**Part 1**

## **Evaluating the Thyroid Gland and Its Diseases**

**1**

Evren Bursuk *University of İstanbul* 

*Turkey* 

**Introduction to Thyroid: Anatomy and Functions** 

As it is known the endocrine system together with the nervous system enables other systems in the body to work in coordination with each other and protect homeostasis using hormones. Hormones secreted by the endocrine system are carried to target organs and

The thyroid gland is among the most significant organs of the endocrine system and has a weight of 15-20g. It is soft and its colour is red. This organ is located between the C5-T1 vertebrae of columna vertebralis, in front of the trachea and below the larynx. It is comprised of two lobes (lobus dexter and lobus sinister) and the isthmus that binds them together (Figure 1a). Capsule glandular which is internal and external folium of thyroid

Hyoid bone

Larynx

Thyroid gland

Isthmus

Trachea

**1. Introduction** 

**2. Anatomy** 

cause affect through receptors.

Fig. 1a. The thyroid gland anatomy

### **Introduction to Thyroid: Anatomy and Functions**

Evren Bursuk *University of İstanbul Turkey* 

#### **1. Introduction**

As it is known the endocrine system together with the nervous system enables other systems in the body to work in coordination with each other and protect homeostasis using hormones. Hormones secreted by the endocrine system are carried to target organs and cause affect through receptors.

#### **2. Anatomy**

The thyroid gland is among the most significant organs of the endocrine system and has a weight of 15-20g. It is soft and its colour is red. This organ is located between the C5-T1 vertebrae of columna vertebralis, in front of the trachea and below the larynx. It is comprised of two lobes (lobus dexter and lobus sinister) and the isthmus that binds them together (Figure 1a). Capsule glandular which is internal and external folium of thyroid

Fig. 1a. The thyroid gland anatomy

Introduction to Thyroid: Anatomy and Functions 5

With these transcription factors working together, follicular cell growth and the development of such thyroid-specific proteins as TSH receptor and thyroglobulin is commenced. If any mutation occurs in these transcription factors, babies are born with hypothyroidism due to thyroid agenesis or insufficient secretion of thyroid hormones. (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Scanlon, 2001;

The fundamental functional unit of the thyroid gland is the follicle cells and their diameter is in the range of 100-300 µm. Follicle cells in the thyroid gland create a lumen, and there exists a protein named thyroglobulin that they synthesize in the colloid in this lumen (Figure 2a-b). The apical part of these follicle cells make contact with colloidal lumen and its basal part with blood circulation through rich capillaries. Thus, thyroid hormones easily pass into circulation and can reach target tissues. Parafollicular-c cells secreting a hormone called calcitonin that affects the calcium metabolism also exist in this gland (Di Lauro & De

Colloid Flat cell

Snell, 1995; Utiger, 1997).

Fig. 2a. Thyroid follicule cell in the inactive state

Fig. 2b. Thyroid follicule cell in the active state

Parafollicular cell

gland is wrapped up by a fibrosis capsule named thyroid. The thyroid gland is nourished by a thyroidea superior that is the branch of a. carotis external and a. thyroid inferior that is the branch of a. subclavia (Figure 1b) (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Snell, 1995; Utiger, 1997).

In addition, there are 4 parathyroid glands in total, two of which are on the right and the other two are on the left in between capsule foliums and behind the thyroid gland lobes (Figure 1b) (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Snell, 1995; Utiger, 1997).

Fig. 1b. The thyroid gland anatomy with vessels

#### **3. Embryology and histology**

The thyroid gland develops from the endoderm by a merging of the 4th pouch parts of the primitive pharynx and tongue base median line in the 3rd gestational week. By fetus organifying iodine in the 10th gestational week and commencing the thyroid hormone synthesis, T4 (L-thyroxin) and TSH (thyroid stimulating hormone) can be measured in fetal blood. Due to the fact that hormone and thyroglobulin syntheses in fetal thyroid increase in the 2nd trimester, an increase is also observed in T4 and TSH amounts. In addition, the development of fetal hypothalamus contributes to the synthesizing of TRH (thyroid releasing hormone) and thus TSH increase. While TRH can be passed from mother to fetus through the placenta, TSH cannot. T3 (3,5,3'-triiodo-L-thyronine) begins increasing at the end of the 2nd trimester and is detected in fetal blood in small amounts. Its synthesis increases after birth.

The development of the thyroid gland is controlled by thyroid transcription factor 1 (TTF-1 or its other name NKX2A), thyroid transcription factor 2 (TTF-2 or FKHL15) and paired homeobox-8 (PAX-8). (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Scanlon, 2001; Snell, 1995; Utiger, 1997).

gland is wrapped up by a fibrosis capsule named thyroid. The thyroid gland is nourished by a thyroidea superior that is the branch of a. carotis external and a. thyroid inferior that is the branch of a. subclavia (Figure 1b) (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti &

In addition, there are 4 parathyroid glands in total, two of which are on the right and the other two are on the left in between capsule foliums and behind the thyroid gland lobes (Figure 1b) (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996;

The thyroid gland develops from the endoderm by a merging of the 4th pouch parts of the primitive pharynx and tongue base median line in the 3rd gestational week. By fetus organifying iodine in the 10th gestational week and commencing the thyroid hormone synthesis, T4 (L-thyroxin) and TSH (thyroid stimulating hormone) can be measured in fetal blood. Due to the fact that hormone and thyroglobulin syntheses in fetal thyroid increase in the 2nd trimester, an increase is also observed in T4 and TSH amounts. In addition, the development of fetal hypothalamus contributes to the synthesizing of TRH (thyroid releasing hormone) and thus TSH increase. While TRH can be passed from mother to fetus through the placenta, TSH cannot. T3 (3,5,3'-triiodo-L-thyronine) begins increasing at the end of the 2nd trimester and is detected in fetal blood in small amounts. Its synthesis increases after birth.

The development of the thyroid gland is controlled by thyroid transcription factor 1 (TTF-1 or its other name NKX2A), thyroid transcription factor 2 (TTF-2 or FKHL15) and paired homeobox-8 (PAX-8). (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

Singer, 1997; Mc Gregor, 1996; Snell, 1995; Utiger, 1997).

Superior thyroid

Larynx

Thyroid gland Isthmus

Trachea Inferior thyroid

artery

Fig. 1b. The thyroid gland anatomy with vessels

artery

Gregor, 1996; Scanlon, 2001; Snell, 1995; Utiger, 1997).

**3. Embryology and histology** 

Snell, 1995; Utiger, 1997).

With these transcription factors working together, follicular cell growth and the development of such thyroid-specific proteins as TSH receptor and thyroglobulin is commenced. If any mutation occurs in these transcription factors, babies are born with hypothyroidism due to thyroid agenesis or insufficient secretion of thyroid hormones. (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Scanlon, 2001; Snell, 1995; Utiger, 1997).

The fundamental functional unit of the thyroid gland is the follicle cells and their diameter is in the range of 100-300 µm. Follicle cells in the thyroid gland create a lumen, and there exists a protein named thyroglobulin that they synthesize in the colloid in this lumen (Figure 2a-b). The apical part of these follicle cells make contact with colloidal lumen and its basal part with blood circulation through rich capillaries. Thus, thyroid hormones easily pass into circulation and can reach target tissues. Parafollicular-c cells secreting a hormone called calcitonin that affects the calcium metabolism also exist in this gland (Di Lauro & De

Fig. 2a. Thyroid follicule cell in the inactive state

Fig. 2b. Thyroid follicule cell in the active state

Introduction to Thyroid: Anatomy and Functions 7

aminoacids. While the most synthesized hormone in thyroid gland is T4, the most efficient hormone is T3. (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed &

*1st stage* is the obtaining of iodine by active transport to thyroid follicle cells by utilizing Na+/I- symporter pump. Starting and acceleration of this transport is under the control of TSH. Organification increases as the iodine concentration of the cell rises, however, this pump slows down and stops after a point. For this reason, it is believed that a concentrationdependent autocontrol mechanism exists at this level. This stage of the synthesis that is the iodine transport can be inhibited by single-value anions such as perchlorate, pertechnetate, and thiocyanate. Pertechnetate (99mm) is also used in thyroid gland imaging due to its characteristic of being radioactive (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

*2nd stage* is oxidation of iodine by NADPH dependent thyroperoxidase enzyme in the presence of H2O2 which, at this stage, occurs in follicular lumen. The drugs propylthiouracil and methimazole inhibit this step (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

*3rd stage* is the binding of oxidized iodine with thyroglobulin tyrosine residues. This is called iodization of tyrosine or organification. Thus, monoiodotyrosine (MIT) or diiodotyrosine (DIT) is synthesized. These are the inactive thyroid hormone forms (Figure 3) (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

Fig. 3. Chemical structures of tyrosine, monoiodothyronine, and diiodothyronine

Pangaro, 1995; Utiger, 1997). Basely, thyroid hormone synthesis occurs in 4 stages:

Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Scanlon, 2001; Snell, 1995; Utiger, 1997).

#### **4. Physiology**

The thyroid gland synthesizes and secretes T3 and T4 hormones and these hormones play an important role in the functioning of the body.

#### **4.1 Iodine metabolism**

Chemicals in the organism are divided into two as organic and inorganic according to their carbon contents. Organic compounds always contain carbon and have covalent bonds. Carbohydrates, fats, proteins, nucleic acids, enzymes, and adenosine triphosphate (ATP) are the organic compounds. Inorganic compounds have simple structures and do not contain carbons except for carbon dioxide (CO2) and bicarbonate ion (HCO3-1). They contain ionic and covalent bonds in their structures. Water, acid, base, salt, and minerals are the inorganic forms. Iodine that is a trace element important for life is among these minerals and is the fundamental substance for thyroid hormones (T3 and T4) synthesis. Iodine exists in 3 forms in the circulation. The first one is inorganic iodine (I-) and is about 2-10 µg/L. Secondly, it exists sparingly in organic compounds before going into the thyroid hormone structure. And the third is the most important one and it is present as bound to protein in thyroid hormones (35-80 µg/L). About 59% and 65%, respectively, of the molecular weights of T3 and T4 hormones are comprised of iodine. This accounts for 30% of iodine in the body. The remaining iodine (approximately 70%) exists in a way disseminated to other tissues such as mammary glands, eyes, gastric mucosa, cervix, and salivary glands, and it bears great importance for the functioning of these tissues (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

The daily intake is recommended by the United States Institute of Medicine as in the range of 110-130 µg for babies up to 12 months, 150 µg for adults, 220 µg for pregnant women, and 290 µg for women in lactation (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

Iodine is taken into the body oral. Among the foods that contain iodine are seafood, iodinerich vegetables grown in soil, and iodized salt. For this reason, iodine intake geographically differs in the world. Places that are seen predominantly to have iodine deficiency are icy mountainous areas and daily iodine intake in these places is less than 25 µg. Hence, diseases due to iodine deficiency are more common in these geographies. Cretinism in which mental retardation is significant was first identified in the Western Alps (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995 Utiger, 1997).

#### **4.2 Thyroid hormone synthesis**

Iodine absorbed from the gastrointestinal system immediately diffuses in extracellular fluid. T3 and T4 hormones are fundamentally formed by the addition of iodine to tyrosine

Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Scanlon, 2001; Snell, 1995;

The thyroid gland synthesizes and secretes T3 and T4 hormones and these hormones play an

Chemicals in the organism are divided into two as organic and inorganic according to their carbon contents. Organic compounds always contain carbon and have covalent bonds. Carbohydrates, fats, proteins, nucleic acids, enzymes, and adenosine triphosphate (ATP) are the organic compounds. Inorganic compounds have simple structures and do not contain carbons except for carbon dioxide (CO2) and bicarbonate ion (HCO3-1). They contain ionic and covalent bonds in their structures. Water, acid, base, salt, and minerals are the inorganic forms. Iodine that is a trace element important for life is among these minerals and is the fundamental substance for thyroid hormones (T3 and T4) synthesis. Iodine exists in 3 forms in the circulation. The first one is inorganic iodine (I-) and is about 2-10 µg/L. Secondly, it exists sparingly in organic compounds before going into the thyroid hormone structure. And the third is the most important one and it is present as bound to protein in thyroid hormones (35-80 µg/L). About 59% and 65%, respectively, of the molecular weights of T3 and T4 hormones are comprised of iodine. This accounts for 30% of iodine in the body. The remaining iodine (approximately 70%) exists in a way disseminated to other tissues such as mammary glands, eyes, gastric mucosa, cervix, and salivary glands, and it bears great importance for the functioning of these tissues (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti

The daily intake is recommended by the United States Institute of Medicine as in the range of 110-130 µg for babies up to 12 months, 150 µg for adults, 220 µg for pregnant women, and 290 µg for women in lactation (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer,

Iodine is taken into the body oral. Among the foods that contain iodine are seafood, iodinerich vegetables grown in soil, and iodized salt. For this reason, iodine intake geographically differs in the world. Places that are seen predominantly to have iodine deficiency are icy mountainous areas and daily iodine intake in these places is less than 25 µg. Hence, diseases due to iodine deficiency are more common in these geographies. Cretinism in which mental retardation is significant was first identified in the Western Alps (Di Lauro & De Felice, 2001; Dillmann, 2004; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al.,

Iodine absorbed from the gastrointestinal system immediately diffuses in extracellular fluid. T3 and T4 hormones are fundamentally formed by the addition of iodine to tyrosine

2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995 Utiger, 1997).

& Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

**4.2 Thyroid hormone synthesis** 

Utiger, 1997).

**4. Physiology** 

**4.1 Iodine metabolism** 

important role in the functioning of the body.

aminoacids. While the most synthesized hormone in thyroid gland is T4, the most efficient hormone is T3. (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997). Basely, thyroid hormone synthesis occurs in 4 stages:

*1st stage* is the obtaining of iodine by active transport to thyroid follicle cells by utilizing Na+/I- symporter pump. Starting and acceleration of this transport is under the control of TSH. Organification increases as the iodine concentration of the cell rises, however, this pump slows down and stops after a point. For this reason, it is believed that a concentrationdependent autocontrol mechanism exists at this level. This stage of the synthesis that is the iodine transport can be inhibited by single-value anions such as perchlorate, pertechnetate, and thiocyanate. Pertechnetate (99mm) is also used in thyroid gland imaging due to its characteristic of being radioactive (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

*2nd stage* is oxidation of iodine by NADPH dependent thyroperoxidase enzyme in the presence of H2O2 which, at this stage, occurs in follicular lumen. The drugs propylthiouracil and methimazole inhibit this step (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

*3rd stage* is the binding of oxidized iodine with thyroglobulin tyrosine residues. This is called iodization of tyrosine or organification. Thus, monoiodotyrosine (MIT) or diiodotyrosine (DIT) is synthesized. These are the inactive thyroid hormone forms (Figure 3) (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

Fig. 3. Chemical structures of tyrosine, monoiodothyronine, and diiodothyronine

*4th stage* is the coupling and T3 and T4 are synthesized from MIT and DIT (Figure 4).

$$\text{MIIT} \vdash \text{DIT} \to \text{T} \flat \tag{1}$$

Introduction to Thyroid: Anatomy and Functions 9

and T4 facilitated diffusion (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor,

Not all hormones separated from thyroglobulin can pass to the blood. Such iodotyronines as MIT and DIT cannot leave the cell and are reused as deiodonized. In addition, T3 is formed from a certain amount of T4 again by deiodonization. These reactions occur in the thyroid follicular cell and the enzyme catalyzing these reactions, in other words, deiodinizations is dehalogenase. Through this deiodinization, about 50% of iodine in the thyroglobulin structure is taken back and can be reused. Iodine deficiency in individuals lacking this enzyme, and correspondingly, hypothyroid goiter is observed. Such patients are given iodine replacement treatment (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor,

When thyroid hormones pass into circulation, all become inactive by reversibly binding to carrier proteins that are synthesized in the liver. While those being bound to proteins prevent a vast amount of hormones to be excreted in the urine, it also acts as a depository. Thus, free, in other words, active hormone exists in blood only as much as is needed. The main carrier proteins are thyroxin-binding globulin (TBG), thyroxin-binding prealbumin (transthyretin, TTR) and serum albumin (Table 1) (Benvenga, 2005; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo

TBG is the most bound protein by thyroid hormones. Its molecular weight is 54 kDa and is has the least concentration among others in circulations. The hormone that binds to this protein the most is T4 and is about 75% of T4 hormone. This is responsible for the diffusion of T4 hormone in extracellular fluid in large amounts. However, T3 is bound in fewer amounts. While TBG rise increases total T3 and total T4, it does not affect free T3 and T4 (Benvenga, 2005; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed &

And TTR has a weight of 55kDa and has a lower rate of binding although its plasma concentration is less than TBG, and this value is more or less around 1/100 (Benvenga, 2005; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed &

Serum albumin is a protein with a molecule weight of 65kDa and has a lower rate of binding even though its plasma concentration is the highest (Benvenga, 2005; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo

Due to the fact that T3 binds to fewer proteins, it is more active in intracellular region. While they become free when needed because of the fact that the affinity of carrier proteins is more to T4, the half-life of T4 is about six days, whereas the half-life of T3 is less than one day. T3 is

Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

1996; Reed & Pangaro, 1995; Utiger, 1997).

1996; Reed & Pangaro, 1995; Utiger, 1997).

**4.4 Thyroid hormone transport** 

Pangaro, 1995; Utiger, 1997).

Pangaro, 1995; Utiger, 1997).

$$\text{DIT} \text{+DT} \to \text{T} \tag{2}$$

Fig. 4. Chemical structures of triiodothyronine, thyroxin, and revers T3

In addition to synthesizing this way, the T3 hormone is also created by the metabolization of T4.

Almost the entire colloid found in each thyroid follicle lumen is thyroglobulin. Thyroglobulin that contains 70% of thyroid protein content is a glycoprotein with a molecular weight of 660 kDa. Each thryoglobulin molecule has 70 tyrosine aminoacids and contains 6 MIT, 4 DIT, 2 T4, and 0.2 T3 residues. Thyroglobulin synthesis is TSHdependent and occurs in the granulose endoplasmic reticulum of the follicle cells of the thyroid gland. The synthesized thyroglobulin is transported to the apical section of the cell and passes to the follicular lumen through exocytose, and then joins thyroid hormone synthesis (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

#### **4.3 Thyroid hormone secretion**

Thyroid hormones are stocked in the colloid of follicle cells lumen in a manner bound to thyroglobulin. With TSH secretion, apical microvillus count increases and colloid droplet is caught by microtubules and taken back to the apex of the follicular cell through pinocytosis. Lysosomes approach these colloidal pinocytic vesicles containing thyroglobulin and thyroid hormones. These vesicles bind with lysosomes and form fagolysosomes. Lysosomal proteases are activated while these fagolysosomes move towards the basal cell, and thus, thyroglobulin is hydrolyzed. Tyrosine formed as a result of this reaction is excreted by T3

MIT+DIT T 3 (1)

DIT+DIT T <sup>4</sup> (2)

*4th stage* is the coupling and T3 and T4 are synthesized from MIT and DIT (Figure 4).

Fig. 4. Chemical structures of triiodothyronine, thyroxin, and revers T3

of T4.

Pangaro, 1995; Utiger, 1997).

**4.3 Thyroid hormone secretion** 

In addition to synthesizing this way, the T3 hormone is also created by the metabolization

Almost the entire colloid found in each thyroid follicle lumen is thyroglobulin. Thyroglobulin that contains 70% of thyroid protein content is a glycoprotein with a molecular weight of 660 kDa. Each thryoglobulin molecule has 70 tyrosine aminoacids and contains 6 MIT, 4 DIT, 2 T4, and 0.2 T3 residues. Thyroglobulin synthesis is TSHdependent and occurs in the granulose endoplasmic reticulum of the follicle cells of the thyroid gland. The synthesized thyroglobulin is transported to the apical section of the cell and passes to the follicular lumen through exocytose, and then joins thyroid hormone synthesis (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed &

Thyroid hormones are stocked in the colloid of follicle cells lumen in a manner bound to thyroglobulin. With TSH secretion, apical microvillus count increases and colloid droplet is caught by microtubules and taken back to the apex of the follicular cell through pinocytosis. Lysosomes approach these colloidal pinocytic vesicles containing thyroglobulin and thyroid hormones. These vesicles bind with lysosomes and form fagolysosomes. Lysosomal proteases are activated while these fagolysosomes move towards the basal cell, and thus, thyroglobulin is hydrolyzed. Tyrosine formed as a result of this reaction is excreted by T3

and T4 facilitated diffusion (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

Not all hormones separated from thyroglobulin can pass to the blood. Such iodotyronines as MIT and DIT cannot leave the cell and are reused as deiodonized. In addition, T3 is formed from a certain amount of T4 again by deiodonization. These reactions occur in the thyroid follicular cell and the enzyme catalyzing these reactions, in other words, deiodinizations is dehalogenase. Through this deiodinization, about 50% of iodine in the thyroglobulin structure is taken back and can be reused. Iodine deficiency in individuals lacking this enzyme, and correspondingly, hypothyroid goiter is observed. Such patients are given iodine replacement treatment (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

#### **4.4 Thyroid hormone transport**

When thyroid hormones pass into circulation, all become inactive by reversibly binding to carrier proteins that are synthesized in the liver. While those being bound to proteins prevent a vast amount of hormones to be excreted in the urine, it also acts as a depository. Thus, free, in other words, active hormone exists in blood only as much as is needed. The main carrier proteins are thyroxin-binding globulin (TBG), thyroxin-binding prealbumin (transthyretin, TTR) and serum albumin (Table 1) (Benvenga, 2005; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

TBG is the most bound protein by thyroid hormones. Its molecular weight is 54 kDa and is has the least concentration among others in circulations. The hormone that binds to this protein the most is T4 and is about 75% of T4 hormone. This is responsible for the diffusion of T4 hormone in extracellular fluid in large amounts. However, T3 is bound in fewer amounts. While TBG rise increases total T3 and total T4, it does not affect free T3 and T4 (Benvenga, 2005; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

And TTR has a weight of 55kDa and has a lower rate of binding although its plasma concentration is less than TBG, and this value is more or less around 1/100 (Benvenga, 2005; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

Serum albumin is a protein with a molecule weight of 65kDa and has a lower rate of binding even though its plasma concentration is the highest (Benvenga, 2005; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

Due to the fact that T3 binds to fewer proteins, it is more active in intracellular region. While they become free when needed because of the fact that the affinity of carrier proteins is more to T4, the half-life of T4 is about six days, whereas the half-life of T3 is less than one day. T3 is

Introduction to Thyroid: Anatomy and Functions 11

Deiodinase I or 2 Deiodinase 5'-DIII

triiodothyronine (T3) reverse T3 (rT3)

3, 3,5 Triiodothyronine 3, 3' 5' Triiodothyronine

L-thyroxin (T4) 3,5,3',5'-tetra iodothyronine

Synthesis and secretions need to be kept at a certain level in order for the liveliness of thyroid hormones to be maintained. In this respect, the most important mechanism in controlling the synthesis and secretion of thyroid hormones is the hypothalamushypophysis-thyroid axis. Another one is the autocontrol mechanism that is dependent on iodine concentration as noted earlier (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

Hormone synthesis and secretion of the thyroid gland is under the strict control of this axis. This event begins with TRH synthesis in the hypothalamus. TRH is carried from the hypothalamus to the hypophysis through portal circulation, and TSH hormone is secreted here following the interaction with TRH receptors in the hypophysis front lobe. TSH is then transferred by blood and stimulates the thyroid gland, and thus, thyroid hormone synthesis and secretion begins. However, if thyroid hormone and synthesis is too large an amount, the feedback system is activated and TSH and TRH are suppressed (Figure 6) (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005;

Fig. 6. Controlling of thyroid hormone secretion by the hypothalamus-hypothyroidism-

Thyroid Secretes T3 and T4

Hypothalamus Secretes TRH

Pituitary lobus Secretes TSH

 (-) 

 (-) 

Fig. 5. Effects of deiodinase enzymes

5'-DI or 5'-DII

**4.6 Controlling the thyroid hormone synthesis and secretion** 

Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Utiger, 1997).

**4.6.1 Hypothalamus-hypophysis-thyroid axe** 

Scanlon, 2001; Utiger, 1997).

thyroid axis

more active since T4 binds to cytoplasmic proteins when they enter the cell are going to affect (Benvenga, 2005; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).


Table 1. Comparison of the binding of thyroid hormones to carrier proteins

#### **4.5 Thyroid hormone metabolism**

A 100 µg thyroid hormone is secreted from the thyroid gland and most of these hormones are T4. About 40% of T4 turn into T3 which is 3 times stronger in periphery, especially in the liver and kidney with deiodinase enzymes (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

Metabolically, in order for active T3 to form, deiodination needs to occur in region 5' of tyrosine. Instead, if it occurs in the 5th atom of inner circle, metabolically inactive reverse triiodothyronine (rT3) is formed. Three types of enzymes that are Selenoenzyme 5' deiodinase type I (5'-DI), the type II5' iodothyronine deiodinase (5'-DII) and the 5, or inner circle deiodinase type III (5-DIII) catalyze these deiodinations (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

5'-DI enzyme is especially found in the liver, kidneys, and thyroid, and 5'-DII enzyme exists in the brain, hypophysis, placenta, and keratinocytes. 5'-DIII is found in the brain, placenta, and epidermis. Both 5'-DI and 5'DII enzymes allow T4 to transform into active T3; but with one difference, that is, while 5'- DI enzyme provides the formed T3 to plasma, T3 formed by 5'-DII enzyme stays in the tissue and regulates local concentration. This enzyme is regulated by increases and decreases in thyroid hormones. For instance, hyperthyroidism inhibits enzyme and blocks the transformation from T4 to T3 in such tissues as the brain and hypophsis. Transformation from T4 to T3 is affected by such changes in the organism as hunger, systemic disease, acute stress, iodine contrating agents, and drugs such as propiltiourasil, propranolol, amiodaron, and glicocortikoid, but is not affected by metrmazol. 5'-DIII enzyme transforms T4 into metabolically inactive reverse T3 (rT3) (Figure 5) (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997). As mentioned earlier, 40% of T4 is used for the formation of T3. This constitutes 90% of T3. Only 10% of T3 is formed directly. Also, 40% of T4 is used for the formation of reverse T3 (rT3). The remaining 20% is excreted with urine or feces.

more active since T4 binds to cytoplasmic proteins when they enter the cell are going to affect (Benvenga, 2005; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996;

(TBG) 54 Lowest Highest

prealbumin ( TTR) 55 Higher Lower Albumin 65 Highest Lowest

A 100 µg thyroid hormone is secreted from the thyroid gland and most of these hormones are T4. About 40% of T4 turn into T3 which is 3 times stronger in periphery, especially in the liver and kidney with deiodinase enzymes (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer,

Metabolically, in order for active T3 to form, deiodination needs to occur in region 5' of tyrosine. Instead, if it occurs in the 5th atom of inner circle, metabolically inactive reverse triiodothyronine (rT3) is formed. Three types of enzymes that are Selenoenzyme 5' deiodinase type I (5'-DI), the type II5' iodothyronine deiodinase (5'-DII) and the 5, or inner circle deiodinase type III (5-DIII) catalyze these deiodinations (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti

5'-DI enzyme is especially found in the liver, kidneys, and thyroid, and 5'-DII enzyme exists in the brain, hypophysis, placenta, and keratinocytes. 5'-DIII is found in the brain, placenta, and epidermis. Both 5'-DI and 5'DII enzymes allow T4 to transform into active T3; but with one difference, that is, while 5'- DI enzyme provides the formed T3 to plasma, T3 formed by 5'-DII enzyme stays in the tissue and regulates local concentration. This enzyme is regulated by increases and decreases in thyroid hormones. For instance, hyperthyroidism inhibits enzyme and blocks the transformation from T4 to T3 in such tissues as the brain and hypophsis. Transformation from T4 to T3 is affected by such changes in the organism as hunger, systemic disease, acute stress, iodine contrating agents, and drugs such as propiltiourasil, propranolol, amiodaron, and glicocortikoid, but is not affected by metrmazol. 5'-DIII enzyme transforms T4 into metabolically inactive reverse T3 (rT3) (Figure 5) (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997). As mentioned earlier, 40% of T4 is used for the formation of T3. This constitutes 90% of T3. Only 10% of T3 is formed directly. Also, 40% of T4 is used for the

Plasma

concentration Levels of binding

Reed & Pangaro, 1995; Utiger, 1997).

**4.5 Thyroid hormone metabolism** 

thyroxin-binding

thyroxin-binding

Proteins Molecular weight

(kDa)

Table 1. Comparison of the binding of thyroid hormones to carrier proteins

1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

& Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997).

formation of reverse T3 (rT3). The remaining 20% is excreted with urine or feces.

Fig. 5. Effects of deiodinase enzymes

#### **4.6 Controlling the thyroid hormone synthesis and secretion**

Synthesis and secretions need to be kept at a certain level in order for the liveliness of thyroid hormones to be maintained. In this respect, the most important mechanism in controlling the synthesis and secretion of thyroid hormones is the hypothalamushypophysis-thyroid axis. Another one is the autocontrol mechanism that is dependent on iodine concentration as noted earlier (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Utiger, 1997).

#### **4.6.1 Hypothalamus-hypophysis-thyroid axe**

Hormone synthesis and secretion of the thyroid gland is under the strict control of this axis. This event begins with TRH synthesis in the hypothalamus. TRH is carried from the hypothalamus to the hypophysis through portal circulation, and TSH hormone is secreted here following the interaction with TRH receptors in the hypophysis front lobe. TSH is then transferred by blood and stimulates the thyroid gland, and thus, thyroid hormone synthesis and secretion begins. However, if thyroid hormone and synthesis is too large an amount, the feedback system is activated and TSH and TRH are suppressed (Figure 6) (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

Fig. 6. Controlling of thyroid hormone secretion by the hypothalamus-hypothyroidismthyroid axis

Introduction to Thyroid: Anatomy and Functions 13

Despite these effects, TSH does not affect the transformation from T4 to T3 in the periphery. Although TSH secretion is stimulated by TRH and estradiol, it is inhibited by somatostatine, dopamine, T3, T4, and glucocorticoids. While α 1 adrenergics demonstrates inhibiting effects, α2 adrenergics are stimulators (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor,

Changes in iodine concentrations in follicular cells of thyroid gland affect the iodine transport and form an autoregulation (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997). Thyroid hormone synthesis is inhibited as the iodine amount increases in follicles, however, synthesis increases as the amount decreases. Wolf Chaikoff effect in which excessive iodine stops the thyroid hormone synthesis may also be mentioned. This effect is especially observed when individuals with hyperthyroidism take antithyroid along with iodine and become euthyroid (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996;

In addition, the sensitivity of the thyroid gland also increases through a development of a response to TSH, although TSH does not have a stimulating effect in iodine deficiency. Along with the increase in sensitivity, follicular cells in the gland reach hypertrophy and hyperplasia, and increase the weight of the gland and create goiter. The effects of TSH decrease as the response to TSH decreases with the rise in iodine (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997). In this case, all of the effects, such as binding of iodine, thyroid hormone synthesis, secretion of thyroglobulin into colloid, taking colloid back to cell by endocytosis, entrapment of iodine, and cell hypertrophy are decreased. However, blood flow to the thyroid glands is reduced. Iodine supplement before thyroid surgery is for the purpose of reducing the blood flow in the thyroid gland. (Dillmann, 2004; Dunn,

1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

The effects of TSH may be divided into three.

a. **Effects occurring within minutes**;






b. **Effects occurring within hours**;

**4.6.2 Autoregulation of the thyroid** 




The thyrotrophin-releasing hormone (TRH) is a tripeptide synthesized in periventricular nucleus in the hypothalamus. The structure of TRH formed by the repetition of -Glu-H.5- Pro-Gly- series 6 times in the beginning turns into pyroglutamyl histidylprolinamide at the end of synthesis. As noted earlier, TRH is carried to the front hypophysis through hypophyseal portal system and provides the secretion of TSH from thyrotrope cells (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

There are receptors specific to TRH on the surfaces of these cells. When TRH makes contact with these receptors, Gq protein is activated, and it then activates the phosphalipase C enzyme, fractionates membrane phospholipids and forms diacylglycerol (DAG) and inositole triphosphate (IP3). These are secondary mesengers and cause the secretion of Ca+2 via IP3 from endoplasmic reticulum, and DAG activates protein kinase C. The effect of TRH on TSH is provided through these secondary messengers (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

TRH also increases the secretions of growth hormone (GH), follicle stimulating hormone (FSH), and prolactin (PRL). While the TRH secretion is increased by noradrenaline, somatostatin and serotonin inhibits it. (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

The thyrotropin-stimulating hormone (TSH) is a hormone that has a glycoprotein structure comprised of α and β subunits and synthesized in 5% basophilic thyrotrope cells of frontal hypophysis. α subunit is almost the same as that found in such hormones as human chorionic gonadotropin (HCG), luteinizing hormone (LH), and follicle stimulating hormone (FSH). It is believed that the task of this subunit is the stimulation of adenilate cyclase that provides the formation of cAMP secondary precursor. β subunit is completely different to other hormones and is related with receptor specificity. Therefore, TSH is active when it possesses both subunits (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

TSH activates Gs protein when it merges with the receptor in the membrane of thyroid gland follicle cell, and thus, the adenilat cyclase enzyme is activated as well. When this enzyme becomes activated, it increases the secondary messenger cAMP. Along with stimulating protein kinase A enzymes, it causes the development of thyroid follicular cell and the synthesis of thyroid hormone (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

TSH is metabolized in kidneys and liver. It is released as pulsatile and demonstrates circadian rhythm, which means that the secretion begins at night, reaches a maximum at midnight, and decreases all day long (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

The effects of TSH may be divided into three.


12 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

The thyrotrophin-releasing hormone (TRH) is a tripeptide synthesized in periventricular nucleus in the hypothalamus. The structure of TRH formed by the repetition of -Glu-H.5- Pro-Gly- series 6 times in the beginning turns into pyroglutamyl histidylprolinamide at the end of synthesis. As noted earlier, TRH is carried to the front hypophysis through hypophyseal portal system and provides the secretion of TSH from thyrotrope cells (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995;

There are receptors specific to TRH on the surfaces of these cells. When TRH makes contact with these receptors, Gq protein is activated, and it then activates the phosphalipase C enzyme, fractionates membrane phospholipids and forms diacylglycerol (DAG) and inositole triphosphate (IP3). These are secondary mesengers and cause the secretion of Ca+2 via IP3 from endoplasmic reticulum, and DAG activates protein kinase C. The effect of TRH on TSH is provided through these secondary messengers (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban,

TRH also increases the secretions of growth hormone (GH), follicle stimulating hormone (FSH), and prolactin (PRL). While the TRH secretion is increased by noradrenaline, somatostatin and serotonin inhibits it. (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

The thyrotropin-stimulating hormone (TSH) is a hormone that has a glycoprotein structure comprised of α and β subunits and synthesized in 5% basophilic thyrotrope cells of frontal hypophysis. α subunit is almost the same as that found in such hormones as human chorionic gonadotropin (HCG), luteinizing hormone (LH), and follicle stimulating hormone (FSH). It is believed that the task of this subunit is the stimulation of adenilate cyclase that provides the formation of cAMP secondary precursor. β subunit is completely different to other hormones and is related with receptor specificity. Therefore, TSH is active when it possesses both subunits (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996;

TSH activates Gs protein when it merges with the receptor in the membrane of thyroid gland follicle cell, and thus, the adenilat cyclase enzyme is activated as well. When this enzyme becomes activated, it increases the secondary messenger cAMP. Along with stimulating protein kinase A enzymes, it causes the development of thyroid follicular cell and the synthesis of thyroid hormone (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

TSH is metabolized in kidneys and liver. It is released as pulsatile and demonstrates circadian rhythm, which means that the secretion begins at night, reaches a maximum at midnight, and decreases all day long (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

2005; Scanlon, 2001; Utiger, 1997).


Despite these effects, TSH does not affect the transformation from T4 to T3 in the periphery.

Although TSH secretion is stimulated by TRH and estradiol, it is inhibited by somatostatine, dopamine, T3, T4, and glucocorticoids. While α 1 adrenergics demonstrates inhibiting effects, α2 adrenergics are stimulators (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

#### **4.6.2 Autoregulation of the thyroid**

Changes in iodine concentrations in follicular cells of thyroid gland affect the iodine transport and form an autoregulation (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997). Thyroid hormone synthesis is inhibited as the iodine amount increases in follicles, however, synthesis increases as the amount decreases. Wolf Chaikoff effect in which excessive iodine stops the thyroid hormone synthesis may also be mentioned. This effect is especially observed when individuals with hyperthyroidism take antithyroid along with iodine and become euthyroid (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

In addition, the sensitivity of the thyroid gland also increases through a development of a response to TSH, although TSH does not have a stimulating effect in iodine deficiency. Along with the increase in sensitivity, follicular cells in the gland reach hypertrophy and hyperplasia, and increase the weight of the gland and create goiter. The effects of TSH decrease as the response to TSH decreases with the rise in iodine (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997). In this case, all of the effects, such as binding of iodine, thyroid hormone synthesis, secretion of thyroglobulin into colloid, taking colloid back to cell by endocytosis, entrapment of iodine, and cell hypertrophy are decreased. However, blood flow to the thyroid glands is reduced. Iodine supplement before thyroid surgery is for the purpose of reducing the blood flow in the thyroid gland. (Dillmann, 2004; Dunn,

Introduction to Thyroid: Anatomy and Functions 15

Thyroid hormones accelerate mRNA synthesis in mitochondria by acting with intrinsic receptors in mitochondria inner and outer membranes and increases protein production. Due to these proteins produced here in mitochondria being respiratory chain proteins such as NADPH dehydrogenase, cytochrome-c-oxidase, and cytochrome reductase, the respiratory chain accelerates as the synthesis of these enzymes increases, and thus, ATP synthesis and oxygen consumption also increases. Therefore, it may be noted that ATP synthesis is dependent on thyroid hormone stimulation. In addition, the number of mitochondria increases due to the increase in mitochondria activity parallel to mitochondria protein synthesis (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed &

Protein synthesis causes an increase in enzyme synthesis by increasing with the effect of thyroid hormones, and this affects the passage by increasing the production of transport enzymes in the cell membrane. Among these enzymes, the Na+- K+- ATPase pump provides Na+ to exit and K+ to enter by using ATP, thus, the rate of metabolism also increases (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995;

Another membrane enzyme Ca+2\_ATPase acts more in the circulation system as intracellular Ca+2 decreases when this enzyme operates (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer,

Among the effects of thyroid is the effect it has on growth. This hormone has both specific and general effects on growth. Thyroid hormones are necessary for normal growth and muscle development. While children with hypothyroidism are shorter due to early epiphysis closure, children with hyperthyroidism are taller compared to their peers. Another important effect of the thyroid hormone is its contribution to the pre- and postnatal development of the brain. When in the mother's uterus, if the fetus cannot synthesize and secrete sufficient thyroid hormone and it is not replaced, growth and development retardation occurs in both pre- and post-natal periods (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995). Normal serum levels are Total T4 5-12µg/dl, Total T3 80-200ng/dl, Free T4 0,9-2ng/dl and free T3 0,2-0,5ng/dl, respectively. If a thyroid hormone test is conducted on the baby after birth and hormone treatment is started immediately, a completely normal child is developed and a dramatic difference between early and late detection of the disease is clearly observed.

