG concentration.

Estimation of FT4 and FT3 Based on TBG Measurements. Since most T4 and T3 in serum are bound to TBG, their free concentration can be calculated from their binding affinity constants to TBG and molar concentrations of hormones and TBG. ^109,110 A simpler calculation of the T4/TBG and T3/TBG ratios yields values that are similar to but less accurate than the FT4I and FT3I, respectively. ¹⁰⁶

Two-step Immunoassays. In these assays, the free hormone is first immunoextracted by a specific bound antibody (first step), frequently fixed to the tube (coated tube). ^111,112 After washing, labeled tracer is added and allowed to equilibrate between the unoccupied sites on the antibody and those of serum thyroid hormone-binding proteins. The free hormone concentration will be inversely related to the antibody bound tracer and values are determined by comparison to a standard curve. Values obtained with this technique are generally comparable to those determined with the direct methods. They are more likely to differ in the presence of circulating inhibitors of protein binding and in sera from patients with non-thyroidal illness.

Analog (One-Step) Immunoassays. In these assays, a labeled analog of T4 or T3 directly competes with the endogenous free hormone for binding to antibodies. ¹¹³ In theory, these analogs are not bound by the thyroid hormone binding proteins in serum. However, various studies have found significant protein binding to the variant albumin-like protein, ^113a to transthyretin and to iodothyronine autoantibodies. ¹¹⁴ This results in discrepant values to other assays in a number of conditions including non-thyroidal illness, pregnancy and in individuals with familial dysalbuminemic hyperthyroxinemia (FDH). ^113a A growing number of commercial kits is available some of which have been modified to minimize these problems, ^113b . Nonetheless, their accuracy remains controversial, although such comercial methods are being increasingly adopted in the routine clinical chemistry laboratory. ¹¹²

Considerations in Selection of Methods for the Estimation of Free Thyroid Hormone Concentration. None of the available methods for the estimation of the free hormone concentration in serum is infallible in the evaluation of the thyroid hormone-dependent metabolic status. Each test possesses inherent advantages and disadvantages depending upon specific physiologic and pathologic circumstances. For example, methods based on the measurement of the total thyroid hormone and TBG concentrations cannot be used in patients with absent TBG due to inherited TBG deficiency. Under such circumstances, the concentration of free thyroid hormone is dependent upon the interaction of the hormone with serum proteins that normally play a negligible role (TTR and albumin). When alterations of thyroid hormone binding do not equally affect T4 and T3, discrepant results of FT4I are obtained when using labeled T4 or T3 in the in vitro uptake test. For example, euthyroid patients with the inherited albumin variant (FDH) or having endogenous antibodies with greater affinity for T4 will have high TT4 but a normal T3U test which will result in an overestimation of the calculated FT4I. In such instances, calculation of the FT4I from a T4U test may provide more accurate results. Conversely, reduced overall binding affinity for T4 which affects T3 to a lesser extent will underestimate the FT4I derived from a T3U test. Similarly, use of the T4U and T3U for estimation of the free hormone concentration, is satisfactory in the presence of alterations in TBG concentration but not alterations of the affinity of TBG for the hormone. ^116,117

Methods based on equilibrium dialysis are most appropriate in the estimation of the free thyroid hormone level in patients with all varieties of abnormal binding to serum proteins provided the true concentration of total hormone has been accurately determined. All methods for the estimation of the FT4 concentration may give either high or low values in patients with severe nonthyroidal illness. ^{96-100 , 119 , 120} This has been attributed to the presence of inhibitors of thyroid hormone binding to serum proteins as well as to the various adsorbents used in the test procedures. ^121,122 Some of these inhibitors have been postulated to leak from the tissues of the diseased patient. ^123,124 Such discrepancies are even more pronounced during transient states of hyperthyroxinemia or hypothyroxinemia associated with acute illness, after withdrawal of treatment with thyroid hormone and in acute changes in TBG concentration (see Chapters 5 and 16 ).

The contribution of various drugs that interfere with binding of thyroid hormone to serum proteins or with the in vitro tests should also be taken into account in the choice and interpretation of tests (see Table 5-2 ). Although the free thyroid hormone concentration in serum seems to determine the amount of hormone available to body tissues, factors that govern their uptake, transport to the nucleus and functional interactions with nuclear receptors ultimately determine their biological effects.

Measurements of Iodine-Containing Hormone Precursors and Products of Degradation

The last two decades have witnessed the development of RIAs for the measurement of a number of naturally occurring, iodine-containing substances that possess little if any thyromimetic activity. Some of these substances are products of T4 and T3 degradation in peripheral tissues. Others are predominantly, if not exclusively, of thyroidal origin. Since they are devoid of significant metabolic activity, measurement of their concentration is of value only in the research setting in detecting abnormalities in the metabolism of thyroid hormone in peripheral tissues, as well as defects of hormone synthesis and secretion.

3,3',5'-Triiodothyronine or Reverse T3 (rT3). rT3 is principally a product of T4 degradation in peripheral tissues (see Chapter 3). It is also secreted by the thyroid gland, but the amounts are practically insignificant. ¹²⁶ Thus, measurement of rT3 concentration in serum reflects both tissue supply and metabolism of T4 and identifies conditions that favor this particular pathway of T4 degradation.

When total rT3 (TrT3) is measured in unextracted serum, a competitor of rT3 binding to serum proteins must be added. ¹²⁷ Several chemically related compounds may cross-react with the antibodies. The strongest cross-reactivity is observed with 3,3'-T2 but this does not present a serious methodologic problem because of its relatively low levels in human serum. Though cross-reactivity with T3 and T4 is lesser, these compounds are more often the cause of rT3 overestimation due to their relative abundance, particularly in thyrotoxicosis. ¹²⁸ Free fatty acids interfere with the measurement of rT3 by RIA. ¹²⁹

The normal range in adult serum for TrT3 is 14-30 ng/dl (0.22 - 0.46 nmol/L) although varying values have been reported. It is elevated in subjects with high TBG and in some individuals with FDH. ¹³² Serum TrT3 levels are normal in hypothyroid patients treated with T4, indicating that peripheral T4 metabolism is an important source of circulating rT3. ^{126 , 133} Values are high in thyrotoxicosis and low in untreated hypothyroidism. High values are normally found in cord blood and in newborns. ^133,134

With only a few exceptions, notably uremia, serum TrT3 concentrations are elevated in all circumstances that cause low serum T3 levels in the absence of obvious clinical signs of hypothyroidism. These conditions include, in addition to the newborn period, a variety of acute and chronic nonthyroidal illnesses, calorie deprivation, and the influence of a growing list of clinical agents and drugs (see Table 5-3 ).

Current clinical application of TrT3 measurement in serum is in the differential diagnosis of conditions associated with alterations in serum T3 and T4 concentrations when thyroid gland and metabolic abnormalities are not readily apparent.

The dialyzable fraction of rT3 in normal adult serum is 0.2 - 0.32%, or approximately the same as that of T3. The corresponding serum FrT3 concentration is 50 - 100 pg/dl (0.77 - 1.5 pmol/L). In the absence of gross TBG abnormalities, variations in serum FrT3 concentration closely follow those of TrT3. ¹⁰¹

3,5-Diiodothyronine (3,5-T2). The normal adult range for total 3,5-T2 in serum measured by direct RIAs is 0.20 - 0.75 ng/dl (3.8 - 14 pmol/L). ¹³⁵ That 3,5-T2 is derived from T3 is supported by the observations that conditions associated with high and low serum T3 levels have elevated and reduced serum concentrations of 3,5-T2, respectively. ¹³⁶ Thus, high serum 3,5-T2 levels have been reported in hyperthyroidism, and low levels in serum of hypothyroid patients, newborns, during fasting, and in patients with liver cirrhosis.

3,3'-Diiodothyronine (3,3'-T2). Normal concentrations in adults probably range from 1 to 8 ng/dl (19 - 150 pmol/L). ¹³⁷ Levels are clearly elevated in hyperthyroidism and in the newborn. Values have been found to be either normal or depressed in nonthyroidal illnesses, ¹³⁷ in agreement with the demonstration of reduced monodeiodination of rT3 to 3,3'-T2. ¹³⁸ In vivo turnover kinetic studies and measurement of 3,3'-T2 in serum after the administration of T3 and rT3 have clearly shown that 3,3'-T2 is the principal metabolic product of these two triiodothyronines.

3',5'-Diiodothyronine (3',5'-T2). Reported concentrations in serum of normal adults have a mean overall range of 1.5 - 9.0 ng/dl (30 - 170 pmol/L). ^139,140 The substances that principally cross react in the assay are rT3, 3,3-LT2 and 3-T1. Values are high in hyperthyroidism and in the newborn. ^139,140 Being the derivative of rT3 monodeiodination, ¹³⁹ 3',5'-T2 levels are elevated in serum during fasting ^140,141 and in chronic illnesses ¹³³ in which the level of the rT3 precursor is also high. Administration of dexamethasone also produces an increase in the serum 3',5'-T2 level. ¹³⁹

3'-Monoiodothyronine (3'-T1). The concentration of 3'-T1 in serum of normal adults, measured by RIA, has been reported to range from 0.6 to 2.3 ng/dl (15 - 58 pmol/L) ¹³³ and from <0.9 to 6.8 ng/dl (<20 - 170 pmol/L). Its two immediate precursors, 3,3,'-T2 and 3',5'-T2 are the main cross-reactants in the RIA. Serum levels are very high in hyperthyroidism and low in hypothyroidism. The concentration of 3'-T1 in serum is elevated in all conditions associated with high rT3 levels, including newborns, nonthyroidal illness, and fasting. ¹³⁴ This finding is not surprising since the immediate precursor at 3'-T1 is 3',5'-T2, ¹⁴² a product of rT3 deiodination, which is also present in serum in high concentration under the same circumstances. The elevated serum levels of 3'-T1 in renal failure are attributed to decreased clearance since the concentrations of its precursors are not increased.

3-Monoiodothyronine (3-T1). Experience with the measurement of 3-T1 in serum is limited. Normal values in serum of adult humans using 3H labeled 3-T1 in a specific RIA ranged from <0.5 - 7.5 ng/dl (<13 - 190 pmol/L). ¹⁴³ The mean concentration of 3-T1 in serum of thyrotoxic patients and in cord blood was significantly higher. 3-T1 appears to be a product of in vivo deiodination of 3,3'-T2.

Tetraiodothyroacetic Acid (TETRAC or T4A) and Triiodothyroacetic Acid (TRIAC or T3A). The iodoamino acids T4A and T3A, products of deamination and oxidative decarboxylation of T4 and T3, respectively, have been detected in serum by direct RIA measurements. ^{21 , 76 , 144} Reported mean concentrations in the serum of healthy adults have been 8.7 ng/dl ¹⁴⁴ and 2.6 ng/dl (range, 1.6 - 3.0 ng/dl or 26 - 48 pmol/L)) ²¹ for T3A and 28 ng/dl (range <8 - 60 mg/dl or <105 - 800 pmol/L) ⁷⁶ for T4A. Serum T4A levels are reduced during fasting and in patients with severe illness, ¹⁴⁵ although the percentage of conversion of T4 to T4A is increased. ^{20 , 146} The concentration of serum T3A remains unchanged during the administration of replacement doses of T4 and T3. ²¹ It has been suggested that intracellular rerouting of T3 to T3A during fasting is responsible for the maintenance of normal serum TSH levels in the presence of low T3 concentrations. ¹⁴⁷

3,5,3'-T3 Sulfate (T3S). A RIA procedure to measure T3S in ethanol extracted serum samples is available. ²² Concentrations in normal adults range from 4-10 ng/dl (50-125 pmol/L). Although the principal source of T3S is T3, and the former binds to TBG, values are high in newborns and low in pregnancy. This suggests different rates of T3S generation or metabolism in mother and fetus. T3S values are high in thyrotoxicosis and in nonthyroidal illness.

Diiodotyrosine (DIT) and Monoiodotyrosine (MIT). Although RIA methods for the measurement of DIT and MIT have been developed, due to limited experience, their value in clinical practice remains unknown. Early reports gave a normal mean value for DIT in serum of normal adults of 156 ng/dl (3.6 nmol/L), ¹⁴⁸ with progressive decline due to refinement of techniques to values as low as 7 ng/dl with a range of 1 - 23 ng/dl (0.02 - 0.5 nmol/L). ¹⁴⁹ Thus, the normal range for MIT of 90 - 390 ng/dl (2.9 - 12.7 nmol/L) ¹⁵⁰ is undoubtedly an overestimation. Iodotyrosine that has escaped enzymatic deiodination in the thyroid gland appears to be the principal source of DIT in serum. Iodothyronine degradation in peripheral tissues is probably a minor source of iodotyrosines since administration of large doses of T4 to normal subjects produces a decline rather than an increase in the serum DIT level. ¹⁴⁹ DIT is metabolized to MIT in peripheral tissues. Serum levels of DIT are low during pregnancy and high in cord blood.

Thyroglobulin (Tg). RIA methods were those first used routinely for measurement of Tg in serum, ¹⁵¹ , although other assays methods employing IRMA, ICMA, and ELISA technology have been reported ^151a-d and are gaining increasing popularity. They are specific and, depending upon the sensitivity of the assay, capable of detecting Tg in the serum of approximately 90% of the euthyroid healthy adults. When antisera are used in high dilutions, there is virtually no cross-reactivity with iodothyronines or iodotyrosines. Results obtained from the analysis of sera containing Tg autoantibodies may be inaccurate, depending upon the antiserum employed. ¹⁵² The presence of thyroid peroxidase antibodies does not interfere with the Tg RIA. Despite the relaibility of measurements of serum Tg, it is clear that different assay methods may result in values discrepant by up to 30%, even though refernce preparations are available. ^152a Typically, IMA methods underestimate the serum Tg value, while RIA methods overestimate it, so it is essential that clinical decisions are based upon serial measurements using the same assay.

Tg concentrations in serum of normal adults range from <1 to 25 ng/ml (<1.5 - 38 pmol/L), with mean levels of 5 - 10 ng/ml. ^{151 , 153-155} On a molar basis, these concentrations of Tg are minute relative to the circulating iodothyronines; 5,000-fold lower than the corresponding concentration of T4 in serum. Values tend to be slightly higher in women than in men. ¹⁵¹ In the neonatal period and during the third trimester of pregnancy, mean values are approximately 4- and 2-fold higher. ^154,156 They gradually decline throughout infancy, childhood and adolescence. ¹⁵⁷ The positive correlation between the levels of serum Tg and TSH indicates that pituitary TSH regulates the secretion of Tg.

Elevated serum Tg levels reflect increased secretory activity by stimulation of the thyroid gland or damage to thyroid tissue, whereas values below or at the level of detectability indicate a paucity of thyroid tissue or suppressed activity. Tg levels in a variety of conditions affecting the thyroid gland have been reviewed ¹⁵⁸ and are listed in Table 6-6.

Interpretation of a serum Tg value should take into account the fact that Tg concentrations may be high under normal physiologic conditions or altered by drugs. Administration of iodine and antithyroid drugs increase the serum Tg level, as do states associated with hyperstimulation of the thyroid gland by TSH or other substances with thyroid-stimulating activity. This is due to increased thyroidal release of Tg rather than changes in its clearance. ¹⁵⁹ Administration of TRH and TSH also transiently increases the serum level of Tg. ¹⁶⁰ Trauma to the thyroid gland, such as that occurring during diagnostic and therapeutic procedures including percutaneous needle biopsy, surgery, or 131I therapy, can produce a striking, although short-lived, elevation in the Tg level in serum. ^{154 , 161,162} Pathological processes with destructive effect on the thyroid gland also produce transient, though more prolonged increases. ¹⁶³ Tg is undetectable in serum after total ablation of the thyroid gland as well as in normal persons receiving suppressive doses of thyroid hormone. ¹⁵⁸ It is thus a useful test in the differential diagnosis of thyrotoxicosis factitia, ¹⁶⁴ especially when transient thyrotoxicosis with a low RAIU or suppression of thyroidal RAIU by iodine are alternative possibilities.

The most striking elevations in serum Tg concentrations have been observed in patients with metastatic differentiated nonmedullary thyroid carcinoma even after total surgical and radioiodide ablation of all normal thyroid tissue. ^{154 , 165} It usually persists despite full thyroid hormone suppressive therapy, suggesting excessive autonomous release of Tg by the neoplastic cells. The determination is thus of particular value in the follow-up and management of metastatic thyroid carcinomas, particularly when they fail to concentrate radioiodide. ^{153 , 165} Follow-up of such patients with sequential serum Tg determinations helps the early detection of tumor recurrence or growth and the assessment of the efficacy of treatment. Measurement of serum Tg is also useful in patients with metastases, particularly to bone, in whom there is no evidence of a primary site and thyroid malignancy is being considered in the differential diagnosis. ^{154 , 165} On the other hand, serum Tg levels are of no value in the differential diagnosis of primary thyroid cancer because levels may be within the normal range in the presence of differentiated thyroid cancer and high in a variety of benign thyroid diseases. ^{153,155 , 165} Whether early detection of recurrent thyroid cancer after initial ablative therapy could be achieved by serum Tg measurement without cessation of hormone replacement therapy is debated because Tg secretion by the tumor is modulated by TSH and is suppressed by the administration of thyroid hormone. ^166-168 Detectable serum thyroglobulin during thyroid hormone suppression reliably indicated the presence residual or recurrent disease.

Tg levels are high in the early phase of subacute thyroiditis. ¹⁶³ Declining serum Tg levels during the course of antithyroid drug treatment of patients with Graves' disease may indicate the onset of a remission. ^{162 , 169} Tg may be undetectable in the serum of neonates with dyshormonogenetic goiters due to defects in Tg synthesis ¹⁷⁰ but are very high in some hypothyroid infants with thyromegaly or ectopy. ¹⁷¹ Measurement of serum Tg in hypothyroid neonates is useful in the differentiation of infants with complete thyroid agenesis from those with hypothyroidism due to other causes, and thus in most cases obviates the need for the diagnostic administration of radioiodide. ^{171 , 172}

Measurement of Thyroid Hormone and Its Metabolites in Other Body Fluids and in Tissues

Clinical experience with measurement of thyroid hormone and its metabolites in body fluids other than serum and in tissues is limited for several reasons. Analyses carried out in urine and saliva do not appear to give additional information, not obtained from measurements carried out in serum. Amniotic fluid, cerebrospinal fluid, and tissues are less readily accessible for sampling. Their likely application in the future will depend on information they could provide beyond that obtained from similar analyses in serum.

Urine

Because thyroid hormone is filtered in the urine predominantly in free form, measurement of the total amount excreted over 24 hours offers an indirect method for the estimation of the free hormone concentration in serum. The 24-hour excretion of T4 in normal adults ranges from 4 to 13 µg and from 1.8 to 3.7 µg, depending upon whether total or only conjugated T4 is measured. Corresponding normal ranges for T3 are 2.0 - 4.0 µg and 0.4 - 1.9 µg. ^173-175 Striking seasonal variations have been shown for the urinary excretion of both hormones, with a nadir during the hot summer months, in the absence of significant changes in serum TT4 and TT3. As expected, values are normal in pregnancy and in nonthyroidal illnesses, and are high in thyrotoxicosis and low in hypothyroidism. ^{174 , 175} The test may not be valid in the presence of gross proteinuria and impairment of renal function. ¹⁷⁶

Amniotic Fluid (AF)

All iodothyronines measured in blood have also been detected in AF. With the exception of T3, 3,3'-T2 and 3'-T2, the concentration at term is lower than that in cord serum. ^{139,140 , 142 , 177-179} This fact cannot be fully explained by the low TBG concentration in AF. Although the source of iodothyronines in AF is unknown, the general pattern more closely resembles that found in the fetal than in the maternal circulation.

The TT4 concentration in AF average 0.5 µg/dl (65 nmol/L) with a range of 0.15 - 1.0 µg/dl and is thus very low when compared to values in maternal and cord serum. ^177-179 The FT4 concentration is, however, twice as high in AF relative to serum. The TT3 concentration is also low relative to maternal serum being on the average 30 ng/dl (0.46 nmol/L) in both AF and cord serum. ¹⁷⁹ rT3, on the other hand, is very high in AF, on average 330 ng/dl (5.1 nmol/L) during the first half of gestation, declining precipitously at about the 30th week of gestation to an average of 85 ng/dl (1.3 nmol/L) which is also found at term. ^178,179

Cerebrospinal Fluid (CSF)

T4, T3, and rT3 concentrations have been measured in human CSF. ^180-182 The concentrations of both TT4 and TT3 are approximately 50-fold lower than those found in serum. However, the concentrations of these iodothyronines in free form are similar to those in serum. In contrast, the level of TrT3 in CSF is only 2.5-fold lower than that of serum, whereas that of FrT3 is 25-fold higher. This is probably due to the presence in CSF of a larger proportion of TTR which has high affinity for rT3. ¹⁸¹ All the thyroid hormone-binding proteins present in serum are also found in CSF, although in lower concentrations. ¹⁸¹ The concentrations of TT4 and FT4 are increased in thyrotoxicosis and depressed in hypothyroidism. Severe nonthyroidal illness gives rise to increased TrT3 and FrT3 levels. ¹⁸²

Milk

TT4 concentration in human milk is of the order of 0.03 - 0.5 µg/dl. ¹⁸³ Analytical artifacts were responsible for the much higher values formerly reported. ^183,184 TT3 concentrations range from 10 to 200 ng/dl (015 - 3.1 nmol/L). ^184,185 The concentration of TrT3 ranges from 1 - 30 ng/dl (15 - 460 pmol/L). ¹⁸⁴ Thus, it is unlikely that milk would provide a sufficient quantity of thyroid hormone to alleviate hypothyroidism in the infant.