Thyroid hormones carry out their metabolic effects by carbohydrates, fat and protein

When the effects of thyroid hormones on carbohydrate metabolism are observed, it is established that it is both anabolic and catabolic. As a result of thyroid hormones increasing

metabolisms, vitamins, basal metabolic rate and its effect on body weight.

1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Pangaro, 1995; Utiger, 1997; Usala, 1995).

Utiger, 1997; Usala, 1995).

**4.8.2 Effects on growth** 

**4.8.3 Metabolic effects** 

2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005; Scanlon, 2001; Utiger, 1997).

#### **4.7 Occurrence of the thyroid hormone effect**

Thyroid hormone receptors exist within the cell. Most of these receptors are in the nucleus and show more affinity to T3. Due to the fact that T4 binds more to carrier proteins and exists more in extracellular region, it passes inside the cell, in other words, intracellular amount of T4 is lesser. When they pass to the intracellular section, very few of them are free for receptors after they are bound to proteins. However, T3 already exists more in intracellular section due to it binding to fewer amount of carrier proteins and receptors show more affinity to T3 due to being free. As a result, T3 is 3-8 times more potent compared to T4. The reason for this difference in effect is that T4 transforms into T3 while T4 exists in high amounts; the actual efficient one is T3 (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Thyroid hormones easily pass through the cell membrane due to being lipid soluble and T3 immediately binds to thyroid hormone receptor in nucleus. Thyroid hormone receptors are of two types as α (TR α) and β (TRβ). Although these receptors generally exist in all tissues, they differ in effects. While TR α is more efficient in the brain, kidneys, heart, muscles and gonads, TRβ is more efficient in liver and hypophysis. TR α and β are bind to a special DNA sequence that has thyroid response elements (TREs). Receptors bind and activate by retinoic acid X (RXRs) receptors. They either stimulate transcription or inhibit it due to regulatory mechanisms in the target gene. When the transcription starts, various mRNAs are synthesized, and various proteins are synthesized by going through translation in ribosomes that are present in cell cytoplasm. Also, enzymes in the protein structure are synthesized and some of these play an active role in the formation of thyroid hormone effects (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

#### **4.8 Effects of thyroid hormones**

The effects of thyroid hormones are varying. It can be divided into 4 as cellular level, and effects on growth, metabolism, and on systems.

#### **4.8.1 Effects of thyroid hormones at the cellular level**

The general cellular effect is the aforementioned T3 synthesizing various proteins in which enzymes are also included by transcription and then translation in ribosomes in cytoplasm after interacting with receptor in nucleus. While, on one hand, protein synthesis increases, and on the other, a rise occurs in catabolism, and thus basal metabolism increases. Cell metabolism shows an increase of 60-100% when thyroid hormones are oversecreted (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Santiseban, 2005;

Thyroid hormone receptors exist within the cell. Most of these receptors are in the nucleus and show more affinity to T3. Due to the fact that T4 binds more to carrier proteins and exists more in extracellular region, it passes inside the cell, in other words, intracellular amount of T4 is lesser. When they pass to the intracellular section, very few of them are free for receptors after they are bound to proteins. However, T3 already exists more in intracellular section due to it binding to fewer amount of carrier proteins and receptors show more affinity to T3 due to being free. As a result, T3 is 3-8 times more potent compared to T4. The reason for this difference in effect is that T4 transforms into T3 while T4 exists in high amounts; the actual efficient one is T3 (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

Thyroid hormones easily pass through the cell membrane due to being lipid soluble and T3 immediately binds to thyroid hormone receptor in nucleus. Thyroid hormone receptors are of two types as α (TR α) and β (TRβ). Although these receptors generally exist in all tissues, they differ in effects. While TR α is more efficient in the brain, kidneys, heart, muscles and gonads, TRβ is more efficient in liver and hypophysis. TR α and β are bind to a special DNA sequence that has thyroid response elements (TREs). Receptors bind and activate by retinoic acid X (RXRs) receptors. They either stimulate transcription or inhibit it due to regulatory mechanisms in the target gene. When the transcription starts, various mRNAs are synthesized, and various proteins are synthesized by going through translation in ribosomes that are present in cell cytoplasm. Also, enzymes in the protein structure are synthesized and some of these play an active role in the formation of thyroid hormone effects (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed &

The effects of thyroid hormones are varying. It can be divided into 4 as cellular level, and

The general cellular effect is the aforementioned T3 synthesizing various proteins in which enzymes are also included by transcription and then translation in ribosomes in cytoplasm after interacting with receptor in nucleus. While, on one hand, protein synthesis increases, and on the other, a rise occurs in catabolism, and thus basal metabolism increases. Cell metabolism shows an increase of 60-100% when thyroid hormones are oversecreted (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995;

Scanlon, 2001; Utiger, 1997).

**4.7 Occurrence of the thyroid hormone effect** 

Pangaro, 1995; Utiger, 1997; Usala, 1995).

effects on growth, metabolism, and on systems.

**4.8.1 Effects of thyroid hormones at the cellular level** 

**4.8 Effects of thyroid hormones** 

Utiger, 1997; Usala, 1995).

Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Thyroid hormones accelerate mRNA synthesis in mitochondria by acting with intrinsic receptors in mitochondria inner and outer membranes and increases protein production. Due to these proteins produced here in mitochondria being respiratory chain proteins such as NADPH dehydrogenase, cytochrome-c-oxidase, and cytochrome reductase, the respiratory chain accelerates as the synthesis of these enzymes increases, and thus, ATP synthesis and oxygen consumption also increases. Therefore, it may be noted that ATP synthesis is dependent on thyroid hormone stimulation. In addition, the number of mitochondria increases due to the increase in mitochondria activity parallel to mitochondria protein synthesis (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Protein synthesis causes an increase in enzyme synthesis by increasing with the effect of thyroid hormones, and this affects the passage by increasing the production of transport enzymes in the cell membrane. Among these enzymes, the Na+- K+- ATPase pump provides Na+ to exit and K+ to enter by using ATP, thus, the rate of metabolism also increases (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Another membrane enzyme Ca+2\_ATPase acts more in the circulation system as intracellular Ca+2 decreases when this enzyme operates (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

#### **4.8.2 Effects on growth**

Among the effects of thyroid is the effect it has on growth. This hormone has both specific and general effects on growth. Thyroid hormones are necessary for normal growth and muscle development. While children with hypothyroidism are shorter due to early epiphysis closure, children with hyperthyroidism are taller compared to their peers. Another important effect of the thyroid hormone is its contribution to the pre- and postnatal development of the brain. When in the mother's uterus, if the fetus cannot synthesize and secrete sufficient thyroid hormone and it is not replaced, growth and development retardation occurs in both pre- and post-natal periods (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995). Normal serum levels are Total T4 5-12µg/dl, Total T3 80-200ng/dl, Free T4 0,9-2ng/dl and free T3 0,2-0,5ng/dl, respectively. If a thyroid hormone test is conducted on the baby after birth and hormone treatment is started immediately, a completely normal child is developed and a dramatic difference between early and late detection of the disease is clearly observed.

#### **4.8.3 Metabolic effects**

Thyroid hormones carry out their metabolic effects by carbohydrates, fat and protein metabolisms, vitamins, basal metabolic rate and its effect on body weight.

When the effects of thyroid hormones on carbohydrate metabolism are observed, it is established that it is both anabolic and catabolic. As a result of thyroid hormones increasing

Introduction to Thyroid: Anatomy and Functions 17

riboflavin, B12, folic acid and ascorbic acid (vitamin C) are predominantly used as co-factors. Therefore, deficiencies of these vitamins are common in cases with hyperthyroidism. In addition, vitamin D deficiency is also observed in these individuals due to an increase in excessive consumption and clearance. Also, thyroid hormones are necessary for carotene from food to be transformed into vitamin A. Vitamin A transformation does not occur in cases with hypothyroidism due to thyroid hormone deficiency and carotene is deposited under the skin giving it a yellow color. (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995). Vitamin D deficiency is present in these cases due to a problem in A, E, and cholesterol metabolism. Thus, vitamin

Another effect of thyroid hormones is the acceleration of basal metabolism. As noted before, thyroid hormones increase the oxygen consumption and thus ATP synthesis by rising the count and activity of mitochondria. Thyroid hormones increase oxygen consumption except for the adult brain, testicles, uterus, lymph nodes, spleen, and front hypophysis. In addition, the increase of such enzymes as Na+- K+- ATPase, and Ca+- ATPase contribute to it. Also, lipid catabolism lends to it. A high level of temperature is produced as a result (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996;

A protein called thermogenin in brown adipose tissue is uncoupled, that is, ATP production and e- - transport chain are separated from each other. An excessive temperature occurs as a result. All these effects provide acceleration of basal metabolism. The overworking thyroid gland increases the basal metabolism by 60-100% (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti &

Due to the increase in basal metabolism, a decrease is observed in body weight. Thyroid hormones greatly reduce the fat deposit. Weight loss is observed in cases with hyperthyroidism although appetite increases in cases with hyperthyroidism. However, in cases with hypothyroidism, basal metabolism deceleration and weight gain occur in cases with hypothyroidism (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996;

The effect of thyroid hormones on circulation systems is predominantly through catecholamine. Thyroid hormones increase the β adrenergic receptor count without affecting catecholamine secretion. This causes an increase in heart rate, cardiac output, stroke volume, and peripheral vasodilation. Peripheral vasodilation causes the skin to be warm and humid. Warm and humid skin, sweating, and restlessness due to increased sympathetic activity are observed in cases with hyperthyroidism. However, the opposite is seen in hypothyroidism. The β adrenergic receptor count is decreased. In relation to this, heart rate, cardiac output, and stroke volume is also decreased and cold, dry skin is observed due to peripheral vasoconstriction. In a study by Bursuk et al., it was established

Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

supplement is necessary in both hypothyroidism and hyperthyroidism cases.

Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

**4.8.4 Effect of thyroid hormones on systems** 

the enzyme synthesis due to protein synthesis in cells, enzymes in carbohydrate metabolism also increase their activities. Thus, thyroid hormones increase the entrance of glucose into the cell, absorption of glucose from the gastrointestinal system, both glycolysis and gluconeogenesis, and secondarily, insulin secretion (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

The effect of thyroid hormone on fat metabolism are both anabolic and catabolic. Thyroid hormones have an especially lipolysis effect on adipose tissue ,and free fatty acid concentrations in plasma increase with the said effect, and in addition, fatty acid oxidation also increases (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995). While, as a result of these effects, an increase is expected in the amounts of cholesterol and triglyceride, in contrast, their levels in blood are established to be low. This occurs due to two reasons. Firstly, thyroid hormones (especially T3) cause an increase in receptor synthesis specific to LDL and cholesterol in liver, bind to lipoproteins, and decrease the triglyceride level in blood. Secondly, thyroid hormones accelerate the transformation of triglyceride to cholesterol with their effect. Cholesterol reaching the liver is used in the production of bile and the produced bile is excreted from the intestines with feces. Consequently, there occurs a decrease in adipose tissue, cholesterol and triglyceride in blood, and an increase in free fatty acids when thyroid hormone is oversecreted. The opposite occurs in individuals with hyperthyroidism. In a study by Bursuk et al., it was established by comparing the body composition in control, hypothyroidism, and hyperthyroidism groups with the bioelectrical impedance analysis method that body fat percentage and the amount decreased in cases with hyperthyroidism while they increased in cases with hypothyroidism (Bursuk et al., 2010; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

As previously noted, thyroid hormones show an anabolic effect by increasing the protein syntheses and a catabolic effect by increasing the destruction when oversecreted. Thyroid hormones also regulate aminoacid transport due to the need for aminoacids in order to increase the protein synthesis. They also provide the synthesis for proteins specific to cell growth. Thyroid hormones provide a normal growth of the baby by increasing the syntheses of insulin-like factors in fetal period (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Hormones that provide growth and development are also under the control of thyroid hormones. As mentioned before, hypothyroidism causes growth-development retardation and can be reversed by hormone replacement treatment when diagnosed early. In hyperthyroidism in which thyroid hormones are oversecreted, muscle atrophies are observed as a result of an increase in protein catabolism (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Most of the enzymes need vitamins as co-factors in order to produce an effect. The need for the co-factor of thyroid hormones increases parallel to enzyme synthesis. Thiamine,

the enzyme synthesis due to protein synthesis in cells, enzymes in carbohydrate metabolism also increase their activities. Thus, thyroid hormones increase the entrance of glucose into the cell, absorption of glucose from the gastrointestinal system, both glycolysis and gluconeogenesis, and secondarily, insulin secretion (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti &

The effect of thyroid hormone on fat metabolism are both anabolic and catabolic. Thyroid hormones have an especially lipolysis effect on adipose tissue ,and free fatty acid concentrations in plasma increase with the said effect, and in addition, fatty acid oxidation also increases (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995). While, as a result of these effects, an increase is expected in the amounts of cholesterol and triglyceride, in contrast, their levels in blood are established to be low. This occurs due to two reasons. Firstly, thyroid hormones (especially T3) cause an increase in receptor synthesis specific to LDL and cholesterol in liver, bind to lipoproteins, and decrease the triglyceride level in blood. Secondly, thyroid hormones accelerate the transformation of triglyceride to cholesterol with their effect. Cholesterol reaching the liver is used in the production of bile and the produced bile is excreted from the intestines with feces. Consequently, there occurs a decrease in adipose tissue, cholesterol and triglyceride in blood, and an increase in free fatty acids when thyroid hormone is oversecreted. The opposite occurs in individuals with hyperthyroidism. In a study by Bursuk et al., it was established by comparing the body composition in control, hypothyroidism, and hyperthyroidism groups with the bioelectrical impedance analysis method that body fat percentage and the amount decreased in cases with hyperthyroidism while they increased in cases with hypothyroidism (Bursuk et al., 2010; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997;

As previously noted, thyroid hormones show an anabolic effect by increasing the protein syntheses and a catabolic effect by increasing the destruction when oversecreted. Thyroid hormones also regulate aminoacid transport due to the need for aminoacids in order to increase the protein synthesis. They also provide the synthesis for proteins specific to cell growth. Thyroid hormones provide a normal growth of the baby by increasing the syntheses of insulin-like factors in fetal period (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

Hormones that provide growth and development are also under the control of thyroid hormones. As mentioned before, hypothyroidism causes growth-development retardation and can be reversed by hormone replacement treatment when diagnosed early. In hyperthyroidism in which thyroid hormones are oversecreted, muscle atrophies are observed as a result of an increase in protein catabolism (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti

Most of the enzymes need vitamins as co-factors in order to produce an effect. The need for the co-factor of thyroid hormones increases parallel to enzyme synthesis. Thiamine,

& Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

riboflavin, B12, folic acid and ascorbic acid (vitamin C) are predominantly used as co-factors. Therefore, deficiencies of these vitamins are common in cases with hyperthyroidism. In addition, vitamin D deficiency is also observed in these individuals due to an increase in excessive consumption and clearance. Also, thyroid hormones are necessary for carotene from food to be transformed into vitamin A. Vitamin A transformation does not occur in cases with hypothyroidism due to thyroid hormone deficiency and carotene is deposited under the skin giving it a yellow color. (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995). Vitamin D deficiency is present in these cases due to a problem in A, E, and cholesterol metabolism. Thus, vitamin supplement is necessary in both hypothyroidism and hyperthyroidism cases.

Another effect of thyroid hormones is the acceleration of basal metabolism. As noted before, thyroid hormones increase the oxygen consumption and thus ATP synthesis by rising the count and activity of mitochondria. Thyroid hormones increase oxygen consumption except for the adult brain, testicles, uterus, lymph nodes, spleen, and front hypophysis. In addition, the increase of such enzymes as Na+- K+- ATPase, and Ca+- ATPase contribute to it. Also, lipid catabolism lends to it. A high level of temperature is produced as a result (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

A protein called thermogenin in brown adipose tissue is uncoupled, that is, ATP production and e- - transport chain are separated from each other. An excessive temperature occurs as a result. All these effects provide acceleration of basal metabolism. The overworking thyroid gland increases the basal metabolism by 60-100% (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Due to the increase in basal metabolism, a decrease is observed in body weight. Thyroid hormones greatly reduce the fat deposit. Weight loss is observed in cases with hyperthyroidism although appetite increases in cases with hyperthyroidism. However, in cases with hypothyroidism, basal metabolism deceleration and weight gain occur in cases with hypothyroidism (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

#### **4.8.4 Effect of thyroid hormones on systems**

The effect of thyroid hormones on circulation systems is predominantly through catecholamine. Thyroid hormones increase the β adrenergic receptor count without affecting catecholamine secretion. This causes an increase in heart rate, cardiac output, stroke volume, and peripheral vasodilation. Peripheral vasodilation causes the skin to be warm and humid. Warm and humid skin, sweating, and restlessness due to increased sympathetic activity are observed in cases with hyperthyroidism. However, the opposite is seen in hypothyroidism. The β adrenergic receptor count is decreased. In relation to this, heart rate, cardiac output, and stroke volume is also decreased and cold, dry skin is observed due to peripheral vasoconstriction. In a study by Bursuk et al., it was established

Introduction to Thyroid: Anatomy and Functions 19

The thyroid also affects response to stimulants. When this hormone is excessively secreted, muscle fatigue occurs due to protein catabolism increase. The most typical symptom of hyperthyroidism is a faint muscle tremor. Such a tremor happening 10-15 times per second, occurs due to increase in activity of neuronal synapses in medulla spinalis regions that control muscle tone, and differs from tremors in Parkinson's disease. This tremor demonstrates the effects of thyroid hormones on central nervous system (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995;

As mentioned above, muscle fatigue is observed in hyperthyroidism due to the accelerating effect of the thyroid hormone on protein catabolism. However, the excessive stimulant effect of this hormone on synapses leads to sleeplessness. In hypothyroidism, a sleepy state exists (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995;

Thyroid hormones play an important role in the development of the central nervous system. They are also responsible for the myelinization of the nerves. If there is thyroid hormone deficiency in fetus, it causes neuronal developmental disorders in the brain, myelinization retardation, decrease in vascularization, retardation in deep tendon reflexes, cerebral hypoxy due to decrease in cerebral blood flow, mental retardation, and lethargy. In cases with hyperthyroidism, the opposite occurs and hyperirritability, anxiety, and sleeplessness are observed in these children (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor,

Thyroid hormones produce an effect by merging with their specific receptors in membrane and nuclei of hemopoietic stem cells. After T3 and T4 hormones bind with a receptor, erythroid stem cells go through mitosis and accelerate erythropoiesis. With the protein synthesis they caused to occur in these precursor cells, they provide the synthesis of enzymes at the beginning and at the end of hemoglobin synthesis (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

In addition, when tissues are left without oxygen with the consumption of oxygen thanks to thyroid hormone effect, they stimulate the kidney and increase erythropoietin synthesis and secretion. Erythropoietin then stimulates the bone marrow and accelerates erythropoiesis. While polycythemia is not observed in patients with hyperthyroidism, anemia is quite prevalent among cases with hypothyroidism. Blood levels of cases with hyperthyroidism are generally within normal limits (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

In a study by Bursuk et al., it has been established by measuring and comparing blood parameters and blood viscosity in control, hypothyroidism, and hyperthyroidism groups that blood viscosity was increased in cases with hypothyroidism due to blood count parameters being higher compared to cases with hyperthyroidism, blood lipids and fibrinogen were higher in cases with hypothyroidism, and in addition, blood viscosity

Utiger, 1997; Usala, 1995).

Utiger, 1997; Usala, 1995)..

1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

by measuring and comparing the stroke volume, cardiac output, heart index, and blood flow in control, hypothyroidism, and hyperthyroidism groups with the bioelectrical impedance analysis method that these parameters significantly increased in cases with hyperthyroidism while they decreased in cases with hypothyroidism (Bursuk et al., 2010; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

In addition, as metabolism products also increase due to an increase in oxygen consumption when thyroid hormones are oversecreted, vasodilation occurs in periphery. Thus, blood flow increases, and cardiac output can be observed to be 60% more than normal. The thyroid hormone also raises the heart rate due to its direct increasing effect on heart stimulation (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Thyroid hormones increase the contraction of heart muscles only when they raise it in small amounts. When thyroid hormones are oversecreted, a significant decrease occurs in muscle strength, and even myocardial infarction is observed in severely thyrotoxic patients (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Due to large amounts of oxygen thyroid hormones use during their increasing protein synthesis, hence the enzyme synthesis, and ATP synthesis as well, carbon dioxide amount is also increased. As a result of the carbon dioxide increase affecting the respiratory center of the brain, hyperventilation, that is, the rise in inhalation frequency and deepening of respiration is observed (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

While appetite and food consumption increases, an increase has also been observed in digestive system fluids, secretions, and movements. Frequently, diarrhea occurs when the thyroid hormone is excessively secreted. In contrast, constipation is observed in the case of hypothyroidism (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

When the effects of thyroid hormones on the skeletal system are checked, the first thing that needs to be examined is their effect on bones. The activities of osteoblast and osteoclast that are the main cells of bone structure increase parallel to thyroid hormones. In normal individuals, thyroid hormones possess direct proliferative effect on osteoblasts. In cases with hyperthyroidism, a decrease develops in the cortex of the bones due to increase in osteoclastic activities. Thus, the risk of post-menopausal osteoporosis development increases in these patients. While, in physiological cases, thyroid hormone creates an osteoblastic effect, it produces an osteoporotic effect in hyperthyroidism (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

by measuring and comparing the stroke volume, cardiac output, heart index, and blood flow in control, hypothyroidism, and hyperthyroidism groups with the bioelectrical impedance analysis method that these parameters significantly increased in cases with hyperthyroidism while they decreased in cases with hypothyroidism (Bursuk et al., 2010; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995;

In addition, as metabolism products also increase due to an increase in oxygen consumption when thyroid hormones are oversecreted, vasodilation occurs in periphery. Thus, blood flow increases, and cardiac output can be observed to be 60% more than normal. The thyroid hormone also raises the heart rate due to its direct increasing effect on heart stimulation (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed &

Thyroid hormones increase the contraction of heart muscles only when they raise it in small amounts. When thyroid hormones are oversecreted, a significant decrease occurs in muscle strength, and even myocardial infarction is observed in severely thyrotoxic patients (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995;

Due to large amounts of oxygen thyroid hormones use during their increasing protein synthesis, hence the enzyme synthesis, and ATP synthesis as well, carbon dioxide amount is also increased. As a result of the carbon dioxide increase affecting the respiratory center of the brain, hyperventilation, that is, the rise in inhalation frequency and deepening of respiration is observed (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996;

While appetite and food consumption increases, an increase has also been observed in digestive system fluids, secretions, and movements. Frequently, diarrhea occurs when the thyroid hormone is excessively secreted. In contrast, constipation is observed in the case of hypothyroidism (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed &

When the effects of thyroid hormones on the skeletal system are checked, the first thing that needs to be examined is their effect on bones. The activities of osteoblast and osteoclast that are the main cells of bone structure increase parallel to thyroid hormones. In normal individuals, thyroid hormones possess direct proliferative effect on osteoblasts. In cases with hyperthyroidism, a decrease develops in the cortex of the bones due to increase in osteoclastic activities. Thus, the risk of post-menopausal osteoporosis development increases in these patients. While, in physiological cases, thyroid hormone creates an osteoblastic effect, it produces an osteoporotic effect in hyperthyroidism (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti

& Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Utiger, 1997; Usala, 1995).

Utiger, 1997; Usala, 1995).

Pangaro, 1995; Utiger, 1997; Usala, 1995).

Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Pangaro, 1995; Utiger, 1997; Usala, 1995).

The thyroid also affects response to stimulants. When this hormone is excessively secreted, muscle fatigue occurs due to protein catabolism increase. The most typical symptom of hyperthyroidism is a faint muscle tremor. Such a tremor happening 10-15 times per second, occurs due to increase in activity of neuronal synapses in medulla spinalis regions that control muscle tone, and differs from tremors in Parkinson's disease. This tremor demonstrates the effects of thyroid hormones on central nervous system (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

As mentioned above, muscle fatigue is observed in hyperthyroidism due to the accelerating effect of the thyroid hormone on protein catabolism. However, the excessive stimulant effect of this hormone on synapses leads to sleeplessness. In hypothyroidism, a sleepy state exists (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995)..

Thyroid hormones play an important role in the development of the central nervous system. They are also responsible for the myelinization of the nerves. If there is thyroid hormone deficiency in fetus, it causes neuronal developmental disorders in the brain, myelinization retardation, decrease in vascularization, retardation in deep tendon reflexes, cerebral hypoxy due to decrease in cerebral blood flow, mental retardation, and lethargy. In cases with hyperthyroidism, the opposite occurs and hyperirritability, anxiety, and sleeplessness are observed in these children (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Thyroid hormones produce an effect by merging with their specific receptors in membrane and nuclei of hemopoietic stem cells. After T3 and T4 hormones bind with a receptor, erythroid stem cells go through mitosis and accelerate erythropoiesis. With the protein synthesis they caused to occur in these precursor cells, they provide the synthesis of enzymes at the beginning and at the end of hemoglobin synthesis (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

In addition, when tissues are left without oxygen with the consumption of oxygen thanks to thyroid hormone effect, they stimulate the kidney and increase erythropoietin synthesis and secretion. Erythropoietin then stimulates the bone marrow and accelerates erythropoiesis. While polycythemia is not observed in patients with hyperthyroidism, anemia is quite prevalent among cases with hypothyroidism. Blood levels of cases with hyperthyroidism are generally within normal limits (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

In a study by Bursuk et al., it has been established by measuring and comparing blood parameters and blood viscosity in control, hypothyroidism, and hyperthyroidism groups that blood viscosity was increased in cases with hypothyroidism due to blood count parameters being higher compared to cases with hyperthyroidism, blood lipids and fibrinogen were higher in cases with hypothyroidism, and in addition, blood viscosity

Introduction to Thyroid: Anatomy and Functions 21

menorrhagia, and polymenorrhea are observed due to sex steroid deficiency and excessive prolactin in cases with hypothyroidism (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc

Anatomy, histology and physiology of thyroid have been addressed in this chapter. In its physiology, its hormone synthesis, metabolism, effect generation mechanism and effects on the body has been explained. While mentioning these effects, the relationship between thyroid diseases and blood hemorheology has also been referred and relationship between disease groups (hyperthyroids and hypothyroids) has been analysed comparatively with

Benvenga, S.(2005). Peripheral hormone metabolism thyroid hormone transport proteins

Bursuk, E., Gulcur, H. & Ercan, M. (2010). The significance of body impedance and blood

Dillmann, W.H. (2004). The thyroid, In: *Cecil Textbook of* Medicine, Goldman, L.&Ausrello,

Dunn, J.T. (2001). Biosynthesis and secretion of thyroid hormones, In: *Endocrinology*,

Ganong, W.F. (1997). *Review of Medical Physiology* (eighteenth edition), Appleton&Lange, 0-

Guyton, A.C. & Hall, JE. (2006). *Textbook of Medical Physiology* (eleventh edition), Elsevier

Jameson, J.L. & Weetman, A.P. (2010). Disorders of the thyroid gland, In: *Harrison's* 

Larsen, P.R., Davies, T.F., Schlumberger, M.J. & Hay, I.D. (2003). Thyroid physiology and

Lo Presti, J.S. & Singer, P.A. (1997). Physiology of thyroid hormone synthesis, secretion, and

Williams&Wilkins Company, 0-7817-5047-4, Philadelphia.

Company, 0-7216-7840-8, Philadelphia.

Sanders, 0-7216-0240-1, Philadelphia.

353), Saunders, 0-7216-9184-6, Philadelphia.

7840-8, Philadelphia.

8385-8443-8, Stamford.

07-174147-7, New York.

Philadelphia.

D., pp. (1391-1411), Saunders, Philadelphia.

and the physiology of hormone binding, In: *Werner&Ingbar's The Thyroid a Fundamental and clinical Text*, Braverman, LE.&Utiger, RD., pp. (97-105), Lippincott

viscosity measurements in thyroid diseases, Proceedings of Biomedical Engineering Meeting (BIYOMUT), 15th National, 978-1-4244-6380-0, Antalya, April 2010 (http://ieeexplore.ieee.org/xpls/abs\_all.jsp?arnumber=5479828&tag=1). Di Lauro, R. & De Felice, M. (2001). Basic Physiology anatomy development, In:

*Endocrinology*, DeGroot, LJ.&Jameson, JL., pp. (1268-1275), W.B. Saunders

DeGroort, LJ.,&Jameson, JL., pp. (1290-1298), W.B. Saunders Company, 0-7216-

*Endocrinology*, Jameson, JL., pp. (62-69), The McGraw-Hill Companies, Inc., 978-0-

diagnostic evaluation of patients with thyroid disorders, In: *Williams Textbook of Endocrinology*, Larsen, PR., Kronenberg, HM., Melmed, S.&Polonsky, KS., pp. (331-

transport, In: *Thyroid Disease Endocrinology, Surger, Nuclear Medicine and Radiotherapy.* Falk, SA, pp. (29-39), Lippincott-Raven Publishers, 0-397-51705-X,

Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

**5. Conclusion** 

these parameters.

**6.References** 

was increased in cases with hypothyroidism due to high plasma viscosity (Bursuk et al., 2010; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Thyroid hormones regulate the actions of other endocrine hormones in order to accelerate basal metabolism. These hormones increase the absorption of glucose in gastrointestinal system, glucose reception into cells, and both glycolysis and gluconeogenesis by producing an effect on insulin and glucagon. Thyroid hormones enable the increase of insulin through secondary mechanism by occasionally rising blood sugar (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Due to the fact that both thyroid hormones and growth hormones are necessary for normal somatic growth, thyroid hormones increase the synthesis and secretion of growth hormone and growth factors (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Also, another effect is produced on prolactin. During hypothyroidism, TRH secretion stimulates prolactin secretion, and while galactorrhea and amenorrhea is observed in females, gynecomastia and impotence is found in males. The inhibiting effect of dopamine is of utmost importance in regulating the secretion of prolactin secretion (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Due to the fact that thyroid hormones regulate the secretion and use of all steroid hormones adrenal gland deficiency with such findings as lack of libido, impotence, amenorrhea, menorrhagia, and polymerrhea is observed in cases with hypothyroidism. Another cause for findings related to these sex steroids may be excessive prolactin (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Thyroid hormones affect bone metabolism in parallel with parathormone. Estrogen, vitamin D3, TGF-β, PGE2, parathormone (PTH), and all of the thyroid hormones are necessary for osteoblastic activity (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

As noted earlier, thyroid hormones increase β adrenergic receptor count. Adrenaline and noradrenaline interact with these receptors and accelerates basal metabolism, stimulates the nervous system, and speeds up the circulation system just as in the effect of thyroid hormones (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

For a normal sexual development and life, thyroid hormones are necessary. The reason for this is that thyroid hormones increase the use and secretion of sex steroids, and in addition, affect prolactin secretion. Lack of libido, impotence, gynecomastia, amenorrhea, menorrhagia, and polymenorrhea are observed due to sex steroid deficiency and excessive prolactin in cases with hypothyroidism (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

#### **5. Conclusion**

20 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

was increased in cases with hypothyroidism due to high plasma viscosity (Bursuk et al., 2010; Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed &

Thyroid hormones regulate the actions of other endocrine hormones in order to accelerate basal metabolism. These hormones increase the absorption of glucose in gastrointestinal system, glucose reception into cells, and both glycolysis and gluconeogenesis by producing an effect on insulin and glucagon. Thyroid hormones enable the increase of insulin through secondary mechanism by occasionally rising blood sugar (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti

Due to the fact that both thyroid hormones and growth hormones are necessary for normal somatic growth, thyroid hormones increase the synthesis and secretion of growth hormone and growth factors (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996;

Also, another effect is produced on prolactin. During hypothyroidism, TRH secretion stimulates prolactin secretion, and while galactorrhea and amenorrhea is observed in females, gynecomastia and impotence is found in males. The inhibiting effect of dopamine is of utmost importance in regulating the secretion of prolactin secretion (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997;

Due to the fact that thyroid hormones regulate the secretion and use of all steroid hormones adrenal gland deficiency with such findings as lack of libido, impotence, amenorrhea, menorrhagia, and polymerrhea is observed in cases with hypothyroidism. Another cause for findings related to these sex steroids may be excessive prolactin (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Thyroid hormones affect bone metabolism in parallel with parathormone. Estrogen, vitamin D3, TGF-β, PGE2, parathormone (PTH), and all of the thyroid hormones are necessary for osteoblastic activity (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996;

As noted earlier, thyroid hormones increase β adrenergic receptor count. Adrenaline and noradrenaline interact with these receptors and accelerates basal metabolism, stimulates the nervous system, and speeds up the circulation system just as in the effect of thyroid hormones (Dillmann, 2004; Dunn, 2001; Ganong, 1997; Guyton & Hall, 1997; Jameson & Weetman, 2010; Larsen et al., 2003; Lo Presti & Singer, 1997; Mc Gregor, 1996; Reed &

For a normal sexual development and life, thyroid hormones are necessary. The reason for this is that thyroid hormones increase the use and secretion of sex steroids, and in addition, affect prolactin secretion. Lack of libido, impotence, gynecomastia, amenorrhea,

& Singer, 1997; Mc Gregor, 1996; Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Pangaro, 1995; Utiger, 1997; Usala, 1995).

Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Reed & Pangaro, 1995; Utiger, 1997; Usala, 1995).

Pangaro, 1995; Utiger, 1997; Usala, 1995).

Usala, 1995).

Anatomy, histology and physiology of thyroid have been addressed in this chapter. In its physiology, its hormone synthesis, metabolism, effect generation mechanism and effects on the body has been explained. While mentioning these effects, the relationship between thyroid diseases and blood hemorheology has also been referred and relationship between disease groups (hyperthyroids and hypothyroids) has been analysed comparatively with these parameters.

#### **6.References**


**2**

Anne Charrié

*France* 

**The Thyroglobulin: A Technically Challenging** 

**the Follow-Up of Differentiated Thyroid Cancer** 

The thyroglobulin (Tg) is a normal secretory product of the thyroid gland. Tg is stored in the follicular light of the thyroid where it constitutes the majority of colloid proteins. It is the

This glycoprotein of high molecular weight (660 kDa) is constituted by two identical subunits bound by disulphide-bridges. Each sub-unit contains 2 749 amino acids (Malthiery & Lissitzky, 1987 and Van de Graf et al., 1997). Its gene is situated on the chromosome 8 and different isoforms of Tg are secreted by alternative splicing. This molecule is heterogeneous by its degree of iodination (0.2 to 1.0%), of glycosylation, and by its contents in oses and in sialic acid (8 to 10%). The epitopic map of Tg revealed approximately about forty antigenic determinants, twelve epitopes grouped together in six domains (Piechaczyck et al., 1985). The central region of the Tg molecule is in majority immunoreactive (Henry et al, 1990).

Tg is not confined in the follicle, some molecules are co-secreted with thyroid hormones by a complex process which can modify it. Any conformational change entails a different antigenicity because some epitopes can be masked or on the contrary be exposed. Molecular forms of Tg found in the serum of patients with differentiated thyroid cancer correspond to dimeric Tg. It is little iodized and presents a change of the glycosylation (Sinadinovic et al., 1992 and Druetta et al., 1998)*.* The heterogeneousness of Tg in the thyroid gland is increased in the cancer (Persani et al., 1998) and the changes of its conformation modifies its immunoreactivity (Kohno et al., 1985). All these structural characteristics are very important to know and can give some explanations about differences between Tg assays. It is not surprising to notice differences between Tg assays which use monoclonal antibodies by definition very specific. The follow-up of the differentiated thyroid cancers is the essential indication of the dosage of the serum Tg. Tg signs the presence of normal or pathological thyroid tissue. It is not possible to differentiate the normal tissue of the cancerous tissue thanks to serum Tg value. One reference point is mentioned in the laboratory medicine practice guidelines (Baloch et al., 2003): one gram of normal thyroid releases about 1µg/L Tg into the circulation when the serum thyroid stimulating hormone (TSH) is normal and 0.5 µg/L if the TSH value is suppressed below 0.1 mUI/L*.* Since its concentration is correlated

**1. Introduction** 

place of synthesis and storage of thyroid hormones.

*Lyon University, INSERM U1060, CarMeN laboratory and CENS, Univ Lyon-1* 

**Assay for a Marker of Choice During** 

*Laboratory of Nuclear Technics and Biophysic, Hospices Civils de Lyon* 


### **The Thyroglobulin: A Technically Challenging Assay for a Marker of Choice During the Follow-Up of Differentiated Thyroid Cancer**

Anne Charrié

*Lyon University, INSERM U1060, CarMeN laboratory and CENS, Univ Lyon-1 Laboratory of Nuclear Technics and Biophysic, Hospices Civils de Lyon France* 

#### **1. Introduction**

22 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

Mc Gregor, A.M. (1996). The thyroid gland and disorders of thyroid function, In: *Oxford* 

Reed, L. & Pangaro, L.N. (1995). Physiology of the thyroid gland I: synthesis and release,

Santiseban, P. (2005). Development and anatomy of the hypothalamic – pituitary – thyroid

Scanlon, M.F. (2001). Thyrothropin releasing hormone and thyrothropin stimulating

Snell, R.S. (1995). *Clinical Anatomy for my students* (fifth edition), Little, Brown and Company,

Usala, S.J. (1995). Physiology of the thyroid gland II: reseptors, postreceptor events, and

Utiger, R.D. (1997). Disorders of the thyroid gland, In: *Textbook of Intecnal Medrane*, Kelley, WN., pp. (2204–2219), Lippincott – Raven Publishers, 0-397-51540-5, Philadelphia.

1621), Oxford University Press, 0-19-262707-4, Oxford, Vol. 2.

Saunders Company, 0-7216-7840-8, Philadelphia.

0-397-51404-2, Philadelphia.

Philadelphia.

Philadelphia.

0-316-80135-6, Boston.

*Fextbook of Medicine,* Weatherall, DJ., Ledingham, JGG. & Warrell, DA, pp. (1603–

iodine metabolism, and binding and transport, In: *Principles and Practice of Endocrinology and Metabolism,* Becher, KL., pp. (285-291), J.B. Lippincott Company,

axis, In: *Werner&Ingbar's The Thyroid a Fundamental and Clinical Text*, Braverman, LE.,&Utiger, RD., pp. (8-23), Lippincot Williams&Wilkins Company, 0-7817-5047-4,

hormone, In: *Endocrinology*, DeGroot, LJ.&Jameson, JL., pp. (1279-1286), W.B.

hormone resistance syndromes, In: *Principle and Practice of Endocrinology and Metabolism*, Becker, KL., pp. (292-298), J.B. Lippincott Company, 0-397-51404-2,

The thyroglobulin (Tg) is a normal secretory product of the thyroid gland. Tg is stored in the follicular light of the thyroid where it constitutes the majority of colloid proteins. It is the place of synthesis and storage of thyroid hormones.

This glycoprotein of high molecular weight (660 kDa) is constituted by two identical subunits bound by disulphide-bridges. Each sub-unit contains 2 749 amino acids (Malthiery & Lissitzky, 1987 and Van de Graf et al., 1997). Its gene is situated on the chromosome 8 and different isoforms of Tg are secreted by alternative splicing. This molecule is heterogeneous by its degree of iodination (0.2 to 1.0%), of glycosylation, and by its contents in oses and in sialic acid (8 to 10%). The epitopic map of Tg revealed approximately about forty antigenic determinants, twelve epitopes grouped together in six domains (Piechaczyck et al., 1985). The central region of the Tg molecule is in majority immunoreactive (Henry et al, 1990).