Saliva

It has been suggested that only the free fraction of small nonpeptide hormones which circulate predominantly bound to serum proteins would be transferred to saliva and that their measurement, in this easily accessible body fluid, would provide a simple and direct means to determine their free concentration in blood. This hypothesis was confirmed for steroid hormones, ¹⁸⁶ not tightly bound to serum proteins. Levels of T4 in saliva range from 4.2 - 35 ng/dl (54 - 450 pmol/L) and do not correlate with the concentration of free T4 in serum. ¹⁸⁷ This finding is, in part, due to the transfer of T4 bound to the small but variable amounts of serum proteins that reach the saliva.

Effusions

TT4 measured in fluid obtained from serous cavities bears a direct relationship to the protein content and the serum concentration of T4. Limited experience with Tg measurement in pleural effusions from patients with thyroid cancer metastatic to lungs suggests that it may be of diagnostic value. ¹⁶⁵

Tissues

Since the response to thyroid hormone is expressed at the cell level, it is logical to assume that hormone concentration in tissues should correlate best with its action. Methods for extraction, recovery, and measurement of iodothyronines from tissues have been developed but, for obvious reasons, data from thyroid hormone measurements in human tissues are limited. Preliminary work has shown that under several circumstances, hormonal levels in tissues such as liver, kidney, and muscle usually correlate with those found in serum. ¹⁸⁸

Measurements of T3 in cells most accessible for sampling in humans, namely, red blood cells gave values of 20 - 45 ng/dl (0.31 - 0.69 nmol/L) or one-fourth those found in serum. ¹⁸⁹ They are higher in thyrotoxicosis and lower in hypothyroidism.

The concentrations of all iodothyronines have been measured in thyroid gland hydrolysates. ^{18 , 133 , 139} In normal glands, the molar ratios relative to the concentration of T4 are on average as follows: T4/T3 = 10; T4/rT3 = 80; T4/3,5'-T2 = 1,400; T4/3,3'-T2 = 350; T4/3',5'-T2 = 1,100; and T4/3'-T1 = 4,400. Information concerning the content of iodothyronines in hydrolysates of abnormal thyroid tissue is limited, and the diagnostic value of such measurements has not been established.

Measurement of Tg in metastatic tissue obtained by needle biopsy may be of value in the differential diagnosis, especially when the primary site is unknown and the histological diagnosis is not conclusive.

Tests Assessing the Effects of Thyroid Hormone on Body Tissues

Thyroid hormone regulates a variety of biochemical reactions in virtually all tissues. Thus, ideally, the adequacy of hormonal supply should be assessed by the tissue responses rather than by parameters of thyroid gland activity or serum hormone concentration which are several steps removed from the site of thyroid hormone action. Unfortunately, the tissue responses (metabolic indices) are nonspecific because they are altered by a variety of physiologic and pathologic mechanisms unrelated to thyroid hormone deprivation or excess. The following review of biochemical and physiologic changes mediated by thyroid hormone has a dual purpose: (1) to outline some of the changes that may be used as clinical tests in the evaluation of the metabolic status, and (2) to point out the changes in various determinations commonly used in the diagnosis of a variety of nonthyroidal illnesses, which may be affected by the concomitant presence of thyroid hormone deficiency or excess.

Basal Metabolic Rate (BMR)

The BMR has a long history in the evaluation of thyroid function. It measures the oxygen consumption under basal conditions of overnight fast and rest from mental and physical exertion. Since standard equipment for the measurement of BMR may not be readily available, it can be estimated from the oxygen consumed over a timed interval by analysis of samples of expired air. ¹⁹⁰ The test indirectly measures metabolic energy expenditure or heat production.

Results are expressed as the percentage of deviation from normal after appropriate corrections have been made for age, sex, and body surface area. Low values are suggestive of hypothyroidism, and high values reflect thyrotoxicosis. The various nonthyroidal illnesses and other factors affecting the BMR, including sources of errors, have been reviewed. ¹⁹¹ Although this test is no longer a part of the routine diagnostic armamentarium, it is still useful in research.

Deep Tendon Reflex Relaxation Time (Photomotogram)

Delay in the relaxation time of the deep tendon reflexes, visible to the experienced eye, occurs in hypothyroidism. Several instruments have been devised to quantitate various phases of the Achilles tendon reflex. Although normal values vary according to the phase of the tendon reflex measured, the apparatus used and individual laboratory standards, the approximate adult normal range for the half-relaxation time is 230-390 msec. Diurnal variation, differences with sex, and changes with age, cold exposure, fever, exercise, obesity, and pregnancy have been reported. However, the main reason for the failure of this test as a diagnostic measure of thyroid dysfunction is the large overlap with values obtained in euthyroid patients and alterations caused by nonthyroidal illnesses. ¹⁹²

Tests Related to Cardiovascular Function

Thyroid hormone induced changes in the cardiovascular system can be measured by noninvasive techniques. One such test measures the time interval between the onset of the electrocardiographic QRS complex (Q) and the arrival of the pulse wave at the brachial artery, detected by the Korotkoff sound (K) at the antecubital fossa. ¹⁹³ Related tests which determine the systolic time interval (STI) measure the preejection period (PEP), obtained by subtraction of the left ventricular ejection time (LVET) from the total electromechanical systole (Q-A2). ¹⁹⁴ The left ventricular ejection time (LVET) which is also affected by the thyroid status can be measured by the M mode echocardiogram ¹⁹⁵ (Figure 6-5). The PEP/LVET ratio is also useful in the assessment of thyroid hormone action in the cardiovascular system. ¹⁹⁶ As with other tests of thyroid hormone action, the principal deficiency of these measurements is their alteration in a variety of nonthyroidal illnesses.

Figure 6-5: Simultaneous tracings of electrocardiogram (ECG), phonocardiogram, carotid pulse and echocardiogram. Measurements of the systolic pre-ejection period (PEP), isovolemic contraction time (ICT), left ventricular ejection time (LVET) and isovolumic relaxation time (IVRT) are indicated. (From I Kline, The thyroid, L.E. Braverman & R.D. Utiger (eds). 1991, J.B. Lippincot Co.)

Miscellaneous Biochemical and Physiologic Changes Related to the Action of Thyroid Hormone on Peripheral Tissues

Thyroid hormone affects the function of a variety of peripheral tissues. Thus, hormone deficiency or excess may alter a number of determinations used in the diagnosis of illnesses unrelated to thyroid hormone dysfunction. Knowledge of the determinations which may be affected by thyroid hormone is important in the interpretation of laboratory data (Table 6-7).

Measurement of Substances Absent in Normal Serum

Tests that measure substances present in the circulation only under pathologic circumstances do not provide information on the level of thyroid gland function. They are of value in establishing the cause of the hormonal dysfunction or thyroid gland pathology.

Thyroid Autoantibodies

The humoral antibodies most commonly measured in clinical practice are directed against thyroglobulin (Tg) or thyroid cell microsomal (MC) proteins. The latter is principally represented by the thyroid peroxidase (TPO). ^296-298 More recently, immunoassays have been developed using purified and recombinant TPO. ^{299, 299a, 299b} Other circulating immunoglobulins, which are less frequently used as diagnostic markers, are those directed against a colloid antigen, T4 and T3. Antibodies against nuclear components are not tissue specific. Immunoglobulins possessing the property of stimulating the thyroid gland will be discussed in the next section.

A variety of techniques have been developed for the measurement of Tg and MC antibodies. These procedures include a competitive binding radioassay, complement fixation reaction, ³⁰⁰ tanned red cell agglutination assay, ³⁰¹ the Coon's immunofluorescent technique, ³⁰² enzyme-linked immunosorbent assay. ^{299 , 303} Although the competitive binding radioassay ^304,305 is a sensitive test, agglutination methods combine sensitivity and simplicity and have now largely superceded other methods. Current commercial kits utilize synthetic gelatin beads rather than red cells. ^305a

In the assay of Tg and MC antibodies by hemagglutination (TgHA and MCHA), particulate material is coated with either human Tg or solubilized thyroid MC proteins (TPO) and exposed to serial dilutions of the patient's serum. Agglutination of the coated particulates occurs in the presence of antibodies specific to the antigen attached to their surface. To detect false-positive reactions, it is important to include a blank for each sample using uncoated particles. Because of the common occurrence of prozone or blocking phenomenon, it is necessary to screen all serum samples through at least six consecutive two-fold dilutions. ³⁰⁶ Results are expressed in terms of the highest serum dilution, or titer, showing persistent agglutination. The presence of immune complexes, particularly in patients with high serum Tg levels, may mask the presence of Tg antibodies. Assays for the measurement of such Tg-anti-Tg immune complexes have been developed. ³⁰⁷

Normally, the test response is negative but results may be positive in up to 10% of the adult population. The frequency of positive test results is higher in women and with advancing age. The presence of thyroid autoantibodies in the apparently healthy population is thought to represent subclinical autoimmune thyroid disease rather than false-positive reactions. Nonetheless, it is difficult to compare results from such studies since some laboratories using agglutination methods report low titres (1/100-1/400) as positive. It is important when reporting values that a method-specific normal range is utilized and assays calibrated against internationally available refernce preparations. The availability of such preparations allows the reporting of results in International Units. ^305a TPO antibodies are detectable in approximately 95% of patients with Hashimoto's thyroiditis and 85% of those with Graves' disease, irrespective of the functional state of the thyroid gland. Similarly, Tg antibodies are positive in about 60 and 30% of adult patients with Hashimoto's thyroiditis and Graves' disease, respectively. ^{305,306 , 308,309} Tg antibodies are less frequently detected in children with autoimmune thyroid disease. ³¹⁰ Although higher titers are more common with Hashimoto's thyroiditis, quantitation of the antibody titer carries little diagnostic implication. The tests are of particular value in the evaluation of patients with atypical or selected manifestations of autoimmune thyroid disease (ophthalmopathy and dermopathy). Positive antibody titers are predicative of post partum thyroiditis. ³¹¹ Low antibody titers occur transiently in some patients after an episode of subacute thyroiditis. ³¹² There is no increased incidence of thyroid autoantibodies in patients with multinodular goiter, thyroid adenomas, or secondary hypothyroidism. In some patients with Hashimoto's thyroiditis and undetectable thyroid autoantibodies in their serum, intrathyroidal lymphocytes have been demonstrated to produce TPO antibodies.

Other antibodies directed against thyroid components or other tissues have been described in the serum of some patients with autoimmune thyroid disease. They are less frequently measured, and their diagnostic value in thyroid disease has not been fully evaluated. Circulating antibodies capable of binding T4 and T3 have also been demonstrated in patients with autoimmune thyroid diseases which may interfere with the measurement of T4 and T3 by RIA techniques. ^{38,39 , 314}

Antibodies reacting with nuclear components, which are not tissue specific, and with cellular components of parietal cells and adrenal, ovarian, and testicular tissues are more commonly encountered in patients with autoimmune thyroid disease. ³¹⁵ Their presence reflects the frequency of coexistence of several autoimmune disease processes in the same patient (see Chapter 7 ).

Thyroid-Stimulating Immunoglobulins (TSI)

A large number of names have been given to tests which measure abnormal ?-globulins present in the serum of some patients with autoimmune thyroid disease, in particular Graves' disease. ³¹⁷ The interaction of these unfractionated immunoglobulins with thyroid follicular cells usually results in a global stimulation of thyroid gland activity and only rarely causes inhibition. It has been recommended that these assays all be called TSH receptor antibodies (TRAb) with a phrase "measured by .................. assay" to identify the type of method used for their determination. ¹⁰⁶ The tests will be described under three general categories: (1) those measuring the thyroid stimulating activity using in vivo or in vitro bioassays; (2) tests based on the competition of the abnormal immunoglobulin with binding of TSH to its receptor; and (3) measurement of thyroid growth promoting activity of immunoglobulins. Tests employ both human and animal tissue material or cell lines.

Thyroid-Stimulation Assays.

The earliest assays employed various modifications of the McKenzie mouse bioassay. ^318,319 The abnormal ?-globulin with TSH-like biological properties has relatively longer in vivo activity, hence its name, long-acting thyroid stimulator (LATS). The assay measures the LATS induced release of thyroid hormone from the mouse thyroid gland prelabeled with radioiodide. The presence of LATS in serum is pathognomonic of Graves' disease. However, depending upon the assay sensitivity, a variable percent of untreated patients will show a positive LATS response. LATS may be found in the serum of patients with Graves' disease even in the absence of thyrotoxicosis. Although it is more commonly present in patients with ophthalmopathy, especially when accompanied by pretibial myxedema, ³²⁰ LATS does not appear to correlate with the presence of Graves' disease, its severity, or course of complications. LATS crosses the placenta and may be found transiently in newborns from mothers possessing the abnormal ? globulin. ³²¹

Attempts to improve the ability to detect thyroid stimulating antibodies (TSAb) in autoimmune thyroid disease lead to the development of several in vitro assays using animal as well as human thyroid tissue. The ability of the patient's serum to stimulate endocytosis in fresh human thyroid tissue is measured by direct count of intracellular colloid droplets formed. Using such a technique, human thyroid stimulator (HTS) activity has been demonstrated in serum samples from patients with Graves' disease that were devoid of LATS activity measured by the standard mouse bioassay. ³²² TSAb can be detected by measuring the accumulation of cyclic adenosine monophosphate (cAMP) or stimulation of adenylate cyclase activity in human thyroid cell cultures and thyroid plasma membranes, respectively. ³²³ Accumulation of cAMP in the cultured rat thyroid cell line FRTL5 has also been used as an assay for TSAb. ³²⁴ Stimulation of release of T3 from human ³²⁵ and porcine ³²⁶ thyroid slices is another form of in vitro assay for TSAb. An in vitro bioassay using a cytochemical technique depends upon the ability of thyroid-stimulating material to increase lysosomal membrane permeability to a chromogenic substrate, leucyl-ß-naphthylamide, which then reacts with the enzyme naphthylamidase. Quantitation is by scanning and integrated microdensitometry. ³²⁷

The cloning of the TSH receptor ^328,329 lead to the development of an in vitro assay of TSab using cell lines that express the recombinant TSH receptor. ^330,331 This assay, based on the generation if cAMP, is specific for the measurement of human TSH receptor antibodies that have thyroid stimulating activity and thus contrasts with assays based on binding to the TSH receptor (see below) that cannot distinguish between antibodies with thyroid-stimulating and TSH-blocking activity. Accordingly, the recombinant human TSH receptor assay measures antibodies relevant to the pathogenesis of autoimmune thyrotoxicosis and is more sensitive than formerly used TSab assays. ^331a For example, 94% of serum samples were positive for TSab compared to 74% when the same samples were assayed using FRTL5 cells. ³³²

Thyrotropin-Binding Inhibition Assays. The principal of binding-inhibition assays dates to the discovery of another class of abnormal immunoglobulins in patients with Graves' disease; those which neutralize the bioactivity of LATS tested in the mouse. ³³³ This material, known as LATS protector (LATS-P), is species specific having no biologic effect on the mouse thyroid gland but capable of stimulating the human thyroid. ³³⁴ The original assay was cumbersome, limiting its clinical application.

Techniques used currently, which may be collectively termed radioreceptor assays, are based on the competition of the abnormal immunoglobulins and TSH for a common receptor-binding site on thyroid cells. The test is akin in principle to the radioligand assays, in which a natural membrane receptor takes the place of the binding proteins or antibodies. Various sources of TSH-receptors are employed including, human thyroid cells, ³³⁵ their particulate or solubilized membrane, ^336,337 and cell membranes from porcine thyroids ³³⁸ or from guinea pig fat cells ³³⁹ or recombinant human TSH receptor expressed in mammalian cells. ³⁴⁰ Since the assays do not directly measure thyroid-stimulating activity, the abnormal immunoglobulins determined have been given variety of names, such as thyroid binding inhibitory immunoglobulins (TBII) or antibodies (TBIAb) and thyrotropin-displacing immunoglobulins (TDI). This type of assay has indicated that not all the antibodies detected do stimulate the thyroid, and some are inhibitory. Even using modern techniques, ^305a the presence of inhibitory antibody is less sensitive and specific for Graves' disease than the presence of styimulatory antibody activity. ³³¹ The stimulatory and inhibitory effects can be differentiated only by functional assays, typically measuring the production of cyclic AMP.

Thyroid Growth-Promoting Assays.

Assays have been also developed that measure the growth promoting activity of abnormal immunoglobulins. One such assay is based on the staining by the Feulgen reaction of nuclei from guinea pig thyroid cells in S-phase. ³⁴¹ Another assay measures the incorporation of 3H-thymidine into DNA in FRTL cells. ³⁴² Whether the thyroid growth stimulating immunoglobulins (TGI) measured by these assays represent a population of immunoglobulins distinct from that with stimulatory functional activity remains a subject of active debate.

Clinical Applications. Measurement of abnormal immunoglobulins that interact with thyroid tissue by any of the methods described above is not indicated as a routine diagnostic test for Graves' disease. It is useful, however, in a few selected clinical conditions: (1) in the differential diagnosis of exophthalmos, particularly unilateral exophthalmos, when the origin of this condition is otherwise not apparent; the presence of TSI would obviate the necessity to undertake more complex diagnostic procedures described elsewhere; ³⁴³ (2) in the differential diagnosis of pretibial myxedema, or other forms of dermopathy, when the etiology is unclear and it is imperative that the cause of the skin lesion be ascertained; (3) in the differentiation of Graves' disease from toxic nodular goiter, when both are being considered as the possible cause of thyrotoxicosis, when other tests such as thyroid scanning and thyroid autoantibody tests have been inconclusive, and particularly when such a distinction would play a role in determining the course of therapy; (4) when non-autoimmune thyrotoxicosis is suspected in a patient with hyperthyroidism and diffuse or nodular goiter ^344,345 ; (5) in Graves' disease during pregnancy, when high maternal levels of TSAb are a warning for the possible occurrence of neonatal thyrotoxicosis; (6) in neonatal thyrotoxicosis, where serial TSAb determinations showing gradual decrease may be helpful to distinguish between intrinsic Graves' disease in the infant and transient thyrotoxicosis resulting from passive transfer of maternal TSAb. ^{321 , 346} Some investigators have found the persistence of TSAb's to be predicative of the relapse of Graves' thyrotoxicosis following a course of antithyroid drug therapy. ³⁴⁷

Other Substances with Thyroid-Stimulating Activity

Some patients with trophoblastic disease develop hyperthyroidism as a result of the production and release of a thyroid stimulator which has been termed molar or trophoblastic thyrotropin or big placental TSH. ³⁴⁸ It is likely that the thyroid-stimulating activity in patients with trophoblastic disease is entirely due to the presence of high levels of human chorionic gonadotropin (hCG). ³⁵⁰ Thus, the RIA of hCG can be useful in the differential diagnosis of thyroid dysfunction.

Exophthalmos-Producing Substance (EPS)

A variety of tests have been developed for measuring exophthalmogenic activity in serum. ^351-354 Although a great uncertainty still exists regarding the pathogenesis of thyroid associated eye disease, the role of the immune system appears to be central. Exophthalmogenic activity has also been detected in IgG fractions of some patients with Graves' ophthalmopathy. The role of assays to detect specific antibodies is discussed further in Chapter 7 .

Tests of Cell-Mediated Immunity (CMI)

Delayed hypersensitivity reactions to thyroid antigens are present in autoimmune thyroid diseases (see Chapters 7 ). CMI was measured in several ways: (1) the migration inhibition test, which measured the inhibition of migration of sensitized leukocytes when exposed to the sensitizing antigen; (2) the lymphotoxic assay, which measured the ability of sensitized lymphocytes to kill target cells when exposed to the antigen; (3) the blastogenesis assay, which scored the formation of blast cells after exposure of lymphocytes to a thyroid antigen; and (4) thymus-dependent (T) lymphocyte subset quantitation utilizing monoclonal antibodies. More recently, measures of T-cell proliferation, determined by uptake of 3Hthymidine, has become the standard test of CMI employed in the research setting. ^{354a, 354b} The tests require fresh leukocytes from the patient, are variable in their response, and are difficult to perform.

Anatomic and Tissue Diagnoses

The purpose of the procedures described in this section is to evaluate the anatomic features of the thyroid gland, localize and determine the nature of abnormal areas and eventually provide a pathologic or tissue diagnosis. All of these tests are performed in vivo.

Thyroid Scintiscanning

Normal and abnormal thyroid tissue can be externally imaged by three scintiscanning methods: (1) with radionuclides that are concentrated by normal thyroid tissues such as iodide isotopes, and 99mTc given as the pertechnetate ion; (2) by administration of radiopharmaceutical agents which are preferentially concentrated by abnormal thyroid tissues; and (3) fluorescent scanning, which uses an external source of 241Am and does not require administration of radioactive material. Each has specific indications, advantages, and disadvantages.