Tg is not confined in the follicle, some molecules are co-secreted with thyroid hormones by a complex process which can modify it. Any conformational change entails a different antigenicity because some epitopes can be masked or on the contrary be exposed. Molecular forms of Tg found in the serum of patients with differentiated thyroid cancer correspond to dimeric Tg. It is little iodized and presents a change of the glycosylation (Sinadinovic et al., 1992 and Druetta et al., 1998)*.* The heterogeneousness of Tg in the thyroid gland is increased in the cancer (Persani et al., 1998) and the changes of its conformation modifies its immunoreactivity (Kohno et al., 1985). All these structural characteristics are very important to know and can give some explanations about differences between Tg assays. It is not surprising to notice differences between Tg assays which use monoclonal antibodies by definition very specific. The follow-up of the differentiated thyroid cancers is the essential indication of the dosage of the serum Tg. Tg signs the presence of normal or pathological thyroid tissue. It is not possible to differentiate the normal tissue of the cancerous tissue thanks to serum Tg value. One reference point is mentioned in the laboratory medicine practice guidelines (Baloch et al., 2003): one gram of normal thyroid releases about 1µg/L Tg into the circulation when the serum thyroid stimulating hormone (TSH) is normal and 0.5 µg/L if the TSH value is suppressed below 0.1 mUI/L*.* Since its concentration is correlated

The Thyroglobulin: A Technically Challenging Assay

Association (Cooper et al., 2006).

**2.3 Interference by TgAb** 

immunometric methods.

for a Marker of Choice During the Follow-Up of Differentiated Thyroid Cancer 25

measuring human pool sera over 6 to 12 months (compatible deadline with the follow-up of the patients) with at least 2 batchs of reagents and 2 instrument calibrations (Baloch et al., 2003). The pools of serum used for this profile have to be TgAb negative. It must be repeated to the scientists how to verify the functional sensitivity of a Tg assay and not to take that given by the manufacturer. Analogous to TSH, Tg assay functional sensitivity permits a generational classification of Tg assays. Most current assays are actually first generation with a functional sensitivity about 0.5 to 1.0µg/L. The functional sensitivity is of a big importance to determine the «detectable Tg < institutional cut-off » mentioned in the European Consensus (Pacini et al., 2006) specially in the flow chart for the follow-up after initial treatment (6 to 12 months) and recombinant human thyrotropin (rhTSH). For example some authors (Kloos & Mazzaferri, 2005) considered a thyroglobulin cutoff level of 2.0µg/L highly sensitive for identifying persistent tumor after rhTSH stimulation in patients who had TSH-suppressed thyroglobulin undetectable with an assay functional sensitivity value of 1µg/L. This cut-off is also mentioned in the recommendations of the American Thyroid

In the European consensus, supersensitive Tg assays which have a higher sensitivity but at the expense of a much lower specificity are not currently recommended for routine use. Nevertheless some current assays are second generation with a ten-fold better functional sensitivity. An insufficient functional sensitivity is at the origin of most of false-negative results corresponding to an authentic recurrence of the disease with a value of Tg given undetectable. With a more sensitive second generation assay it would be possible to detect responses that will be undetectable with a first generation assay. At present this very low functional sensitivity for certain cases of dosages could allow to replace rhTSH stimulated Tg testing for the patients at low risk by a simple dosage of second generation Tg. Low risk patients are those with well-differentiated papillary or follicular thyroid cancer, patient age <45 years, thyroid tumor size ranging from 1 cm to <4 cm in diameter, no extension of the tumor beyond the thyroid capsule, no lymph-node involvement and no distant metastases

This type of analytical problem is completely characteristic of Tg assays and exists in no other immunoassay. It is connected to the fact that Tg is a major auto-antigen. All the actually methods are prone to interference by TgAb (Mariotti et al., 1995). The combined use of judiciously selected monoclonal antibodies directed against antigenic domains of Tg not recognized by most TgAb allowed to develop a Tg assay with minimal interference from TgAb (Marquet et al., 1996). In every case the presence of TgAb that mask certain epitopes can lead to underestimation of the Tg concentrations with the actuals

We are however unable to evaluate the true interference of these TgAb: it is known that in some patients few Tgab can induce a major interference while in some others a lot of TgAb induce only a smooth interference. Everything depends on the affinity of these antibodies which we do not estimate. The various consensus recommend to measure antibodies by a enough sensitive method in a systematic way with any dosage of Tg. At first it had been suggested realizing a test of recovery to estimate the importance of the interference but this one was abandoned because of a bad standardization of the protocol

(Schlumberger et al., 2007; Smallridge et al., 2007; Schlumberger et al., 2011).

with the size rather than with the nature of nodule of the thyroid gland, Tg is not used for the diagnosis of the thyroid cancer. Routine preoperative measurement of serum Tg for initial evaluation of thyroid nodules is not recommended (Cooper et al., 2006).

#### **2. Thyroglobulin assay in serum**

Serum Tg measurement is a technically challenging assay for a marker of choice during the follow-up of differentiated thyroid cancer. The use of the Tg assays requires a good knowledge of the technical difficulties. The quality of current Tg assay methods varies and influences the clinical utility of this test. All techniques are today immunometric assays with isotopic signal or not. Several methodological problems must be taken into account: standardization, functional sensitivity, precision, hook effects, interference by heterophile antibodies and interference by Tg antibodies (TgAb) (Spencer et al., 1996). Precision and hook effects are two parameters which are usual in biology when markers are used in the follow-up of cancer. Every laboratory scientist knows that it is sometimes better to measure stored serum samples from the patient in the same run as the current specimen to better appreciate the variability of the marker during the time. As regards the hook effect it is careful either to use a technique in 2 steps or to dilute systematically the serum suspected of very high values of Tg. Heterophile antibodies may cause falsely elevated serum Tg levels as in al immunometric assays. It is possible to reduce this interference by using heterophile blocking tubes when these antibodies are suspected (Preissner et al., 2003). Even if some solutions were studied for the other problems (standardisation, functional sensitivity and interference by TgAb) all persist always for more than fifteen years and guidelines have been published (Baloch et al., 2003; Pacini et al., 2006; Borson-Chazot et al., 2008).

#### **2.1 Standardisation**

Different guidelines and consensus (Baloch et al., 2003; Pacini et al., 2006; Borson-Chazot et al., 2008) recommended the use of the European human reference material CRM 457 (Feldt-Rasmussen U et al., 1996). Even if the use of this standard doesn't resolve all problems between different techniques it will be a minimal consensus that manufacturers would follow to get a homogenous basis of standardisation. The CRM 457 is produced from normal thyroid tissue. Now we know that tissular Tg is not strictly the one which circulates in the blood (Schulz et al., 1989). The ideal standard would be a preparation of thyroglobulin extracted from the blood. Because of a too small quantity of circulating Tg the manufacturing of such a reference was not possible. The actual recommendation is to use 1:1 CRM 457 standardisation. The configuration of the Tg molecule is not enough taken into account in the various Tg methods.

#### **2.2 Functional sensitivity**

Since Tg measurements have to detect very small amount of thyroid tissue, it is absolutely necessary to determine the sensitivity of the Tg assays. The definition of the functional sensitivity was established by Spencer for the TSH (Spencer et al., 1996 a). The same concept can be applied to Tg (Spencer et al, 1996 b): it is the Tg value that can be measured with 20% between-run coefficient of variation (CV), using a 1:1 CRM 457 standardisation. The proposed protocol is similar for Tg with the establishment of a profile of precision measuring human pool sera over 6 to 12 months (compatible deadline with the follow-up of the patients) with at least 2 batchs of reagents and 2 instrument calibrations (Baloch et al., 2003). The pools of serum used for this profile have to be TgAb negative. It must be repeated to the scientists how to verify the functional sensitivity of a Tg assay and not to take that given by the manufacturer. Analogous to TSH, Tg assay functional sensitivity permits a generational classification of Tg assays. Most current assays are actually first generation with a functional sensitivity about 0.5 to 1.0µg/L. The functional sensitivity is of a big importance to determine the «detectable Tg < institutional cut-off » mentioned in the European Consensus (Pacini et al., 2006) specially in the flow chart for the follow-up after initial treatment (6 to 12 months) and recombinant human thyrotropin (rhTSH). For example some authors (Kloos & Mazzaferri, 2005) considered a thyroglobulin cutoff level of 2.0µg/L highly sensitive for identifying persistent tumor after rhTSH stimulation in patients who had TSH-suppressed thyroglobulin undetectable with an assay functional sensitivity value of 1µg/L. This cut-off is also mentioned in the recommendations of the American Thyroid Association (Cooper et al., 2006).

In the European consensus, supersensitive Tg assays which have a higher sensitivity but at the expense of a much lower specificity are not currently recommended for routine use. Nevertheless some current assays are second generation with a ten-fold better functional sensitivity. An insufficient functional sensitivity is at the origin of most of false-negative results corresponding to an authentic recurrence of the disease with a value of Tg given undetectable. With a more sensitive second generation assay it would be possible to detect responses that will be undetectable with a first generation assay. At present this very low functional sensitivity for certain cases of dosages could allow to replace rhTSH stimulated Tg testing for the patients at low risk by a simple dosage of second generation Tg. Low risk patients are those with well-differentiated papillary or follicular thyroid cancer, patient age <45 years, thyroid tumor size ranging from 1 cm to <4 cm in diameter, no extension of the tumor beyond the thyroid capsule, no lymph-node involvement and no distant metastases (Schlumberger et al., 2007; Smallridge et al., 2007; Schlumberger et al., 2011).

#### **2.3 Interference by TgAb**

24 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

with the size rather than with the nature of nodule of the thyroid gland, Tg is not used for the diagnosis of the thyroid cancer. Routine preoperative measurement of serum Tg for

Serum Tg measurement is a technically challenging assay for a marker of choice during the follow-up of differentiated thyroid cancer. The use of the Tg assays requires a good knowledge of the technical difficulties. The quality of current Tg assay methods varies and influences the clinical utility of this test. All techniques are today immunometric assays with isotopic signal or not. Several methodological problems must be taken into account: standardization, functional sensitivity, precision, hook effects, interference by heterophile antibodies and interference by Tg antibodies (TgAb) (Spencer et al., 1996). Precision and hook effects are two parameters which are usual in biology when markers are used in the follow-up of cancer. Every laboratory scientist knows that it is sometimes better to measure stored serum samples from the patient in the same run as the current specimen to better appreciate the variability of the marker during the time. As regards the hook effect it is careful either to use a technique in 2 steps or to dilute systematically the serum suspected of very high values of Tg. Heterophile antibodies may cause falsely elevated serum Tg levels as in al immunometric assays. It is possible to reduce this interference by using heterophile blocking tubes when these antibodies are suspected (Preissner et al., 2003). Even if some solutions were studied for the other problems (standardisation, functional sensitivity and interference by TgAb) all persist always for more than fifteen years and guidelines have been published (Baloch et al., 2003; Pacini et

Different guidelines and consensus (Baloch et al., 2003; Pacini et al., 2006; Borson-Chazot et al., 2008) recommended the use of the European human reference material CRM 457 (Feldt-Rasmussen U et al., 1996). Even if the use of this standard doesn't resolve all problems between different techniques it will be a minimal consensus that manufacturers would follow to get a homogenous basis of standardisation. The CRM 457 is produced from normal thyroid tissue. Now we know that tissular Tg is not strictly the one which circulates in the blood (Schulz et al., 1989). The ideal standard would be a preparation of thyroglobulin extracted from the blood. Because of a too small quantity of circulating Tg the manufacturing of such a reference was not possible. The actual recommendation is to use 1:1 CRM 457 standardisation. The configuration of the Tg molecule is not enough taken into

Since Tg measurements have to detect very small amount of thyroid tissue, it is absolutely necessary to determine the sensitivity of the Tg assays. The definition of the functional sensitivity was established by Spencer for the TSH (Spencer et al., 1996 a). The same concept can be applied to Tg (Spencer et al, 1996 b): it is the Tg value that can be measured with 20% between-run coefficient of variation (CV), using a 1:1 CRM 457 standardisation. The proposed protocol is similar for Tg with the establishment of a profile of precision

initial evaluation of thyroid nodules is not recommended (Cooper et al., 2006).

**2. Thyroglobulin assay in serum** 

al., 2006; Borson-Chazot et al., 2008).

account in the various Tg methods.

**2.2 Functional sensitivity** 

**2.1 Standardisation** 

This type of analytical problem is completely characteristic of Tg assays and exists in no other immunoassay. It is connected to the fact that Tg is a major auto-antigen. All the actually methods are prone to interference by TgAb (Mariotti et al., 1995). The combined use of judiciously selected monoclonal antibodies directed against antigenic domains of Tg not recognized by most TgAb allowed to develop a Tg assay with minimal interference from TgAb (Marquet et al., 1996). In every case the presence of TgAb that mask certain epitopes can lead to underestimation of the Tg concentrations with the actuals immunometric methods.

We are however unable to evaluate the true interference of these TgAb: it is known that in some patients few Tgab can induce a major interference while in some others a lot of TgAb induce only a smooth interference. Everything depends on the affinity of these antibodies which we do not estimate. The various consensus recommend to measure antibodies by a enough sensitive method in a systematic way with any dosage of Tg. At first it had been suggested realizing a test of recovery to estimate the importance of the interference but this one was abandoned because of a bad standardization of the protocol

The Thyroglobulin: A Technically Challenging Assay

**3.2 The volume** 

**3.3 The Tg method** 

liquid (Leenhardt et al., 2011).

**3.4 The results and interpretation** 

and not a concentration of Tg in the LN.

solution supplemented with serum albumin (Borel et al., 2008).

rinse can be poured into the same tube (Leenhardt et al., 2011).

for a Marker of Choice During the Follow-Up of Differentiated Thyroid Cancer 27

the matrix to measure samples (Wild, 2005). We think that it is much better to use the Tgfree medium of the test kit to avoid bias in the determination of thyroglobulin in FNA wash samples (Bournaud et al., 2010). But for practical use the saline solution is often used and so it is recommended in the French good practice guide for cervical ultrasound scan and echoguided techniques (Leenhardt et al., 2011) to check for the absence of matrix effect in the usual assay method. It is possible to validate the use of saline solution by comparing the results of Tg immunoreactivity obtained with Tg-free solution, saline solution and saline

The quantity of the liquid used to rinse the needle varies between 0.5 to 1.0mL but is in general 1.0mL. All content of the needle is carefully removed by washing with from one to three pumping depending of the operator. Borel et al. (2008) shows that a triple pumping action of the 1 mL liquid through the needle was sufficient to wash out 97% of Tg out of the needle. If the needle has to be inserted several times into the same lymph node, the needle

The Tg method is the same used for the Tg serum assay. The problem of the interference by TgAb is however different. The presence of TgAb in fine needle aspiration biopsy washout can result of blood contamination when they are present or of active lymph node synthesis (Boi et al., 2006). But this interference seams to have small effect on the result of FNA-Tg. An explanation of this could be that the excessive high concentration of Tg is able to saturate TgAb binding sites. So it is not recommended to assay TgAb in the rinsing

Another interference could be also evoked: the contamination with serum Tg. It seems that FNA-Tg is not affected by the circulating serum levels. In 2008 Borel et al calculate that the maximal contamination of FNA-Tg by serum Tg varied from 0.003 to 0.012% what is not significant. He measured also albumin in the LN washout to evaluate the contamination by plasma proteins. He concluded that serum Tg did not interfere in results of FNA-Tg and specially in negative controls (not thyroidectomized) who had undetectable FNA-Tg values.

The expression of the results varies according to studies. Some authors (Baskin, 2004; Boi et al, 2006; Kim et al, 2009) use the unit µg/L (or ng/ml), others (Pacini et al., 1992; Cignarelli et al., 2003; Borel et al., 2008; Bournaud et al., 2010) use µg/FNA. It is more suitable to use this type of result which reflects only the quantity of Tg present in the needle after rinsing

We find here again the problem of functional sensitivity of the Tg method which directly affect the cut-off value. In the first study (Pacini et al., 1992), the cut-off value was 21.7µg/L but the functional sensitivity was only 3µg/L. This cut-off value was established as equal to the mean plus two standard deviations of the FNA-Tg values in patients with negative cytology. Other authors used the same type of cut-off (Cignarelli et al., 2003; Baskin, 2004;

(Spencer et al, 1996c). When there is presence of TgAb and if Tg is found undetectable, its value is not interpretable. When the value of Tg is dosable with presence of antibodies, the returned value is then a "minimal" value knowing that she could be more raised in the absence of antibody. After thyroidectomy, TgAb will decrease and disappear in patients with remission but these antibodies may persist during 2-3 years after disappearance of Tg (Chiovato et al., 2003). During the follow-up of some patients persistence or reappearance of circulating TgAb may be regarded as an indicator of disease. More recently Spencer even concluded that TgAb trends can be used as a surrogate tumor marker in differentiated thyroid cancer in preference to Tg measurement, provided that the same method is used.

#### **3. Thyroglobulin in fine needle aspiration biopsy**

After surgery for differentiated thyroid cancer, cervical ultrasound is recommended to evaluate the thyroid bed and central and lateral cervical nodal compartments should be performed at 6 and 12 months and then annually for at least 3-5 years, depending on the patients' risk for recurrent disease and thyroglobulin status (Cooper et al., 2006). At present numerous studies describe the utility to look for thyroglobulin measurements in fine-needle aspiration biopsies (FNA-Tg) of lymph node (LN) during the follow-up of differentiated thyroid carcinoma. Although most patients have a long term survival rate, 5 to 20% of them will develop recurrence during follow-up, primarily in the cervical lymph nodes. An accurate distinction between metastatic and reactive benign lymph nodes (BLN) is essential in the management of thyroid cancer prior to surgery; it is necessary to specify the extent of surgery and identify early cervical relapse.

Cytological examination of fine-needle aspiration cytology (FNA-C) the reference method for the diagnosis of thyroid nodules has also been, until recently, the best method to diagnose a cervical LN in subjects with suspicion of thyroid cancer or patients followed for thyroid neoplasia. However, sensitivity of FNA-C is far from excellent, varying from 75 to 85% and altered by a high rate of non-diagnostic samples. Pacini was the first author who showed in 1992 high concentrations of thyroglobulin in metastatic LN of thyroid carcinoma. Although the performance of FNA-Tg is now well established, some methodological factors may influence the results and threshold value remains controversial. The first step is how to obtain the material from the fine needle aspiration. Ultrasound-guided fine-needle aspiration biopsy is carried out by a trained operator with a fine needle, preferably 25 to 27 gauges. After aspiration, the needle is rinsed.

#### **3.1 The middle**

The middle used to rinse the needle is variable according to the teams; it can be either physiological saline solution or a liquid supplied by the laboratory (assay buffer or Tg-free serum). Two studies show that some parasite effects are present in the dosage: some "noise" in the Tg assay was described by Baskin et al. (2004). Snozek et al. (2007) demonstrate with a recovery test (after an overload of exogenous Tg) that the values of Tg are 25% higher with the saline solution than with a serous matrix with his Tg assay. The nature of the buffer may have an influence on the conformation of proteins and affect antibody binding. The most important matrix effect is that due to the matrix used to prepare the calibration curve and the matrix to measure samples (Wild, 2005). We think that it is much better to use the Tgfree medium of the test kit to avoid bias in the determination of thyroglobulin in FNA wash samples (Bournaud et al., 2010). But for practical use the saline solution is often used and so it is recommended in the French good practice guide for cervical ultrasound scan and echoguided techniques (Leenhardt et al., 2011) to check for the absence of matrix effect in the usual assay method. It is possible to validate the use of saline solution by comparing the results of Tg immunoreactivity obtained with Tg-free solution, saline solution and saline solution supplemented with serum albumin (Borel et al., 2008).

#### **3.2 The volume**

26 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

(Spencer et al, 1996c). When there is presence of TgAb and if Tg is found undetectable, its value is not interpretable. When the value of Tg is dosable with presence of antibodies, the returned value is then a "minimal" value knowing that she could be more raised in the absence of antibody. After thyroidectomy, TgAb will decrease and disappear in patients with remission but these antibodies may persist during 2-3 years after disappearance of Tg (Chiovato et al., 2003). During the follow-up of some patients persistence or reappearance of circulating TgAb may be regarded as an indicator of disease. More recently Spencer even concluded that TgAb trends can be used as a surrogate tumor marker in differentiated thyroid cancer in preference to Tg measurement, provided that

After surgery for differentiated thyroid cancer, cervical ultrasound is recommended to evaluate the thyroid bed and central and lateral cervical nodal compartments should be performed at 6 and 12 months and then annually for at least 3-5 years, depending on the patients' risk for recurrent disease and thyroglobulin status (Cooper et al., 2006). At present numerous studies describe the utility to look for thyroglobulin measurements in fine-needle aspiration biopsies (FNA-Tg) of lymph node (LN) during the follow-up of differentiated thyroid carcinoma. Although most patients have a long term survival rate, 5 to 20% of them will develop recurrence during follow-up, primarily in the cervical lymph nodes. An accurate distinction between metastatic and reactive benign lymph nodes (BLN) is essential in the management of thyroid cancer prior to surgery; it is necessary to specify the extent of

Cytological examination of fine-needle aspiration cytology (FNA-C) the reference method for the diagnosis of thyroid nodules has also been, until recently, the best method to diagnose a cervical LN in subjects with suspicion of thyroid cancer or patients followed for thyroid neoplasia. However, sensitivity of FNA-C is far from excellent, varying from 75 to 85% and altered by a high rate of non-diagnostic samples. Pacini was the first author who showed in 1992 high concentrations of thyroglobulin in metastatic LN of thyroid carcinoma. Although the performance of FNA-Tg is now well established, some methodological factors may influence the results and threshold value remains controversial. The first step is how to obtain the material from the fine needle aspiration. Ultrasound-guided fine-needle aspiration biopsy is carried out by a trained operator with a fine needle, preferably 25 to 27

The middle used to rinse the needle is variable according to the teams; it can be either physiological saline solution or a liquid supplied by the laboratory (assay buffer or Tg-free serum). Two studies show that some parasite effects are present in the dosage: some "noise" in the Tg assay was described by Baskin et al. (2004). Snozek et al. (2007) demonstrate with a recovery test (after an overload of exogenous Tg) that the values of Tg are 25% higher with the saline solution than with a serous matrix with his Tg assay. The nature of the buffer may have an influence on the conformation of proteins and affect antibody binding. The most important matrix effect is that due to the matrix used to prepare the calibration curve and

the same method is used.

**3. Thyroglobulin in fine needle aspiration biopsy** 

surgery and identify early cervical relapse.

gauges. After aspiration, the needle is rinsed.

**3.1 The middle** 

The quantity of the liquid used to rinse the needle varies between 0.5 to 1.0mL but is in general 1.0mL. All content of the needle is carefully removed by washing with from one to three pumping depending of the operator. Borel et al. (2008) shows that a triple pumping action of the 1 mL liquid through the needle was sufficient to wash out 97% of Tg out of the needle. If the needle has to be inserted several times into the same lymph node, the needle rinse can be poured into the same tube (Leenhardt et al., 2011).

#### **3.3 The Tg method**

The Tg method is the same used for the Tg serum assay. The problem of the interference by TgAb is however different. The presence of TgAb in fine needle aspiration biopsy washout can result of blood contamination when they are present or of active lymph node synthesis (Boi et al., 2006). But this interference seams to have small effect on the result of FNA-Tg. An explanation of this could be that the excessive high concentration of Tg is able to saturate TgAb binding sites. So it is not recommended to assay TgAb in the rinsing liquid (Leenhardt et al., 2011).

Another interference could be also evoked: the contamination with serum Tg. It seems that FNA-Tg is not affected by the circulating serum levels. In 2008 Borel et al calculate that the maximal contamination of FNA-Tg by serum Tg varied from 0.003 to 0.012% what is not significant. He measured also albumin in the LN washout to evaluate the contamination by plasma proteins. He concluded that serum Tg did not interfere in results of FNA-Tg and specially in negative controls (not thyroidectomized) who had undetectable FNA-Tg values.

#### **3.4 The results and interpretation**

The expression of the results varies according to studies. Some authors (Baskin, 2004; Boi et al, 2006; Kim et al, 2009) use the unit µg/L (or ng/ml), others (Pacini et al., 1992; Cignarelli et al., 2003; Borel et al., 2008; Bournaud et al., 2010) use µg/FNA. It is more suitable to use this type of result which reflects only the quantity of Tg present in the needle after rinsing and not a concentration of Tg in the LN.

We find here again the problem of functional sensitivity of the Tg method which directly affect the cut-off value. In the first study (Pacini et al., 1992), the cut-off value was 21.7µg/L but the functional sensitivity was only 3µg/L. This cut-off value was established as equal to the mean plus two standard deviations of the FNA-Tg values in patients with negative cytology. Other authors used the same type of cut-off (Cignarelli et al., 2003; Baskin, 2004;

The Thyroglobulin: A Technically Challenging Assay

*Cytojournal* Vol.5, pp.1-5.

Vol.91, N0.4, pp.1364-9.

nodes. *Thyroid,* Vol. 14, pp.959-63.

*Lab Med*, Vol.48, N0.8, pp.1171-7.

pp.109-42

cytology. *Thyroid* Vol.13, N012, pp.1163-7.

for a Marker of Choice During the Follow-Up of Differentiated Thyroid Cancer 29

Baloch, ZW.; Barroeta, JE.; Walsh, J.; Gupta, PK.; Livolsi, VA.; Langer, JE & Mandel, SJ.

Baskin, HJ. (2004).Detection of recurrent papillary thyroid carcinoma by thyroglobulin

Boi, F.; Baghino, G.; Atzeni, F.; Lai, ML.; Faa, G. & Mariotti, S. (2006). The diagnostic value

Borel, AL.; Boizel, R.; Faure, P.; Barbe, G.; Boutonnat, J.; Sturm, N.; Seigneurin, D.; Bricault,

history of differentiated thyroid cancer. *Eur J Endocrinol,* Vol. 158, pp.691-8. Borson-Chazot, F.; Bardet, S.; Bournaud, C.; Conte-Devolx, B.; Corone, C.; D'Herbomez, M.;

monitoring of thyroid disease. *Thyroid,* Vol. 13, pp.3-126.

Laboratory medicine practice guidelines. Laboratory support for the diagnosis and

(2008). Utility of thyroglobulin measurement in fine needle aspiration biopsy specimens of lymph nodes in the diagnosis of recurrent thyroid carcinoma.

assessment in the needle washout after fine-needle aspiration of suspicious lymph

for differentieted thyroid carcinoma metastases of thyroglobulin (Tg) measurement in washout fluid from fine-needle aspiration biopsy of neck lymph nodes is maintained in the presence of circulating anti-Tg antibodies. *J Clin Endocrinol Metab,* 

I.; Caravel, JP.; Chaffanjon, P. & Chabre, O. (2008). Significance of low levels of thyroglobulin in fine needle aspirates from cervocal lymph nodes of patients with a

Henry, JF.; Leenhardt, L.; Peix, JL.; Schlumberger, M.; Wemeau, JL.; Baudin, E.; Berger, N.; Bernard, MH.; Calzada-Nocaudie, M.; Caron, P.; Catargi, B.; Chabrier, G.; Charrié, A.; Franc, B.; Hartl, D.; Helal, B.; Kerlan, V.; Kraimps, JL.; Leboulleux, S.; Le Clech, G.; Menegaux, F.; Orgiazzi, J.; Perie, S.; Raingeard, I.; Rodien, P.; Rohmer, V.; Sadoul, JL.; Schwartz, C.; Tenenbaum, F.; Toubert, ME.; Tramalloni, J.; Travagli, JP. & Vaudrey, C. (2008). Guidelines for the management of differentiated thyroid carcinoma of vesicular origin. *Ann Endocrinol (Paris)* Vol. 69, pp.472-86. Bournaud, C; Charrié, A.; Nozières, C.; Chikh, K.; Lapras, V.; Denier, ML.; Paulin, C.;

Decaussin-Petrucci, M.; Peix, JL.; Lifante, JC.; Cornu, C.; Giraud, C.; Orgiazzi, J. & Borson-chazot, F. (2010). Thyroglobulin measurement in fine-needle aspirates of lymph nodes in patients with differentiated thyroid cancer: a simple definition of the threshold value, with emphasis on potential pitfalls of the method. *Clin Chem* 

& Pinchera, A. (2003). A disappearance of humoral thyroid autoimmunity after

(2003). Diagnostic utility of thyroglobulin detection in fine-needle aspiration of cervical cystic metastatic lymph nodes from papillary thyroid cancer with negative

McIver, B.; Sherman, SI. & Tuttle, RM. (2006). Management guidelines for patients with thyroid nodules and differentiated thyroid cancer. *Thyroid* Vol.16, No.2,

J.; Gomes, I.; Pereira, H. Real, O.; Figueiredo, P.; Campos, B. & Valido, F. (2007). Thyroglobulin detection in fine-needle aspirates of cervical lymph nodes: a

Chiovato, I.; Iatrofa, F.; Braverman, LE. ; Pacini, F.; Capezzone, M.; Masserini, I.; Grasso, L.

complete removal of thyroid antigens. *Ann Intern Med* Vol.139, pp.346-51. Cignarelli, M.; Ambrosi, A.; Marino, A.; Lamacchia, O.; Campo, M.; Picca, G. & Giorgino, F.

Cooper,DS.; Doherty, GM.; Haugen, BR.; Kloos, RT.; Lee, SL.; Mandel, SJ.; Mazzaferri, EL.;

Cunha, N.; Rodrigues, F.; Curado, F.; Ilheu, O.; Cruz, C.; Naidenov, P.; Rascao, MJ.; Ganho,

Boi et al., 2006). In other studies of the literature threshold are sometimes the functional sensitivity (Cunha et al., 2007; Snozek et al., 2007) or study of sensitivity and specificity and choice of the better cut-off with a Receiver Operating Characteristic Curve (ROC) (Bournaud et al., 2010; Giovanella et al., 2011). For others again the FNA-Tg is compared with serum Tg: when the FNA-Tg value is greater than serum Tg value the LN is considered as metastasis (Uruno et al., 2005; Sigstad et al., 2007). However there is no correlation between serous Tg and FNA-Tg (Frasoldati et al., 1999). Kim et al. (2009) tested different threshold and propose a combination: the threshold values for FNA-Tg levels should be >10ng/ml if the serum Tg level or the mean plus two standard deviation in node-negative patients is not available for reference. Finally the French consensus (Leenhardt et al., 2011) recommends: Tg <1ng/FNA: normal result, Tg between 1 and 10 ng/FNA: to be compared with the results from cytology and Tg> 10 ng/FNA: suggest the presence of tumoral tissue.

FNA-Tg levels are significantly lower in subjects with metastatic poorly differentiated thyroid carcinoma than in subjects with differentiated thyroid cancer (Cignarelli et al., 2003) and may be nil (Boi et al., 2006), causing "false negatives" values.

Conversely FNA-Tg is particularly usefull for the diagnosis of LN metastasis when these LN have cystic changes (Cignarelli et al., 2003; Baloch et al., 2008). FNA-Tg is more sensitive for detecting metastasis when compared with FNA cytology (FNA-C) alone and allows the accurate diagnosis for samples with non conclusive cytology (Giovanella et al., 2011). For patients who received therapy with 131I the delay between the treatment and FNA has to be enough long (more than 3 months) to allow definitive destruction of the metastatic LN because FNA-Tg value can be false-positive. Sensitivity of FNA-Tg in the different studies are comprise between 84% (Frasoldati et al., 1999) and 100% (Pacini et al., 1992; Snozek et al., 2007; Cunha et al., 2007; Sigstad et al., 2007). When FNA-Tg is combined with FNA-C 100% sensitivity and 100% specificity can be obtained (Bournaud et al., 2010; Giovanella et al., 2011). So FNA-C should remain combined with FNA-Tg (Leenhardt et al., 2011).

#### **4. Conclusion**

It seems that we can again progress in the evolution of the dosage of Tg in terms of quality. We underlined here the importance of analytical quality for a highly strategic parameter in the decision tree of the follow-up of differentiated thyroid cancer: the thyroglobulin. During these periods of great changes in laboratories with automation we have to remember ourselves another guideline: "choose a method Tg on the basis of its characteristics of performance not the costs". The biologist has to know all the difficulties of Tg assays to argue the choice of his method, to guarantee the quality of the dosage and to avoid serious medical errors especially in the follow-up of differentiated thyroid carcinoma. A good laboratory-physician dialogue is more than ever of great importance.

#### **5. References**

Baloch, Z.; Carayon, P.; Conte-Devolx, B.; Demers, L.M.; Feldt-Rasmussen, U.; Henry, J.F.; LiVosli, V.A.; Niccoli-Sire, P.; John, R.; Ruf, J.; Smyth, P.P.; Spencer, C.A.; Stockigt, J.R. & Guidelines Committee, National Academy of Clinical Biochemistry.(2003).

Boi et al., 2006). In other studies of the literature threshold are sometimes the functional sensitivity (Cunha et al., 2007; Snozek et al., 2007) or study of sensitivity and specificity and choice of the better cut-off with a Receiver Operating Characteristic Curve (ROC) (Bournaud et al., 2010; Giovanella et al., 2011). For others again the FNA-Tg is compared with serum Tg: when the FNA-Tg value is greater than serum Tg value the LN is considered as metastasis (Uruno et al., 2005; Sigstad et al., 2007). However there is no correlation between serous Tg and FNA-Tg (Frasoldati et al., 1999). Kim et al. (2009) tested different threshold and propose a combination: the threshold values for FNA-Tg levels should be >10ng/ml if the serum Tg level or the mean plus two standard deviation in node-negative patients is not available for reference. Finally the French consensus (Leenhardt et al., 2011) recommends: Tg <1ng/FNA: normal result, Tg between 1 and 10 ng/FNA: to be compared with the

results from cytology and Tg> 10 ng/FNA: suggest the presence of tumoral tissue.

2011). So FNA-C should remain combined with FNA-Tg (Leenhardt et al., 2011).

laboratory-physician dialogue is more than ever of great importance.

and may be nil (Boi et al., 2006), causing "false negatives" values.

**4. Conclusion** 

**5. References** 

FNA-Tg levels are significantly lower in subjects with metastatic poorly differentiated thyroid carcinoma than in subjects with differentiated thyroid cancer (Cignarelli et al., 2003)

Conversely FNA-Tg is particularly usefull for the diagnosis of LN metastasis when these LN have cystic changes (Cignarelli et al., 2003; Baloch et al., 2008). FNA-Tg is more sensitive for detecting metastasis when compared with FNA cytology (FNA-C) alone and allows the accurate diagnosis for samples with non conclusive cytology (Giovanella et al., 2011). For patients who received therapy with 131I the delay between the treatment and FNA has to be enough long (more than 3 months) to allow definitive destruction of the metastatic LN because FNA-Tg value can be false-positive. Sensitivity of FNA-Tg in the different studies are comprise between 84% (Frasoldati et al., 1999) and 100% (Pacini et al., 1992; Snozek et al., 2007; Cunha et al., 2007; Sigstad et al., 2007). When FNA-Tg is combined with FNA-C 100% sensitivity and 100% specificity can be obtained (Bournaud et al., 2010; Giovanella et al.,

It seems that we can again progress in the evolution of the dosage of Tg in terms of quality. We underlined here the importance of analytical quality for a highly strategic parameter in the decision tree of the follow-up of differentiated thyroid cancer: the thyroglobulin. During these periods of great changes in laboratories with automation we have to remember ourselves another guideline: "choose a method Tg on the basis of its characteristics of performance not the costs". The biologist has to know all the difficulties of Tg assays to argue the choice of his method, to guarantee the quality of the dosage and to avoid serious medical errors especially in the follow-up of differentiated thyroid carcinoma. A good

Baloch, Z.; Carayon, P.; Conte-Devolx, B.; Demers, L.M.; Feldt-Rasmussen, U.; Henry, J.F.;

LiVosli, V.A.; Niccoli-Sire, P.; John, R.; Ruf, J.; Smyth, P.P.; Spencer, C.A.; Stockigt, J.R. & Guidelines Committee, National Academy of Clinical Biochemistry.(2003). Laboratory medicine practice guidelines. Laboratory support for the diagnosis and monitoring of thyroid disease. *Thyroid,* Vol. 13, pp.3-126.


The Thyroglobulin: A Technically Challenging Assay

*Endocrinol* Vol.154, pp.787-803.

*Invest* Vol.21 No.Suppl 4, pp.4.

*Endocrinol Metab* Vol.88, pp.3069-74.

men. *Eur J Clin Invest* Vol.19, pp.459-63.

assay. *Diagn Cytopathol* Vol.35, pp.761-7.

*Clin Endocrinol Metab*, Vol. 92, pp. 4278-81.

*Endocrinol.* Vol.100, pp.124-8.

pp.1401-4.

Vol.4, pp.361-7.

pp.625-8.

for a Marker of Choice During the Follow-Up of Differentiated Thyroid Cancer 31

Pacini, F. Schlumberger, M.; Dralle, H.; Elisei, R.; Smit, JWA.; Wiersinga, W.& the European

Persani, L.; Ferrari, M.; Borgato, S.; Bestagno, M.; Faglia, G. & Beck Peccoz, P. (1998).

Piechaczyck, M.; Chardès, T.; Cot, C.; Pau, B. & Bastide, JM. (1985). Production and

Preissner, CM.; O'Kane, DJ.; Singh, RJ.; Morris, JC. & Grebe, SK. (2003). Phantoms in the

Schlumberger, M.; Hitzel, A.; Toubert, ME.; Corone, C.; Troalen, F.; Schlageter, MH.;

Schlumberger, M.; Borget, I.; Nascimento, C.; Brassard, M. & Leboulleux S (2001). Treatment

Schultz, R.; Bethauser, H.; Stempka, L.; Heilig, B.; Moll, A. & Hufner, M. (1989). Evidence for

Sigstad, E.; Heilo, A.; Paus, E.; Holgersen, K.; Groholt, KK.; Jorgensen, LH.; Bogsrud, TV.;

Sinadinovic, J.; Cvejic, D.; Savin, S.; Jancic-Zuguricas, M. & Micic, JV. (1992). Altered

Smallridge, RC.; Meek, SE.; Morgan, MA.; Gates, GS.; Fox, TP.; Grebe, S. & Fatourechi, V.

Snozek, CL.; Chambers, EP.; Reading, CC. ; Sebo, TJ.; Sistrunk, JW. ; Singh, RJ. & Grebe, SK.