The physical properties, dosages, and radiation delivered by the most commonly used radioisotopes are listed in Table 6-2 . The choice of scanning agents depends on the purpose of the scan, the age of the patient, and the equipment available. Radioiodide scans cannot be performed in patients who have recently ingested iodine-containing compounds. 123I and 99mTcO4- are the radionuclides of choice because of the low radiation exposure. ^355-357 Iodine-131 is still used for the detection of functioning metastatic thyroid carcinoma by total body scanning.

Radioiodide and 99mPertechnetate Scans. 99mTcO4- is concentrated, and all iodide isotopes are concentrated and bound, by thyroid tissue. Depending upon the isotope used, scans are carried out at different times after administration: 20 minutes for 99mTcO4-, 4 or 24 hours for 123I-; 24 hours for 125I- and 131I-; and 48, 72, and 96 hours when 131I- is used in the search for metastatic thyroid carcinoma. The appearance of the normal thyroid gland on scan may be best described as a narrow-winged butterfly. Each "wing" represents a thyroid lobe, which in the adult measures 5 ? 1 cm in length and 2.3 ? 0.5 cm in width. ³⁵⁸ Common variants include the absence of a connecting isthmus, a large isthmus, asymmetry between the two lobes, and trailing activity extending to the cricoid cartilage (pyramidal lobe). The latter is more commonly found in conditions associated with diffuse thyroid hyperplasia. Occasionally, collection of saliva in the esophagus during 99mTcO4- scanning may simulate a pyramidal lobe, but this artifact can be eliminated by drinking water.

The indications for scanning are listed in Table 6-8 . In clinical practice, scans are most often requested for evaluation of the functional activity of solitary nodules. Normally, the isotope is homogeneously distributed throughout both lobes of the thyroid gland. This distribution occurs in the enlarged gland of Graves' disease and may be seen in Hashimoto's thyroiditis. A mottled appearance may be noted in Hashimoto's thyroiditis and can occasionally be seen in Graves' disease especially after therapy with radioactive iodide. Irregular areas of relatively diminished and occasionally increased uptake are characteristic of large multinodular goiters. The traditional nuclear medicine jargon classifies nodules as "hot", "warm," and "cold," according to their isotope-concentrating ability relative to the surrounding normal parenchyma (Figure 6-6). Hot, or hyperfunctioning, nodules are typically benign, although the presence of malignancy has been reported. ^359,360 Cold, or hypofunctioning, nodules may be solid or cystic. Some may prove to be malignant, but the great majority are benign. This differentiation cannot be made by scanning. ^{77 , 361} Occasionally, a nodule which is functional on a 99mTcO4- scan will be found to be cold on an iodine scan; this pattern is found with both benign and malignant nodules. The scan is of particular value in identifying autonomous thyroid nodules since the remainder of the gland is suppressed. Search for functioning thyroid metastases is best accomplished using 2-10 mCi of 131I after ablation of the normal thyroid tissue and cessation of hormone therapy to allow TSH to increase above the upper limit of normal. Recent studies have addressed the question of whether recombinant human TSH allows scanning without requiring cessation of hormone therapy. ³⁶² Uptake is also found outside the thyroid gland in patients with lingual thyroids and in the rare ovarian dermoid tumor containing functioning thyroid tissue.

Figure 6-6. Thyroid Scans. (a) Normal thyroid imaged with 123I. (b) Cold nodule in the right lobe imaged by 99mTc. (c) Elderly woman with obvious multinodular goiter and the corresponding radioiodide scan on the right.

The scan can be used as an adjunct during TSH stimulation and T3 suppression tests to localize suppressed normal thyroid tissue or autonomously functioning areas, respectively (see below). Applications other than those listed in Table 6-8 are of doubtful benefit and are rarely justified considering the radiation exposure, expense, and inconvenience. 123I single photon emission computed tomography (SPECT) may also be useful in the evaluation of thyroid abnormalities. ³⁶³

Other Isotope Scans. Because most test procedures, short of direct microscopic examination of thyroid tissue, fail to detect thyroid malignancy with any degree of certainty, efforts have been made to find other radioactive materials that would hopefully be of diagnostic use. Several such agents that are concentrated by metabolically active tissues, irrespective of whether they have iodide-concentrating ability, have been tried. However, despite claims to the contrary, they have either had only limited value or their diagnostic usefulness has not been fully evaluated. These agents include 75Se methionine, 125Ce, 67Ga, citrate, 32P, pyrophosphate 99mTc, and 201Thallium. ³⁶⁴

Scanning with 131I-labeled anti-TG for the detection of occult metastatic thyroid malignancy that fails to concentrate 131I showed early promising results. ³⁶⁵ However, the procedure has not proved clinically useful.

Ultrasonography

Ultrasonography, or echography, is used to outline the thyroid gland and to characterize lesions differing in density from the surrounding tissue. The technique differentiates interphases of different acoustic densities, using sound frequencies in the megahertz range that are above the audible range. A transducer fitted with a piezoelectric crystal produces and transmits the signal and receives echo reflections. Interfaces of different acoustic densities reflect dense echoes, liquid transmits sound without reflections, and air-filled spaces do not transmit the ultrasound. ³⁶⁸

One of the most useful applications of the ultrasonogram is the differentiation of solid from cystic lesions. ^368,369 Purely cystic lesions are entirely sonolucent, whereas solid lesions produce multiple echoes due to multiple sonic interphases. Many lesions, however, are mixed (solid and cystic) called complex lesions. Some tumors may have the same acoustic characteristics as the surrounding normal tissue thus, escaping echographic detection. While high-resolution ultrasonography can detect thyroid nodules of the order of few millimeters, ³⁷⁰ lesions need to be larger than 0.5 cm to allow differentiation between solid and cystic structures. A sonolucent pattern is frequently noted in glands with Hashimoto's thyroiditis, but this has also been described in multinodular glands and in patients with Graves' disease. ^{368 , 371 , 372}

Because sonography localizes the position as well as the depth of lesions, the procedure has been used to guide the needle during aspiration biopsy. ³⁷³ In complex lesions, the sonographic guiding insures sampling from the solid portion of the nodule. With experience and proper calibration, sonography can be used for the estimation of thyroid gland size. ^374,375 Several recent reports have described treatment of toxic nodules by the injection of alcohol under sonographic guidance. ³⁷⁶ Although ultrasonography has found virtually the same applications as scintiscanning, claims that the former may differentiate benign from malignant lesions are unfounded. Also, ultrasonography cannot be used for the assessment of substernal goiters because of interference from overlying bone.

The procedure is simple and painless, and at the frequencies of sound used, do not produce tissue damage. Since it does not require the administration of isotopes, it can be safely used in children and during pregnancy. Also, because the procedure is independent of iodine-concentrating mechanisms, it is valuable in the study of suppressed glands.

X-Ray Procedures

A simple X-ray film of the neck and upper mediastinum may provide valuable information regarding the location, size, and effect of goiter on surrounding structures. X-rays may show an asymmetric goiter, an intrathoracic extension of the gland, and displacement or narrowing of the trachea. If there is any suggestion of posterior extension of the mass, it is useful to take films during the swallow of X-ray contrast material. The soft tissue X-ray technique may disclose calcium deposits. Large deposits in flakes or rings are typical of an old multinodular goiter, whereas foci of finely stippled flecks of calcium are suggestive of papillary adenocarcinoma.

Information, not related to anatomic abnormalities of the thyroid gland may be obtained from X-ray studies. In children with a history of hypothyroidism, an X-ray film of the hand to determine the bone age could aid in estimating the onset and duration of thyroid dysfunction. ^294,295 Hypothyroidism leads to retardation in bone age and in infants produces a dense calcification of epiphyseal plates most easily seen at the distal end of the radius. Long-standing myxedema produces pituitary hypertrophy which, especially in children but also in adults, causes enlargement of the sella turcica demonstrable on imaging of the pituitary region.

Computed Tomography (CT) and Magnetic Resonance Imaging (MRI). These techniques provide useful information on the location and architecture of the thyroid gland as well as its relationship to surrounding tissues. ³⁷⁸ They are, however, too costly relative to other procedures which provide similar information. An important application of CT is the assessment and delineation of obscure mediastinal masses and large substernal goiters. ³⁷⁹ The necessity to infuse iodine containing contrast agents limits the application of CT in patients being considered for radioiodide therapy. CT and MRI have found firm application in another area of thyroid diseases, namely, in the evaluation of ophthalmopathy ³⁴³ and mediastinal masses. ³⁷⁹

Other Procedures

A barium swallow may be useful in evaluating impingement of a goiter on the esophagus, while a flow volume loop ³⁸⁰ may be useful in documenting functional impingement on the upper airway.

Biopsy of the Thyroid Gland

Histologic examination of thyroid tissue for diagnostic purposes requires some form of an invasive procedure. The biopsy procedure depends on the intended type of microscopic examination. Core biopsy for histologic examination of tissue with preservation of architecture is obtained by closed needle or open surgical procedure; aspiration biopsy is performed to obtain material for cytologic examination.

Core Biopsy. Closed core biopsy is an office procedure carried out under local anesthesia. A large (about 15-gauge) cutting needle of the Vim-Silverman type is most commonly used. ³⁸⁴ The needle is introduced under local anesthesia through a small skin nick and firm pressure is applied over the puncture site for 5-10 minutes after withdrawal of the needle. In experienced hands, complications are rare, but may include transient damage to the laryngeal nerve, puncture of the trachea, laryngospasm, jugular vein phlebitis, and bleeding.385 Because of the fear of disseminating malignant cells, biopsy was restricted for many years to the differential diagnosis of diffuse benign diseases. With the improvement of cytology and biopsy techniques, open biopsy carried out under local or general anesthesia has been virtually abandoned. ³⁸⁵

Percutaneous Fine Needle Aspiration (FNA). The development of more sophisticated staining techniques for cytologic examination, the realization that fear of tumor dissemination along the needle tract was not well founded, and especially the high diagnostic accuracy of the technique are responsible for the increasing popularity of percutaneous fine needle aspiration. ^{385 , 388,388a,388b}

The procedure is exceedingly simple and safe. The patient lays supine, with the neck hyperextended by placing a small pillow under the shoulders. Local anesthesia is usually not required. The skin is prepared with an antiseptic solution. The lesion, fixed between two gloved fingers, is penetrated with a fine (22- to 27-gauge) needle attached to a syringe. Suction is then applied while the needle is moved within the nodule. A non-suction technique using capillary action has also been developed. The small amount of aspirated material, usually contained within the needle or its hub, is applied to glass slides and spread. Some slides are air dried and others are fixed before staining. As biopsy of small nodules may be technically more difficult, the use of ultrasound to guide the needle is preferred. ^{373 , 376} It is important that the slides be properly prepared, stained and read by a cytologist experienced in the interpretation of material from thyroid gland aspirates.

The yield of false-positive and false-negative results is variable from one center to another, but both are acceptably low. Various centers have reported that the accuracy of this technique in distinguishing benign from malignant lesions may be as high as 95%. ^{385 , 388} In one clinic in which the procedure is used routinely, the number of patients operated upon decreased by one-third, whereas the percentage of thyroid carcinomas among the patients who underwent surgery doubled. ³⁸⁹ When results are suggestive of a follicular neoplasia, surgery is required as follicular adenoma cannot be differentiated from follicular cancer by cytology alone. As the sample obtained may not always be representative of the lesion, surgical treatment is indicated for lesions highly suspicious of being malignant on clinical grounds. Other uses of aspiration biopsy include presumed lymphoma or invasive anaplastic carcinoma when biopsy may spare the patient an unnecessary neck exploration. Another application of needle aspiration is in the confirmation and treatment of thyroid cysts and autonomous thryoid nodules. ^389a

Evaluation of the Hypothalamic-Pituitary-Thyroid Axis

The development of an RIA for the routine measurement of TSH in serum and the availability of synthetic TRH ^390,391 have placed increased reliance on tests assessing the hypothalamic-pituitary control of thyroid function. These tests allow the diagnosis of mild and subclinical forms of thyroid dysfunction, and provide a means to differentiate between primary, pituitary (secondary) or hypothalamic (tertiary), thyroid gland failure.

Thyrotropin (TSH)

The routine measurement of TSH in clinical practice used initially RIA techniques. These first generation assays had a sensitivity level of 1 mU/L which did not allow the separation of normal from reduced values. A major problem with early TSH RIAs was cross-reactivity with gonadotropins (LH, FSH, and hCG) sharing with TSH a common a-subunit. ³⁹⁹ Nevertheless, even older RIA methods for measurement of pituitary TSH correlated well with values obtained using bioassay techniques. ⁴⁰¹ Another uncommon source of error is the presence in the serum sample of heterophilic antibodies induced by vaccination with materials contaminated with animal serum, ⁴⁰² or endogenous TSH antibodies. ⁴⁰³ RIA techniques for measurement of TSH in dry blood spots on filter paper are used for the screening of neonatal hypothyroidism. ³³

Newer techniques have been developed using multiple antibodies to produce a "sandwich" type assay in which one antibody (usually directed against the a subunit) serves to anchor the TSH molecule and an other (usually monoclonal antibodies directed against the ß subunit) is either radioiodinated (Immunoradiometric assay, IRMA) or is conjugated with an enzyme (Immunoenzymometric, IEMA) or a chemiluminescent compound (Chemiluminescent assay, ICMA). ^{112 , 404} In these assays, the signal should be directly related to the amount of the ligand present rather than being inversely related as in RIAs measuring the bound tracer. ⁴⁰⁵ This results in decreased background "noise" and a greater sensitivity, decreased interference from related compounds as well as an expanded useful range. ^{112 , 404 , 406} Initial improvements of the TSH assay resulted in assays with sensitivity limit of 0.1 mU/L, a normal range of approximately 0.5 - 4.5 mU/L and the ability to distinguish between low and normal TSH values. Recently, commercial assays have been developed with even higher sensitivity limit of 0.005 - 0.01 mU/L and a similar normal range but an expanded range between the lower limit of normal and the lower limit of sensitivity. ^407,408

The nomenclature for differentiating these various assays has not been standardized with manufacturers applying various combinations of "high(ly)", "ultra" and "sensitive". It has been recommended that the sensitivity limit be used in defining the assays with the early radioimmunoassays detecting values ?1 mU/L designated "first generation assays", those with a lower sensitivity limit of 0.1 mU/L designated as "second generation assays" and those with a lower sensitivity limit of ? 0.01 mU/L designated as "third generation assays". ¹¹² The determination of the appropriate sensitivity level has also been controversial. Some define it based on the level with a coefficient of variation less than 20% and others as the lowest level which can be reliably differentiated from the zero TSH standard.112,406 At a minimum, for a TSH assay to be considered "sensitive", the overlap of TSH values in sera from clinically hyperthyroid and euthyroid individuals should be less than 5% and preferably less than 1%. ¹¹²

In a number of these "third generation" assays, TSH detected in clinically toxic patients and elevated values found in euthyroid subjects were not confirmed when the samples were measured in other assays. In some cases, this has been attributed to the presence of antibodies directed against the animal immunoglobulins used in the assay. ^409-411 These act to bind the anchoring and detecting antibodies and lead to an over-estimation of TSH. In some cases, this effect may be blocked by the addition of an excess of non-specific immunoglobulin of the same species. ⁴¹¹

TSH appears abruptly in the pituitary and serum of the fetus at midgestation, and can also be detected in amniotic fluid. ^{51 , 412,413} The mean TSH level is higher in cord than in maternal blood. A substantial increase, to levels several fold above the upper range in adults, is observed during the first half-hour of life. ⁴¹³ Levels decline to near the normal adult range by the third day of life. Minimal changes reported to occur during adult life and in early adolescence ⁴¹⁴ have no significant effect on the overall range of normal. In the absence of pregnancy, no significant sex differences have been observed. Although early studies failed to show diurnal TSH variation, ⁴¹⁵ significantly higher values have been recorded during the late evening and early night which are partially inhibited by sleep. ⁴¹⁶ This diurnal rhythm of TSH is superimposed upon continuous high-frequency, low-amplitude variations. The nocturnal TSH surge persists in patients with mild primary hypothyroidism, ^417,418 and is abolished in hypothalamic hypothyroidism ^{417 , 419} and in some patients during fasting ⁴²⁰ and with non-thyroidal illness. ^421,422 It is enhanced by oral contraceptives, ⁴²³ and is abolished by high levels of glucocorticoids. ⁴²⁴ The presence of seasonal variation has not been a uniform finding, but it is unlikely to affect the clinical interpretation of serum values. ⁴²⁵ Various types of stressful stimuli have no significant effect on the basal serum TSH level, except for a rise during surgical hypothermia in infants but not in adults. ⁴²⁶ Various stimuli, such as administration of insulin, vasopressin, glucagon, bacterial pyrogens, arginine, prostaglandins, and chlorpromazine, which elicit in normal humans a secretory response of some pituitary hormones, have no effect on serum TSH. However, administration of any of a growing list of drugs has been found to alter the basal concentration of serum TSH and/or its response to exogenous TRH (see Table 5-4 ).

In the presence of a normally functioning hypothalamic-pituitary system, there is an inverse correlation between the serum concentration of FT4 and TSH. Changes in the serum concentration of TT4 as a result of TBG abnormalities, or drugs competing with T4 binding to TBG, have no effect on the level of serum TSH. The pituitary is exquisitely sensitive to both minimal decreases and increases in thyroid hormone concentration, with a logarithmic change in TSH levels in response to changes in T4 ^{404 , 408 , 427 , 428} (Figure 6-7) Thus, serum TSH levels should be elevated in patients with primary hypothyroidism and low or undetectable in thyrotoxicosis. Indeed, in the absence of hypothalamic pituitary disease, illness or drugs, TSH is an accurate indicator of thyroid hormone status and the adequacy of thyroid hormone replacement. ^{404 , 429}

Figure 6-7. Correlation of the serum TSH concentration and the free thyroxine index (FT4I) in three individuals given increasing doses of L-T4. Note the logarithmic correlation between TSH and FT4I and the variable individual requirement of free T4 to normalize the TSH level. Normal ranges are included in the heavy lined box and those for subjects on L-T4 replacement in the light liquid box. (From D. Sarne and S. Refetoff, Endocrinology, L.J. DeGroot (ed). 1995, Grune & Straton Inc.)

In patients with primary hypothyroidism of whatever cause, levels may reach 1,000 µU/ml or higher. The magnitude of serum TSH elevation grossly correlates with the severity and in part with the duration of thyroid hormone deficiency. ^430,431 TSH concentrations above the upper limit of normal have been observed in the absence of clinical symptoms and signs of hypothyroidism and in the presence of serum T4 and T3 levels well within the normal range. ^{430 , 432} This condition is most commonly encountered in patients developing hypothyroidism due to Hashimoto's thyroiditis or with limited ability to synthesize thyroid hormone because of prior thyroid surgery, radioiodide treatment, or severe iodine deficiency. ^{430 , 433} There is disagreement on whether such patients have subclinical hypothyroidism or a "compensated state" in which euthyroidism is maintained by chronic stimulation of a reduced amount of functioning thyroid tissue through hypersecretion of TSH. Transient hypothyroidism, may occur in some infants during the early neonatal period. ⁴³⁴ There are two circumstances in which the usual reverse relationship between the serum level of TSH and T4 is not maintained in patients with proven primary hypothyroidism. Treatment with replacement doses of T4 may normalize or even produce serum levels of thyroid hormone above the normal range before the high TSH levels have reached the normal range. ^{404 , 431 , 435} This is particularly true in patients with severe or long-standing primary hypothyroidism who may require three to six months of hormone replacement before TSH levels are fully suppressed. Conversely, serum TSH concentration may remain low or normal for up to five weeks after withdrawal of thyroid hormone replacement when serum levels of T4 and T3 have already declined to values well below the lower range of normal. ^{404 , 436} Causes for discrepancies between TSH and free T4 and T3 levels are listed in  Table 6-9 .

At this time, it is uncertain as to what TSH level is appropriate for suppressive thyroid hormone therapy. The frequency with which patients have subnormal, but detectable, TSH values depends on both the population studied and the sensitivity of the assay (Figure 6-8, below). Using an assay with a sensitivity limit of 0.1 mU/L, 3 to 4% of hospitalized patients have been noted to have a subnormal TSH. ^{432 , 437} When patients with an undetectable TSH in such an assay were re-evaluated in an assay with a sensitivity limit of 0.005 mU/L, 3 of 77 (4%) with thyrotoxicosis and 32 of 37 (86%) with non-thyroidal illness or on drugs were found to have a subnormal but detectable TSH level. ⁴⁰⁷ Thus, the more sensitive the assay, the more likely that patients with clinical thyrotoxicosis will have undetectable serum TSH while those with illness will have a subnormal but detectable level. However, with progressively more sensitive assays, the likelihood of a clinically toxic patient to have a detectable TSH will increase, and if patients on suppressive therapy are treated until the TSH is undetectable, the more likely they will have symptoms of thyrotoxicosis.

Fig ure 6-8. The effect of serum TSH assay sensitivity on the discrimination of euthyroid subject (Euth) from those with thyrotoxicosis (Toxic). (From C. Spencer, Clinical Diagnostics, Eastman Kodak Co., 1992).