Spencer, C.; Takeuchi, M. & Kazarosyan, M. (1996), (a) Current status and performance

thyroid cancer patients. *J Clin Endocrinol Metab* Vol.92, pp.82-7.

thyroid cancer. *J Clin Endocrinol Metab* Vol.92, pp.2487-95.

diagnosis of metastasis differentiated thyroid cancer. *J Clin Endocrinol Metab* Vol.74,

Thyroid Cancer Taskforce (2006). European consensus for the management of patients with differentiated thyroid carcinoma of the follicular epithelium. *Eur J* 

Concanavalin A affinity chromatography can distinguish serum thyroglobulin (Tg) from normal subjects or patients with differentiated thyroid cancer*. J Endocrinol* 

characterisation of monoclonal antibodies against human thyroglobulin *Hybridoma*

assay tube: heterophile antibody interferences in serum thyroglobulin assays. *J Clin* 

Claustrat, F.; Koscielny, S.; Taieb, D.; Toubeau, M.; Bonichon, F.; Borson-Chazot, F.; Leenhardt, L.; Schwartz, C.; Dejax, C.; Brenot-Rossi, I.; Torlontano, M.; Tenenbaum, F.; Bardet, S.; Bussière, F.; Girard, JJ.; Morel, O.; Schneegans, O.; Schlienger, JL.; Prost, A.; So, D.; Archambaud, F.; Ricard, M. & Benhamou, E. (2007). Comparison of seven serum thyroglobulin assays in the follow-up of papillary and follicular

and follow-up of low-risk patients with thyroid cancer. *Nat Rev Endocrinol* Vol7,

immunological differences between circulating and tissue-derived thyroglobulin in

Berner, A. & Bjoro, T. (2007). The usefulness of detecting thyroglobulin in fineneedle aspirates from patients with neck lesions using a sensitive thyroglobulin

terminal glycosylation of thyroglobulin in papillary thyroid carcinoma. *Exp Clin* 

(2007). Monitoring thyrogloulin in a sensitive immunoassay has comparable sensitivity to recombinant human TSH stimulated thyroglobulin in follow-up of

(2007). Serum thyroglobulin, high resolution ultrasound and lymph node thyroglobulin in diagnosis of differentiated thyroide carcinoma nodal metastases. *J* 

goals for serum thyrotropin (TSH) assays. *Clin Chem* Vol42, pp.140-45 (b) Current

technique for the diagnosis of metastatic differentiated thyroid cancer. *Eur J Endocrinol* Vol.157, pp.101-7.


Druetta, L.; Croizet, K.; Bornet, H. & Rousset, B. (1998). Analyses of the molecular forms of

Feldt-Rasmussen, U.; Profilis, C.; Colinet, E.; Black, E.; Bornet, H.; Bourdoux, P.; Carayon, P.;

Herle, AJ. & De Vijlder, JJ. (1996) (a). Human thyroglobulin reference material (CRM 457).

Frasoldati, A.; Toschi, E.; Zini, M.; Flora, M.; Caroggio, A.; Dotti, C. & Valcavi, R. (1999).

Henry, M.; Malthiery, Y.; Zanelli, E. & Charvet, B. (1990). Epitope mapping of human

Kim, MJ.; Kim, EK.; Kim, BM.; Kwak, JY.; Lee, EJ.; Park, CS.; Cheong, WY. & Nam, KH.

Kloos, RT. & Mazzaferri, EL. (2005). A single recombinant human thyrotropin-stimulated

Leenhardt, L.; Borson-Chazot, F.; Calzada, M.; Carnaille, B.; Charrié, A.; Cochand-Priolet,

Malthiery, Y. & Lissitzky, S. (1987). Primary structure of human thyroglobulin deduced

Mariotti, S.; Barbesino, G.; Caturegli, P.; Marino, M.; Manetti, L.; Pacini, F.; Centoni, R. &

Pacini, F.; Fugazzola, I.; Lippi, F.; Ceccarelli, C.; Centoni, R. & Miccoli, P. (1992). Detection of

cancer of vesicular origin. *Ann Endocrinol* Vol. 72, pp.173-97.

obtainable goal? *J Clin Endocrinol Metab* Vol*.*90, pp.5566-75.

*Endocrinol* Vol.157, pp.101-7.

*(Paris)* Vol.54, pp.343-8.

pp.105-11.

pp.145-51.

Vol.145, pp.3692-8.

Vol.61, pp.343-50.

pp.491-8.

Western blot. *Eur J Endocrinol*. Vol.139, pp.498-507.

G.; Santos, A.; Schlumberger, M.; Seidel, C.; Van

technique for the diagnosis of metastatic differentiated thyroid cancer. *Eur J* 

serum thyroglobulin from patients with Graves' disease, subacute thyroiditis or differentiated thyroid cancer by velocity sedimentation on sucrose gradient and

Ericsson, UB.; Koutras, DA.; Lamas de Leon, L.; DeNayer, P.; Pacini, F.; Palumbo,

1st Part: Assessment of homogeneity, stability and immunoreactivity. *Ann Biol Clin (Paris)* Vol.54, pp.337-42. (b) a human thyroglobulin reference material (CRM 457). 2nd Part: Physicochemical characterization and certification. *Ann Biol Clin* 

Role of thyroglobulin measurement in fine-needle aspiartion biopsies of cervical lymph nodes in patients with differantiated thyroid cancer. *Thyroid*. Vol.9,

thyroglobulin Heterogeneous Recognition by thyroid pathologic sera. *J Immunol*

(2009). Thyroglobulin measurement in fine-needle aspirate washouts: the criteria for node dissection for patients with thyroid cancer. *Clin Endocrinol* Vol.70,

serum thyroglobulin measurement predicts differentiated thyroid carcinoma metastases three to five years later*. J Clin Endocrinol Metab.* Vol.90, pp.5047-57. Kohno, Y.; Tarutani, O.; Sakata, S. & Nakajima, H. (1985).Monoclonal antibodies to

thyroglobulin elucidate differences in protein structure of thyroglobulin in healthy individuals and those with papillary adenocarcinoma. *J Clin Endocrinol Metab*

B. ; Cao, CD.; Leboulleux, S.; Le CLech, G.; Mansour, G.; Menegaux, F.; Monpeyssen, H.; Orgiazzi, J.; Rouxel, A.; Sadoul, JL.; Schlumberger, M.; Tramalloni, J.; Tranquart, F.; Wemeau, JL.; (2011).Good practice guide for cervical ultrasound scan and echo- guided techniques in treating differentiated thyroid

from the sequence of its 8448-base complementary DNA *Eur J Biochem* Vol.165,

Pinchera, A. (1995). Assay of thyroglobulin in serum with Tg antibodies: un

thyroglobulin in fine needle aspirates of nonthyroidal neck masses: a clue to

diagnosis of metastasis differentiated thyroid cancer. *J Clin Endocrinol Metab* Vol.74, pp.1401-4.


**3**

Carles Zafon

*Spain* 

**Papillary Thyroid Microcarcinoma –**

It has been broadly demonstrated that there has been a dramatic, worldwide, increase in the incidence of papillary thyroid carcinoma (PTC). Leenhardt *et al.* [2004] showed that there was approximately a 10-fold increase in the ratio of thyroid cancer for the cohort born in 1978 compared to those born in 1928. Davies & Welch [2006] found that the incidence of thyroid cancer in the United States had more than doubled from 1973 to 2002 and that this augmentation was virtually entirely due to an increase in PTC. However, it is uncertain whether this increase is a real phenomenon, or whether it is simply due to an increased rate of detection. Practices for management of thyroid diseases were deeply modified over the past few decades. The wide availability of ultrasonography (US) and fine needle aspiration biopsy (FNAB), as well as the improved accuracy of histopathological examination of surgical samples (that is the thinness of the anatomical slice of the thyroid specimen) are indicated as causes of this so-called spreading of the epidemic [Grodski & Delbridge, 2008]. Furthermore, the characteristics of PTCs, especially

According to the World Health Organization, papillary microcarcinoma (PTMC) of the thyroid is defined as a papillary carcinoma measuring 1 cm or less [Lloyd et al., 2004]. PTMC is not recognized as a specific entity in the Tumor, Node and Metastasis (TNM) classification, and it is included in the T1 category, which has tumors as large as 2 cm.

The aim of the present article is to highlight how PTMC is changing the classical point of view of PTC and how, in the next few years, we must be able to incorporate the new

Several authors described a temporal trend toward decreasing tumor size in PTC. Chow *et al.* [2003] found that the percentage of PTMC has increased from 11.9% of all PTCs before the year 1980 to 25.5% in the decade 1990-1999. In an epidemiologic study carried out in a Brazilian region, Cordioli *et al.* [2009] reported that the average size of thyroid tumors

**1. Introduction** 

its size at diagnosis, have changed over time.

phenotypic characteristics of PTC in the staging systems.

**2. PTMC has changed the classical features of PTC** 

**Do Classical Staging Systems** 

*Dept. of Endocrinology, Vall d'Hebron University Hospital* 

*Autonomous University of Barcelona, Barcelona* 

**Need to Be Changed?** 

status and performance goals for serum thyroglobulin assays. *Clin Chem* Vol.42, pp.164-73 (c) Recoveries cannot be used to authenticate thyroglobulin (Tg) when sera contain Tg autoantibodies. *Clin Chem* Vol.42, pp.661-3.


### **Papillary Thyroid Microcarcinoma – Do Classical Staging Systems Need to Be Changed?**

Carles Zafon *Dept. of Endocrinology, Vall d'Hebron University Hospital Autonomous University of Barcelona, Barcelona Spain* 

#### **1. Introduction**

32 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

Uruno, T.; Miyauchi, A.; Shimizu, K.; Tomoda, C.; Takamura, Y.; Ito, Y.; Miya, A.;

Van de Graf, SA.; Pauws, E.; de Vijlder, JJ. & Ris-Stalpers, C. (1997). The revised 8307 base

Wild, D. (Ed). (2005). *The immunoassay handbook*, Third edition, Elsevier Ltd Publisher, ISBN-

sera contain Tg autoantibodies. *Clin Chem* Vol.42, pp.661-3.

cancer. *World J Surg* Vol.29, pp.483-5.

10: 0080445268, London

cells. *Eur J Endocrinol* Vol.136, pp.508-15.

status and performance goals for serum thyroglobulin assays. *Clin Chem* Vol.42, pp.164-73 (c) Recoveries cannot be used to authenticate thyroglobulin (Tg) when

Kobayashi, K.; Matsuzukz, F.; amino, N. & Kuma, K. (2005). Usefulness of thyroglobulin measurement in fine-needle aspiration biopsy specimens for diagnosing cervical lymph node metastasis in patients with papillary thyroid

pair coding sequence of human thyroglobulin transiently expressed in eukaryotic

It has been broadly demonstrated that there has been a dramatic, worldwide, increase in the incidence of papillary thyroid carcinoma (PTC). Leenhardt *et al.* [2004] showed that there was approximately a 10-fold increase in the ratio of thyroid cancer for the cohort born in 1978 compared to those born in 1928. Davies & Welch [2006] found that the incidence of thyroid cancer in the United States had more than doubled from 1973 to 2002 and that this augmentation was virtually entirely due to an increase in PTC. However, it is uncertain whether this increase is a real phenomenon, or whether it is simply due to an increased rate of detection. Practices for management of thyroid diseases were deeply modified over the past few decades. The wide availability of ultrasonography (US) and fine needle aspiration biopsy (FNAB), as well as the improved accuracy of histopathological examination of surgical samples (that is the thinness of the anatomical slice of the thyroid specimen) are indicated as causes of this so-called spreading of the epidemic [Grodski & Delbridge, 2008]. Furthermore, the characteristics of PTCs, especially its size at diagnosis, have changed over time.

According to the World Health Organization, papillary microcarcinoma (PTMC) of the thyroid is defined as a papillary carcinoma measuring 1 cm or less [Lloyd et al., 2004]. PTMC is not recognized as a specific entity in the Tumor, Node and Metastasis (TNM) classification, and it is included in the T1 category, which has tumors as large as 2 cm.

The aim of the present article is to highlight how PTMC is changing the classical point of view of PTC and how, in the next few years, we must be able to incorporate the new phenotypic characteristics of PTC in the staging systems.

#### **2. PTMC has changed the classical features of PTC**

Several authors described a temporal trend toward decreasing tumor size in PTC. Chow *et al.* [2003] found that the percentage of PTMC has increased from 11.9% of all PTCs before the year 1980 to 25.5% in the decade 1990-1999. In an epidemiologic study carried out in a Brazilian region, Cordioli *et al.* [2009] reported that the average size of thyroid tumors

Papillary Thyroid Microcarcinoma – Do Classical Staging Systems Need to Be Changed? 35

better than the other. However, the TNM staging system, employed by the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (UICC) is

PTMCs are considered a subset of PTCs that behave more benign. They follow an indolent course and carry an excellent prognosis. Distant metastases and mortality rates were reported to be less than 0.5% [Hay et al., 2008; Roti et al., 2008]. However, some authors suggest that there exists a subgroup of PTMCs that can be aggressive, requiring therapeutic management similar to larger tumors [Page et al., 2009]. Unfortunately, within this set of patients, prognostic factors have not been well defined. However, in recent years some specific markers for aggressiveness were identified, including sizes larger than 5 mm, multifocality, capsular invasion, tumor extension beyond the parenchyma, lymph node involvement, tumor non-incidentally discovered, and the extent of primary surgery [Küçük et al., 2007; S. Le et al., 2008; Mercante et al., 2009; Paget et al., 2009; Pelizzo et al., 2006; Roti et al, 2006]. Probably, three of the most accepted factors are multifocality, lymph node

Multiple foci were reported in approximately 7-56% of PTMCs [Dietlein et al., 2005; Hay et al., 2008; J. Lee et al., 2006; Roti et al., 2008]. A number of clinical studies showed that patients with ≥ two foci had a higher recurrence rate and cancer mortality than those with unifocal PTMCs [Baudin et al., 1998; Hay et al., 2008; J. Lin et al., 2009]. Moreover, multifocality is an independent risk factor for metastases [Gülben et al., 2008]. Hence,

PTMCs also showed a high incidence of regional lymph node metastasis, occurring in 12-- 64% of patients [Besic et al., 2008; Choi et al, 2008; Chung et al., 2009; J. Lee et al., 2006; S. Lee et al., 2008; Y. Lim et al., 2009; Roh et al., 2008]. It was demonstrated that cases with positive lymph nodes had a higher risk of recurrence [Chow et al., 2003]. Kim et al. [2008] found that lateral cervical node metastasis was the most powerful independent predictor

More than 70% of PTMCs are diagnosed incidentally (in specimens of the thyroid removed for benign thyroid disease) [Chow et al., 2003; Roti et al., 2008]. It has been suggested that clinical and biological behaviors may differ between incidental and non-incidental PTMCs [Barbaro et al., 2005; Chow et al., 2003; J. Lin et al., 2008]. Overt tumors are associated with a higher incidence of multicentricity, extrathyroidal involvement, lymphovascular invasion, higher stage, risk of relapse, and death [Besic et al., 2009; Chow et al., 2003; J. Lin et al., 2008;

The knowledge of the molecular basis of cancer has changed dramatically, and what is more important, the accuracy of the diagnosis has changed as well [Chan, 2000]. The diagnosis of cancer in pathology is mainly based on the morphology of tissues and cells. Immunohistochemistry allows us to detect molecules in these tissues, including cell components, cell products, tumor markers or molecules, which help to predict the tumor

multifocal PTMCs have been considered to have a poor prognosis.

Lo et al., 2006; Noguchi et al., 2008; Pisanu et al., 2009].

**4. Immunohistological markers** 

currently the most widely used.

**3.1 PTMC in the staging systems** 

metastasis, and the mode of diagnosis.

of clinical recurrence.

decreased from 1.51 cm in the year 2000 to 1.02 cm in the year 2005. Moreover, in 2000 36.9% of cancers were smaller than 1 cm, whereas in 2005 PTMC accounted for 61.48% of all thyroid carcinomas. In the USA there was a 49% increase in the incidence of PTC, consisting of cancers measuring 1 cm or smaller [Davies & Welch, 2006]. In a large study Hay *et al.* [2008] found that PTMC represented 31% of the total patients with PTC. Additionally, during the decade 1945-1954 PTMC accounted for only 19% of the total patients with PTC, whereas in the decade 1995-2004 the percentage rose to 35%. Leenhardt *et al.* [2004] showed that the proportion of PTMC among cancers, which were operated on, increased form 18.4% in the period 1883 to 1987 to 43.1% in the period 1998 to 2001. Furthermore, in the most recent literature, especially those that analyzed cases from the last decade, PTMC comprises almost half of all papillary cases [D. Lim et al., 2007; Pakdaman et al., 2008].

#### **3. Staging systems**

There exist different scoring systems currently used to stratify patients with differentiated thyroid carcinoma (DTC). With the identification of certain clinicopathological parameters, associated with indolent or aggressive tumor behavior, patients may be separated in to risk groups based on these parameters, such as age, gender, size of tumor, and cancer extension. Consequently, treatment and follow-up decisions should be based on the analysis of these risk groups. Although they are broadly accepted, prognostic significance of the scoring systems is limited for several reasons [Sherman, 1999]. For example, all the systems are based on retrospective studies and the vast majority of them were published more than 20 years ago using historical cases. Thus, the age, grade, extent, size (AGES) scoring system was verified in a cohort of subjects with papillary thyroid carcinoma treated in the Mayo Clinic from 1946 through 1970 [Hay et al., 1987]. The age, distant metastases, extent, and size (AMES) staging proposal was developed in a controlled study of 821 patients with differentiated thyroid carcinoma (including both PTC and follicular thyroid carcinoma, FTC) between 1941 and 1980 [Cady et al., 1988]. The Clinical Class staging system, proposed by deGroot, was based on 269 patients with PTC treated during the interval of 1968-1980 [DeGroot et al., 1990]. The Ohio State University (OSU) study first enrolled 1355 patients (including PTC and FTC), treated between 1950 and 1993 [Mazzaferri & Jhiang, 1994].

It is interesting to note that treatment of PTC has significantly changed from those early years. Radioiodine ablation was introduced some years later. In the aforementioned cohort study of the Mayo Clinic only 3% of patients underwent postoperative ablation [Hay et al, 1987]. Moreover, the utilization of thyroglobulin levels as a tumor marker was introduced in 1975 [Van Herle & Uller, 1975]. Tubiana et al.[1985] showed that patients treated after 1960 had a better outcome than patients treated earlier, though they did not differ in age, histological characteristics, sex ratio or incidence of palpable lymph nodes. In addition, it has been said that most of the scoring systems do not take in to consideration the clinical status of the patient or the treatment procedure [Duntas & Grab-Duntas, 2006]. Moreover, it was proposed that different staging systems should be evaluated and validated independently for PTC and FTC [Lang et al., 2007a]. Finally, PTMCs are excluded from some studies [Schindler et al., 1991].

Some authors compared the utility of several staging systems in their series of patients, with the aim to find out the one that is the most predictive [Kingma et al., 1991; Lang et al., 2007b; Passler et al., 2003; Voutilainen et al, 2003]. Results do not confirm that any of them are better than the other. However, the TNM staging system, employed by the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (UICC) is currently the most widely used.

#### **3.1 PTMC in the staging systems**

34 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

decreased from 1.51 cm in the year 2000 to 1.02 cm in the year 2005. Moreover, in 2000 36.9% of cancers were smaller than 1 cm, whereas in 2005 PTMC accounted for 61.48% of all thyroid carcinomas. In the USA there was a 49% increase in the incidence of PTC, consisting of cancers measuring 1 cm or smaller [Davies & Welch, 2006]. In a large study Hay *et al.* [2008] found that PTMC represented 31% of the total patients with PTC. Additionally, during the decade 1945-1954 PTMC accounted for only 19% of the total patients with PTC, whereas in the decade 1995-2004 the percentage rose to 35%. Leenhardt *et al.* [2004] showed that the proportion of PTMC among cancers, which were operated on, increased form 18.4% in the period 1883 to 1987 to 43.1% in the period 1998 to 2001. Furthermore, in the most recent literature, especially those that analyzed cases from the last decade, PTMC comprises

There exist different scoring systems currently used to stratify patients with differentiated thyroid carcinoma (DTC). With the identification of certain clinicopathological parameters, associated with indolent or aggressive tumor behavior, patients may be separated in to risk groups based on these parameters, such as age, gender, size of tumor, and cancer extension. Consequently, treatment and follow-up decisions should be based on the analysis of these risk groups. Although they are broadly accepted, prognostic significance of the scoring systems is limited for several reasons [Sherman, 1999]. For example, all the systems are based on retrospective studies and the vast majority of them were published more than 20 years ago using historical cases. Thus, the age, grade, extent, size (AGES) scoring system was verified in a cohort of subjects with papillary thyroid carcinoma treated in the Mayo Clinic from 1946 through 1970 [Hay et al., 1987]. The age, distant metastases, extent, and size (AMES) staging proposal was developed in a controlled study of 821 patients with differentiated thyroid carcinoma (including both PTC and follicular thyroid carcinoma, FTC) between 1941 and 1980 [Cady et al., 1988]. The Clinical Class staging system, proposed by deGroot, was based on 269 patients with PTC treated during the interval of 1968-1980 [DeGroot et al., 1990]. The Ohio State University (OSU) study first enrolled 1355 patients (including PTC and FTC), treated between 1950 and 1993 [Mazzaferri & Jhiang, 1994].

It is interesting to note that treatment of PTC has significantly changed from those early years. Radioiodine ablation was introduced some years later. In the aforementioned cohort study of the Mayo Clinic only 3% of patients underwent postoperative ablation [Hay et al, 1987]. Moreover, the utilization of thyroglobulin levels as a tumor marker was introduced in 1975 [Van Herle & Uller, 1975]. Tubiana et al.[1985] showed that patients treated after 1960 had a better outcome than patients treated earlier, though they did not differ in age, histological characteristics, sex ratio or incidence of palpable lymph nodes. In addition, it has been said that most of the scoring systems do not take in to consideration the clinical status of the patient or the treatment procedure [Duntas & Grab-Duntas, 2006]. Moreover, it was proposed that different staging systems should be evaluated and validated independently for PTC and FTC [Lang et al., 2007a]. Finally, PTMCs are excluded from

Some authors compared the utility of several staging systems in their series of patients, with the aim to find out the one that is the most predictive [Kingma et al., 1991; Lang et al., 2007b; Passler et al., 2003; Voutilainen et al, 2003]. Results do not confirm that any of them are

almost half of all papillary cases [D. Lim et al., 2007; Pakdaman et al., 2008].

**3. Staging systems** 

some studies [Schindler et al., 1991].

PTMCs are considered a subset of PTCs that behave more benign. They follow an indolent course and carry an excellent prognosis. Distant metastases and mortality rates were reported to be less than 0.5% [Hay et al., 2008; Roti et al., 2008]. However, some authors suggest that there exists a subgroup of PTMCs that can be aggressive, requiring therapeutic management similar to larger tumors [Page et al., 2009]. Unfortunately, within this set of patients, prognostic factors have not been well defined. However, in recent years some specific markers for aggressiveness were identified, including sizes larger than 5 mm, multifocality, capsular invasion, tumor extension beyond the parenchyma, lymph node involvement, tumor non-incidentally discovered, and the extent of primary surgery [Küçük et al., 2007; S. Le et al., 2008; Mercante et al., 2009; Paget et al., 2009; Pelizzo et al., 2006; Roti et al, 2006]. Probably, three of the most accepted factors are multifocality, lymph node metastasis, and the mode of diagnosis.

Multiple foci were reported in approximately 7-56% of PTMCs [Dietlein et al., 2005; Hay et al., 2008; J. Lee et al., 2006; Roti et al., 2008]. A number of clinical studies showed that patients with ≥ two foci had a higher recurrence rate and cancer mortality than those with unifocal PTMCs [Baudin et al., 1998; Hay et al., 2008; J. Lin et al., 2009]. Moreover, multifocality is an independent risk factor for metastases [Gülben et al., 2008]. Hence, multifocal PTMCs have been considered to have a poor prognosis.

PTMCs also showed a high incidence of regional lymph node metastasis, occurring in 12-- 64% of patients [Besic et al., 2008; Choi et al, 2008; Chung et al., 2009; J. Lee et al., 2006; S. Lee et al., 2008; Y. Lim et al., 2009; Roh et al., 2008]. It was demonstrated that cases with positive lymph nodes had a higher risk of recurrence [Chow et al., 2003]. Kim et al. [2008] found that lateral cervical node metastasis was the most powerful independent predictor of clinical recurrence.

More than 70% of PTMCs are diagnosed incidentally (in specimens of the thyroid removed for benign thyroid disease) [Chow et al., 2003; Roti et al., 2008]. It has been suggested that clinical and biological behaviors may differ between incidental and non-incidental PTMCs [Barbaro et al., 2005; Chow et al., 2003; J. Lin et al., 2008]. Overt tumors are associated with a higher incidence of multicentricity, extrathyroidal involvement, lymphovascular invasion, higher stage, risk of relapse, and death [Besic et al., 2009; Chow et al., 2003; J. Lin et al., 2008; Lo et al., 2006; Noguchi et al., 2008; Pisanu et al., 2009].

#### **4. Immunohistological markers**

The knowledge of the molecular basis of cancer has changed dramatically, and what is more important, the accuracy of the diagnosis has changed as well [Chan, 2000]. The diagnosis of cancer in pathology is mainly based on the morphology of tissues and cells. Immunohistochemistry allows us to detect molecules in these tissues, including cell components, cell products, tumor markers or molecules, which help to predict the tumor

Papillary Thyroid Microcarcinoma – Do Classical Staging Systems Need to Be Changed? 37

Some studies failed to identify independent prognostic factors, arguing that to distinguish PTC on the basis of size alone may be clinically irrelevant [Arora et al., 2009; Sugino et al., 1998]. Moreover classical scoring systems seem to be less accurate when the PTC is of a smaller size. Additionally, the role of age, as the paradigm of prognostic factors, remains

Most reports in the literature show that older patients with PTC have a worse prognosis. In DTC age is the most important factor and this parameter is included in the TNM staging system as well as in the vast majority of the other scores. Older age is especially significant in patients with advanced tumors [Pelizzo et al., 2005]. However, once again, though articles recognize the age factor, most of them are retrospective studies that include cases without current standardized therapeutic protocols. Moreover, few reports specifically analyze the behavior of thyroid cancer in the elderly. Vini et al. [2003] studied the biological behavior in 111 patients with DTC, who were older than 70. The authors found that older age was an important risk factor for overall survival. It is noteworthy that only 52% of patients had PTC, total thyroidectomy was performed in only 41% of cases and postoperative radioiodine was administered in the 72%. Furthermore, investigators showed that the probability of survival changed significantly according to the decade in which the patient was treated. Thus, median survival improved from 4.7 years before 1970, to more than 10 years after 1990 [Vini et al., 2003]. J. Lin et al. [2000] analyzed thyroid cancer in patients age 60 or older. Less than half of all the cases were papillary. They concluded that one important difference with respect to younger subjects

The increased aggressiveness of PTC in elderly patients may be attributed to a variety of factors. It is assumed that older subjects have tumors with a higher percentage of histological types with less favorable prognosis [Hundahl et al., 1998]. Also, effectiveness of radioiodine therapy decreases in the elderly. Schlumberger et al. [1996] found that metastases of DTC uptake iodine in 90% of patients less than 40 years of age and in 56% of patients over 40. Mihailovic et al. [2009] found that age is related with the radiodione avidity of distant metastases. Moreover, aged patients show a higher rate of large tumors. Biliotti et al. [2006] found that in subjects, who were older than 70, with thyroid tumors > 2 cm in diameter, the survival rates were markedly lower than rates among patients with a tumor diameter of < 2 cm. Other factors have been proposed such as the sexual hormone status and the impaired immune response, which accompanies older individuals [Haymart, 2009a]. Accordingly, it appears that thyroid cancer in the elderly and in younger patients

Some reports also demonstrate the importance of age in PTMCs [J. Lin et al, 2005]. H. Lin & Bhattacharyya [2009] examined the Surveillance, Epidemiology and End Results (SEER) registry, a database from the National Cancer Institute of the USA. The authors analyzed 7,818 cases of PTMC, which presented without local or distant metastasis. They found that only an increased age at diagnosis predicted decreased disease-specific survival. In a recent report, Elisei et al. [2010] showed that though patients diagnosed during the last

**5. The age factor** 

to be established.

was the delay in the diagnosis.

**5.1 Age in PTMC** 

could have a different behavior [Biliotti et al, 2006].

behavior. The immunological reaction that takes place with this technique has remarkable sensitivity and specificity and it is applicable to routinely processed tissues, including fixed tissues. A great advantage of immunohistochemistry is the fact that we can simultaneously visualize the morphology of the cells and the immunostaining, so that we can locate the antigen we are detecting, in a particular subcellular localization or in a specific subtype of cells. Another advantage of the technique is that it is applicable to several types of material including tissue sections and cytological specimens [Chess & Hajdu, 1986].

In PTC, immunohistochemistry could be a useful tool to help not only in identifying the subset of patients at high risk, but also in those cases with no clear histological diagnosis [Rezk & Khan, 2005]. Several novel markers were tested, but, unfortunately, none of them were proved to be useful enough in clinical diagnosis [Asa, 2005]. At present, it is thought that their utility depends on the use of a panel of markers that include various combinations of them [Zafon et al., 2010]. For example, simultaneous immunohistochemical expression of HBME-1 and galectin-3 differentiates papillary carcinomas from hyperfunctioning lesions of the thyroid [Rossi et al, 2006].

Also in PTMC several possible immunohistological markers were proposed to assess the biological aggressiveness of the cancer [Boucek et al., 2009; Cvejic et al., 2008; Khoo et al., 2002; D. Lim et al., 2007] (table). Some authors compared molecular expression in PTMCs and PTCs of larger size. For example, Cvejic et al [2009] reported differences in the expression of the apoptotic molecule Bax and in the ratio Bcl-2/Bax between PTMC and larger tumors. Batistatou *et al* [2008] found a negative correlation between E-cadherin and dysadherin expression and the tumor size. Other authors attempted to define molecular characteristics of aggressiveness. For instance, D. Lim et al. [2007] showed that the absence of EGFR expression was correlated with extrathyroid extension and lymph node metastases. Lantsov et al. [2005] found a significant association between Cyclin D1 expression and both tumor size and lymph node metastases. Khoo et al. [2002] obtained similar results. Finally, Ito et al. [2005] reported that expression of proliferating markers such as Ki-67, Cyclin D1 and the retinoblastoma gene product (pRb) increased in PTMCs with clinically apparent metastases.


Table 1. Correlation (+ positive, - negative, 0 no correlation) between molecular markers and papillary thyroid microcarcinoma features.

### **5. The age factor**

36 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

behavior. The immunological reaction that takes place with this technique has remarkable sensitivity and specificity and it is applicable to routinely processed tissues, including fixed tissues. A great advantage of immunohistochemistry is the fact that we can simultaneously visualize the morphology of the cells and the immunostaining, so that we can locate the antigen we are detecting, in a particular subcellular localization or in a specific subtype of cells. Another advantage of the technique is that it is applicable to several types of material

In PTC, immunohistochemistry could be a useful tool to help not only in identifying the subset of patients at high risk, but also in those cases with no clear histological diagnosis [Rezk & Khan, 2005]. Several novel markers were tested, but, unfortunately, none of them were proved to be useful enough in clinical diagnosis [Asa, 2005]. At present, it is thought that their utility depends on the use of a panel of markers that include various combinations of them [Zafon et al., 2010]. For example, simultaneous immunohistochemical expression of HBME-1 and galectin-3 differentiates papillary carcinomas from hyperfunctioning lesions of

Also in PTMC several possible immunohistological markers were proposed to assess the biological aggressiveness of the cancer [Boucek et al., 2009; Cvejic et al., 2008; Khoo et al., 2002; D. Lim et al., 2007] (table). Some authors compared molecular expression in PTMCs and PTCs of larger size. For example, Cvejic et al [2009] reported differences in the expression of the apoptotic molecule Bax and in the ratio Bcl-2/Bax between PTMC and larger tumors. Batistatou *et al* [2008] found a negative correlation between E-cadherin and dysadherin expression and the tumor size. Other authors attempted to define molecular characteristics of aggressiveness. For instance, D. Lim et al. [2007] showed that the absence of EGFR expression was correlated with extrathyroid extension and lymph node metastases. Lantsov et al. [2005] found a significant association between Cyclin D1 expression and both tumor size and lymph node metastases. Khoo et al. [2002] obtained similar results. Finally, Ito et al. [2005] reported that expression of proliferating markers such as Ki-67, Cyclin D1 and the retinoblastoma gene product (pRb) increased in PTMCs

**Molecule Size Aggressiveness Reference**  E-Cadherin **--** Batistatou *et al* [2008] Dysadherin **--** Batistatou *et al* [2008] Bcl-2/Bax ratio **+** Cvejic *et al* [2009] MUC4 **+** Nam *et al*. [2011]

Ki-67 **+** Ito *et al*. [2005] pRb **+** Ito *et al*. [2005] S100A4 **+** Min *et al*. [2008] EGFR **--** D. Lim *et al*. [2007] Galectin-3 **0** Cvejic *et al* [2005] Table 1. Correlation (+ positive, - negative, 0 no correlation) between molecular markers and

**+ +** Lantsov *et al*. [2005]

**+** Khoo *et al*. [2002] **+** Ito *et al*. [2005]

including tissue sections and cytological specimens [Chess & Hajdu, 1986].

the thyroid [Rossi et al, 2006].

with clinically apparent metastases.

papillary thyroid microcarcinoma features.

Cyclin- D1

Some studies failed to identify independent prognostic factors, arguing that to distinguish PTC on the basis of size alone may be clinically irrelevant [Arora et al., 2009; Sugino et al., 1998]. Moreover classical scoring systems seem to be less accurate when the PTC is of a smaller size. Additionally, the role of age, as the paradigm of prognostic factors, remains to be established.

Most reports in the literature show that older patients with PTC have a worse prognosis. In DTC age is the most important factor and this parameter is included in the TNM staging system as well as in the vast majority of the other scores. Older age is especially significant in patients with advanced tumors [Pelizzo et al., 2005]. However, once again, though articles recognize the age factor, most of them are retrospective studies that include cases without current standardized therapeutic protocols. Moreover, few reports specifically analyze the behavior of thyroid cancer in the elderly. Vini et al. [2003] studied the biological behavior in 111 patients with DTC, who were older than 70. The authors found that older age was an important risk factor for overall survival. It is noteworthy that only 52% of patients had PTC, total thyroidectomy was performed in only 41% of cases and postoperative radioiodine was administered in the 72%. Furthermore, investigators showed that the probability of survival changed significantly according to the decade in which the patient was treated. Thus, median survival improved from 4.7 years before 1970, to more than 10 years after 1990 [Vini et al., 2003]. J. Lin et al. [2000] analyzed thyroid cancer in patients age 60 or older. Less than half of all the cases were papillary. They concluded that one important difference with respect to younger subjects was the delay in the diagnosis.

The increased aggressiveness of PTC in elderly patients may be attributed to a variety of factors. It is assumed that older subjects have tumors with a higher percentage of histological types with less favorable prognosis [Hundahl et al., 1998]. Also, effectiveness of radioiodine therapy decreases in the elderly. Schlumberger et al. [1996] found that metastases of DTC uptake iodine in 90% of patients less than 40 years of age and in 56% of patients over 40. Mihailovic et al. [2009] found that age is related with the radiodione avidity of distant metastases. Moreover, aged patients show a higher rate of large tumors. Biliotti et al. [2006] found that in subjects, who were older than 70, with thyroid tumors > 2 cm in diameter, the survival rates were markedly lower than rates among patients with a tumor diameter of < 2 cm. Other factors have been proposed such as the sexual hormone status and the impaired immune response, which accompanies older individuals [Haymart, 2009a]. Accordingly, it appears that thyroid cancer in the elderly and in younger patients could have a different behavior [Biliotti et al, 2006].

#### **5.1 Age in PTMC**

Some reports also demonstrate the importance of age in PTMCs [J. Lin et al, 2005]. H. Lin & Bhattacharyya [2009] examined the Surveillance, Epidemiology and End Results (SEER) registry, a database from the National Cancer Institute of the USA. The authors analyzed 7,818 cases of PTMC, which presented without local or distant metastasis. They found that only an increased age at diagnosis predicted decreased disease-specific survival. In a recent report, Elisei et al. [2010] showed that though patients diagnosed during the last

Papillary Thyroid Microcarcinoma – Do Classical Staging Systems Need to Be Changed? 39

The rising incidence of PTMC demands the identification of specific prognostic factors for cancers measuring 1.0 cm or less, to differentiate those truly aggressive neoplasms from the clinically insignificant tumors. For that, it is mandatory to reevaluate the classic prognostic scores with the aim to define their usefulness in the management of PTMC. To date, the clinical significance of many of these variables is yet to be established. As a consequence, there is no agreement about the optimal treatment of smaller tumors [Küçük et al, 2007]. Whereas some authors argue for an aggressive approach, others suggest that no further treatment is needed after lobectomy or thyroidectomy. Moreover, some even propose observation, without surgical treatment [Ito et al, 2003]. In the next few years, we will need to improve the role of the staging systems in accordance with the new phenotypic characteristics of PTC. Finally, age, as a prognostic factor, must be cautiously

Arora, N.; Turbendian, H.; Kato, M.; Moo, T.; Zarnegar, R. & Fahey III, T. (2009). Papillary

Asa, S. (2005). The role of immunohistochemical markers in the diagnosis of follicularpatterned lesions of the thyroid. *Endocrine Pathology*, Vol. 16, No. 4, pp. 295-309. Barbaro, D.; Simi, U.; Meucci, G.; Orsini, P. & Pasquini, C. (2005). Thyroid papillary cancers:

Batistatou, A.; Charalabopoulos, K.; Nakanishi, Y.; Vagianos, C.; Hirohashi, S.; Agnantis, N.

Baudin, E.; Travagli, J.; Ropers, J.; Mancusi, F.; Bruno-Bossio, G.; Caillou, B.; Cailleaux, A.;

Besic, N.; Pilko, G.; Petric, R.; Hocevar, M. & Zgajnar, J. (2008). Papillary thyroid

Besic, N.; Zgajnar, J.; Hocevar, M. & Petric, R. (2009). Extent of thyroidectomy and

Boucek, J.; Kastner, J.; Skrivan, J.; Grosso, E.; Gibelli, B.; Giugliano, G. & Betka, J. (2009). Occult thyroid carcinoma. *Acta Otorhinolaryngologica Italica*, Vol. 29, No. 6, pp. 296-304. Cady, B. & Rossi, R. (1988). An expanded view of risk-group definition in differentiated

institution experience. *Annals of Surgical Oncology*, Vol. 16, pp. 920-928. Biliotti, G.; Martini, F.; Vezzosi, V.; Seghi, P.; Tozzi, F.; Castagnoli, A.; Basili, G. & Peri, A.

years of age. *Journal of Surgical Oncology*, Vol. 93, pp. 194-198.

thyroid carcinoma. *Surgery*, Vol. 104, No. 6, pp. 947-953.

they different diseases? *Clinical Endocrinology*, Vol. 63, pp. 577-581.

thyroid carcinoma and microcarcinoma: is there a need to distinguish the two?

microcarcinoma and carcinoma, incidental cancers and non-incidental cancers - are

& Scopa, C. (2008). Differential expression of dysadherin in papillary thyroid carcinoma and microcarcinoma: correlation with E-cadherin. *Endocrine Pathology*,

Lumbroso, J.; Parmentier, C. & Schlumberger, M. (1998). Microcarcinoma of the thyroid gland. The Gustave-Roussy institute experience. *Cancer*, Vol. 83, No. 3, pp.

microcarcinoma: prognostic factors and treatment. *Journal of Surgical Oncology*, Vol.

lymphadenectomy in 254 patients with papillary thyroid microcarcinoma: A single-

(2006). Specific features of differentiated thyroid carcinoma in patients over 70

**6. Conclusions** 

**7. References** 

interpreted in PTCs less than 1 cm.

*Thyroid,* Vol. 19, No. 5, pp. 473-477.

Vol. 19, No. 3, pp. 197-202.

553-559.

97, pp. 221-225.

two decades have smaller tumors, older age still represents the most important prognostic factor for survival.

However, despite the fact that older age is a universally identified poor prognostic factor in PTC, other investigators failed to find that age affects the outcome of patients with PTMC. In the aforementioned report of Chow et al. [2003], the authors found that, in PTMC, age was not a significant factor in predicting disease recurrence or survival. Gülben et al. [2008] found that mean age was higher in patients with lymph node metastases but the difference was not significant. In the large study reported by Pakdaman et al. [2008] investigators showed that the prevalence of PTMC was higher in patients 45 years and older, than in patients under 45. However, age was not related with multifocality, bilaterality and extrathyroid extension, risk factors shown to increase recurrences. Of particular interest is the recent article of Besic et al. [2009], which reported that in PTMC, lymph node metastases were more common in patients over 45 years of age. The same authors also showed that there was no correlation between the duration of the disease-free interval and the age of patients [2008]. Moreover, in an adjusted model, Noguchi et al. [2008] found that age was not a risk factor for recurrence in PTMC. Mercante et al. [2009], in their large study of 445 cases demonstrated that age was not a significant risk factor for neck recurrence or distant metastasis. Another study reported that patients with lymph node metastasis were younger than those without lymph node metastases [Chung et al., 2009]. Y. Lim et al. [2009] also found that in patients under 45 there was a higher incidence of ipsilateral central lymph node metastases. Previously, Baudin et al. [1998] described that patients with non-incidental PTMC were significantly younger. Non-incidental diagnosis was proposed as a criterion for a poor outcome. Another study reported that patients with PTMC were significantly older that patients with larger tumors. Moreover, in the PTMC group lymph node metastasis at diagnosis was correlated with a younger age [Tzvetov et al., 2009]. Jacquot-Laperrière et al. [2007] found that age did not become a prognostic factor for the risk of metastatic spread.

In the meta-analysis carried out by Roti et al. [2008] a younger age (< 45 years) was significantly associated with cancer recurrence. Haymart et al. [2009b] found that patients who received radioiodine ablation were younger that those not receiving this treatment. Recently, we reported that PTMCs in older patients were associated with less multifocality, bilaterality, fewer lymphadenectomies and a decreased rate of non-incidental tumors than in younger patients [Zafon et al, 2011].

In summary, several data suggest that age is not a significant factor in predicting disease recurrence or survival for PTMC. On the contrary, some reports suggest that younger age could be a worse prognostic factor. It is conceivable that in older patients there exist two different forms of PTMC. One form is the "clinical PTMC" which behaves as PTC. The second form is a "silent PTMC," a tumor incidentally discovered that will never be apparent and that may be in concordance with the occult carcinoma detected in thyroid glands from autopsies. In this regard, it is interesting to note that gender distribution of PTMC found in autopsies shows differences as compared to clinical papillary tumors [Kovács et al, 2005]. It is well established that the incidence of PTC in women is significantly higher than that in men (with a female to male ratio greater than 2 to 1) [Yao et al, 2011]. However, several authors have not found any significant gender-related differences in PTMC found at autopsies [Lang et al, 1988; Neuhold et al, 2001; Kovács et al, 2005].

#### **6. Conclusions**

38 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

two decades have smaller tumors, older age still represents the most important prognostic

However, despite the fact that older age is a universally identified poor prognostic factor in PTC, other investigators failed to find that age affects the outcome of patients with PTMC. In the aforementioned report of Chow et al. [2003], the authors found that, in PTMC, age was not a significant factor in predicting disease recurrence or survival. Gülben et al. [2008] found that mean age was higher in patients with lymph node metastases but the difference was not significant. In the large study reported by Pakdaman et al. [2008] investigators showed that the prevalence of PTMC was higher in patients 45 years and older, than in patients under 45. However, age was not related with multifocality, bilaterality and extrathyroid extension, risk factors shown to increase recurrences. Of particular interest is the recent article of Besic et al. [2009], which reported that in PTMC, lymph node metastases were more common in patients over 45 years of age. The same authors also showed that there was no correlation between the duration of the disease-free interval and the age of patients [2008]. Moreover, in an adjusted model, Noguchi et al. [2008] found that age was not a risk factor for recurrence in PTMC. Mercante et al. [2009], in their large study of 445 cases demonstrated that age was not a significant risk factor for neck recurrence or distant metastasis. Another study reported that patients with lymph node metastasis were younger than those without lymph node metastases [Chung et al., 2009]. Y. Lim et al. [2009] also found that in patients under 45 there was a higher incidence of ipsilateral central lymph node metastases. Previously, Baudin et al. [1998] described that patients with non-incidental PTMC were significantly younger. Non-incidental diagnosis was proposed as a criterion for a poor outcome. Another study reported that patients with PTMC were significantly older that patients with larger tumors. Moreover, in the PTMC group lymph node metastasis at diagnosis was correlated with a younger age [Tzvetov et al., 2009]. Jacquot-Laperrière et al. [2007] found that age did not become a prognostic factor for the risk of metastatic spread.

In the meta-analysis carried out by Roti et al. [2008] a younger age (< 45 years) was significantly associated with cancer recurrence. Haymart et al. [2009b] found that patients who received radioiodine ablation were younger that those not receiving this treatment. Recently, we reported that PTMCs in older patients were associated with less multifocality, bilaterality, fewer lymphadenectomies and a decreased rate of non-incidental tumors than in

In summary, several data suggest that age is not a significant factor in predicting disease recurrence or survival for PTMC. On the contrary, some reports suggest that younger age could be a worse prognostic factor. It is conceivable that in older patients there exist two different forms of PTMC. One form is the "clinical PTMC" which behaves as PTC. The second form is a "silent PTMC," a tumor incidentally discovered that will never be apparent and that may be in concordance with the occult carcinoma detected in thyroid glands from autopsies. In this regard, it is interesting to note that gender distribution of PTMC found in autopsies shows differences as compared to clinical papillary tumors [Kovács et al, 2005]. It is well established that the incidence of PTC in women is significantly higher than that in men (with a female to male ratio greater than 2 to 1) [Yao et al, 2011]. However, several authors have not found any significant gender-related differences in PTMC found at

autopsies [Lang et al, 1988; Neuhold et al, 2001; Kovács et al, 2005].

factor for survival.

younger patients [Zafon et al, 2011].

The rising incidence of PTMC demands the identification of specific prognostic factors for cancers measuring 1.0 cm or less, to differentiate those truly aggressive neoplasms from the clinically insignificant tumors. For that, it is mandatory to reevaluate the classic prognostic scores with the aim to define their usefulness in the management of PTMC. To date, the clinical significance of many of these variables is yet to be established. As a consequence, there is no agreement about the optimal treatment of smaller tumors [Küçük et al, 2007]. Whereas some authors argue for an aggressive approach, others suggest that no further treatment is needed after lobectomy or thyroidectomy. Moreover, some even propose observation, without surgical treatment [Ito et al, 2003]. In the next few years, we will need to improve the role of the staging systems in accordance with the new phenotypic characteristics of PTC. Finally, age, as a prognostic factor, must be cautiously interpreted in PTCs less than 1 cm.

#### **7. References**


Papillary Thyroid Microcarcinoma – Do Classical Staging Systems Need to Be Changed? 41

Hay, I.; Grant, C.; Taylor, W. & McConahey, W. (1987). Ipsilateral lobectomy versus bilateral

Hay, I.; Hutchinson, M.; Gonzalez-Losada, T.; McIver, B.; Reinalda, M.; Grant, C.; Thompson,

Haymart, M.; Cayo, M. & Chen, H. (2009b). Papillary thyroid microcarcinoma: big decisions for a small tumor. *Annals of Surgical Oncology*, Vol. 16, No. 11, pp. 3132-3139. Hundahl, S.; Fleming, I.; Fremgen, A. & Menck, H. (1998). A National Center Data Base

Ito, Y.; Uruno, T.; Nakano, K.; Takamura, Y.; Miya, A.; Kobayashi, K.; Yokozawa, T.;

Ito, Y.; Uruno, T.; Takamura, Y.; Miya, A.; Kobayashi, K.; Matsuzuka, F.; Kuma, K. &

on immunohistochemical examination. *Oncology*, Vol. 68, No. 2-3, pp. 87-96. Jacquot-Laperrière, S.; Timoshenko, A.; Dumollard, J.; Peoc'h, M.; Estour, B.; Martin, C. &

factors. *European Archives of Otorhinolaryngology*, Vol. 264, No. 8, pp. 935-939. Khoo, M.; Ezzat, S.; Freeman, J. & Asa, S. (2002). Cyclin D1 protein expression predicts

Kim, T. Hong, S.; Kim, J.; Kim, W.; Gong, G.; Ryu, J.; Kim, W.; Yun, S. & Shong, Y. (2008).

Kingma, G.; van der Bergen, H. & de Vries, J. (1991). Prognostic scoring systems in

Kovács, G.; Gonda, G.; Vadász, G.; Ludmány, E.; Uhrin, K.; Görömbey, Z.; Kovács, L.;

Küçük, N.; Tari, P.; Tokmak, E. & Aras, G. (2007). Treatment for microcarcinoma of the thyroid - clinical experience. *Clinical Nuclear Medicine*, Vol. 32, No. 4, pp. 279-281. Lang, B.; Lo, C.; Chan, W.; Lam, K. & Wan, K. (2007a). Prognostic factors in papillary and

Lang, B.; Lo, C.; Chan, W & Lam, K. (2007b). Staging systems for papillary thyroid carcinoma. A review and comparison. *Annals of Surgery*, Vol. 245, No. 3, pp. 366-378.

cases observed in a 60-year period. *Surgery*, Vol. 144, No. 6; pp. 980-988. Haymart, M. (2009a). Understanding the relationship between age and thyroid cancer.

1088-1095.

pp. 1810-1813.

*Cancer*, Vol. 8, No. 296.

Vol. 43, No. 3, pp. 63-66.

intake. *Thyroid*, Vol. 15, No. 2, pp. 152-157.

*Oncology*, Vol. 14, No. 2, pp. 730-738.

*Oncologist*, Vol. 14, pp. 216-221.

Vol. 83, No. 12, pp. 2638-2648.

thyroid. *Thyroid*, Vol. 13, No. 4, pp. 381-387.

lobar resection in papillary thyroid carcinoma: A retrospective analysis of surgical outcome using a novel prognostic scoring system. *Surgery*, Vol. 102, No. 6, pp.

G.; Sebo, T. & Goellner, J. (2008). Papillary thyroid microcarcinoma: A study of 900

report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985-1995. *Cancer*,

Matsuzuka, F.; Kuma, S.; Kuma, K. & Miyauchi, A. (2003). An observation trial without surgical treatment in patients with papillary microcarcinoma of the

Miyauchi, A. (2005). Papillary microcarcinomas of the thyroid with preoperatively detectable lymph node metastasis show significantly higher aggressive characteristics

Prades, J. (2007). Papillary thyroid microcarcinoma: incidence and prognostic

metastatic behavior in thyroid papillary microcarcinomas but is not associated with gene amplification. *Journal of Clinical Endocrinology and Metabolism*, Vol. 87, No. 4,

Prognostic parameters for recurrence of papillary thyroid microcarcinoma. *BMC* 

differentiated thyroid carcinoma: which is the best? *Netherlands Journal of Surgery*,

Hubina, E.; Bodó, M.; Góth, M. & Szabolcs, I. (2005). Epidemiology of thyroid microcarcinoma found in autopsy series conducted in areas of different iodine

follicular thyroid carcinoma: their implications for cancer staging. *Annals of Surgical* 


Chan, J. (2000). Advances in immunohistochemistry: impact on surgical pathology practice.

Chess, Q. & Hajdu, S. (1986). The role of immunoperoxidase staining in diagnostic cytology.

Choi, S.; Kim, T.; Lee, J.; Shong, Y.; Cho, K.; Ryu, J.; Lee, J.; Roh, J & Kim, S. (2008). Is routine

Chow, S.; Law, S.; Chan, J.; Au, S.; Yau, S. & Lau, W. (2003). Papillary microcarcinoma of the

Chung, Y.; Kim, J.; Bae, J.; Song, B.; Kim, J.; Jeon, H.; Jeong, S.; Kim, E. & Park, W. (2009).

Cvejic, D.; Selemetjev, S.; Savin, S.; Paunovic, I.; Petrovic, I. & Tatic, S. (2008). Apoptosis and

thyroid tumours. *European Journal of Histochemistry*, Vol. 53, No. 2, pp. 65-71. Davies, L. & Welch, H. (2006). Increasing incidence of thyroid cancer in the United States, 1973- 2002. *Journal of the American Medical Association*, Vol. 295, No. 18, pp. 2164-2167. DeGroot, L.; Kaplan, E.; McCormick, M. & Straus, F. (1990). Natural history, treatment, and

Dietlein, M.; Luyken, W.; Schicha, H. & Larena-Avellaneda, A. (2005). Incidental multifocal

Duntas, L. & Grab-Duntas, B. (2006). Risk and prognostic factors for differentiated thyroid cancer. *Hellenic Journal of Nuclear Medicine*, Vol. 9, No. 3, pp. 156-162. Elisei, R.; Molinaro, E.; Agate, L.; Bottici, V.; Masserini, L.; Ceccarelli, C.; Lippi, F.; Grasso, L.;

Grodski, S. & Delbridge, L. (2008). An update on papillary microcarcinoma. *Current Opinion* 

Gülben, K.; Berberoglu, U.; Çelen, O. & Mersin, H. (2008). Incidental papillary

therapeutic lymph node dissection. *Thyroid*, Vol. 19, No. 3, pp. 241-246. Cordioli, M.; Canalli, M. & Coral, M. (2009). Increase incidence of thyroid cancer in

central neck dissection necessary for the treatment of papillary thyroid microcarcinoma? *Clinical and Experimental Otorhinolaryngology*, Vol. 1, No. 1, pp. 41-45.

thyroid - prognostic significance of lymph node metastasis and multifocality.

Lateral lymph node metastasis in papillary thyroid carcinoma: results of the

Florianopolis, Brazil: comparative study of diagnosed cases in 2000 and 2005. *Arquivos Brasileiros de Endocrinologia e Metabologia*, Vol. 53, No. 4, pp. 453-460. Cvejic, D.; Savin, S.; Petrovic, I.; Paunovic, I.; Tatic, S.; Krgovic, K. & Havelka, M. (2005).

Galectin-3 expression in papillary microcarcinoma of the thyroid. *Histopathology*,

proliferation related molecules (Bcl-2, Bax, p53, PCNA) in papillary microcarcinoma versus papillary carcinoma of the thyroid. *Pathology*, Vol. 40, No. 5, pp. 475-480. Cvejic, D.; Selemetjev, S.; Savin, S.; Paunovic, I. & Tatic, S. (2009). Changes in the balance

between proliferation and apoptosis during the progression of malignancy in

course of papillary thyroid carcinoma. *Journal of Clinical Endocrinology and* 

papillary microcarcinomas of the thyroid: is subtotal thyroidectomy combined with radioiodine ablation enough? *Nuclear Medicine Communications*, Vol. 26, No. 1, pp 3-8.

Basolo, F.; Bevilacqua, G.; Miccoli, P.; Di Coscio, G.; Vitti, P.; Pacini, F. & Pinchera, A. (2010). Are the clinical and pathological features of differentiated thyroid carcinoma really changed over the last 35 years? Study on 4187 patients from a single Italian institution to answer this question. *Journal of Clinical Endocrinology and* 

microcarcinoma of the thyroid - factors affecting lymph node metastasis.

*Seminars in Diagnostic Pathology*, Vol. 17, No. 3, pp. 170-177.

*Acta Cytologica*, Vol. 30, No. 1, pp. 1-7.

*Cancer*, Vol. 98, No. 1, pp. 31-40.

Vol. 47, No. 2, pp. 209-214.

*Metabolism*, Vol. 71, No. 2, pp. 414-424.

*Metabolism*, Vol 95, No. 4, pp. 1516-1527.

*Langenbeck's Archives of Surgery*, Vol. 393, pp. 25-29.

*in Oncology*, Vol. 21, pp. 1-4.


Papillary Thyroid Microcarcinoma – Do Classical Staging Systems Need to Be Changed? 43

Mihailovic, J.; Stefanovic, L.; Malesevic, M. & Markoski, B. (2009). The importance of age

Min, H.; Choer, G.; Kim, S.; Park, Y.; Park do, J.; Youn, Y.; Park, S.; Cho, B. & Park, S. (2008).

Neuhold, N.; Kaiser, H. & Kaserer, K. (2001). Latent carcinoma of the thyroid in Austria: a

Noguchi, S.; Yamashita, H.; Uchino, S. & Watanabe, S. (2008). Papillary microcarcinoma.

Page, C.; Biet, A.; Boute, P.; Cuvelier, P. & Strunski, V. (2009). "Aggressive papillary" thyroid

Pakdaman, M.; Rochon, L.; Gologan, O.; Tamilia, M.; Garfield, N.; Hier, M.; Black, M. &

Passler, C.; Prager, G.; Scheuba, C.; Kaserer, K.; Zettinig, G. & Niederle, B. (2003). Application

Pelizzo, M.; Boschin, I.; Toniato, A.; Piotto, A.; Bernante, P.; Pagetta, C.; Rampin, L. &

Pisanu, A.; Reccia, I.; Nardello, O. & Uccheddu, A. (2009). Risk factors for nodal metastasis

Rezk, S. & Khan, A. (2005). Role of immunohistochemistry in the diagnosis and progression

Roh, J.; Kim, J. & Park, C. (2008). Central cervical nodal metastasis from papillary thyroid

Rossi, E.; Raffaelli, M.; Miraglia, A.; Lombardi, C.; Vecchio, F. & Fadda, G. (2006).

with iodine substitution. *Annals of Surgery*, Vol. 237, No. 2, pp. 227-234. Pelizzo, M.; Toniato, A.; Boschin, I.; Piotto, A.; Bernante, P.; Pagetta, C.; Palazzi, M.; Maria

*Medicine Communications*, Vol. 26, No. 11, pp. 965-968.

*and Molecular Morphology*, Vol. 13, No. 3, pp. 256-264.

*Surgical Oncology*, Vol. 15, No. 9, pp. 2482-2486.

*Histopathology*, Vol. 48, No. 7, pp. 795-800.

systematic autopsy study. *Endocrine Pathology*, vol. 12, pp. 23 – 31.

*World Journal of Surgery*, Vol. 32, pp. 747-753.

with distant metastases. *Thyroid*, Vol. 19, No. 3, pp. 227-232.

pp. 745-750.

1959-1963.

139, No. 5, pp 718-722.

Vol. 32, No. 10, pp. 1144-1148.

*Surgery*, Vol. 33, No. 3, pp. 460-468.

over radioiodine avidity as a prognostic factor in differentiated thyroid carcinoma

S100A4 expression is associated with lymph node metastasis in papillary microcarcinoma of the thyroid. *Modern Pathology*, Vol. 21, No. 6, pp. 748-755. Nam, K.; Noh, T.; Chung, S.; Lee, S.; Lee, M.; Hong, S.; Chung, W.; Lee, E. & Park, C. (2011).

Expression of the membrane mucins MUC4 and MUC15, potential markers of malignancy and prognosis, in papillary thyroid carcinoma. *Thyroid*, Vol. 21, No. 7,

microcarcinoma. *European Archives of Otorhinolaryngology*, Vol. 266, No. 12, pp.

Payne, R. (2008). Incidence and histopathological behavior of papillary microcarcinomas: Study of 429 cases. *Otolaryngology Head and Neck Surgery*, Vol.

of staging systems for differentiated thyroid carcinoma in an endemic goiter region

Guolo, A.; Preo, P.; Nibale, O. & Rubello, D. (2005). Locally advanced differentiated thyroid carcinoma: a 35-year mono-institutional expericence in 280 patients. *Nuclear* 

Rubello, D. (2006). Papillary thyroid microcarcinoma (PTMC): prognostic factors, management and outcome in 403 patients. *European Journal of Surgical Oncology*;

and recurrence among patients with papillary thyroid microcarcinoma: differences in clinical relevance between nonincidental and incidental tumors. *World Journal of* 

of follicular epithelium-derived thyroid carcinoma. *Applied Immunohistochemistry* 

microcarcinoma: pattern and factors predictive of nodal metastasis. *Annals of* 

Simultaneous immunohistochemical expression of HBME-1 and galectin-3 differentiates papillary carcinomas from hyperfunctioning lesions of the thyroid.


Lang, W.; Borrusch, H. & Bauer, L. (1988). Evaluation of 1020 sequential autopsies. *American* 

Lantsov, D.; Meirmanov, S.; Nakashima, M.; Kondo, H.; Saenko, V.; Naruke, Y.; Namba, H.;

Lee, S.; Le, S.; Jin, S.; Kim, J. & Rho, Y. (2008). Predictive factors for central compartment

Leenhardt, L.; Grosclaude, P. & Chérié-Challine, L. (2004). Increased incidence of thyroid

of papillary thyroid microcarcinoma. *Thyroid*, Vol. 17, No. 9, pp. 883-888. Lim, Y.; Choi, E.; Yoon, Y.; Kim, E. & Koo, B. (2009). Central lymph node metastases in

Lin, H & Bhattacharyya, N. (2009). Survival impact of treatment options for papillary microcarcinoma of the thyroid. *Laryngoscope*, Vol. 119, No. 10, pp. 1983-1987. Lin, J.; Chao, T.; Chen, S.; Weng, H & Lin, K. (2000). Characteristics of thyroid carcinomas in aging patients. *European Journal of Clinical Investigation*, Vol. 30, No. 2, pp. 147-153. Lin, J.; Chen, S.; Chao, T.; Hsueh, C. & Weng, H. (2005). Diagnosis and therapeutic strategy for papillary thyroid microcarcinoma. *Archives of Surgery*, Vol. 140, pp. 940-945. Lin, J.; Kuo, S.; Chao, T. & Hssue, C. (2008). Incidental and nonincidental papillary thyroid microcarcinoma. *Annals of Surgical Oncology*, Vol. 15, No. 8, pp. 2287-2292. Lin, J.; Chao, T.; Hsueh, C. & Kuo, S. (2009). High recurrent rate of multicentric papillary thyroid carcinoma. *Annals of Surgical Oncology*, Vol. 16, No.9, 2609-2616. Lloyd, R.; De Lellis, R.; Heitz, P. & Eng, C. (2004). World Health Organization classification

Ito, M.; Abrosimov, A.; Lushnikov, E.; Sekine, I. & Yamashita, S. (2005). Cyclin D1 overexpression in thyroid papillary microcarcinoma: its association with tumor size and aberrant beta-catenin expression. *Histopathology*, Vol. 47, No. 3, pp. 248-256. Lee, J.; Rhee, Y.; Ahn, C.; Cha, B.; Kim, K.; Lee, H.; Kim, S.; Park, C. & Lim, S. (2006).

Frequent, Aggressive behaviors of thyroid microcarcinoma in korean patients.

lymph node metastasis in thyroid papillary microcarcinoma. *Laryngoscope*, Vol. 118,

carcinoma in France: a true epidemic or thyroid nodule management effects? Report from the French thyroid cancer committee. *Thyroid*, Vol. 14, No. 12, pp. 1056-1060. Lim, D.; Baek, K.; Lee, Y.; Park, W.; Kim, M.; Kang, M.; Jeon, H.; Lee, J.; Yun-Cha, B.; Lee, K.;

Son, H. & Kang, S. (2007). Clinical, Histopathological, and molecular characteristics

unilateral papillary thyroid microcarcinoma. *British Journal of Surgery*, Vol. 96, pp.

of tumors: Pathology and genetics of tumors of the endocrine organs. Lyon, France:

difference between clinically overt and occult tumors? *World Journal of Surgery*, Vol.

on papillary and follicular thyroid cancer. *American Journal of Medicine*, Vol. 97, No.

Valcavi, R. & Barbieri, V. (2009). Prognostic factors affecting neck lymph node recurrence and distant metastasis in papillary microcarcinoma of the thyroid:

Lo, C.; Chan, W.; Lang, B.; Lam, K. & Wan, K. (2006). Papillary microcarcinoma: is there any

Mazzaferri, E. & Jhiang, S. (1994). Long-term impact of initial surgical and medical therapy

Mercante, G.; Frasoldati, A.; Pedroni, C.; Formisano, D.; Renna, L.; Piana, S.; Gardini, G.;

results of a study in 445 patients. *Thyroid*, Vol. 19, No. 7, pp. 707-716.

*Journal of Clinical Pathology*, Vol. 90, pp. 72 – 76.

*Endocrine Journal*, Vol. 53, No. 5. pp. 627-632.

No. 4, pp. 659-662.

253-257.

IARC Press.

30, pp. 759-766.

5, pp. 418-428.


**4** 

*Italy* 

**Thyroid Neoplasm** 

Augusto Taccaliti, Gioia Palmonella, Francesca Silvetti and Marco Boscaro *Politecnic University of Marche, Ancona* 

Thyroid gland comprises 2 types of cells: Follicular cells (or thyrocytes) which produce and secrete thyreoglobulin and thyroid hormones, thyroxine (T4) and triiodothyronine (T3) and Parafollicular cells (or C cells), secrete calcitonin. Papillary Thyroid Carcinoma (PTC) and Follicular Thyroid Carcinoma (FTC) are tumors originating by thyrocytes and are referred as Differentiated Thyroid Carcinomas (DTCs). Anaplastic Thyroid Carcinoma (ATC) is the undifferentiate tumor which may arises from DTCs or may be undifferentiated to origin. Medullary Thyroid Carcinoma (MTC), is the tumor arising to C cell. Rare tumors of nonepithelial thyroid origin are lymphoma, fibrosarcoma, squamous cell carcinoma, malignant

Thyroid tumor represent about 1% of all human malignancy and about 90% of all endocrine tumor. PTC represents about 90% and FTC the 10% of DTCs. The annual incidence of thyroid cancer has been reported to range between 1.2 and 2.6 cases per 100.000 in men and 2.0-3.8 cases per 100.000 in women. Recently epidemiological studies have shown an increased incidence of DTCs in worldwide (Kosary, 2007; Kilfoy et al., 2009). PTC and microPTC (size < 1cm) represents the cancer prevalently increased. Reasons of increased incidence are not completely understood and controversies exist whether this increase is real or only apparent due to an increase in diagnostic activity. Probably the increased incidence may reflect the increased detection of small tumors through the use of imaging, particularly ultrasonografy (US), and increased use of fine needle aspiration citology (FNAC). Mortality records in the SEER database from 1997-2006 show relatively stable or slightly improved mortality rates for thyroid cancer (Edwards et al, 2010). However, over the same period, SEER mortality rates measured in terms of relative survival show reduced mortality rates in

Ionizing radiations interacting with DNA produce mutations. thyroid was a tissue sensitive to ionizing radiation as demonstrate by the increased incidence in thyroid cancer after

**1. Introduction** 

**2.1 Epidemiology** 

**2.2 Risk factors** 

**2.2.1 Ionizing radiation** 

teratoma and metastasis of other tumors.

women respect to in men (Kosary, 2007).

**2. Differentiated thyroid carcinomas (DTCs)** 


### **Thyroid Neoplasm**

Augusto Taccaliti, Gioia Palmonella, Francesca Silvetti and Marco Boscaro *Politecnic University of Marche, Ancona Italy* 

#### **1. Introduction**

44 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

Roti, E.; Rossi, R.; Trasforini, G.; Bertelli, F.; Ambrosio, M.; Busutti, L.; Pearce, E.; Braverman,

Roti, E.; Uberti, E.; Bondanelli, M. & Braverman, L. (2008). Thyroid papillary

Schindler, A.; van Melle, G.; Evequoz, B. &, Scazziga, B. (1991). Prognostic factors in papillary carcinoma of the thyroid. *Cancer*, Vol. 68, No. 2, pp. 324-330. Schlumberger, M.; Challeton, C.; De Vathaire, F.; Travagli, J. ; Gardet, P. ; Lumbroso, J.;

Sherman, S. (1999). Toward a standard clinicopathologic staging approach for differentiated thyroid carcinoma. *Semininars in Surgical Oncology*, Vol. 16, No. 1, pp. 12-15. Sugino, K.; Ito, K.J.; Ozaki, O.; Mimura, T.; Iwasaki, H. & Ito, K. (1998). Papillary

Tubiana, M.; Schlumberger, M.; Rougier, P.; Laplanche, A.; Benhamou, E.; Gardet, P.; Caillou,

Van Herle, A. & Uller, R. (1975). Elevated serum thyroglobulin. A marker of metastases in

Vini, L.; Hyer, S.; Marshall, J.; A´Hern, R. & Harmer, C. (2003). Long-term results in elderly patients with differentiated thyroid carcinoma. *Cancer*, Vol. 97, No.11, pp. 2736-2742. Voutilainen, P.; Siironen, P.; Franssila, K.; Sivula, A.; Haapiainen, R. & Haglund, C. (2003).

Yao, R.; Chiu, C.; Strugnell, S.; Gill, S. & Wiseman, S. (2011). Gender differences in thyroid cancer. *Expert Review of Endocrinology and Metabolism*, Vol. 6, No. 2, pp. 215 – 243. Zafon, C.; Castellvi, J. & Obiols, G. (2010). Usefulness of the immunohistochemical analysis of

carcinoma. *Journal of Nuclear Medicine*, Vol. 37, No. 4, pp. 598-605.

macroscopic disease. *Thyroid*, Vol. 19, No. 5, pp. 487-494.

*Anticancer Research*, Vol. 23, No. 5b, pp. 4283-4288.

of larger size. *Journal of Thyroid Research*, ID 639156.

*Endocrinology and Metabolism*, Vol. 91, No. 6, pp. 2171-2178.

*Endocrinology*, Vol. 159, pp. 659-673.

7, pp. 445-448.

L. & Uberti, E. (2006). Clinical and histological characteristics of papillary thyroid microcarcinoma: results of a retrospective study in 243 patients. *Journal of Clinical* 

microcarcinoma: a descriptive and meta-analysis study. *European Journal of* 

Francese, C.; Fontaine, F.; Ricard, M. & Parmentier, C. (1996). Radioactive iodine treatment and external radiotherapy for lung and bone metastases from thyroid

microcarcinoma of the thyroid. *Journal of Endocrinological Investigation*, Vol. 21, No.

B.; Travagli, J. & Parmentier, C. (1985). Long-term results and prognostic factors in patients with differentiated thyroid carcinoma. *Cancer*, Vol. 55, No. 4, pp. 794-804. Tzvetov, G.; Hirsch, D.; Shraga-Slutzky, I.; Weinstein, R.; Manistersky, Y.; Kalmanovich, R.;

Lapidot, M.; Grozinsky-Glasberg, S.; Singer, J.; Sulkes, J.; Shimon, I. & Benbassat, C. (2009). Well-differentiated thyroid carcinoma: comparison of microscopic and

differentiated thyroid carcinomas. *Journal of Clinical Investigation*, Vol. 56, pp. 272-277.

AMES, MACIS and TNM prognostic classifications in papillary thyroid carcinoma.

several molecular markers in the characterization of papillary thyroid carcinoma with initial lymph node mestastasis. *Endocrinologia y Nutricion*, Vol. 57, No. 4, pp. 165-169. Zafon, C.; Baena, J.; Castellvi, J.; Obiols, G.; Monroy, G. & Mesa, J. (2011). Differences in the

form of presentation between papillary microcarcinomas and papillary carcinomas

Thyroid gland comprises 2 types of cells: Follicular cells (or thyrocytes) which produce and secrete thyreoglobulin and thyroid hormones, thyroxine (T4) and triiodothyronine (T3) and Parafollicular cells (or C cells), secrete calcitonin. Papillary Thyroid Carcinoma (PTC) and Follicular Thyroid Carcinoma (FTC) are tumors originating by thyrocytes and are referred as Differentiated Thyroid Carcinomas (DTCs). Anaplastic Thyroid Carcinoma (ATC) is the undifferentiate tumor which may arises from DTCs or may be undifferentiated to origin. Medullary Thyroid Carcinoma (MTC), is the tumor arising to C cell. Rare tumors of nonepithelial thyroid origin are lymphoma, fibrosarcoma, squamous cell carcinoma, malignant teratoma and metastasis of other tumors.

#### **2. Differentiated thyroid carcinomas (DTCs)**

#### **2.1 Epidemiology**

Thyroid tumor represent about 1% of all human malignancy and about 90% of all endocrine tumor. PTC represents about 90% and FTC the 10% of DTCs. The annual incidence of thyroid cancer has been reported to range between 1.2 and 2.6 cases per 100.000 in men and 2.0-3.8 cases per 100.000 in women. Recently epidemiological studies have shown an increased incidence of DTCs in worldwide (Kosary, 2007; Kilfoy et al., 2009). PTC and microPTC (size < 1cm) represents the cancer prevalently increased. Reasons of increased incidence are not completely understood and controversies exist whether this increase is real or only apparent due to an increase in diagnostic activity. Probably the increased incidence may reflect the increased detection of small tumors through the use of imaging, particularly ultrasonografy (US), and increased use of fine needle aspiration citology (FNAC). Mortality records in the SEER database from 1997-2006 show relatively stable or slightly improved mortality rates for thyroid cancer (Edwards et al, 2010). However, over the same period, SEER mortality rates measured in terms of relative survival show reduced mortality rates in women respect to in men (Kosary, 2007).

#### **2.2 Risk factors**

#### **2.2.1 Ionizing radiation**

Ionizing radiations interacting with DNA produce mutations. thyroid was a tissue sensitive to ionizing radiation as demonstrate by the increased incidence in thyroid cancer after

Thyroid Neoplasm 47

DTCs frequently have somatic mutations that constitutively activated the mitogenactivated protein kinase (MAPK) pathway and PI3K-AKT pathway. These pathways include cell surface receptors such as RET and NTK, and intracellular signal transduceds , RAS gene and kinases RAF. Ultimately, this leads to increased nuclear translocation of phosphorylated MAPK and altered transcriptional regulation of target genes. Although the characteristic genetic alterations in PTC are all capable of activating the MAPK pathway, the histological phenotype and the expression profile are not identical between the different genetic alterations suggesting that other pathways such as the phosphoinositide-3-kinase (PI3K), protein kinase C and Wnt signalling pathways may be

Tyrosine kinase receptors function as receptors for many growth factors and carry growth signals into the cell through tyrosine autophosphorylation and the initiation of kinase cascades. Tyrosine kinase receptors implicated on thyroid oncogenesis include RET and

1. RET: RET proto-oncogene is a 21-exons gene located on the proximal long arm of chromosome 10 that encodes a tyrosine kinase receptor. It is involved in the regulation of growth, survival, differentiation, and migration of cells of neural crest origin. It is not normally expressed in the follicular cell. The interaction extracellular ligand-binding domain and RET receptor leads the activation of a serine/threonine kinase pathway including RAS, BRAF and MAPK. This ultimately leads to a proliferative signal as well

RET/PTC rearrangement: Rearrangements of RET gene in papillary thyroid carcinoma (PTC) are known as RET/PTC. Low-level expression may be seen in non-malignant follicular cells especially in Hashimoto's thyroiditis (Nikiforov, 2006). Although more than 10 rearrangements have been described, RET/PTC1 (60–70%), RET/PTC2 (20–30%), and RET/PTC3 (10%) account for most of the rearrangements found in PTC. Other RET/PTC rearrangements are rare (Santoro et al., 2006). In each of these rearrangements, the upstream (5´) component of a "housekeeping" (or ubiquitously expressed) gene drives the expression of the tyrosine kinase domain of RET. Two of the most common rearrangement types are RET/PTC1 and RET/PTC3. Both type of rearrangement paracentric intrachromosomal inversions, as all fusion partners reside on the long arm of chromosome 10. By contrast, RET/PTC2 and nine more RET/PTC rearrangements are all intrachromosomal rearrangements formed by RET fusion to genes located on different chromosomes. In the adult population, the RET rearrangements have been found in 2.6% to 34% of PTC. This variation is due to true differences in the prevalence of this alteration in PTC in specific age group in individuals exposed to ionizing radiation. Other causes might be represented by heterogeneous distribution of this rearrangements within the cancer and the various sensibilities of the detention methods used. In the pediatric population RET/PTC1 and RET/PTC3 have been found in up to 80% of the cases. These mutations are found in children exposed to radiation after the Chernobyl nuclear accident or to external irradiation for treatment of benign diseases of the head and neck. There are evidences that RET/PTC

as inhibiting apoptosis and increasing genetic instability.

**2.5 Somatic genetic alterations** 

variously involved (figure 1).

TRK:

**2.5.1 Thyrosine kinase receptors** 

Chernobyl accident. Children and young people were affected by DTCs and PTC was the histotype prevalently. Moreover, WHO has reported that newborn and children below 5 years old have high risk to develop thyroid cancer respect to adolescent and adults (Papadopoulou et al., 2009; Williams, 2008).

#### **2.2.2 Iodine intake**

Iodine is essential for T4 and T3 production. iodine deficient or inadequate intake induces low t4 levels and TSh increase and chronic tsh stimuli promotes growth goiter and nodules. thyroid nodules represent a clinical condition to develop thyroid cancer and especially FTC histotype. Studies have show a reduction of PTC/FTC ratio in iodine deficient area (Farahati et al., 2004; Lind et al., 1998).

#### **2.3 Familial thyroid cancer syndromes**

Non Medullary Familial Thyroid Cancer represents a rare disease where thyroid cancer is the only manifestation or thyroid cancer may represent a component of a complex syndrome.

Familial thyroid cancer PTC may have familial factors in 3.2 and 6.2%of cases. in fact, has been reported an increased incidence in relatives of patients with PTC of 4-10 fold (Pal et all, 2001). Some studies have found an association between altered telomere length (TL) and cancer phenotype (Capezzone et al, 2011). In these cases, the tumors are PTC with onset at an earlier age, with reversed gender distribution and with a more aggressive phenotype (Hemminki et al., 2005) with anticipation of neoplasia.

#### **2.4 Familial tumour syndromes with thyroid cancer**


Chernobyl accident. Children and young people were affected by DTCs and PTC was the histotype prevalently. Moreover, WHO has reported that newborn and children below 5 years old have high risk to develop thyroid cancer respect to adolescent and adults

Iodine is essential for T4 and T3 production. iodine deficient or inadequate intake induces low t4 levels and TSh increase and chronic tsh stimuli promotes growth goiter and nodules. thyroid nodules represent a clinical condition to develop thyroid cancer and especially FTC histotype. Studies have show a reduction of PTC/FTC ratio in iodine deficient area (Farahati

Non Medullary Familial Thyroid Cancer represents a rare disease where thyroid cancer is the only manifestation or thyroid cancer may represent a component of a complex syndrome.

Familial thyroid cancer PTC may have familial factors in 3.2 and 6.2%of cases. in fact, has been reported an increased incidence in relatives of patients with PTC of 4-10 fold (Pal et all, 2001). Some studies have found an association between altered telomere length (TL) and cancer phenotype (Capezzone et al, 2011). In these cases, the tumors are PTC with onset at an earlier age, with reversed gender distribution and with a more aggressive phenotype

1. Polyposis coli and Gardner's syndrome: Familial Adenomatous Polyposis (FAP) and Gardner's syndrome are inherited diseases characterized by colonic polyposis the former, and osteomas, lipomas and fibromas plus colonic polyposis the latter. Both syndromes show 5–10-fold increase in the incidence of PTC and tumor is multicentric. Germline mutations of tumor suppressor gene APC (Adenomatosis Polyposis Coli) are described in both syndromes. PTC in familial polyposis syndromes often harbours RET/PTC rearrangements (see below) in addition to the

2. Cowden's disease: Patients with Cowden's disease have breast carcinoma and hamartomas. Cowden's disease is caused by germ-line mutations in the phosphatase and tensin homologue (PTEN) tumour suppressor gene inheritance in autosomal dominant pattern. Cowden's disease increases the incidence of follicular tumours of the thyroid, but the incidence is hard to estimate (Hemmings, 2003). Papillary cancer is also

3. Carney complex: The Carney complex consists of spotty skin pigmentation, myxomas, schwannomas, pigmented nodular adrenal hyperplasia, pituitary and testicular tumors and an increased incidence of follicular adenoma and FC. It is due to a mutation in the type 1 alpha regulatory subunit of the protein kinase A (PRKAR1A) which leads to constitutively activated protein kinase A (PKA) (Boikos & Stratakis, 2006) PRKAR1A mutations have been found in sporadic thyroid tumors and are more common in FC

(Papadopoulou et al., 2009; Williams, 2008).