A persistent absence of a reverse correlation between serum thyroid hormone and TSH concentration has a very different connotation. A low serum level of thyroid hormone without clear elevation of the serum TSH concentration is suggestive of trophoprivic hypothyroidism, especially when associated with obvious clinical stigmata of hypothyroidism. ⁴³³ An inherited defect of the TSH receptor has been shown to produce marked persistent hyperthyrotropinemia in the presence of normal thyroid hormone levels. ⁴³⁸ In some cases, a mild elevation of the serum TSH level measured by RIA is probably due to the presence of immunoreactive TSH with reduced biologic activity. ³⁹⁷ Distinction between pituitary and hypothalamic hypothyroidism can be made on the basis of the TSH response to the administration of TRH (see below).

In another group of pathologic conditions, serum TSH levels may not be suppressed despite a clear elevation of serum free thyroid hormone levels. Because such a finding is incompatible with a normal thyroregulatory control mechanism of the pituitary, which is preserved in the more common forms of thyrotoxicosis, it has been termed inappropriate secretion of TSH. ⁴³⁹ It implicitly suggests a defective feedback regulation of TSH. When associated with the classical clinical and metabolic changes of thyrotoxicosis, it is usually due to TSH-secreting pituitary adenoma or isolated pituitary resistance to the feedback suppression by thyroid hormone. ⁴³⁹ The existence of hypothalamic hyperthyroidism can be questioned. ⁴⁴⁰ Precise diagnosis requires further studies, including radiologic examination of the pituitary gland and a TRH test. In addition, the presence of high circulating levels of the a-subunit of pituitary glycoprotein hormones (a-SU), giving rise to a disproportionately high a-SU/TSH molar ratio in serum, is characteristic, if not pathognomonic, of TSH-secreting pituitary tumors. ^{439 , 441} Normal, and occasionally high serum TSH levels, associated with a clear elevation in serum FT4 and FT3 but no clear clinical evidence of hypothyroidism or symptoms and signs suggestive of both thyroid hormone deficiency and excess are typical of resistance to thyroid hormone (RTH) ⁴⁴² (see Chapter 16 ).

Although TSH has been implicated in the pathogenesis of simple, nontoxic goiter, unless hypothyroidism supervenes or iodide deficiency is very severe, TSH levels are characteristically normal. Elevated TSH levels may occur in the presence of normal thyroid hormone levels and apparent euthyroidism in nonthyroidal diseases ^{437 , 443} (see also Chapter 5 ) and with primary adrenal failure. ⁴⁴⁴ A more common occurrence in severe acute and chronic illnesses is a normal or low serum TSH concentration despite low levels of T3 and even low T4 levels. ^{407 , 429 , 445} TSH values may be transiently elevated during the recovery phase.446 Various hypotheses to explain these anomalous findings have been proposed, but a satisfactory explanation is not at hand.

A specific RIA for the ß subunits of human TSH is also available but has not found clinical application. ⁴⁴⁷

Thyrotropin-Releasing Hormone (TRH)

TRH. The hypothalamic tripeptide TRH (protirelin) plays a central role in the regulation of pituitary TSH secretion. ^{391 , 419} It is thus not surprising that attempts have been made to measure its concentration in a variety of body fluids, with the purpose of deriving information relevant to the function of the thyroid gland in health and in disease. Several methods have been used for quantitation of TRH, ^448-451 but for many reasons, measurement in humans has failed to provide information of diagnostic value. These include, high dilution of TRH by the time it reaches the systemic circulation, rapid enzymatic degradation and ubiquitous tissue distribution. ^{448 , 450,451} Mean serum TSH levels of 5 and 6 pg/ml have been reported. It is uncertain whether measurements carried out in urine truly represent TRH. ⁴⁴⁹

TRH Test.

The TRH test measures the increase of pituitary TSH in serum in response to the administration of synthetic TRH. The magnitude of the TSH response to TRH is modulated by the thyrotroph response to active thyroid hormone and is thus almost always proportional to the concentration of free thyroid hormone in serum. The response is exquisitely sensitive to minor changes in the level of circulating thyroid hormones, which may not be detected by direct measurement. ^427,428 A direct correlation between basal serum TSH values and the maximal response to TRH has been observed even in the absence of thyroid hormone abnormalities, suggesting that there may be a fine modulation of pituitary sensitivity to TRH in the euthyroid state. ⁴⁵²

TRH normally stimulates pituitary prolactin secretion and, under certain pathologic conditions, the release of GH and ACTH. ³⁹¹ Accordingly, the test has been used for the assessment of a variety of endocrine functions, some unrelated to the thyroid. In clinical practice, the TRH test is used mainly (1) to assess the functional integrity of the pituitary thyrotrophs and thus to aid in differentiating hypothyroidism due to intrinsic pituitary disease from hypothalamic dysfunction and (2) in the diagnosis of mild thyrotoxicosis when results of other tests are equivocal, and (3) in the differential diagnosis of inappropriate TSH secretion, in particular when a TSH-secreting adenoma is suspected.

TRH is effective when given intravenously as a bolus or by infusion, ^{414 , 453} intramuscularly, ⁴⁵⁴ or orally ⁴⁵⁵ in single or repeated doses. Doses as small as 6 µg can elicit a significant TSH response, and there is a linear correlation between the incremental changes in serum TSH concentrations and the logarithm of the administered TRH dose. ⁴¹⁴ The standard test uses a single TRH dose of 400 µg/1.73 m2 body surface area, given by rapid intravenous injection. Serum is collected before and at 15 minutes and then at 30 minute intervals over 120-180 minutes although many clinicians chose to obtain a single post-injection sample at 15, 20 or 30 minutes. In normal persons there is a prompt increase in serum TSH, with a peak level at 15-40 minutes, which is, on the average, 16 µU/ml, or fivefold the basal level. The decline is more gradual, with a return of serum TSH to the preinjection level by three to four hours. ^{414 , 453} Results can be expressed in terms of the peak level of TSH achieved, the maximal increment above the basal level (?TSH), the peak TSH value expressed as a percentage of the basal value, or the integrated area of the TSH response curve. Determination of TSH before and 30 minutes after the injection of TRH provides information concerning the presence or absence of TSH responsiveness but cannot detect delayed or prolonged responses.

The stimulatory effect of TRH is specific for pituitary TSH, its free a- and ß- subunits, ⁴⁴⁷ and prolactin. Under normal circumstances, no significant changes are observed in the serum levels of other pituitary hormones ⁴⁵⁶ or potential thyroid stimulators. ⁴⁵⁷ Responsiveness is present at birth, ⁴⁵⁸ is greater in women than in men, particularly in the follicular phase of the menstrual cycle, ⁴⁵⁹ and may be blunted in older men, ^{414 , 454,455} but this is not a consistent finding. ⁴⁶⁰ On the average, the magnitude of the response is greater at 11 P.M. than at 11 A.M., ⁴⁵² in accordance with the diurnal pattern of the basal TSH level which correlates to its response to TRH. Repetitive administration of TRH to the same subject at daily intervals causes a gradual obtundation of the TSH response, ⁴⁵³ presumably due to the increase in thyroid hormone concentration ⁴⁶¹ and also in part due to TSH "exhaustion". ⁴⁶² However, more than one hour must elapse between the increase in thyroid hormone concentration and TRH administration for inhibition of the TSH response to occur. A number of drugs (see Table 5-4 ) and nonendocrine diseases (see Chapter 5 ) may affect to various extents the magnitude of the response.

TRH-induced secretion of TSH is followed by a release of thyroid hormone that can be detected by direct measurement of serum TT4 and TT3 concentrations. ¹⁶⁰ Peak levels are normally reached approximately four hours after the administration of TRH and are accompanied by an increase in serum Tg concentration. The incremental rise in serum TT3 is relatively greater, and the peak is, on the average, 50% above the basal level. Measurement of changes in serum thyroid hormone concentration after the administration of TRH has been proposed as an adjunctive test and is useful in the evaluation of the integrity of the thyroid gland or bioactivity of endogenous TSH. ⁴⁶³ Increase in RAIU is minimal and occurs only with high doses of TRH given orally. ⁴⁵⁵

Side effects from the intravenous administration of TRH, in decreasing order of frequency, include nausea, flushing or a sensation of warmth, desire to micturate, peculiar taste, light-headedness or headache, dry mouth, urge to defecate, and chest tightness. They are usually mild, begin within a minute after the injection of TRH, and last for a few seconds to several minutes. A transient rise in blood pressure has been observed on occasion, but there are no other changes in vital signs, urine analysis, blood count, or routine blood chemistry tests. ^{456 , 464} The occurrence of circulatory collapse is exceedingly rare. ⁴⁶⁵

The test provides a means to distinguish between secondary (pituitary) and tertiary (hypothalamic) hypothyroidism ( Fig. 6-9 ). Although the diagnosis of primary hypothyroidism can be easily confirmed by the presence of elevated basal serum TSH levels, secondary and tertiary hypothyroidism are typically associated with TSH levels that are low or normal. On occasion the serum TSH concentration may be slightly elevated due to the secretion of biologically less potent molecules, ³⁹⁷ but it remains inappropriately low for the degree of thyroid hormone deficiency. Differentiation between secondary and tertiary hypothyroidism cannot be made with certainty without the TRH test. A TSH response is suggestive of a hypothalamic disorder, and a failure to respond is compatible with intrinsic pituitary dysfunction. ⁴⁶⁶ Furthermore, the typical TSH response curve in hypothalamic hypothyroidism shows a delayed peak with a prolonged elevation of serum TSH before return to the basal value (Figure 6-9). The lack of a TSH response in association with normal prolactin stimulation may be due to isolated pituitary TSH deficiency. ⁴⁶⁷ Caution should be exercised in the interpretation of test results after withdrawal of thyroid hormone replacement or after treatment of thyrotoxicosis when, despite a low serum thyroid hormone concentration, TSH may remain low and not respond to TRH for several weeks. ^{404 , 433 , 436 , 468}

Figure 6-9. Typical serum TSH responses to the administration of a single intravenous bolus of TRH at time 0 in various conditions. The normal response is represented by the shaded area. Data used for this figure are the average of several studies. (From S. Refetoff, Endocrinology, L.J. DeGroot (ed). 1979, Grune & Straton Inc.)