**2.3 Familial thyroid cancer syndromes** 

(Hemminki et al., 2005) with anticipation of neoplasia.

**2.4 Familial tumour syndromes with thyroid cancer** 

APC deletion (Cetta et al., 1998).

found with increased frequency.

than follicular adenoma.

**2.2.2 Iodine intake** 

et al., 2004; Lind et al., 1998).

#### **2.5 Somatic genetic alterations**

DTCs frequently have somatic mutations that constitutively activated the mitogenactivated protein kinase (MAPK) pathway and PI3K-AKT pathway. These pathways include cell surface receptors such as RET and NTK, and intracellular signal transduceds , RAS gene and kinases RAF. Ultimately, this leads to increased nuclear translocation of phosphorylated MAPK and altered transcriptional regulation of target genes. Although the characteristic genetic alterations in PTC are all capable of activating the MAPK pathway, the histological phenotype and the expression profile are not identical between the different genetic alterations suggesting that other pathways such as the phosphoinositide-3-kinase (PI3K), protein kinase C and Wnt signalling pathways may be variously involved (figure 1).

#### **2.5.1 Thyrosine kinase receptors**

Tyrosine kinase receptors function as receptors for many growth factors and carry growth signals into the cell through tyrosine autophosphorylation and the initiation of kinase cascades. Tyrosine kinase receptors implicated on thyroid oncogenesis include RET and TRK:

1. RET: RET proto-oncogene is a 21-exons gene located on the proximal long arm of chromosome 10 that encodes a tyrosine kinase receptor. It is involved in the regulation of growth, survival, differentiation, and migration of cells of neural crest origin. It is not normally expressed in the follicular cell. The interaction extracellular ligand-binding domain and RET receptor leads the activation of a serine/threonine kinase pathway including RAS, BRAF and MAPK. This ultimately leads to a proliferative signal as well as inhibiting apoptosis and increasing genetic instability.

RET/PTC rearrangement: Rearrangements of RET gene in papillary thyroid carcinoma (PTC) are known as RET/PTC. Low-level expression may be seen in non-malignant follicular cells especially in Hashimoto's thyroiditis (Nikiforov, 2006). Although more than 10 rearrangements have been described, RET/PTC1 (60–70%), RET/PTC2 (20–30%), and RET/PTC3 (10%) account for most of the rearrangements found in PTC. Other RET/PTC rearrangements are rare (Santoro et al., 2006). In each of these rearrangements, the upstream (5´) component of a "housekeeping" (or ubiquitously expressed) gene drives the expression of the tyrosine kinase domain of RET. Two of the most common rearrangement types are RET/PTC1 and RET/PTC3. Both type of rearrangement paracentric intrachromosomal inversions, as all fusion partners reside on the long arm of chromosome 10. By contrast, RET/PTC2 and nine more RET/PTC rearrangements are all intrachromosomal rearrangements formed by RET fusion to genes located on different chromosomes. In the adult population, the RET rearrangements have been found in 2.6% to 34% of PTC. This variation is due to true differences in the prevalence of this alteration in PTC in specific age group in individuals exposed to ionizing radiation. Other causes might be represented by heterogeneous distribution of this rearrangements within the cancer and the various sensibilities of the detention methods used. In the pediatric population RET/PTC1 and RET/PTC3 have been found in up to 80% of the cases. These mutations are found in children exposed to radiation after the Chernobyl nuclear accident or to external irradiation for treatment of benign diseases of the head and neck. There are evidences that RET/PTC

Thyroid Neoplasm 49

in PTC. BRAF mutations occur in 30–60% of PTC and are 100% specific. BRAF point mutations are rarely associated with radiation-induced PTC. A paracentric inversion involving BRAF (AKAP9-BRAF) was found in 11% of post Chernobyl tumors BRAF and RET/PTC are mutually exclusive, not occurring in the same tumor. The presence of BRAF mutations appears to be associated with poor prognosis, reduced iodine uptake and failure of radioiodine ablation. Poorly differentiated and ATC harbour BRAF mutations, but not RET/PTC rearrangements, suggesting that BRAF mutations may predispose PTC to de-

Three RAS genes, H-RAS, K-RAS, and N-RAS, synthesize a family of 21-kDa proteins that play an important role in tumorigenesis. Their function is to convey signals originating from tyrosine kinase membrane receptors to a cascade of mitogen-activated protein kinases (MAPK). This activates the transcription of target genes involved in cell proliferation, survival, and apoptosis. The RAS proteins exist in two different forms: an inactive form that is bound to guanosine diphosphate (GDP) and an active form that exhibits guanosine triphosphatase (GTPase) activity. Oncogenic RAS activation results from point mutations, affecting the GTP-binding domain (codons 12 or 13) in exon 1 or the GTPase domain (codon 61) in exon 2, which fix the protein in the activated state and thus resulting in chronic stimulation of downstream targets, genomic instability, additional mutations, and malignant transformation. Mutations in all three cellular RAS genes have been identified in benign and malignant thyroid tumors. They seem to be common in follicular carcinoma (50%) (Di Cristofaro et al., 2006), PDTC, and ATC and occur less frequently in PTC (<10%) (Meinkoth, 2004). Some studies have shown a similar prevalence of RAS mutations in benign and malignant thyroid neoplasms, suggesting that RAS activation may represent an early event. Other studies have shown that RAS mutations, specifically mutations at codon 61 of N-RAS, are involved with tumor progression and aggressive clinical behavior. The presence of RAS mutations predicted a poor outcome for WDTC independent of tumor stage. Furthermore, they found that PDTC and ATC often harbor multiple RAS mutations. These mutations probably represent an intermediate event in the progression of thyroid

The PAX8 gene encodes a transcription factor essential for the genesis of thyroid follicular cell lineages and regulation of thyroid specific gene expression. The Peroxisome Proliferator-Activated Receptor **γ** (PPAR**γ**) is a member of the nuclear hormone receptor superfamily that includes thyroid hormone, retinoic acid, and androgen and estrogen receptors. The PAX8-PPAR**γ** rearrangement leads to in-frame fusion of exon 7, 8, or 9 of PAX8 on 2q13 with exon 1 of PPARγ on 3p25. It appears as though the PAX8-PPARγ chimeric protein inactivates the wild-type PPARγ, which is a putative tumor suppressor (Ying et al., 2003) As with RAS mutations, PAX8-PPARγ rearrangement has also been shown to be involved in the development of FTC. The PAX8-PPARγ rearrangement is found in FTC (26–63%) and in the follicular variant of PTC, where it occurs in approximately 33% of all tumors (Castro et al., 2006) The role of this rearrangement in the progression

differentiation (Xing et al., 2005a; Nikiforova et al., 2003).

**2.5.3 RAS mutations**

carcinoma.

**2.5.4 PAX8-PPARγ** 

rearrangements represent an early genetic changes leading to the development of PTC (Nikiforov, 2002; Xing, 2005).

Fig. 1. RET receptor with MAP-Kinase and PI3-Kinase-AKT pathways in thyroid cell.

2. Trk: Trk proto-oncogene is located on chromosome 1q22 and encodes a tyrosine kinase receptor for nerve growth factor (27). It is expressed in the neurons in both peripheral and central nervous system, and is involved in the regulation of growth, differentiation and survival of these cells.

**Trk** rearrangements: occur in DTCs with lower prevalence than RET/PTC rearrangements. In thyroid follicular cells, the gene is activated through chromosomal rearrangement, with juxtaposes the intracellular thyrosine kinase domain of NTRK1 to the 5' terminal sequence of different genes. Various genes combine with TRK gene forming chimeric genes. The main ones are tropomyosin gene (TPM3) gene and the translocated gene promoter (TPR). Trk rearrangements have been identified in 2-5% of PTC. (Musholt et al., 2000).

#### **2.5.2 BRAF mutations**

The RAF proteins are serine/threonine kinases involved in intracellular signalling via the MAPK pathway (Davies et al., 2002). Point mutations in BRAF leading to mutant proteins that mimic the conformation of the phosphorylated form and are therefore constitutively activated are involved in several of human malignancies including melanoma, ovarian and colorectal cancers. Mutations in the BRAF gene are the most common genetic alteration seen

rearrangements represent an early genetic changes leading to the development of PTC

**Ligand** 

**RAS**

MAP

BRAF

MEK

**Co-receptor**

**CELL-SURFACE** 

Fig. 1. RET receptor with MAP-Kinase and PI3-Kinase-AKT pathways in thyroid cell.

**GENE TRANSCRIPTION** 

P13-Kinase

rearrangements have been identified in 2-5% of PTC. (Musholt et al., 2000).

2. Trk: Trk proto-oncogene is located on chromosome 1q22 and encodes a tyrosine kinase receptor for nerve growth factor (27). It is expressed in the neurons in both peripheral and central nervous system, and is involved in the regulation of growth, differentiation

**Trk** rearrangements: occur in DTCs with lower prevalence than RET/PTC rearrangements. In thyroid follicular cells, the gene is activated through chromosomal rearrangement, with juxtaposes the intracellular thyrosine kinase domain of NTRK1 to the 5' terminal sequence of different genes. Various genes combine with TRK gene forming chimeric genes. The main ones are tropomyosin gene (TPM3) gene and the translocated gene promoter (TPR). Trk

The RAF proteins are serine/threonine kinases involved in intracellular signalling via the MAPK pathway (Davies et al., 2002). Point mutations in BRAF leading to mutant proteins that mimic the conformation of the phosphorylated form and are therefore constitutively activated are involved in several of human malignancies including melanoma, ovarian and colorectal cancers. Mutations in the BRAF gene are the most common genetic alteration seen

(Nikiforov, 2002; Xing, 2005).

**RET receptor** 

AKT

m-TOR

and survival of these cells.

**Nuclear membrane** 

**2.5.2 BRAF mutations** 

in PTC. BRAF mutations occur in 30–60% of PTC and are 100% specific. BRAF point mutations are rarely associated with radiation-induced PTC. A paracentric inversion involving BRAF (AKAP9-BRAF) was found in 11% of post Chernobyl tumors BRAF and RET/PTC are mutually exclusive, not occurring in the same tumor. The presence of BRAF mutations appears to be associated with poor prognosis, reduced iodine uptake and failure of radioiodine ablation. Poorly differentiated and ATC harbour BRAF mutations, but not RET/PTC rearrangements, suggesting that BRAF mutations may predispose PTC to dedifferentiation (Xing et al., 2005a; Nikiforova et al., 2003).

#### **2.5.3 RAS mutations**

Three RAS genes, H-RAS, K-RAS, and N-RAS, synthesize a family of 21-kDa proteins that play an important role in tumorigenesis. Their function is to convey signals originating from tyrosine kinase membrane receptors to a cascade of mitogen-activated protein kinases (MAPK). This activates the transcription of target genes involved in cell proliferation, survival, and apoptosis. The RAS proteins exist in two different forms: an inactive form that is bound to guanosine diphosphate (GDP) and an active form that exhibits guanosine triphosphatase (GTPase) activity. Oncogenic RAS activation results from point mutations, affecting the GTP-binding domain (codons 12 or 13) in exon 1 or the GTPase domain (codon 61) in exon 2, which fix the protein in the activated state and thus resulting in chronic stimulation of downstream targets, genomic instability, additional mutations, and malignant transformation. Mutations in all three cellular RAS genes have been identified in benign and malignant thyroid tumors. They seem to be common in follicular carcinoma (50%) (Di Cristofaro et al., 2006), PDTC, and ATC and occur less frequently in PTC (<10%) (Meinkoth, 2004). Some studies have shown a similar prevalence of RAS mutations in benign and malignant thyroid neoplasms, suggesting that RAS activation may represent an early event. Other studies have shown that RAS mutations, specifically mutations at codon 61 of N-RAS, are involved with tumor progression and aggressive clinical behavior. The presence of RAS mutations predicted a poor outcome for WDTC independent of tumor stage. Furthermore, they found that PDTC and ATC often harbor multiple RAS mutations. These mutations probably represent an intermediate event in the progression of thyroid carcinoma.

#### **2.5.4 PAX8-PPARγ**

The PAX8 gene encodes a transcription factor essential for the genesis of thyroid follicular cell lineages and regulation of thyroid specific gene expression. The Peroxisome Proliferator-Activated Receptor **γ** (PPAR**γ**) is a member of the nuclear hormone receptor superfamily that includes thyroid hormone, retinoic acid, and androgen and estrogen receptors. The PAX8-PPAR**γ** rearrangement leads to in-frame fusion of exon 7, 8, or 9 of PAX8 on 2q13 with exon 1 of PPARγ on 3p25. It appears as though the PAX8-PPARγ chimeric protein inactivates the wild-type PPARγ, which is a putative tumor suppressor (Ying et al., 2003) As with RAS mutations, PAX8-PPARγ rearrangement has also been shown to be involved in the development of FTC. The PAX8-PPARγ rearrangement is found in FTC (26–63%) and in the follicular variant of PTC, where it occurs in approximately 33% of all tumors (Castro et al., 2006) The role of this rearrangement in the progression

Thyroid Neoplasm 51

**Submandibular** 

**Hyoid Bone** 

**Anterior didastric** 

**gland** 

**Carotid artery** 

**Cricoid cartilage** 

Fig. 2. Shows the lymph nodes in 6 neck clustered in compartments.

respect to PTC. Lung and bone are the most common sites.

FTC represents thyroid cancers in area with insufficient iodine intake. As PTC, FTC occurs 3 times more frequently in women than in men. The mean age range at diagnosis is late in the

1. *Pathology*. Macroscopically, FTC appears as encapsulated nodule. Microscopically, tumor cells may show an increase solid, trabecular or follicular, which may invade the tumor capsule or the surrounding vascular structures. The tumors are divided into minimally invasive and widely invasive lesions depending on the histologic evidence of

2. *Local invasion.* Local invasion can occur as PTC (see Local invasion for Papillary

3. *Cervical and distant metastases.* Cervical metastases might be present at diagnosis. However, the rate of distant metastasis is significantly increased (approximately 20%)

Clinical presentation of thyroid cancer is a thyroid nodule. Suspicious criteria of malignancy are: 1) Age: <20 or> 60 years 2) Sex: male> female; 3) Irradiation of head and/or neck; 4) Family history of papillary carcinoma 5) Rapid growth of the nodule 6) Growth during suppressive therapy with LT4 (L-thyroxine); 7)Fixed, hard consistency 8) Lymphadenopathy

**2.6.2 Follicular carcinoma** 

**Jugular vein** 

**Sternocleidomastoid Spinal accessory nerve**

Carcinoma, above).

**2.7 Diagnosis** 

capsule and vascular invasion.

fifth to sixth decades.

and dedifferentiation of follicular thyroid cancer to PDTC and ATC has not been well defined.

#### **2.5.5 PI3K/AKT mutations**

The PI3K/Akt pathway is a key regulator of cell proliferation and inhibitor of apoptosis. This pathway can be activated by the upstream stimulatory molecules (i.e. RAS), through the loss of function of PTEN protein that normally inhibits PI3K signaling, or as a results of activating mutations or amplification of gene coding for the effectors of this pathway. Inactivating mutations in PTEN are seen in Cowden's disease, a familial tumour syndrome associated with FC. In sporadic FC, the incidence of PTEN mutations is low (7%). Activating mutations of the catalytic subunit of PI3K have been found in small numbers of FC and FA (Wang et al., 2007; Hou et al., 2007).

#### **2.6 Clinical features**

The thyroid carcinoma manifesting as thyroid nodule. Palpable thyroid nodules are present in approximately 4-7% while high-resolution ultrasonography thyroid nodules are described in 19-67% of the general population. Most thyroid nodules are benign and only 5- 10% are malignant. Thyroid nodule of large or small size have the same risk of malignancy. Solitary nodules in patients older more than 60 years and in young patients of less 30 years old are more frequently malignant. Male subjects have more risk of thyroid cancer than women. Rapid growth of a nodule may suggest malignancy.

#### **2.6.1 Papillary carcinoma**

Women develop PTC 3 times more frequently than men do, and the mean age at presentation is 34-40 years.


and dedifferentiation of follicular thyroid cancer to PDTC and ATC has not been well

The PI3K/Akt pathway is a key regulator of cell proliferation and inhibitor of apoptosis. This pathway can be activated by the upstream stimulatory molecules (i.e. RAS), through the loss of function of PTEN protein that normally inhibits PI3K signaling, or as a results of activating mutations or amplification of gene coding for the effectors of this pathway. Inactivating mutations in PTEN are seen in Cowden's disease, a familial tumour syndrome associated with FC. In sporadic FC, the incidence of PTEN mutations is low (7%). Activating mutations of the catalytic subunit of PI3K have been found in small numbers of FC and FA

The thyroid carcinoma manifesting as thyroid nodule. Palpable thyroid nodules are present in approximately 4-7% while high-resolution ultrasonography thyroid nodules are described in 19-67% of the general population. Most thyroid nodules are benign and only 5- 10% are malignant. Thyroid nodule of large or small size have the same risk of malignancy. Solitary nodules in patients older more than 60 years and in young patients of less 30 years old are more frequently malignant. Male subjects have more risk of thyroid cancer than

Women develop PTC 3 times more frequently than men do, and the mean age at

1. *Pathology:* Macroscopically the PTC are whitish nodules, without capsule and with illdefined margins compared to the surrounding thyroid tissue. Microscopically the tumor cells of PTC typically grow with papillae and are characterized by ground-glass nuclei with pseudoinclusios, rare mitosis, and psammoma bodies (in 50% of papillary carcinomas). Beyond this classic PTC other variants of PTC may be present as Oxyphilic, Tall Cell, Columnar cell invade the thyroid capsule and surrounding extrathyroidal structures such as trachea, laryngeal, Follicular variant and diffuse

2. *Local invasion* Cancer can nerves, and airways. In these cases the patient may present

3. *Regional and metastatic disease* PTC spreads to the cervical lymph nodes. Clinically evident lymph node metastases are present in approximately one third of patients at presentation. Microscopic metastases are present in one half. The most common site of lymph node involvement is central compartment (level 6). The jugular lymph node chains (levels 2-4) are the next most common sites of cervical node involvement. Lymph nodes in the posterior triangle of the neck (level 5) may also develop metastases. This finding has important implications on the treatment algorithm for patients in this situation (Figure 2). Approximately 5-10% of patients develop distant metastases.

defined.

**2.5.5 PI3K/AKT mutations** 

(Wang et al., 2007; Hou et al., 2007).

women. Rapid growth of a nodule may suggest malignancy.

with hemoptysis, hoarse voice and dysphagia.

Distant spread of PTC typically affects the lungs and bone

**2.6 Clinical features** 

**2.6.1 Papillary carcinoma** 

presentation is 34-40 years.

sclerosing

Fig. 2. Shows the lymph nodes in 6 neck clustered in compartments.

#### **2.6.2 Follicular carcinoma**

FTC represents thyroid cancers in area with insufficient iodine intake. As PTC, FTC occurs 3 times more frequently in women than in men. The mean age range at diagnosis is late in the fifth to sixth decades.


#### **2.7 Diagnosis**

Clinical presentation of thyroid cancer is a thyroid nodule. Suspicious criteria of malignancy are: 1) Age: <20 or> 60 years 2) Sex: male> female; 3) Irradiation of head and/or neck; 4) Family history of papillary carcinoma 5) Rapid growth of the nodule 6) Growth during suppressive therapy with LT4 (L-thyroxine); 7)Fixed, hard consistency 8) Lymphadenopathy

Thyroid Neoplasm 53

THYR 5.All cases with a diagnosis of malignant neoplasm (papillary, medullary and anaplastic carcinomas, lymphomas and metastasis) are included in this category Operative suggestion. Surgery for differentiated carcinomas. The results of FNAC are very sensitive for the differential diagnosis of benign and malignant nodules although there are limitations: inadequate samples and follicular neoplasia. In these cases the definitive

Near-total or total thyroidectomy is recommended if the primary thyroid carcinoma is >1 cm, multinodular goiter, regional or distant metastases at diagnosis, patient with personal history of radiation therapy to the head and neck, or patient with family history of DTCs. Older age (>45 years) may also be a criterion for recommending near-total or total thyroidectomy even with tumors <1–1.5 cm, because of higher recurrence rates in this age group. Increased extent of primary surgery may improve survival for high-risk patients and

Regional lymph node metastases are frequently at diagnosis ranging from 20 to 90%. Although lymph node metastases in PTC patients are reported no clinically relevance on outcome in low risk patients, recently SEER registry study concluded that cervical lymph node metastases are a poor prognostic factor on survival in patients with FTC and in patients with PTC over 45 years (Zaydfudim et al., 2008). In experienced hands, therapeutic or prophylactic central compartment dissection can be achieved with low morbidity. In addition, selective unilateral paratracheal central compartment node dissection increases the

Postoperative staging for thyroid cancer is used: 1) to permit prognostication for an individual patient with DTC; 2) to tailor decisions regarding postoperative adjunctive therapy, including RAI therapy and TSH suppression, to assess the patient's risk for disease recurrence and mortality; 3) to make decisions regarding the frequency and intensity of follow-up, directing more intensive follow-up towards patients at highest risk; and 4) to enable accurate communication regarding a patient among health care professionals. Varius risk definition for thyroid carcinoma have been evaluated. The American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) tumor-node-metastasis (TNM) classification has been used in clinical practice for DTC. This classification has also been evaluated to determine its utility in discriminating patients who have distinct outcomes. The fifth edition AJCC/UICC TNM classification (1997) was revised as the sixth

In 2009, ATA has developed classes of risk in DTCs patients, to predict risk for recurrence,

diagnosis can be made only by histological examination

**2.8.1 Neartotal or total thyroidectomy** 

low-risk patients (Bilimoria et al., 2007)

proportion of patients who appear disease free.

not death (ATA Surgery Working Group, 2009):

**2.8.2 Lymph node dissection** 

**2.8.3 Risk staging** 

edition in 2002 (Table 1).

**2.8 Treatment** 

#### **2.7.1 Laboratory and thyroid scintigraphy**

TSh evaluation allows to identify hyperfunction nodule. The TSH determination should be performed in nodule with large size > 2.5-3 cm. Tc99 thyroid scintiscan allows to confirm the uptake of large nodule. Hyperfunctioning nodules rarely are malignant, therefore no other diagnostic procedure should be performed.

#### **2.7.2 Thyroid ultrasound (US)**

US is a widespread technique that is used as a first-line diagnostic procedure for detecting and characterizing nodular thyroid disease. US permit to distinguish solid, cystic or mixed nodule, to evaluate ultrasound characteristic as hyperechogenicity , hypoechogenicity or isoechogenicity. the presence of some US aspects in the same thyroid nodule might have a higher likelihood of malignancy. These include: hypoechogenicity, irregular margins, microcalcifications, an absent halo, increased intranodular vascularity (Moon et al., 2008).

Elastography is an emerging and promising sonographic technique that requires additional validation with prospective studies (Machens et al., 2005; Rago et al., 2007).

#### **2.7.3 Fine-needle aspiration cytology (FNAC)**

Fine-needle aspiration cytology (FNAC) is the most accurate and cost-effective method for evaluating thyroid nodules. FNAC is not recommended in all nodules. The presence in the same nodule of 2 or more US characteristics above reported, recommended FNAC.

Cytology results should be included in the following diagnostic categories **(**Fadda et al., 2010**):** THYR 1: non diagnostic; THYR 2: negative for malignant cells; THYR 3: inconclusive /indeterminate (follicular proliferation); THYR 4: suspicion of malignancy; THYR 5: positive for malignant cells

THYR 1:.The "non diagnostic" can be classified as inadequate and/or non representative, depending on technical factor. *Operative suggestion.* FNAC repetition after at least one month from the previous one, according to the clinician's opinion.

THYR 2.This category accounts for 60-75% of all cytologic samples. Operative suggestion. FNAC repetition to reduce the false negative results, .if nodule growing during L-T4 treatment or modified US aspects

THYR 3.This category encompasses all follicular-patterned lesions: About 80% of the TIR 3 diagnoses are benign lesions whereas only 20% of them result as malignant tumors after surgery and histologic examination. Some immunohistochemical markers such as Galectin-3, HBME-1, Cytokeratin 19 may improve the accuracy of the cytologic diagnosis. Operative suggestion. Surgical excision of the lesion and histological examination. The surgical option should be evaluated in the clinical and imaging setting.

THYR 4.It represents an heterogeneous group of lesions. Are included in this category samples without a sufficient amount of malignant cells or without cytological atypias sufficient for a diagnosis of cancer. Operative suggestion. Surgery

TSh evaluation allows to identify hyperfunction nodule. The TSH determination should be performed in nodule with large size > 2.5-3 cm. Tc99 thyroid scintiscan allows to confirm the uptake of large nodule. Hyperfunctioning nodules rarely are malignant, therefore no other

US is a widespread technique that is used as a first-line diagnostic procedure for detecting and characterizing nodular thyroid disease. US permit to distinguish solid, cystic or mixed nodule, to evaluate ultrasound characteristic as hyperechogenicity , hypoechogenicity or isoechogenicity. the presence of some US aspects in the same thyroid nodule might have a higher likelihood of malignancy. These include: hypoechogenicity, irregular margins, microcalcifications, an absent halo, increased intranodular vascularity

Elastography is an emerging and promising sonographic technique that requires additional

Fine-needle aspiration cytology (FNAC) is the most accurate and cost-effective method for evaluating thyroid nodules. FNAC is not recommended in all nodules. The presence in the

Cytology results should be included in the following diagnostic categories **(**Fadda et al., 2010**):** THYR 1: non diagnostic; THYR 2: negative for malignant cells; THYR 3: inconclusive /indeterminate (follicular proliferation); THYR 4: suspicion of malignancy; THYR 5: positive

THYR 1:.The "non diagnostic" can be classified as inadequate and/or non representative, depending on technical factor. *Operative suggestion.* FNAC repetition after at least one month

THYR 2.This category accounts for 60-75% of all cytologic samples. Operative suggestion. FNAC repetition to reduce the false negative results, .if nodule growing during L-T4

THYR 3.This category encompasses all follicular-patterned lesions: About 80% of the TIR 3 diagnoses are benign lesions whereas only 20% of them result as malignant tumors after surgery and histologic examination. Some immunohistochemical markers such as Galectin-3, HBME-1, Cytokeratin 19 may improve the accuracy of the cytologic diagnosis. Operative suggestion. Surgical excision of the lesion and histological examination. The surgical option

THYR 4.It represents an heterogeneous group of lesions. Are included in this category samples without a sufficient amount of malignant cells or without cytological atypias

same nodule of 2 or more US characteristics above reported, recommended FNAC.

validation with prospective studies (Machens et al., 2005; Rago et al., 2007).

**2.7.1 Laboratory and thyroid scintigraphy** 

diagnostic procedure should be performed.

**2.7.3 Fine-needle aspiration cytology (FNAC)** 

from the previous one, according to the clinician's opinion.

should be evaluated in the clinical and imaging setting.

sufficient for a diagnosis of cancer. Operative suggestion. Surgery

**2.7.2 Thyroid ultrasound (US)** 

(Moon et al., 2008).

for malignant cells

treatment or modified US aspects

THYR 5.All cases with a diagnosis of malignant neoplasm (papillary, medullary and anaplastic carcinomas, lymphomas and metastasis) are included in this category Operative suggestion. Surgery for differentiated carcinomas. The results of FNAC are very sensitive for the differential diagnosis of benign and malignant nodules although there are limitations: inadequate samples and follicular neoplasia. In these cases the definitive diagnosis can be made only by histological examination

#### **2.8 Treatment**

#### **2.8.1 Neartotal or total thyroidectomy**

Near-total or total thyroidectomy is recommended if the primary thyroid carcinoma is >1 cm, multinodular goiter, regional or distant metastases at diagnosis, patient with personal history of radiation therapy to the head and neck, or patient with family history of DTCs. Older age (>45 years) may also be a criterion for recommending near-total or total thyroidectomy even with tumors <1–1.5 cm, because of higher recurrence rates in this age group. Increased extent of primary surgery may improve survival for high-risk patients and low-risk patients (Bilimoria et al., 2007)

#### **2.8.2 Lymph node dissection**

Regional lymph node metastases are frequently at diagnosis ranging from 20 to 90%. Although lymph node metastases in PTC patients are reported no clinically relevance on outcome in low risk patients, recently SEER registry study concluded that cervical lymph node metastases are a poor prognostic factor on survival in patients with FTC and in patients with PTC over 45 years (Zaydfudim et al., 2008). In experienced hands, therapeutic or prophylactic central compartment dissection can be achieved with low morbidity. In addition, selective unilateral paratracheal central compartment node dissection increases the proportion of patients who appear disease free.

#### **2.8.3 Risk staging**

Postoperative staging for thyroid cancer is used: 1) to permit prognostication for an individual patient with DTC; 2) to tailor decisions regarding postoperative adjunctive therapy, including RAI therapy and TSH suppression, to assess the patient's risk for disease recurrence and mortality; 3) to make decisions regarding the frequency and intensity of follow-up, directing more intensive follow-up towards patients at highest risk; and 4) to enable accurate communication regarding a patient among health care professionals. Varius risk definition for thyroid carcinoma have been evaluated. The American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) tumor-node-metastasis (TNM) classification has been used in clinical practice for DTC. This classification has also been evaluated to determine its utility in discriminating patients who have distinct outcomes. The fifth edition AJCC/UICC TNM classification (1997) was revised as the sixth edition in 2002 (Table 1).

In 2009, ATA has developed classes of risk in DTCs patients, to predict risk for recurrence, not death (ATA Surgery Working Group, 2009):

Thyroid Neoplasm 55

The TNM classification allows to stratify patients into four classes according to risk of death

**Stage T N M T N M I** Any T Any N M0 T1 N0 M0 **II** Anny T Any N M1 T2 N0 M0 **III** T3 N0 M0

**IVA** T4a N0 M0

**IVB** T4b Any N M0 **IVC** Any T Any N M1

Tuttle (Tuttle et al., 2008a) stratified risk of death into four categories: very low risk, low

 **Very low Low Intermediate High** 

< 45 years

of any size

>45 years

as above

resection Complete resection

Classic PTC > 4cm or extrathyroidal extention, or worrisome histology < 1-2 cm confined to the thyroid

T4a N1a M0

**Age < 45 years Age ≥ 45 years** 

T1 - T3 N1a M0

T1 - T4a N1b M0

Classic PTC > 4 cm or vascular invasion, or extrathyroidal extention, or worrisome histology

Histology in conjunction with age

absent Present or absent Present or

apparent None apparent Present

> 45 years

> 4 cm classic PTC

Worrisome histology > 1-2 cm

Incomplete tumor resection

absent

at 10 years (Table 2).

Table 2. TMN classification

**Age at** 

**Primary tomor**

**Histology** 

**Completennes of resection** 

**Lymph node** 

Table 3. Risk of death.

**Distant** 

risk, intermediate risk and high risk (table 3).

**diagnosis** < 45 years < 45 years

**size** <1 cm 1-4 cm

Classic PTC, confined to the thyroid gland

Complete resection

**involvement** None apparent Present or

**metastasis** None apparent None

Classic PTC, confined to the thyroid gland

Complete



Table 1. TMN 6th edition (IUCC 2002)

1. Low-risk patients have the following characteristics: 1) no local or distant metastases; 2) all macroscopic tumor has been resected; 3) there is no tumor invasion of locoregional tissues or structures; 4) the tumor does not have aggressive histology (e.g., tall cell, insular, columnar cell carcinoma) or vascular invasion; 5) and, if 131I is given, there is no 131I uptake outside the thyroid bed on the first post-treatment whole-body RAI scan

2. Intermediate-risk patients have any of the following:1) microscopic invasion of tumor into the perithyroidal soft tissues at initial surgery; 2) cervical lymph node metastases or 131I uptake outside the thyroid bed on the RxWBS done after thyroid remnant

3. High-risk patients have:1) macroscopic tumor invasion, 2) incomplete tumor resection, 3) distant metastases, and possibly 4) thyroglobulin out of proportion to what is seen on

Any tumor size with minimal extrathyroidal extention (soft perithyroid tissue or

subcutaneous soft tissues, larynx, trachea, esophagus or recurrent laryngeal nerve

T4b Tumor invades prevertebral fascia or encases carotid artery or mediastinal vessels

N1a Metastasis to level IV (pretracheal, paratracheal and prelaryngeal lymph nodes)

N1b Ipsilateral, controlateral or bilateral cervical lymph nodes metastases or superior

ablation or 3) tumor with aggressive histology or vascular invasion

(RxWBS)

the post-treatment scan

T0 Failure to evidence of primary tumor

sternocleidomastoid muscle)

Nx Regional lymph nodes can not be assessed

mediastinal lymph nodes metastases

Mx Distant metastasis can not be assessed

M0 No distant metastasis

M1 Presence of distant metastasis

Table 1. TMN 6th edition (IUCC 2002)

N0 Absence of lymph nodes metastases

N1 Metastasis to regional lymph nodes

T1 Tumor diameter ≤ 2 cm, limited to the thyroid

T3 Tumor diameter > 4 cm, limited to the thyroid

T2 Tumor diameter > 2 cm but < 4 cm, limited to the thyroid

T4a Any tumor size with extension beyond the thyroid capsule to invade


The TNM classification allows to stratify patients into four classes according to risk of death at 10 years (Table 2).

Table 2. TMN classification

Tuttle (Tuttle et al., 2008a) stratified risk of death into four categories: very low risk, low risk, intermediate risk and high risk (table 3).


Table 3. Risk of death.

Thyroid Neoplasm 57

Recent studies demonstrated ablation with lower doses than 100 mCi of I131. In fact the same ablation rate might be performed with 50 (Chianelli et al., 2009) and 30 mCi (Maenpaa et al., 2008) with rhTSh stimuli. These low doses reduce the radiation exposure to the whole body. Body weight or surface area should be evaluated for ablation in pediatric patients

Thyroid hormone suppression therapy has an important role during follow-up blocking the recurrence and metastasis progression. Several reports have shown that L-T4 suppressive treatment has usefull in patients with high-risk decreasing progression, recurrence rates, and cancer-related mortality (Mc Griff et al., 2002; Hovens et al., 2007). On the other hand, in patients with low-risk no significant improvement has been obtained by L-T4 suppressive therapy. The duration of suppression therapy is currently being debated. According to the current guidelines, low-risk patients free at the first follow-up might have replacement L-T4 therapy, with the goal of maintaining serum TSH level within the normal range. On the contrary, high risk patients even free at the follow-up should continue with suppressive L-T4 doses for the high risk of relapse (Jonklaas et al.,

1. Histology Histological characteristics have critical role of patient outcomes. The

 Encapsulated tumor: About 10% of PTC are completely surrounded by a dense fibrous capsule. The prognosis for this subtype is better than unencapsulated PTC. Diffuse sclerosing variant: Occurs in PTC of younger age, the diffuse sclerosing variant constitutes 2% of PTC. Prognosis for this subtype is less favorable than

Oxyphilic (Hürthle) cell type: The oxyphilic (Hürthle) cell type may be more

 Tall-cell carcinoma: Tall-cell carcinoma is a more aggressive form of thyroid carcinoma that differs from the usual form by showing tall columnar cells. Columnar cell carcinoma: Columnar cell carcinoma is a distinctly more aggressive form of PTC that occurs more often in older men and is associated with a poor

FTC is encapsulated, and invasion of the capsule and vessels is the key feature distinguishing follicular carcinomas from follicular adenomas. The subtypes are: 1) minimally invasive: good prognosis is a cancer with very low aggressiveness; widely invasive: cancer with poor prognosis for quick spreading of metastasis; 3) Hurthle-cell (oxyphilic follicular or oncocytic) carcinoma is a cytological variant of FTC with poor

Follicular variant: The follicular variant is less favorable than typical PTC.

(Franzius et al., 2007).

2006; Cooper et al., 2009)

**2.9.1 Tumour factors** 

typical PTC.

prognosis.

outcome

variants of PTC include the following:

aggressive than usual PTC.

**2.9 Prognosis** 

**2.8.5 Levo-thyroxine (L-T4) therapy** 

In accordance with this system, a European Consensus Report (ETA) defined three categories of risk to establish the indication for radioiodine ablation therapy (Pacini et al., 2006):


Recently, has been proposed an 'ongoing risk stratification' which takes into account the response to therapy (Tuttle et al., 2008). Patients can be classified as having an excellent, acceptable or incomplete response to therapy:


#### **2.8.4 Radioiodine ablation**

Surgery is usually followed by the administration of 131I activities aimed at ablating any remnant thyroid tissue and potential microscopic residual tumour. This procedure decreases the risk of locoregional recurrence and facilitates long-term surveillance based on serum Tg measurement and diagnostic radioiodine whole body scan (WBS). In addition the high activity of 131I allows obtaining a highly sensitive post-therapeutic WBS. Radioiodine ablation is recommended for all patients except those at very low risk. FTC and Hurthle cell cancer are generally regarded as higher risk tumors. On the contrary, "minimally invasive FTC", characterized only by capsular invasion, has an excellent prognosis with surgery alone and RAI ablation may not be required. Effective thyroid ablation requires adequate stimulation by TSH. The method of choice for preparation to perform radioiodine ablation is based on:


In accordance with this system, a European Consensus Report (ETA) defined three categories of risk to establish the indication for radioiodine ablation therapy (Pacini et al.,

1. very low-risk: unifocal T1 (<1 cm) N0 M0, no extension beyond the thyroid capsule,

2. low-risk : T1 (>1 cm) or T2 N0 M0 or multifocal T1 N0 M0, or unfavourable histology,

Recently, has been proposed an 'ongoing risk stratification' which takes into account the response to therapy (Tuttle et al., 2008). Patients can be classified as having an excellent,

1. Excellent response (undetectable basal and stimulated Tg, negative AbTg and negative neck US) patients should have a very low risk of recurrence and their long-term follow-

2. Acceptable response (undetectable basal Tg, stimulated Tg <10 ng/ml, trend of Tg in decline, AbTg absent or declining, substantially negative neck US) patients require a closer follow-up reserving additional treatment in the case of evidence of disease

3. Incomplete response (detectable basal and stimulated Tg, trend of Tg stable or rising, structural disease present, persistent or recurrent RAI-avid disease present) patients require continued intensive follow-up with neck ultrasound, cross-sectional imaging, RAI imaging and FDG-PET imaging. The majority of these patients will require additional therapy such as surgical resection, RAI therapy, external beam irradiation

Surgery is usually followed by the administration of 131I activities aimed at ablating any remnant thyroid tissue and potential microscopic residual tumour. This procedure decreases the risk of locoregional recurrence and facilitates long-term surveillance based on serum Tg measurement and diagnostic radioiodine whole body scan (WBS). In addition the high activity of 131I allows obtaining a highly sensitive post-therapeutic WBS. Radioiodine ablation is recommended for all patients except those at very low risk. FTC and Hurthle cell cancer are generally regarded as higher risk tumors. On the contrary, "minimally invasive FTC", characterized only by capsular invasion, has an excellent prognosis with surgery alone and RAI ablation may not be required. Effective thyroid ablation requires adequate stimulation by TSH. The method of choice for preparation to

1. Endogenous TSH elevation: can be achieved by thyroid hormone withdrawal, increasing serum TSH levels >30mU=L in more than 90% of patients. Patients affected by chronic kidney failure, heart failure, panhypopituitarism, ecc...might worse their

2. Administration of recombinant human TSH (rhTSH) rhTSH is helpful in patients with chronic diseases, able to increase tSH levels after L-T4 withdrawal, or intollerant

up will be based on yearly physical examination and suppressed Tg value.

favourable histology], no indication for radioiodine ablation,

3. high-risk: any T3 and T4 or any T, N1, or any M1, definite indication

2006):

probable indication

progression.

and systemic therapies.

perform radioiodine ablation is based on:

hypothyroidism (Tuttle et al., 2008b).

clinical status with thyroid hormone withdrawal

**2.8.4 Radioiodine ablation** 

acceptable or incomplete response to therapy:

Recent studies demonstrated ablation with lower doses than 100 mCi of I131. In fact the same ablation rate might be performed with 50 (Chianelli et al., 2009) and 30 mCi (Maenpaa et al., 2008) with rhTSh stimuli. These low doses reduce the radiation exposure to the whole body. Body weight or surface area should be evaluated for ablation in pediatric patients (Franzius et al., 2007).

#### **2.8.5 Levo-thyroxine (L-T4) therapy**

Thyroid hormone suppression therapy has an important role during follow-up blocking the recurrence and metastasis progression. Several reports have shown that L-T4 suppressive treatment has usefull in patients with high-risk decreasing progression, recurrence rates, and cancer-related mortality (Mc Griff et al., 2002; Hovens et al., 2007). On the other hand, in patients with low-risk no significant improvement has been obtained by L-T4 suppressive therapy. The duration of suppression therapy is currently being debated. According to the current guidelines, low-risk patients free at the first follow-up might have replacement L-T4 therapy, with the goal of maintaining serum TSH level within the normal range. On the contrary, high risk patients even free at the follow-up should continue with suppressive L-T4 doses for the high risk of relapse (Jonklaas et al., 2006; Cooper et al., 2009)

#### **2.9 Prognosis**

#### **2.9.1 Tumour factors**

	- Encapsulated tumor: About 10% of PTC are completely surrounded by a dense fibrous capsule. The prognosis for this subtype is better than unencapsulated PTC.
	- Diffuse sclerosing variant: Occurs in PTC of younger age, the diffuse sclerosing variant constitutes 2% of PTC. Prognosis for this subtype is less favorable than typical PTC.
	- Oxyphilic (Hürthle) cell type: The oxyphilic (Hürthle) cell type may be more aggressive than usual PTC.
	- Follicular variant: The follicular variant is less favorable than typical PTC.
	- Tall-cell carcinoma: Tall-cell carcinoma is a more aggressive form of thyroid carcinoma that differs from the usual form by showing tall columnar cells.
	- Columnar cell carcinoma: Columnar cell carcinoma is a distinctly more aggressive form of PTC that occurs more often in older men and is associated with a poor prognosis.

FTC is encapsulated, and invasion of the capsule and vessels is the key feature distinguishing follicular carcinomas from follicular adenomas. The subtypes are: 1) minimally invasive: good prognosis is a cancer with very low aggressiveness; widely invasive: cancer with poor prognosis for quick spreading of metastasis; 3) Hurthle-cell (oxyphilic follicular or oncocytic) carcinoma is a cytological variant of FTC with poor outcome

Thyroid Neoplasm 59

tumor stage (Garcia-Rostan et al., 2003).

potential for de-differentiation.

Disease-free status comprises all of the following:

**2.9.2 Patient variables** 

**2.10 Follow-up** 

patient is free of disease.

1. no clinical evidence of tumor,

interfering antibodies.

metastases, and is associated with poor prognosis among DTC independently

 RET/PTC: Ret/PTC1 is the most frequent (60–70%), while ret/PTC3 (20–30%) and ret/PTC2 are the least common (10%). RET/PTC rearrangements are associated with PTC that lacks evidence of progression to PDTC or ATC demonstrating a low

1. Age: Age at diagnosis and therapy is a critical predictor of patient outcome; patients aged >45 years have increased recurrence rates and reduced mortality. Children and adolescents (age <20 years) tend to present with higher-stage disease and greater likelihood of locoregional and distant metastases. Despite late-stage presentation of tumors, children generally have excellent survival rates. The exception to this rule is when the disease presents in children aged ≤10 years; in this age group, the disease is

notably more aggressive. Mortality is high in this group (Fugazzola et al., 2004). 2. Gender: As discussed above, mortality rates are higher among men than women even

The aim of the follow-up is the early discovery of persistent disease. Local recurrences develop in the first 5 years mainly, and only in a minority of cases local or distant recurrences develop 20 years after the initial treatment. Thyroid hormone FT3, FT4, TSH, should be evaluated 2-3 months after initial treatment to check the adequacy of LT4 suppressive therapy. At 6–12 months the first follow-up is aimed to ascertain whether the

2. no imaging evidence of tumor (no uptake outside the thyroid bed on the initial posttreatment WBS, or, if uptake outside the thyroid bed had been present, no imaging

3. undetectable serum Tg levels during TSH suppression and stimulation in the absence of

In the absence of Tg antibody the measurement of serum Tg levels is an important modality to monitor patients for residual or recurrent disease. Serum Tg has a high degree of sensitivity and specificity to detect thyroid cancer recurrences during thyroid hormone withdrawal or stimulation using rhTSH. Serum Tg measurements obtained during L-T4suppression therapy may fail to identify patients with relatively small amounts of residual tumor (Hovens et al., 2007). Nevertheless, a single rhTSH-stimulated serumTg<0.5 ng/mL in the absence of anti-Tg antibody has an approximately 98–99.5% likelihood of identifying patients completely free of tumor on follow-up (67). On the contrary, diagnostic-therapeutic procedures should be performed in patients with basal or after rhTSH stimuli Tg detectable values (value>2ng/ml). Diagnostic procedures comprise neck US, diagnostic I131 Whole Body Scan, Computer Tomography. Negative

evidence of tumor on a recent diagnostic scan and neck US), and

DTCs are more frequent in woman. Recurrence rates are also higher in men.

	- BRAF: The presence of a *BRAF* mutation is associated with extrathyroidal invasion, multicentricity, presence of nodal metastases, higher-stage disease, older age at initial presentation, and higher likelihood of recurrent or persistent disease. (Elisei et al., 2008). Further study is needed to clarify this complex issue.
	- RAS: Numerous studies have show that ras mutations define a subset of thyroid carcinoma characterized by aggressive behavior. This is indicated by the close relationship between oncogenic ras and the loss of those histologic features that characterize well-differentiated thyroid tumor phenotypes. Remarkably, oncogenic K-ras correlates with the loss of tumor differentiation, presence of distant

2. Tumour size: Tumor size correlates with outcome in patients with PTC; larger tumours are more likely to present with locoregional and/or distant metastases. However, the risk of recurrent disease and cancer-specific mortality increases linearly

3. Lymph node metastases: Cervical lymph nodes are involved in 20–50% of DTC, particularly PTC, and may be the first presentation. The frequency of micrometastases may approach 90%. Preoperative US identifies suspicious cervical adenopathy in 20–31% of cases, (Stulak et al., 2006). Confirmation of malignancy in lymph nodes with a suspicious sonographic appearance is achieved by US-guided FNA aspiration for cytology. Malignant lymph nodes are much more likely to occur in levels III, IV, and VI than in level II (Figure 2). Controversy exists over the clinical importance of lymph node metastases. Several studies have found no difference in survival between patients with and without lymph node metastases. Other studies have found that their presence leads to an increased risk of recurrence and reduced survival (Lee et al., 2009). The presence of lymph node metastases in patients <45 years has no effect on survival. On the contrary lymph nodes metastasis in patient

4. Extrathyroidal extension: The extension of cancer outside the thyroid into the surrounding tissues may be found in about 30% of patients with PTC (Mazzaferri, 2007). The massive extension out thyroid into the surrounding musculature, oesophagus or trachea is associated with a high-risk of locoregional disease recurrence. It requires massive surgical debulking and may benefit from external beam radiotherapy. Microscopic extension beyond the thyroid capsule is associated with a higher risk of recurrent disease, greater likelihood of lymph node metastases and a higher mortality rate than in patients without such extracapsular spread (Lee et

5. Distant metastases The main cause of death from DTC is distant metastases; fortunately, only 5–10% of patients have distant metastases at initial presentation. Over 50% have lung involvement alone, 25% have bone involvement alone, 20% have both lung and bone involvement, and about 5% develop distant metastases in other sites (Lee & Soh, 2010). Mortality is high with distant disease, with 50% survival at 3.5

6. Oncogenes The study of oncogenes and their ability to predict the clinical behaviour of thyroid cancers has been an exciting and intensely investigated field. Moreover, these researches have resulted in the creation of several new therapeutic agents to

 BRAF: The presence of a *BRAF* mutation is associated with extrathyroidal invasion, multicentricity, presence of nodal metastases, higher-stage disease, older age at initial presentation, and higher likelihood of recurrent or persistent disease. (Elisei

 RAS: Numerous studies have show that ras mutations define a subset of thyroid carcinoma characterized by aggressive behavior. This is indicated by the close relationship between oncogenic ras and the loss of those histologic features that characterize well-differentiated thyroid tumor phenotypes. Remarkably, oncogenic K-ras correlates with the loss of tumor differentiation, presence of distant

et al., 2008). Further study is needed to clarify this complex issue.

≥45 years are associated an increased risk of death.

years. Less frequently are liver metastasis.

target these genetic aberrations.

with tumour size.

al., 2009).

metastases, and is associated with poor prognosis among DTC independently tumor stage (Garcia-Rostan et al., 2003).

 RET/PTC: Ret/PTC1 is the most frequent (60–70%), while ret/PTC3 (20–30%) and ret/PTC2 are the least common (10%). RET/PTC rearrangements are associated with PTC that lacks evidence of progression to PDTC or ATC demonstrating a low potential for de-differentiation.

#### **2.9.2 Patient variables**


#### **2.10 Follow-up**

The aim of the follow-up is the early discovery of persistent disease. Local recurrences develop in the first 5 years mainly, and only in a minority of cases local or distant recurrences develop 20 years after the initial treatment. Thyroid hormone FT3, FT4, TSH, should be evaluated 2-3 months after initial treatment to check the adequacy of LT4 suppressive therapy. At 6–12 months the first follow-up is aimed to ascertain whether the patient is free of disease.

Disease-free status comprises all of the following:


In the absence of Tg antibody the measurement of serum Tg levels is an important modality to monitor patients for residual or recurrent disease. Serum Tg has a high degree of sensitivity and specificity to detect thyroid cancer recurrences during thyroid hormone withdrawal or stimulation using rhTSH. Serum Tg measurements obtained during L-T4suppression therapy may fail to identify patients with relatively small amounts of residual tumor (Hovens et al., 2007). Nevertheless, a single rhTSH-stimulated serumTg<0.5 ng/mL in the absence of anti-Tg antibody has an approximately 98–99.5% likelihood of identifying patients completely free of tumor on follow-up (67). On the contrary, diagnostic-therapeutic procedures should be performed in patients with basal or after rhTSH stimuli Tg detectable values (value>2ng/ml). Diagnostic procedures comprise neck US, diagnostic I131 Whole Body Scan, Computer Tomography. Negative

Thyroid Neoplasm 61

ATC accounts for less than 2% of all thyroid malignancies, it is responsible for 14%-39% of deaths related to malignant thyroid tumors. The female/male ratio is 5 to 1 and the peak of incidence is in the sixth and seventh decades of life. The age at diagnosis of ATC is over 70 years. The incidence of ATC is estimated at 1 to 2 cases per million population per year, and the trend has been decreasing even though the incidence of well-differentiated subtypes

Patients with ATC show goiter in 25% of cases, in 10% a family history of goiter. Therefore, ATC is more common in places with endemic goiter and iodine supplementation has

ATC may derive from DTCs, including PTC, FTC or Hurthle cell or may be "de novo". Several mutations are identified in ATC, some occurring in PTC and FTC (e.g., RAS and BRAF). Late mutations include p53, catenin (cadherin-associated protein), beta 1, and PIK3CA, suggesting that one or more of these mutations contribute to the extremely aggressive behavior of ATC. By contrast, the RET/PTC rearrangements found in childhood and radiation-induced PTCs, and the PAX8/PPARG fusion protein detected in follicular carcinoma, are not observed in poorly differentiated and ATCs. RAS and BRAF are the same described in DTCs. p53 is a tumor suppressor gene located in chromosome 17p that increases the cyclin kinase inhibitor, p21, promoting cell cycle arrest at G1/S. Mutations impair p53 transcriptional activity, and occur in 55% of ATC. Polymorphism in codon 72, identify only in ATC, but in no benign nodules or differentiated thyroid cancers, could be considered as a risk factor (Boltze C, 2002). Other gene as Wnt, a catenin beta 1 gene is involved in signaling and cell–cell adhesion, was detected in ATC and

Clinical manifestation of ATC is a nodule with a rapidly growth, enlarging anterior neck mass, accompanying dysphagia (40%), voice change or hoarseness (40%), and stridor (24%). Regional symptoms included a noticeable lymph node mass (54%) and neck pain (26%). Systemic symptoms include anorexia, weight loss, and shortness of breath with pulmonary metastases. ATC is usually advanced at diagnosis and frequently surgically unresectable. Around 20%-50% of patients present with distant metastases, most often pulmonary, and another 25% develop new metastasis during the rapid course of the disease, lungs (80%),

The median survival rate from 4 to 12 months. On multivariate analysis, distant or metastatic disease, tumor size greater than 7 centimeters, and treatment with surgery with or without radiotherapy were statistically significant prognostic markers(Chen et al.,

bone (6–16%), and brain (5–13%) were the most common sites of metastasis.

(e.g., papillary and follicular) of thyroid cancer has been increasing.

decrease the incidence of ATC in these countries (Besic N, 2010).

**4.1 Epidemiology** 

**4.2 Risk factors** 

**4.3 Genetic alteration** 

PDTC but not in PTC and FTC.

**4.4 Clinical presentation** 

**4.5 Prognosis** 

radiological imagines require a therapeutic dose of I131 to identified recurrences and the subsequent follow-up will be evaluated on post dose scintiscan. PET- TC 18 FDG sholud be performed in all patients with detectable Tg values and negative post therapeutic I 131 dose scintiscan. The identification of PET-TC positive lesions implies the de-differentation of metastasis.

The long-term follow-up differs on class of risk of recurrence. Very low-risk patients should be followed through physical examination, basal serum Tg measurement on substitutive LT4 therapy and neck US yearly if Tg values was undetectable at first follow-up. Low risk, Intermediate Risk and High-risk require Tg evaluation after rhTSH stimuli, neck US. Radiological techniques imagines if Tg increased up the cut-off, as above described. However, the recent introduction of an ultrasensitive Tg assay might reduce the need to perform Tg measurements after rhTSH-stimuli (Robert et al., 2007). About 25% of DTCs shows anti-Tg antibodies that could falsely lower serum Tg in immunometric assays (Cooper et al., 2009). Serial serum anti-Tg antibody quantification using the same methodology may serve as an imprecise surrogate marker of residual normal thyroid tissue or tumor. Patients with anti Tg antibodies should perform follow-up with I 131 Whole Body Scan. Suppressive doses of L-T4 with TSH levels under 0.1 mU/l reduce recurrence or progression of disease only in patients with high risk. Therefore, TSH levels <0.1 mU/l should be maintained until first follow-up and in high risk patients. Patients disease free and very low risk should take replacement doses.

#### **3. Poor differentiate thyroid carcinoma (PDTC)**

PDTC includes tumors of follicular origin that retain sufficient differentiation to produce Tg, histologically show scattered small follicular structures, but generally lack the usual morphologic characteristic of PTC or FTC. PDTC could be considered as an intermediate form of thyroid cancer with a prognosis that falls between DTCs and ATC (Sobrinho-Simoes et al., 2002). It is noteworthy that in the north half of Italy about 15% of thyroid cancer are PDTC whereas in North America the PDTC comprise only 2-3% of thyroid malignancy. These observations demonstrate that environmental factors may play a significant role in the genesis of these lesions, including iodine deficiency. PDTC may occur in a recurrence of a previously treated DTCs or at the time of diagnosis PDTC parts of tumor may show characteristics histological characteristics of PTC more rarely FTC. Clinically PDTC can produce Tg but do not respond to radioactive iodine. Therefore, therapy of PDTC can be inefficient by radioiodine. PDTC with extracapsular extension, massive lymph nodes involvement and no radioiodine up-take benefits of external radiotherapy (RT) after surgery, but no advantage to adjuvant RT to the neck in PDTC with diffuse metastasis. PDTC has poor sensitive to chemotherapy using different drugs as methotrexate, adriamycin, bleomycin and vinblastin. Response ranging from 55 to 70% when chemotherapy is associated to RT.

#### **4. Anaplastic thyroid carcinoma (ATC)**

Anaplastic thyroid carcinoma (ATC) is the most aggressive and lethal form of thyroid cancer with a median survival of 4 to 12 months from the time of diagnosis.

radiological imagines require a therapeutic dose of I131 to identified recurrences and the subsequent follow-up will be evaluated on post dose scintiscan. PET- TC 18 FDG sholud be performed in all patients with detectable Tg values and negative post therapeutic I 131 dose scintiscan. The identification of PET-TC positive lesions implies the de-differentation

The long-term follow-up differs on class of risk of recurrence. Very low-risk patients should be followed through physical examination, basal serum Tg measurement on substitutive LT4 therapy and neck US yearly if Tg values was undetectable at first follow-up. Low risk, Intermediate Risk and High-risk require Tg evaluation after rhTSH stimuli, neck US. Radiological techniques imagines if Tg increased up the cut-off, as above described. However, the recent introduction of an ultrasensitive Tg assay might reduce the need to perform Tg measurements after rhTSH-stimuli (Robert et al., 2007). About 25% of DTCs shows anti-Tg antibodies that could falsely lower serum Tg in immunometric assays (Cooper et al., 2009). Serial serum anti-Tg antibody quantification using the same methodology may serve as an imprecise surrogate marker of residual normal thyroid tissue or tumor. Patients with anti Tg antibodies should perform follow-up with I 131 Whole Body Scan. Suppressive doses of L-T4 with TSH levels under 0.1 mU/l reduce recurrence or progression of disease only in patients with high risk. Therefore, TSH levels <0.1 mU/l should be maintained until first follow-up and in high risk patients. Patients disease free

PDTC includes tumors of follicular origin that retain sufficient differentiation to produce Tg, histologically show scattered small follicular structures, but generally lack the usual morphologic characteristic of PTC or FTC. PDTC could be considered as an intermediate form of thyroid cancer with a prognosis that falls between DTCs and ATC (Sobrinho-Simoes et al., 2002). It is noteworthy that in the north half of Italy about 15% of thyroid cancer are PDTC whereas in North America the PDTC comprise only 2-3% of thyroid malignancy. These observations demonstrate that environmental factors may play a significant role in the genesis of these lesions, including iodine deficiency. PDTC may occur in a recurrence of a previously treated DTCs or at the time of diagnosis PDTC parts of tumor may show characteristics histological characteristics of PTC more rarely FTC. Clinically PDTC can produce Tg but do not respond to radioactive iodine. Therefore, therapy of PDTC can be inefficient by radioiodine. PDTC with extracapsular extension, massive lymph nodes involvement and no radioiodine up-take benefits of external radiotherapy (RT) after surgery, but no advantage to adjuvant RT to the neck in PDTC with diffuse metastasis. PDTC has poor sensitive to chemotherapy using different drugs as methotrexate, adriamycin, bleomycin and vinblastin. Response ranging from 55 to 70% when

Anaplastic thyroid carcinoma (ATC) is the most aggressive and lethal form of thyroid cancer

with a median survival of 4 to 12 months from the time of diagnosis.

of metastasis.

and very low risk should take replacement doses.

chemotherapy is associated to RT.

**4. Anaplastic thyroid carcinoma (ATC)** 

**3. Poor differentiate thyroid carcinoma (PDTC)** 

#### **4.1 Epidemiology**

ATC accounts for less than 2% of all thyroid malignancies, it is responsible for 14%-39% of deaths related to malignant thyroid tumors. The female/male ratio is 5 to 1 and the peak of incidence is in the sixth and seventh decades of life. The age at diagnosis of ATC is over 70 years. The incidence of ATC is estimated at 1 to 2 cases per million population per year, and the trend has been decreasing even though the incidence of well-differentiated subtypes (e.g., papillary and follicular) of thyroid cancer has been increasing.

#### **4.2 Risk factors**

Patients with ATC show goiter in 25% of cases, in 10% a family history of goiter. Therefore, ATC is more common in places with endemic goiter and iodine supplementation has decrease the incidence of ATC in these countries (Besic N, 2010).

#### **4.3 Genetic alteration**

ATC may derive from DTCs, including PTC, FTC or Hurthle cell or may be "de novo". Several mutations are identified in ATC, some occurring in PTC and FTC (e.g., RAS and BRAF). Late mutations include p53, catenin (cadherin-associated protein), beta 1, and PIK3CA, suggesting that one or more of these mutations contribute to the extremely aggressive behavior of ATC. By contrast, the RET/PTC rearrangements found in childhood and radiation-induced PTCs, and the PAX8/PPARG fusion protein detected in follicular carcinoma, are not observed in poorly differentiated and ATCs. RAS and BRAF are the same described in DTCs. p53 is a tumor suppressor gene located in chromosome 17p that increases the cyclin kinase inhibitor, p21, promoting cell cycle arrest at G1/S. Mutations impair p53 transcriptional activity, and occur in 55% of ATC. Polymorphism in codon 72, identify only in ATC, but in no benign nodules or differentiated thyroid cancers, could be considered as a risk factor (Boltze C, 2002). Other gene as Wnt, a catenin beta 1 gene is involved in signaling and cell–cell adhesion, was detected in ATC and PDTC but not in PTC and FTC.

#### **4.4 Clinical presentation**

Clinical manifestation of ATC is a nodule with a rapidly growth, enlarging anterior neck mass, accompanying dysphagia (40%), voice change or hoarseness (40%), and stridor (24%). Regional symptoms included a noticeable lymph node mass (54%) and neck pain (26%). Systemic symptoms include anorexia, weight loss, and shortness of breath with pulmonary metastases. ATC is usually advanced at diagnosis and frequently surgically unresectable. Around 20%-50% of patients present with distant metastases, most often pulmonary, and another 25% develop new metastasis during the rapid course of the disease, lungs (80%), bone (6–16%), and brain (5–13%) were the most common sites of metastasis.

#### **4.5 Prognosis**

The median survival rate from 4 to 12 months. On multivariate analysis, distant or metastatic disease, tumor size greater than 7 centimeters, and treatment with surgery with or without radiotherapy were statistically significant prognostic markers(Chen et al.,

Thyroid Neoplasm 63

Achieving local control is important since death from ATC is usually a consequence of uncontrolled local disease. The indication for RadioTherapy (RT) range from providing palliation to improving survival. RT is used alone or in combined with surgery and chemotherapy. Intensity-modulated radiation therapy (IMRT) based on computerized treatment planning and delivery is able to generate a dose distribution that delivers radiation accurately with sparing of the surrounding normal tissue (Lee et al., 2007). Higher doses of radiation can be given over a shorter time with less toxicity by employing hyperfractionation techniques. Toxicity can be a limiting factor with radiation. Kim and Leeper reported complications particularly, pharyngoesophagitis and tracheitis in their series. Wong also noted skin changes, esophageal toxicity, and radiation myelopathy (Wong et al., 2001). Daily doses of greater than 3 Gy should be cautiously used as it can increase the

PDTC and ATC are poor sensitive to radioiodione, chemotherapy and RT alone or associate. The knowledges on genetic transformation and the intracellular pathway involved in thyroid cancer transformation has permitted to develop target drugs. Therefore, interest arose in the therapeutic potential of target-specific kinase inhibitors for these diseases. Angiogenesis plays a critical role to support tumor cell growth and metastasis, supplying nutrients and oxygen, removing waste products, and facilitating

RET and VEGFR kinases have considerable similarity structural and multitargeted kinase inhibitors are capable of affecting both kinases. A wide variety of kinase inhibitors have entered clinical trials for patients with advanced thyroid cancers, PDTC or ATC. Because of the targeting similarities of many of these agents, common toxicities exist among these

1. Motesanib (AMG706): is an oral, tyrosine kinase inhibitor targeting the VEGF receptors

2. Sorafenib (BAY 43-9006): is an oral, small molecule tyrosin-kinase inhibitor (TKI) and targeting VEGF receptors 2 and 3, RET and BRAF. Like other agents that inhibit BRAF, sorafenib also has been associated with development of cutaneous squamous cell carcinomas in up to 5% of treated patients, and a similar frequency of keratoacanthomas and other premalignant actinic lesions (Dubauskas et al., 2009). In a recent retrospective series, sorafenib therapy was associated with prolongation of median progression-free survival by at least 1 year, compared with patients' rate of disease progression prior to initiation of therapy (Cabanillas et al., 2010). A randomized, placebo-controlled phase III study of sorafenib as first-line therapy for progressive metastatic DTC has been initiated. Although not specifically approved for thyroid carcinomas, sorafenib is being used in selected patients with PDTC and medullary thyroid carcinoma for whom clinical trials are not appropriate

agents, including hypertension, diarrhea, skin rashes, and fatigue.

**4.6.3 Radiation** 

incidence of myelopathy.

**5. New treatments** 

distant metastasis.

1, 2, and 3.

**5.1 Targeting signaling kinases** 

2008). Age, sex, size of the tumor, resectability, and the extent of disease has been shown to affect the course of the disease. Age less than 60 years, female sex, tumor size less than 7 centimeters, were the most favourable prognostic markers (Kim et al., 2007). A recent study from France based on 26 patients with ATC, univariate analysis showed that age above 75, capsular invasion, lymph nodes metastasis, tumor residue after surgery, and lack of multimodal treatment (particularly radiotherapy in patients without tumor residue) are poor prognosistic factors. Multivariate analysis in the same cohort showed age above 75, followed by node invasion, capsular invasion, and female sex to be poor prognosticators (Roche B, 2010).

#### **4.6 Therapeutic approach**

Patients with ATC even in the absence of metastatic disease are considered to have systemic disease at the time of diagnosis. ATC is considered stage IV by the International Union Against Cancer (UICC)—TNM staging and American Joint Commission on Cancer (AJCC) system. Multimodality treatment consisting of surgery when feasible combined with radiation and chemotherapy is generally recommended.

#### **4.6.1 Surgery**

The aim of surgery in ATC, whether it is removal of all gross disease or palliation, remains controversial. Complete resection has been identified as a prognostic factor in several clinical trials. When feasible, surgery must aim at a radical intent. The categories of patients that may be most suitable for this approach are young patients (< 65 years old) with small lesions (< 6 cm) and no distant metastasis. However, surgery also plays an important role for palliation. Partial resection of the tumor followed by radiotherapy and chemotherapy may delay or avoid airway obstruction, although it can improve survival only by a few months (Miccoli et al., 2007). It is theoretically possible that, in selected patients, even in the setting of metastatic disease, surgery may result in an improved quality of life and prevent death from suffocation (Yau et al., 2008). Since surgery alone is not able to control the disease even in patients with small intra-thyroidal masses, adjuvant therapy is always required, and can be administered either with radiotherapy (RT) or chemoradiotherapy.

#### **4.6.2 Systemic treatment**

Chemotherapy: ATC cannot be regarded as a very chemo-sensitive tumor. Doxorubicin is not able to achieve more than a 20% response rate. A study (Shimaoka et al., 1985) has observed that combination chemotherapy based on doxorubicin (60 mg/m2) and cisplatin (40 mg/m2) was more effective than doxorubicin alone and provided a higher complete response rate. More recently, single drug docetaxel was tested as first-line chemotherapy in patients with advanced ATC. In a prospective phase II clinical trial of paclitaxel, showed a remarkable response rate of 53% (Schoenberger et al., 2004). In a preclinical experiment only paclitaxel, gemcitabine and vinorelbine appeared to be active in ATC (Bauer et al., 2003) and the combinations of vinorelbine/gemcitabine and paclitaxel/gemcitabine seemed to act synergistically. These results should receive confirmation in clinical trials.

2008). Age, sex, size of the tumor, resectability, and the extent of disease has been shown to affect the course of the disease. Age less than 60 years, female sex, tumor size less than 7 centimeters, were the most favourable prognostic markers (Kim et al., 2007). A recent study from France based on 26 patients with ATC, univariate analysis showed that age above 75, capsular invasion, lymph nodes metastasis, tumor residue after surgery, and lack of multimodal treatment (particularly radiotherapy in patients without tumor residue) are poor prognosistic factors. Multivariate analysis in the same cohort showed age above 75, followed by node invasion, capsular invasion, and female sex to be poor

Patients with ATC even in the absence of metastatic disease are considered to have systemic disease at the time of diagnosis. ATC is considered stage IV by the International Union Against Cancer (UICC)—TNM staging and American Joint Commission on Cancer (AJCC) system. Multimodality treatment consisting of surgery when feasible combined with

The aim of surgery in ATC, whether it is removal of all gross disease or palliation, remains controversial. Complete resection has been identified as a prognostic factor in several clinical trials. When feasible, surgery must aim at a radical intent. The categories of patients that may be most suitable for this approach are young patients (< 65 years old) with small lesions (< 6 cm) and no distant metastasis. However, surgery also plays an important role for palliation. Partial resection of the tumor followed by radiotherapy and chemotherapy may delay or avoid airway obstruction, although it can improve survival only by a few months (Miccoli et al., 2007). It is theoretically possible that, in selected patients, even in the setting of metastatic disease, surgery may result in an improved quality of life and prevent death from suffocation (Yau et al., 2008). Since surgery alone is not able to control the disease even in patients with small intra-thyroidal masses, adjuvant therapy is always required, and can be administered either with radiotherapy (RT) or

Chemotherapy: ATC cannot be regarded as a very chemo-sensitive tumor. Doxorubicin is not able to achieve more than a 20% response rate. A study (Shimaoka et al., 1985) has observed that combination chemotherapy based on doxorubicin (60 mg/m2) and cisplatin (40 mg/m2) was more effective than doxorubicin alone and provided a higher complete response rate. More recently, single drug docetaxel was tested as first-line chemotherapy in patients with advanced ATC. In a prospective phase II clinical trial of paclitaxel, showed a remarkable response rate of 53% (Schoenberger et al., 2004). In a preclinical experiment only paclitaxel, gemcitabine and vinorelbine appeared to be active in ATC (Bauer et al., 2003) and the combinations of vinorelbine/gemcitabine and paclitaxel/gemcitabine seemed to act

synergistically. These results should receive confirmation in clinical trials.

prognosticators (Roche B, 2010).

radiation and chemotherapy is generally recommended.

**4.6 Therapeutic approach** 

**4.6.1 Surgery** 

chemoradiotherapy.

**4.6.2 Systemic treatment** 

#### **4.6.3 Radiation**

Achieving local control is important since death from ATC is usually a consequence of uncontrolled local disease. The indication for RadioTherapy (RT) range from providing palliation to improving survival. RT is used alone or in combined with surgery and chemotherapy. Intensity-modulated radiation therapy (IMRT) based on computerized treatment planning and delivery is able to generate a dose distribution that delivers radiation accurately with sparing of the surrounding normal tissue (Lee et al., 2007). Higher doses of radiation can be given over a shorter time with less toxicity by employing hyperfractionation techniques. Toxicity can be a limiting factor with radiation. Kim and Leeper reported complications particularly, pharyngoesophagitis and tracheitis in their series. Wong also noted skin changes, esophageal toxicity, and radiation myelopathy (Wong et al., 2001). Daily doses of greater than 3 Gy should be cautiously used as it can increase the incidence of myelopathy.

#### **5. New treatments**

PDTC and ATC are poor sensitive to radioiodione, chemotherapy and RT alone or associate. The knowledges on genetic transformation and the intracellular pathway involved in thyroid cancer transformation has permitted to develop target drugs. Therefore, interest arose in the therapeutic potential of target-specific kinase inhibitors for these diseases. Angiogenesis plays a critical role to support tumor cell growth and metastasis, supplying nutrients and oxygen, removing waste products, and facilitating distant metastasis.

#### **5.1 Targeting signaling kinases**

RET and VEGFR kinases have considerable similarity structural and multitargeted kinase inhibitors are capable of affecting both kinases. A wide variety of kinase inhibitors have entered clinical trials for patients with advanced thyroid cancers, PDTC or ATC. Because of the targeting similarities of many of these agents, common toxicities exist among these agents, including hypertension, diarrhea, skin rashes, and fatigue.


Thyroid Neoplasm 65

5. **Azacytidine and decitabine**  A broad array of tumor suppressor genes is hypermethylated in PTC and FTC leading to their decreased expression, including *PTEN*, tissue inhibitor of metalloproteinase-3, and death-associated protein kinase . A phase II trial of 5-azacytidine monotherapy to restore radioiodine uptake was initiated, but results were never reported. Given the greater potency and tolerance of the azacytidine derivative decitabine, a phase II trial of this latter agent has been underway, evaluating the ability to restore radioiodine uptake in radioiodine-non-avid metastases;

MTC arises from C cells o parafollicular cells which produce and secrete Calcitonin (Ct) . MTC represents about 5-10% of all thyroid cancers and 13.4% of all thyroid-related deaths.

Several biochemical features typical of normal C cells (production of Ct) are retained by neoplastic C cells and represent specific and sensitive diagnostic markers. Ct is a small peptide (32 amino acids) coded by a gene located on the short arm of chromosome 11, the gene codes a second peptide called CT-gene-related peptide (CGRP). Carcinoembryonic antigen (CEA) is produced by neoplastic C cells. There is no close relationship between serum concentrations of CEA and CT. Serum CEA concentration is normal in patients with preclinical MTC and does not increase after pentagastrin stimulation. Measurement of serum CEA concentration is useful during follow-up because high concentrations or rapidly

MTC is mainly in sporadic form, but an hereditary pattern is present in 20–30% of cases, transmitted as an autosomal-dominant trait (Schlumberger & Pacini, 2006).The hereditary form is also referred to as 'multiple endocrine neoplasia type 2' (MEN 2), characterized by MTC in combination with pheochromocytoma and hyperparathyroidism (MEN 2A), or MTC in combination with pheochromocytoma, multiple mucosal neuromas and marfanoid habitus (MEN 2B). The occurrence of familial MTC (FMTC) in the absence of other

Patients with sporadic MTC usually present with a palpable thyroid nodule indistinguishable from any other thyroid nodule. Clinical neck lymph node metastases are detected in at least 50% of patients and may reveal the disease. Metastases outside the neck, in liver, lungs or

Survival rates for MTC are impacted by age of diagnosis and stage of disease

responses were identified in any tumor type.

results of this multicenter trial are expected shortly.

increasing concentrations indicate disease progression.

**6. Medullary thyroid carcinoma (MTC)** 

**6.1 Epidemiology** 

**6.2 Secretory products** 

**6.3 Clinical presentation** 

neoplasias is also possible.

**6.3.1 Sporadic MTC** 

enzymes. An ongoing phase II trial is evaluating the effect of monotherapy with VPA on tumor size and radioiodine uptake in patients with radioiodine-refractory PDTC. One PTC patient had prolonged stable disease beyond 1 year, but no objective

(Waguespack et al., 2009). Compared with patients' rate of disease progression prior to initiation of therapy, sorafenib may prolong progression-free survival in DTC by at least 1 year (Cabanillas et al., 2010). The drug may also be appropriate in selected pediatric cases; in 1 report, treatment with sorafenib yielded a marked response in a child whose lung metastases from PTC were progressing despite radioiodine therapy (Waguespack et al., 2009).


#### **5.2 Other drugs**

Beyond direct inhibitors of angiogenic kinases such as VEGFR, other drugs are capable of either inhibiting angiogenesis or disrupt existing tumor vasculature. Two of these agents, thalidomide and fosbretabulin (combretastatin A4 phosphate), have been of particular interest following reported responses in individual patients with ATC .


3. Sunitinib (SU11248): is an oral, small molecule TKI of all 3 VEGF receptors, RET, and

4. Axitinib (AG-013736): is an oral inhibitor that effectively blocks VEGF-1, -2 and -3. Partial response was seen in patients refractory radioiodine. Currently ongoing is a multicenter, open-label phase II study to determine the efficacy of axitinib in patients with metastatic DTC refractory to doxorubicin, or for whom doxorubicin therapy is

5. Pazopanib: is a potent small molecule inhibitor of all VEGFR subtypes as well as PDGFR. Like axitinib, it has insignificant inhibitory activity against the oncogenic kinases RET, RET/PTC, or BRAF, and therefore its actions are expected to be primarily

6. Gefitinib (ZD1839): is an oral, small molecule inhibitor of the EGF receptor, was initially introduced for treatment of non-small cell lung carcinoma. Because many PDTC and ATC display activated EGFR signaling, and inhibitors have had demonstrated efficacy in preclinical models, an open-label phase II study was initiated, examining the effectiveness of gefitinib in a mixed cohort of thyroid cancer

Beyond direct inhibitors of angiogenic kinases such as VEGFR, other drugs are capable of either inhibiting angiogenesis or disrupt existing tumor vasculature. Two of these agents, thalidomide and fosbretabulin (combretastatin A4 phosphate), have been of particular

1. **Thalidomide and lenalidomide** Thalidomide was found to be an angiogenesis inhibitor decades after it achieved notoriety as a teratogenic cause of neonatal dysmelia. Eligibility was limited to PDTC patients whose measured tumor volumes had increased

2. **Combretastatin** A4 phosphate (CA4P): is a tubulin-binding vascular disrupting agent that inhibits tumor blood flow. In a phase ⎹I⎸trial, one patient with ATC showed a progression-free survival of 30 mo, however, the drug was found to be associated with

3. **Romidepsin** The cyclic peptide romidepsin (previously known as depsipeptide) selectively inhibits four isotypes of histone deacetylases. Toxicities were primarily hematologic, nausea, and vomiting. A phase II trial was initiated in patients with radioiodine-unresponsive, PDTC. Romidepsin induces stable disease and in few subjects exhibited restoration of uptake permitting therapeutic radioiodine

4. **Vorinostat and valproic acid (VPA)** The orally available histone deacetylase (HDAC) inhibitor vorinostat, derived from hydroxamic acid, inhibits all known classes of HDAC

significant cardiovascular side effects at the escalating doses employed.

interest following reported responses in individual patients with ATC .

(Waguespack et al., 2009).

anti-angiogenic in thyroid carcinoma.

patients (Mrozek et al., 2006).

by at least 30% in the past year.

administration.

contraindicated.

**5.2 Other drugs** 

RET/PTC subtypes 1 and 3 (Pasqualetti et al., 2011)

(Waguespack et al., 2009). Compared with patients' rate of disease progression prior to initiation of therapy, sorafenib may prolong progression-free survival in DTC by at least 1 year (Cabanillas et al., 2010). The drug may also be appropriate in selected pediatric cases; in 1 report, treatment with sorafenib yielded a marked response in a child whose lung metastases from PTC were progressing despite radioiodine therapy enzymes. An ongoing phase II trial is evaluating the effect of monotherapy with VPA on tumor size and radioiodine uptake in patients with radioiodine-refractory PDTC. One PTC patient had prolonged stable disease beyond 1 year, but no objective responses were identified in any tumor type.