In the most common forms of thyrotoxicosis, the mechanism of feedback regulation of TSH secretion is intact but is appropriately suppressed by the excessive amounts of thyroid hormone. Thus, both the basal TSH level and its response to TRH are suppressed unless thyrotoxicosis is TSH induced. ^{404 , 407 , 417} With the development of more sensitive TSH assays, the TRH test is generally not needed in the evaluation of a thyrotoxic patient with an undetectable TSH.407 Differential diagnosis of conditions leading to inappropriate secretion of TSH may be aided by the TRH test result. Elevated basal TSH values that do not respond by a further increase to TRH are typical of TSH-secreting pituitary adenomas. ^{439 , 441} Patients with inappropriate secretion of TSH due to resistance to thyroid hormone have a normal or exaggerated TSH response to TRH that, in most instances, is suppressed with supraphysiologic doses of thyroid hormone. ⁴⁴²

Because of the high sensitivity of the pituitary gland to the feedback regulation by thyroid hormone, small changes in the latter profoundly affects the response of TSH to TRH. Thus, patients with non-TSH-induced thyrotoxicosis of the mildest degree have a reduced TSH response to TRH whereas those with primary hypothyroidism exhibit an accentuated response that is prolonged (Figure 6-9, see above). These changes may occur in the absence of clinical or other laboratory evidence of thyroid dysfunction.

The TSH response to TRH, is subnormal or absent in one-third of apparently euthyroid patients with autoimmune thyroid disease, and even members of their family, may not respond to TRH. ^469,470 Most, but not all patients with reduced TSH response to TRH, will also show thyroid activity that is nonsuppressible by thyroid hormone. A common dissociation between these two tests is typified by a normal TRH response in a nonsuppressible patient. This finding is not surprising since patients with nonsuppressible thyroid glands often have limited capacity to synthesize and secrete thyroid hormone, due to prior therapy or partial destruction of their glands by the disease process. Clinically, euthyroid patients, who do not respond to TRH, admittedly have a slight excess of thyroid hormone. It is less easy to reconcile the rare occurrence of TRH unresponsiveness in a patient who is suppressible by exogenous thyroid hormone. It should be remembered, however, that a suppressed pituitary may take a variable amount of time to recover, a phenomenon that may be the basis of such discrepancies. ^{404 , 436 , 468} Despite discrepancies between the results of the TRH and T3 suppression tests, ^469,470 the use of the former is much preferred particularly in elderly patients in whom administration of T3 can produce untoward effects.

Thyroid Suppression Test

The maintenance of thyroid gland activity that is independent of TSH can be demonstrated by the thyroid suppression test. Under normal conditions, administration of thyroid hormone in quantities sufficient to satisfy the body requirement suppresses endogenous TSH resulting in reduction of thyroid hormone synthesis and secretion. Since thyrotoxicosis due to excessive secretion of hormone by the thyroid gland implies that the feedback control mechanism is not operative or has been perturbed, it is easy to understand why under such circumstances the supply of exogenous hormone would also be ineffective in suppressing thyroid gland activity. The test is of particular value in patients who are euthyroid or only mildly thyrotoxic but suspected of having abnormal thyroid gland stimulation or autonomy.

Usually the test is carried out with 100 µg of L-T3 (liothyronine) given daily in two divided doses over a period of 7-10 days. 24 hour RAIU is obtained before and during the last two days of T3 administration.476 Normal persons show a suppression of the RAIU by at least 50% compared to the pre-L-T3 treatment value. No change or lesser reduction is not only typical of Graves' disease but also other form of endogenous thyrotoxicosis, including toxic adenoma, functioning carcinoma, and thyrotoxicosis due to trophoblastic diseases. The presence of nonsuppressibility indicates thyroid gland activity independent of TSH but not necessarily thyrotoxicosis. Euthyroid patients with autonomous thyroid function have a normal TSH response to TRH before the administration of L-T3. However, inhibition of TSH secretion by the exogenous T3 does not suppress the autonomous activity of the thyroid gland. This is the most commonly encountered discrepancy between the results of the two related tests. When the T3 suppression test is used in conjunction with the scintiscan, localized areas of autonomous function can be identified. The test can be carried out without the administration of radioisotopes by measuring serum T4 before and two weeks following the ingestion of L-T3. Although total suppression of T4 secretion never occurs, even after prolonged treatment with L-T3, a reduction by at least 50% is normal. ⁴⁷⁷

Variants of the test have been proposed to reduce the potential risks of L-T3 administration in elderly patients and in those with angina pectoris or congestive heart failure. With the availability of sensitive TSH determinations and the TRH test, which are less dangerous, thyroid suppression tests are no longer indicated.

Specialized Thyroid Tests

A number of specialized tests are available for the evaluation of specific aspects of thyroid hormone biosynthesis, secretion, turnover, distribution, and absorption. Their primary application is of investigative nature. They are only briefly mentioned here for the sake of completeness.

Iodotyrosine Deiodinase Activity

The test involves the intravenous administration of tracer MIT or DIT labeled with radioiodide. Urine, collected over a period of four hours, is analyzed by chromatography or resin column separation. Normally, only 4-8% of the radioactivity is excreted as such; the remainder appears in the urine in the form of iodide. ⁴⁸⁰ Excretion of larger amounts of the parent compound indicates inability to deiodinate iodotyrosine. The test is useful in the diagnosis of a dehalogenase defect (see Chapter 16 ).

Test for Defective Hormonogenesis

After administration of RAI, the isotopically labeled compounds synthesized in the thyroid gland and those secreted into the circulation can be analyzed by immunologic, chromatographic, electrophoretic, and density gradient centrifugation techniques. ⁴⁸¹ Such tests serve to evaluate the synthesis and release of thyroid hormone, as well as to delineate the formation of abnormal iodoproteins.

Iodine Kinetic Studies

The iodine kinetic procedure is used to evaluate overall iodide metabolism and to elucidate the pathophysiology of thyroid diseases. The analysis involves follow-up of the fate of administered radioiodide tracer by measurement of thyroidal accumulation, secretion into blood, and excretion in the urine and feces. ⁴⁸² Double tracer techniques and programs for computer-assisted analysis of data are available.

Absorption of Thyroid Hormone

Failure to achieve normal serum thyroid hormone concentration after administration of replacement doses of thyroid hormone is usually due to poor compliance, occasionally to the use of inactive preparations, and rarely, if ever, to malabsorption. The last can be evaluated by the simultaneous oral and intravenous administration of the hormone labeled with two different iodine isotope tracers. The ratio of the two isotopes in blood is proportional to the net absorbed fraction of the orally administered hormone. ^483,484 Under normal circumstances, approximately 80% of T4 and 95% of T3 administered orally are absorbed. Hypothyroidism and a variety of other unrelated conditions have little effect on the intestinal absorption of thyroid hormones. Absorption may be diminished in patients with steatorrhea, in some cases of hepatic failure, during treatment with cholestyramine, and with diets rich in soybeans. The absorption of thyroid hormone can also be evaluated by the administration of a single oral dose of 100 µg T3 or 1 mg T4, followed by their measurement in blood sampled at various intervals. ^485,486

Turnover Kinetics of T4 and T3

Turnover kinetic studies require the intravenous administration of isotope-labeled tracer T4 or T3. ^487-491 The half-time (t1/2) of disappearance of the hormone is calculated from the rate of decrease in serum trichloroacetic acid precipitable, ethanol extractable, or antibody precipitable isotope counts. Compartmental analysis can be used for the calculation of the turnover parameters. ^488,489 The calculated daily degradation (D) or production rate (PR) is the product of the fractional turnover rate (K), the extrathyroidal distribution space (DS), and the average concentration of the hormone in serum. Noncompartmental analysis may be used for the calculation of kinetic parameters. ⁴⁸⁸ The metabolic clearance rate (MCR) is defined as the dose of the injected labeled tracer divided by the area under its curve of disappearance. The PR is then calculated from the product of the MCR and the average concentration of the respective nonradioactive iodothyronine measured in serum over the period of the study. Simultaneous studies of the T4 and T3 turnover kinetics can be carried out by injection of both hormones, labeled with different iodine isotopes. ^{488 , 490,491}

Average normal values in adults for T4 and T3, respectively, are: t1/2 = 7.0 and 0.8 days; K = 10% and 90% per day; DS = 11 and 30 liters of serum equivalent; MCR = 1.1 and 25 liters/day; and PR = 90 and 25 µg/day.

The hormonal PR is accelerated in thyrotoxicosis and diminished in hypothyroidism. In euthyroid patients with TBG abnormalities, the PR remains normal, since changes in the serum hormone concentration are accompanied by compensatory changes in the fractional turnover rate and the extrathyroidal hormonal pool. ⁴⁹² A variety of nonthyroidal illnesses may alter hormone kinetics ^{491 , 493} (see Chapter 5 ).

Metabolic Kinetics of Thyroid Hormones and Their Metabolites

The kinetics of production of various metabolites of T4 and T3 in peripheral tissues and their further metabolism can be studied. Most methods use radiolabeled iodothyronine tracers injected intravenously. ^489-491 Their disappearance is followed in serum samples obtained at various intervals of time after injection of the tracers by means of chromatographic and immunologic techniques of separation. Kinetic parameters can be calculated by noncompartmental analysis or by two or multiple compartment analysis. Estimates have been made by the differential measurement in urine of the isotopes derived from the precursor and its metabolite. They are in agreement with measurements carried out in serum. ⁴⁹⁴ Conversion rates (CR) of iodothyronines, principally generated in peripheral tissues, can be calculated from the ratio of their PR, and that of their respective precursors. Some iodothyronines, such as T3, are secreted by the thyroid gland as well as generated in peripheral tissues. Studies to calculate the CR require administration of thyroid hormone to block thyroidal secretion. ⁴⁹³

On the average 35% and 45% of T4 are converted to T3 and rT3, respectively, in peripheral tissues. The conversion of T4 to T3 is greatly diminished in a variety of illnesses (see Chapter 5 ) of nonthyroidal origin and in response to many drugs ( Table 5-3 ). Degradation and monodeiodination of iodothyronines can be estimated without the administration of isotopes. They are, however, less accurate. The conversion of T4 to T3 can be estimated semiquantitatively by the measurement of serum TT3 concentration after treatment with replacement doses of T4. ⁴⁹³

Measurement of the Production Rate and Metabolic Kinetics of Other Compounds

The metabolism and PRs of a variety of compounds related to thyroid physiology can be studied using their radiolabeled congeners and application of the general principles of turnover kinetics. Studies of TSH have demonstrated changes related not only to thyroid dysfunction but also associated with age, kidney, and liver disease. ^495,496 Studies of the turnover kinetics of TBG have shown that the slight increases and decreases of serum TBG concentration associated with hypothyroidism and thyrotoxicosis, respectively, are due to changes in the degradation rate of TBG rather than synthesis. ⁴⁹²

Transfer of Thyroid Hormone from Blood to Tissues

Transfer of hormone from blood to tissues can be estimated in vivo by two techniques. A direct method follows the accumulation of the administered labeled hormone tracer by surface counting over the organ of interest. ⁴⁹⁷ An indirect method follows the early disappearance from plasma of the simultaneously administered hormone and albumin, labeled with different radioisotope tracers. ⁴⁹⁸ The difference between the rates of disappearance of the hormone and albumin represents the fraction of hormone that has left the vascular (albumin) space and presumably has entered the tissues.