5. **Azacytidine and decitabine**  A broad array of tumor suppressor genes is hypermethylated in PTC and FTC leading to their decreased expression, including *PTEN*, tissue inhibitor of metalloproteinase-3, and death-associated protein kinase . A phase II trial of 5-azacytidine monotherapy to restore radioiodine uptake was initiated, but results were never reported. Given the greater potency and tolerance of the azacytidine derivative decitabine, a phase II trial of this latter agent has been underway, evaluating the ability to restore radioiodine uptake in radioiodine-non-avid metastases; results of this multicenter trial are expected shortly.

#### **6. Medullary thyroid carcinoma (MTC)**

#### **6.1 Epidemiology**

MTC arises from C cells o parafollicular cells which produce and secrete Calcitonin (Ct) . MTC represents about 5-10% of all thyroid cancers and 13.4% of all thyroid-related deaths. Survival rates for MTC are impacted by age of diagnosis and stage of disease

#### **6.2 Secretory products**

Several biochemical features typical of normal C cells (production of Ct) are retained by neoplastic C cells and represent specific and sensitive diagnostic markers. Ct is a small peptide (32 amino acids) coded by a gene located on the short arm of chromosome 11, the gene codes a second peptide called CT-gene-related peptide (CGRP). Carcinoembryonic antigen (CEA) is produced by neoplastic C cells. There is no close relationship between serum concentrations of CEA and CT. Serum CEA concentration is normal in patients with preclinical MTC and does not increase after pentagastrin stimulation. Measurement of serum CEA concentration is useful during follow-up because high concentrations or rapidly increasing concentrations indicate disease progression.

#### **6.3 Clinical presentation**

MTC is mainly in sporadic form, but an hereditary pattern is present in 20–30% of cases, transmitted as an autosomal-dominant trait (Schlumberger & Pacini, 2006).The hereditary form is also referred to as 'multiple endocrine neoplasia type 2' (MEN 2), characterized by MTC in combination with pheochromocytoma and hyperparathyroidism (MEN 2A), or MTC in combination with pheochromocytoma, multiple mucosal neuromas and marfanoid habitus (MEN 2B). The occurrence of familial MTC (FMTC) in the absence of other neoplasias is also possible.

#### **6.3.1 Sporadic MTC**

Patients with sporadic MTC usually present with a palpable thyroid nodule indistinguishable from any other thyroid nodule. Clinical neck lymph node metastases are detected in at least 50% of patients and may reveal the disease. Metastases outside the neck, in liver, lungs or

Thyroid Neoplasm 67

3. **Familial Medullary Thyroid Carcinoma (FMTC):** FMTC have only hereditary MTC. Clinical presentation of MTC at a later age, 60-70 years old, and a relatively more favourable prognosis respect the others hereditary forms. It is still debated whether FMTC represents a separate syndrome or a variant of MEN 2A in which the genetic component is modified to delay the onset of the array of manifestations typifying the

The predisposing gene for inherited MTC was the RET proto-oncogene localized to centromeric chromosome 10 , identified by genetic linkage analysis in 1987, and germline mutations of were demonstrated in 1993 in MEN 2A, FMTC and MEN 2B. The RET gene is a 21-exon gene that encodes a tyrosine kinase receptor. This membrane-associated receptor is characterised by a cadherin-like region in the extracellular domain, a cysteine-rich region immediately external to the membrane, and an intracellular tyrosine kinase domain. Hereditary MTC is caused by germline autosomal-dominant gain-of-function mutations in the RET proto-oncogene. About 98% of patients with MEN 2 have germline mutations in exons 5, 8, 10, 11, 13, 14, 15 or 16 of the RET gene. Mutations causing MEN 2A affect the cysteine-rich extracellular domain with substitution of a cysteine to another amino acid in exon 10 and, and more commonly (80%), in exon 11. In about 95% of patients with MEN 2B, a single mutation converting methionine to threonine in codon 918 of exon 16 has been identified. It is frequently (>50%) a de-novo mutation in the allele inherited from the patient's father. Other rare intracellular mutations associated with MEN 2B involve exon 15. Rare patients with MEN 2B phenotype have double *RET* mutations. Germline mutations induce different tirosin-kinasi activity. Strong activation of the *RET* proto-oncogene is associated with a more aggressive form of MTC, and mutations providing weaker *RET* activation result in a less aggressive and late-onset form of the disease. On the basis of these findings, the American Thyroid Association (ATA) has recently developed an MTC risk

1. Level D mutations carry the highest risk for MTC. These mutations include codons 883 and 918, and are associated with the youngest age of onset and highest risk of

2. Level C mutations carry a lower, yet still high, risk of aggressive MTC, and include

3. Level B mutations carry a lower risk for aggressive MTC mutations, and include

4. Level A mutations carry the 'least high' risk, and include *RET* gene mutations in codons 768, 790, 791, 804 and 891. This system may be used to individualise the aggressiveness

Somatic mutations in codon 918 of the *RET* proto-oncogene have been identified in 25–33% of sporadic MTC, and may be associated with a poor outcome compared with sporadic tumours without *RET* mutation (Elisei et al., 2008). Mutations in codons 618, 634, 768, 804

and 883 and partial deletion of the *RET* gene have been identified in a few tumours

stratification based on genotype (Kloos et al., 2009) (Table 4.)

mutations at *RET* codons 609, 611, 618, 620 and 630.

metastases and disease-specific mortality.

mutations in codon 634.

of treatment.

**6.4.2 Somatic mutations** 

MEN 2A syndrome.

**6.4 Genetic alterations 6.4.1 Germline mutations** 

bones, are initially present in 10–20% of cases. Flushing and diarrhea might occur in the presence of liver metastasis. FNAC has made it possible to diagnose MTC prior to surgery. However, cytology may be misleading and, in case of doubt, positive immunocytochemical staining for Ct, Ct measurement in the washout fluid of FNAC or both (Kudo et al., 2007) will confirm the diagnosis. Patients with clinical MTC have elevated basal circulating Ct concentrations. Whenever MTC is suspected, staging and careful clinical screening for pheochromocytoma and hyperparathyroidism should be carried out before surgery.

#### **6.3.2 Hereditary MTC**


bones, are initially present in 10–20% of cases. Flushing and diarrhea might occur in the presence of liver metastasis. FNAC has made it possible to diagnose MTC prior to surgery. However, cytology may be misleading and, in case of doubt, positive immunocytochemical staining for Ct, Ct measurement in the washout fluid of FNAC or both (Kudo et al., 2007) will confirm the diagnosis. Patients with clinical MTC have elevated basal circulating Ct concentrations. Whenever MTC is suspected, staging and careful clinical screening for

1. **Multiple Endocrine Neoplasia Type 2A (MEN2A):** MEN 2A is a syndrome characterized by MTC, pheochromocytoma and hyperparathyroidism. Clinically MTC develops in about 100% of patients affected by this syndrome. Pheochromocytoma occurs in about 50% of *MEN 2A* patients depending on the type of gene mutation. Hyperparathyroidism occurs in 10–25% of known *MEN 2A* gene carriers with a mutation in codon 634, usually after the third decade of life (Leboulleux et al., 2002). Clinical MTC is rarely observed under 10 years of age; prevalence increases with age, and is 25% at 13 years and about 70% at 70 years. The pentagastrin stimulation test is positive in about 20% of gene carriers at 10 years of age; this increases with age to 50% at 13 years, 65% at 20 years and 95% at 30 years. At present, genetic testing is carried out before 5 years of age in all subjects at risk to establish which individuals are gene carriers. Pheochromocytomas are located in an adrenal gland, and very few cases have been observed in the retroperitoneal region. It is bilateral in 50% of cases, but often after an interval of several years. Pheochromocytoma is almost always benign. Hyperparathyroidism consists of parathyroid hyperplasia, with one or more adenomas in older patients, develops slowly and is usually mild. Clinical and biochemical features do not differ from those seen in sporadic hyperparathyroidism.Cutaneous lichen amyloidosis is a pruritic and hyperpigmented lesion of the skin on the upper portion of the back has been observed in some families with MEN 2A. This lesion may occur early in life and often precedes C-cell disease. Hirschsprung's disease has been observed in a few families with MEN 2A. Patient with multiple endocrine neoplasia type 2A with lichen cutaneous amyloidosis over the interscapular area. The patient reported intense

2. **Multiple Endocrine Neoplasia Type 2B (MEN2B):** MEN2B is a syndrome with MTC, pheochromocytoma, ganglioneuromatosis, marfanoid features and skeletal abnormalities. MTC associated with MEN 2B is the most aggressive form of MTC and occurs early in life, usually before the age of 5 years. It is frequently associated with extension beyond the thyroid capsule, with lymph node and distant metastases at the time of diagnosis. Pheochromocytomas are identified in about one-half of the individuals presenting with the syndrome. Mucosal neuromas are a typical feature of MEN 2B. They occur on the distal portion of the tongue, on the lips, throughout the intestinal tract and eventually in the urinary tract. These patients also have chronic constipation and colonic cramping due to the presence of megacolon disorder. Hypertrophy of corneal nerves is frequent and is evaluated by slit lamp ophthalmic examination. Marfanoid features include long, thin extremities, an altered upper-tolower body ratio and ligament hyperlaxity. Skeletal abnormalities are frequent,

pheochromocytoma and hyperparathyroidism should be carried out before surgery.

**6.3.2 Hereditary MTC** 

pruritus in this area since 3 years of age.

including slipped femoral epiphysis and pectus excavatum.

3. **Familial Medullary Thyroid Carcinoma (FMTC):** FMTC have only hereditary MTC. Clinical presentation of MTC at a later age, 60-70 years old, and a relatively more favourable prognosis respect the others hereditary forms. It is still debated whether FMTC represents a separate syndrome or a variant of MEN 2A in which the genetic component is modified to delay the onset of the array of manifestations typifying the MEN 2A syndrome.

#### **6.4 Genetic alterations**

#### **6.4.1 Germline mutations**

The predisposing gene for inherited MTC was the RET proto-oncogene localized to centromeric chromosome 10 , identified by genetic linkage analysis in 1987, and germline mutations of were demonstrated in 1993 in MEN 2A, FMTC and MEN 2B. The RET gene is a 21-exon gene that encodes a tyrosine kinase receptor. This membrane-associated receptor is characterised by a cadherin-like region in the extracellular domain, a cysteine-rich region immediately external to the membrane, and an intracellular tyrosine kinase domain. Hereditary MTC is caused by germline autosomal-dominant gain-of-function mutations in the RET proto-oncogene. About 98% of patients with MEN 2 have germline mutations in exons 5, 8, 10, 11, 13, 14, 15 or 16 of the RET gene. Mutations causing MEN 2A affect the cysteine-rich extracellular domain with substitution of a cysteine to another amino acid in exon 10 and, and more commonly (80%), in exon 11. In about 95% of patients with MEN 2B, a single mutation converting methionine to threonine in codon 918 of exon 16 has been identified. It is frequently (>50%) a de-novo mutation in the allele inherited from the patient's father. Other rare intracellular mutations associated with MEN 2B involve exon 15. Rare patients with MEN 2B phenotype have double *RET* mutations. Germline mutations induce different tirosin-kinasi activity. Strong activation of the *RET* proto-oncogene is associated with a more aggressive form of MTC, and mutations providing weaker *RET* activation result in a less aggressive and late-onset form of the disease. On the basis of these findings, the American Thyroid Association (ATA) has recently developed an MTC risk stratification based on genotype (Kloos et al., 2009) (Table 4.)


#### **6.4.2 Somatic mutations**

Somatic mutations in codon 918 of the *RET* proto-oncogene have been identified in 25–33% of sporadic MTC, and may be associated with a poor outcome compared with sporadic tumours without *RET* mutation (Elisei et al., 2008). Mutations in codons 618, 634, 768, 804 and 883 and partial deletion of the *RET* gene have been identified in a few tumours

Thyroid Neoplasm 69

Before surgery, all patients with suspicious MTC should undergo a staging work-up. The goal of pre-operative evaluation is to define the extent of disease and to identify the comorbid conditions of hyperparathyroidism, pheochromocytoma or both in the case of hereditary forms. The pre-operative biochemical evaluation should include basal serum Ct, CEA, calcium and 24-h urine collection for metanephrines and normetanephrines determination. Pre-operative imaging, including neck US, should be carried out in all patients; pre-operative chest and neck computed tomography. The primary treatment of both hereditary and sporadic forms of MTC is total thyroidectomy and removal of all neoplastic tissue present in the neck. Several studies have shown that survival in patients with MTC is dependent upon the adequacy of the initial surgical procedure. Multicentric and bilateral MTC is observed in 30% of sporadic cases and in nearly 100% of hereditary cases. The therapeutic option for lymph node surgery should be dictated by the results of presurgical evaluation. Patients with no clinical or imaging evidence of lymph node metastases should undergo prophylactic central compartment (level VI) neck dissection. This strategy will probably include about 30–40% of patients with MTC. In the remaining patients lymph node involvement are documented before surgery. Lymph node metastases may occur in 20–30% of cases with tumours <1 cm in diameter, in 50% of patients with a tumour 1–4 cm in diameter, and in up to 90% in patients with a tumour >4

After total thyroidectomy, replacement thyroxine treatment is given to maintain the serum TSH value into the normal range. Measurement of the serum marker Ct and CEA is of paramount importance in the postsurgical follow-up of patients with MTC because this reflects the presence of persistent or recurrent disease. The half-life of serum Ct is reported to be about 30 h. An undetectable basal serum Ct level after surgery is a strong predictor of complete remission. Complete remission may be further confirmed if the serum Ct level remains undetectable after a provocative (pentagastrin or calcium) test. In this situation, no other diagnostic test is indicated. Serum Ct should be repeated every 6 months for the first 2–3 years and annually thereafter. Patients with biochemical remission after initial treatment have only a 3% chance of recurrence during long-term follow-up. On the contrary, if basal serum Ct is detectable or becomes detectable after stimulation, the patient is not cured. Radiological imagines comprise neck US, because frequently recurrence are in locoregional lymph nodes, FNAC with Ct measurement in the washout fluid should typically be carried out to confirm the diagnosis when US demonstrates suspicious lymph nodes enlargement. Chest CT, abdominal MRI, bone scintigraphy, 18 Fluorodeoxyglucose (FDG) positron emission tomography (PET) and 18- F-dihydroxyphenylalanine (DOPA) PET when there is suspicious of diffuse metastasis. These imaging techniques will be positive when Ct levels are high >150 pg/ml. In patients with serum CT <150 pg/ml, clinical evaluation of disease should be limited to neck US and a careful every 6 months follow up with Ct and CEA determinations (Laure Giraudet et al., 2008). Patients with detectable basal serum Ct and no evidence of disease, long-term surveillance is indicated. Pain by bone metastases rapidly improvements with local RT

**6.5 Therapeutic approach** 

**6.5.1 Initial treatment** 

cm or with a T4 tumour.

**6.5.2 Postoperative management** 


Table 4. MTC risk stratification based on genotype by American Thyroid Association (ATA)

Exon Mutation Phenotype ATA risk level\*

FMTC/MEN 2A FMTC/MEN 2A A A A A

A A A B B B B

B B B B C C A A A

A A A A

A A D D A A A

A D A

A D

B D

FMTC/MEN 2A

FMTC/MEN 2A FMTC/MEN 2A

FMTC/MEN 2A FMTC/MEN 2A FMTC/MEN 2A FMTC/MEN 2A

FMTC/MEN 2A

FMTC/MEN 2A FMTC/MEN 2A FMTC/MEN 2A FMTC/MEN 2A FMTC/MEN 2A FMTC/MEN 2A FMTC/MEN 2A

FMTC/MEN 2A FMTC/MEN 2A FMTC/MEN 2A FMTC/MEN 2A

FMTC/MEN 2A FMTC/MEN 2A

FMTC/MEN 2A MEN 2B FMTC/MEN 2A

FMTC/MEN 2A

FMTC/MEN 2A MEN 2B/MEN 2A

MEN 2B

Table 4. MTC risk stratification based on genotype by American Thyroid Association (ATA)

MEN 2B MEN 2B FMTC FMTC FMTC

FMTC

FMTC

FMTC

5 G321R FMTC/MEN 2A A

8

10

11

13

14

15

13/14 14/15 C515S G533C

R600Q K603E Y606C

532 duplication

C609F/R/G/S/Y C611R/G/F/S/W/Y C618R/G/F/S/Y C620R/G/F/S/W/Y

C630R/F/S/Y D631Y

C634G/F/S/W/Y

C634R

S649L K666E

E768D N776S L790F Y791F

V804L V804M

G819K R833C R844Q

R866W A883F S891A

V804M+V778I V804M+S904C

<sup>16</sup>R912P M918T

V804M+E805K V804M+Y806C

531/9 base pair duplication

633/9 base pair duplication 634/12 base pair duplication

635/insertion ELCR; T636P

#### **6.5 Therapeutic approach**

#### **6.5.1 Initial treatment**

Before surgery, all patients with suspicious MTC should undergo a staging work-up. The goal of pre-operative evaluation is to define the extent of disease and to identify the comorbid conditions of hyperparathyroidism, pheochromocytoma or both in the case of hereditary forms. The pre-operative biochemical evaluation should include basal serum Ct, CEA, calcium and 24-h urine collection for metanephrines and normetanephrines determination. Pre-operative imaging, including neck US, should be carried out in all patients; pre-operative chest and neck computed tomography. The primary treatment of both hereditary and sporadic forms of MTC is total thyroidectomy and removal of all neoplastic tissue present in the neck. Several studies have shown that survival in patients with MTC is dependent upon the adequacy of the initial surgical procedure. Multicentric and bilateral MTC is observed in 30% of sporadic cases and in nearly 100% of hereditary cases. The therapeutic option for lymph node surgery should be dictated by the results of presurgical evaluation. Patients with no clinical or imaging evidence of lymph node metastases should undergo prophylactic central compartment (level VI) neck dissection. This strategy will probably include about 30–40% of patients with MTC. In the remaining patients lymph node involvement are documented before surgery. Lymph node metastases may occur in 20–30% of cases with tumours <1 cm in diameter, in 50% of patients with a tumour 1–4 cm in diameter, and in up to 90% in patients with a tumour >4 cm or with a T4 tumour.

#### **6.5.2 Postoperative management**

After total thyroidectomy, replacement thyroxine treatment is given to maintain the serum TSH value into the normal range. Measurement of the serum marker Ct and CEA is of paramount importance in the postsurgical follow-up of patients with MTC because this reflects the presence of persistent or recurrent disease. The half-life of serum Ct is reported to be about 30 h. An undetectable basal serum Ct level after surgery is a strong predictor of complete remission. Complete remission may be further confirmed if the serum Ct level remains undetectable after a provocative (pentagastrin or calcium) test. In this situation, no other diagnostic test is indicated. Serum Ct should be repeated every 6 months for the first 2–3 years and annually thereafter. Patients with biochemical remission after initial treatment have only a 3% chance of recurrence during long-term follow-up. On the contrary, if basal serum Ct is detectable or becomes detectable after stimulation, the patient is not cured. Radiological imagines comprise neck US, because frequently recurrence are in locoregional lymph nodes, FNAC with Ct measurement in the washout fluid should typically be carried out to confirm the diagnosis when US demonstrates suspicious lymph nodes enlargement. Chest CT, abdominal MRI, bone scintigraphy, 18 Fluorodeoxyglucose (FDG) positron emission tomography (PET) and 18- F-dihydroxyphenylalanine (DOPA) PET when there is suspicious of diffuse metastasis. These imaging techniques will be positive when Ct levels are high >150 pg/ml. In patients with serum CT <150 pg/ml, clinical evaluation of disease should be limited to neck US and a careful every 6 months follow up with Ct and CEA determinations (Laure Giraudet et al., 2008). Patients with detectable basal serum Ct and no evidence of disease, long-term surveillance is indicated. Pain by bone metastases rapidly improvements with local RT

Thyroid Neoplasm 71

Boltze, C.; Roessner, A. & Schneider-Stock, R. (2002). Homozygous proline at codon 72 of

Capezzone, M.; Cantara, S. & Pacini, F. (2011). Telomere Length in Neoplastic and

Castagna, MG.; Brilli, L. & Pacini, F. (2008). Limited value of repeat recombinant thyrotropin

Castro, P.; Rebocho, AP. & Sobrinos-Simoes, M. (2006). PAX8-PPARgamma rearrangement

Cetta, F.; Chiappetta, G. & Fusco, A. (1998). The ret/ptc1 oncogene is activated in familial

Chen, J.; Tward, JD. & Hitchcock, YJ. (2008). Surgery and radiotherapy improves survival in

Chianelli, M.; Todino, V. & Papini, E. (2009). Low dose (2.0 GBq; 54 mCi) radioiodine

Cooper, DS.; Doherty, GM. & Tuttle, RM. (2009). Revised American Thyroid Association

Davies, H.; Bignel GR & Futreal, PA. (2002).Mutations of the BRAF gene in human cancer.

Di Cristofaro, J.; Marcy, M. & De Micco, C. (2006). Molecular genetic study comparing

Dubauskas, Z.; Kunishige, J. & Tannir, N. (2009). Cutaneous squamous cell carcinoma and

Edwards, B.; Ward, E. & Ries, LAG. (2010). Annual report to the nation on the status of

thyroid cancer. Thyroid, Vol. 19, No. 1167–1214, ISNN.

*Nature*, Vol.417, No.6892, pp.949–54, ISSN.

Cancer. *J Clin Endocrinol Metab,* 2011 Aug 24. (Epub ahead of print)

*Endocrinol Metab*, Vol.91, No.1 , pp.213-220, ISSN.

*Metab*. Vol.83, No.3, pp.1003–6, ISSN.

Vol.31, No.5, pp.460–464, ISSN.

Vol. 7, No.1 , pp. 20–23, ISSN.

No.3, pp.544–573, ISSN.

pp.431–436, ISSN.

ISSN.

pp. 6060, ISSN.

ISSN.

p53 as a potential risk factor favoring the development of undifferentiated thyroid carcinoma. *International Journal of Oncology,* Vol.21, No.5 , pp.1151–1154, ISSN. Cabanillas, ME.; Waguespack, SG. & Busaydi, NL. (2010). Treatment with tyrosine kinase

inhibitors for patients with differentiated thyroid cancer. *J Clin Oncol*, Vol.27, N0.6,

Nonneoplastic Tissues of Patients with Familial and Sporadic Papillary Thyroid

(rhTSH)- stimulated thyroglobulin testing in differentiated thyroid carcinoma patients with previous negative rhTSHstimulated thyroglobulin and undetectable basal serumthyroglobulin levels. J Clin Endocrinol Metab, Vol.93, No.1, pp.76–81,

is frequently detected in the follicular variant of papillary thyroid carcinoma. *J Clin* 

adenomatous polyposis-associated thyroid papillary carcinomas*. J. Clin. Endocrinol.* 

patients with anaplastic thyroid carcinoma: analysis of the surveillance, epidemiology, and end results 1983–2002. *American Journal of Clinical Oncology*,

postsurgical remnant ablation in thyroid cancer: comparison between hormone withdrawal and use of rhTSH in low risk patients. *Eur J Endocrinol*, Vol.160, No.3,

management guidelines for patients with thyroid nodules and differentiated

follicular variant versus classic papillary thyroid carcinomas: association of N-ras mutation in codon 61 with follicular variant. Hum. *Pathol,* Vol.37, No.7, pp. 824–30,

inflammation of actinic keratoses associated with sorafenib. *Clin Genitourin Cancer*,

cancer, 1975-2006, featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates. Cancer, Vol.116,

and it can also be useful for brain metastases. In patients with predominant liver involvement, embolisation or chemo-embolisation proved to be efficient for some symptomatic benefit and for partial reduction in tumour mass (Fromigué et al., 2006).

#### **6.5.3 Novel chemotherapy**

Traditional chemotherapy is inefficient in metastatic MTC. New strategies to treat metastates of MTC are being evaluated and include radio-immunotherapy and vaccinebased therapies (Kraeber-Bodere et al., 2009). Improved understanding of MTC molecular oncogenesis has resulted in identification of novel molecular targets for treatment, and there has been recent focus on the use of compounds inhibiting receptors of intracellular kinases. These new therapies primarily target RET oncogene and angiogenesis and have entered clinical trials for metastatic MTC. Partial response rates of up to 30% have been reported in single-agent studies, but prolonged disease stabilization is seen more commonly. The most successful agents target the vascular endothelial growth factor receptors (VEGFRs), with focus on tyrosine kinase inhibitors; these compounds include motesanib diphosphate, vandetanib, sorafenib, and sunitinib (Sherman et al., 2009). In a phase I trial, (Kurzrock et al., 2010) treated patients with metastatic MTC with XL184, an oral inhibitor of MET, VEGFR2, and RET that exhibits anti-angiogenic, antiproliferative, and anti-invasive effects. A phase III trial, comparing XL184 with placebo, is now underway. Wells et al. (Wells et al., 2010) describe an open-label, phase III study that assessed the efficacy of vandetanib, a selective inhibitor of RET, VEGFR, and epidermal growth factor receptor. A total of 30 patients with unresectable locally advanced or metastatic hereditary MTC were enrolled. By response evaluation criteria in solid tumors (RECIST), 20% of patients experienced a confirmed partial response and an additional 53% of patients experienced stable disease at 24 weeks; this yielded a disease control rate of 73%. In addition, vandetanib had a tolerable adverse event profile, as well as significant progression-free survival prolongation when compared to placebo.

#### **7. References**


and it can also be useful for brain metastases. In patients with predominant liver involvement, embolisation or chemo-embolisation proved to be efficient for some symptomatic benefit and for partial reduction in tumour mass (Fromigué et al., 2006).

Traditional chemotherapy is inefficient in metastatic MTC. New strategies to treat metastates of MTC are being evaluated and include radio-immunotherapy and vaccinebased therapies (Kraeber-Bodere et al., 2009). Improved understanding of MTC molecular oncogenesis has resulted in identification of novel molecular targets for treatment, and there has been recent focus on the use of compounds inhibiting receptors of intracellular kinases. These new therapies primarily target RET oncogene and angiogenesis and have entered clinical trials for metastatic MTC. Partial response rates of up to 30% have been reported in single-agent studies, but prolonged disease stabilization is seen more commonly. The most successful agents target the vascular endothelial growth factor receptors (VEGFRs), with focus on tyrosine kinase inhibitors; these compounds include motesanib diphosphate, vandetanib, sorafenib, and sunitinib (Sherman et al., 2009). In a phase I trial, (Kurzrock et al., 2010) treated patients with metastatic MTC with XL184, an oral inhibitor of MET, VEGFR2, and RET that exhibits anti-angiogenic, antiproliferative, and anti-invasive effects. A phase III trial, comparing XL184 with placebo, is now underway. Wells et al. (Wells et al., 2010) describe an open-label, phase III study that assessed the efficacy of vandetanib, a selective inhibitor of RET, VEGFR, and epidermal growth factor receptor. A total of 30 patients with unresectable locally advanced or metastatic hereditary MTC were enrolled. By response evaluation criteria in solid tumors (RECIST), 20% of patients experienced a confirmed partial response and an additional 53% of patients experienced stable disease at 24 weeks; this yielded a disease control rate of 73%. In addition, vandetanib had a tolerable adverse event profile, as well as significant

progression-free survival prolongation when compared to placebo.

*Neuroendocrinology,* Vol.83, No.3-4, pp.189–99, ISNN.

ATA Surgery Working Group. (2009).Consensus Statement on the Terminology and

Bauer, AJ.; Patel, A. & Francis, GL. (2003). Systemic administration of vascular endothelial

Besic, N.; Hocevar, M. & Zgajnar J. (2010). Lower incidence of anaplastic carcinoma after higher iodination of salt in slovenia. *Thyroid*, Vol.20, No.6, pp.623–626, ISSN. Bilimoria, KY.; Bentrem, DJ. & Sturgeon, C. (2007). Extent of surgery affects survival for papillary thyroid cancer. Ann Surg, Vol.246, No.3, pp.375–381, ISSN. Boikos, SA. & Stratakis, CA. (2006). Carney complex: pathology and molecular genetics.

Classification of Central Neck Dissection for Thyroid Cancer. *Thyroid*, Vol.19,

growth factor monoclonal antibody reduces the growth of papillary thyroid carcinoma in a nude mouse model. *Ann Clin Lab Sci*, Vol.33, No.2 , pp.192–199,

**6.5.3 Novel chemotherapy** 

**7. References** 

ISSN.

No.11, pp.1153– 1158.


Thyroid Neoplasm 73

Kloos, RT.; Eng, C. & Wells, SA Jr. (Jun 2009). American Thyroid Association Guidelines

Kosary, CL. (2007). Cancer Survival Among Adults: U.S. SEER Program, 1988-2001, Patient

Kudo, T.; Miyauchi, A. & Hirokawa, M. (2007). Diagnosis of medullary thyroid carcinoma

Kurzrock, R. & Cohen, EE. (2010). Long-term results in a cohort of medullary thyroid cancer

Leboulleux, S.; Travagli JP. & Baudin, E. (2002). Medullary thyroid carcinoma as part of a

Lee, J. & Soh, EY. (2010). Differentiated thyroid carcinoma presenting with distant

Lee, N.; Puri, DR. & Blanco Chao, KS. Intensity-modulated radiation therapy in head and neck cancers: an update. *Head & Neck*, Vol.29, No.4, pp.387–400, ISSN. Lee, SH.; Lee, SS. & Rho, YS. (2008). Predictive factors for central compartment lymph node

Lind, P.; Langsteger, W. & Gomez, I. (1998). Epidemiology of thyroid diseases in iodine

Machens, A,; Holzhausen, HJ. & Dralle, H. (2005). The prognostic value of primary tumor

Maenpaa, HO.; Heikkonen, J. & Joensuu, H. (2008). Low vs. high radioiodine activity to

Mazzaferri, EL. (2007). Management of low-risk differentiated thyroid cancer. *Endocr Pract*,

Meinkoth JL. (2004). Biology of Ras in thyroid cells. *Cancer Treat Res*, Vol.122, pp.131-148,

Miccoli, P.; Materazzi, G. & Berti, P. (2007). New trends in the treatment of undifferentiated

Moon, WJ.; Jung, SL. & Lee, DH. (2008). Benign and malignant thyroid nodules:US

Thyroid Association. *Thyroid*, Vol. 19, No. 6, pp. 565–612, ISSN.

pp.16:3–8, ISSN.

659–662, ISSN.

2273, ISSN.

ISSN.

770, ISSN.

Vol.3, No.4, pp.e1885, ISSN.

Vol. 13, No.5, pp.498–512, ISSN.

Vol.17, No.7, pp. 635–638, ISSN.

Vol.251, No.1, pp. 114–119, ISSN.

*Clin Oncol,* Vol.28, No.15, pp.5502-5505, ISSN.

clinical course. *Cancer*, Vol 94, No.1, pp. 44–50,ISSN.

sufficiency. *Thyroi* , Vol.8, No.12, pp.1179-83, ISSN.

Program, NIH No. 07-6215, Bethesda, MD. *http://www.seer.cancer.gov.* Kraeber-Bodere, F.; Goldenberg, DM. & Barbet, J. (2009). Pretargeted radioimmunotherapy

Task Force, Medullary thyroid cancer: management guidelines of the American

and Tumor Characteristics. In: SEER Survival Monograph. LAG Ries, JL Young, GE Keel, MP Eisner, YD Lin and MJ Horner. U.S., National Cancer Institute, SEER

in the treatment of metastatic medullary thyroid cancer. *Curr Oncol,* Vol.116, No.4,

by calcitonin measurement in fine-needle aspiration biopsy specimens. *Thyroid*,

patients in a phase I study of SL184, an oral inhibitor of MET, VEGFR2, and RET. *J* 

multiple endocrine neoplasia type 2B syndrome: influence of the stage on the

metastasis at initial diagnosis: clinical outcomes and prognostic factors. *Ann Surg* ,

metastasis in thyroid papillary microcarcinoma. *Laryngoscope*, Vol.118, No.4, pp.

size in papillary and follicular thyroid carcinoma. *Cancer*, Vol.103, No.11, pp.2269–

ablate the thyroid after thyroidectomy for cancer: a randomized study. *PLoS One*,

carcinomas of the thyroid. *Langenbecks Arch Surg*, Vol.392, No.4 , pp. 397–404, ISSN.

differentiation multicenter retrospective study. *Radiology*, Vol. 247, No.3, pp.762


Elisei, R.; C. Ugolini, C. & Basolo, F. (2008). BRAF(V600E) mutation and outcome of patients

Elisei, R.; Cosci, B. & Pinchera, A. (Mar 2008) Prognostic significance of somatic RET

Fadda, G.; Basolo, F. &Palombini, L. (2010). Cytological classification of thyroid nodules.

Farahati, J.; Geling, M. &. Reiners, C. (2004). Changing trends of incidence and prognosis of

Franzius, C.; Dietlein, M. & Schober, O. (2007). Procedure guideline for radioiodine therapy

Fromigué, J.; De Baere, T. & Schlumberger, M. (2006). Chemoembolization for liver

Fugazzola, L.; Mannavola, D. & Beck-Peccoz P . (2004). BRAF mutations in an Italian cohort of thyroid cancers, *Clin Endocrinol (Oxf)*, Vol.61, No. 2, pp. 239–243, ISSN. Garcia-Rostan, G.; Zhao, H. & Tallini, G. (2003). ras Mutations Are Associated With

Gomez Segovia, I.; Gallowitsch, HJ. & Lind, P. (2004). Descriptive epidemiology of thyroid

Hemmings, CT. (2003). Thyroid pathology in four patients with Cowden's disease.

Hemminki, K.; Eng, C. & Chen, B. (2005). Familial risks for nonmedullary thyroid cancer. *J.* 

Hou, P.; Liu, D. & Xing M. (2007). Genetic alterations and their relationship in the

Hovens, GC.; Stokkel, MP. & Smit, JW. (2007). Association of serum thyrotropin

Kilfoy, BA.; Zheng, T & Zhang, I. (2009). International patterns and trends in thyroid cancer incidence, 1973–2002. *Cancer Causes Control*, Vol.20, No.5, pp. 525–531, ISSN. Kim, TY.; Kim, KW. & Shong, YK. (2007). Prognostic factors for korean patients with anaplastic thyroid carcinoma. *Head & Neck*, Vol.29, No.8, pp.765–772, ISSN.

phosphatidylinositol 3-kinase/akt pathway in thyroid cancer. *Clin. Cancer Res*,

concentration with recurrence and death in differentiated thyroid cancer. J Clin

study. *J Clin Endocrinol Metab,* Vol. 93, No.3, pp. 682–687, ISSN.

thyroid cancer. *Nuklearmedizin*, Vol.46, No.5, pp.224–231, ISSN.

*Clinical Oncology*, Vol 21, No.17, pp.3226-3235, ISSN.

*Clin. Endocrinol. Metab,* Vol.90, No.10, pp.5747–53, ISSN.

Endocrinol Metab, Vol. 92, No. 7, pp.2610–2615, ISSN.

*Endocrinol Metab*, Vol.93, No.10, pp. 3943–3949.

Vol.12, No.5, pp.405-408, ISSN.

No.2, pp. 141-7, ISSN.

pp. 2496–2499, ISSN.

Vol.14, No.4, pp.277-86, ISSN.

Vol.13,No. 4, pp.1161–70, ISSN.

*Pathology.* Vol.35, No.4, pp.311–4, ISSN.

with papillary thyroid carcinoma: a 15-year median follow-up study. *J Clin* 

oncogene mutations in sporadic medullary thyroid cancer: a 10-year follow-up

Proposal of the SIAPEC-IAP Italian Consensus Working Group. Phatologyca,

thyroid carcinoma in lower Franconia, Germany, from 1981–1995. *Thyroid* , Vol.14,

and 131iodine whole-body scintigraphy in paediatric patients with differentiated

metastases from medullary thyroid carcinoma. *J Clin Endocrinol Metab.* Vol.91, No.7,

Aggressive Tumor Phenotypes and Poor Prognosis in Thyroid Cancer. *Journal of* 

carcinoma in Carinthia, Austria: 1984–2001. Histopathologic features and tumor classification of 734 cases under elevated general iodination of table salt since 1990: population based age-stratified analysis on thyroid carcinoma incidence. *Thyroid,*


Thyroid Neoplasm 75

Sherman SI. (2009). Advances in chemotherapy of differentiated epithelial and medullary thyroid cancers. *J Clin Endocrinol Metab*, Vol.94, No.5, pp.1493–1499, ISSN. Shimaoka, K.; Schoenfeld, DA. & De Conti, R. (1985). A randomized trial of doxorubicin

Sobrinho-Simoes, M.; Sambade, C. & (2002). Poorly differentiated carcinomas of the thyroid

Spencer, CA. (2004). Challenges of serum thyroglobulin (thyroglobulin) measurement in the

Stulak, JM.; Grant, CS. & Charboneau, JW. (2006). Value of preoperative ultrasonography in

Tuttle, RM.; Brokhin, M. & Robbins, RJ. (2008). Recombinant human TSH-assisted

Tuttle, RM.; Leboeuf, R. & Shaha, AR. (2008). Medical management of thyroid cancer: a risk

Waguespack, SG.; Sherman, SI. & Herzog, CE. (2009). The successful use of sorafenib to treat pediatric papillary thyroid carcinoma. *Thyroid*, Vol.19, No.4, pp. 407–412, ISSN. Wang, Y.; Hou, P. & Xing, M. (2007). High prevalence and mutual exclusivity of genetic

Wells, SA.; Gosnell, JE. & Schlumberger, M. (2010). Vandetanib for the treatment of patients

Williams D. (2008). Radiation carcinogenesis: lessons from Chernobyl*. Oncogene.* Vol.27,

Wong, CS.; Van Dyk, J. & Simpson WJ. (1991). Myelopathy following hyperfractionated

Xing M. (2005). BRAF mutation in thyroid cancer. *Endocr Relat Cancer*, Vol.12, No.2, pp.245-

Xing, M.; Westra, WH & Ladenson, PW. (2005). BRAF mutation predicts a poorer clinical

Yau, T.; Lo, CY. & Lang, BH. (2008). Treatment outcomes in anaplastic thyroid carcinoma:

adapted approach. *J Surg Oncol*, Vol.97, No.8, pp.712-716, ISSN.

*Clin. Endocrinol. Metab*, Vol.92, No.6, pp.2387–90, ISSN.

*Oncol*, Vol.28, No.15, pp.767–772, ISSN.

*Oncology*, Vol.20, No.1, pp.3–9, ISSN.

*Cancer*, Vol.56, No.9, pp.2155–2160, ISSN.

Vol.10, No.2, pp.123-131, ISSN.

*Surg*, Vol.141, No.5, pp.489–494ISSN.

pp.3702–3704, ISSN.

No.5, pp.764–770, ISSN.

No.2, pp.S9-18, ISSN.

262, ISSN.

2505, ISSN.

pp.6373–9, ISSN.

versus doxorubicin plus cisplatin in patients with advanced thyroid carcinoma.

gland: a review of the clinicopathologic features of a series of 28 cases of a heterogeneous, clinically aggressive group of thyroid tumors. Int J Surg Pathol,

presence of thyroglobulin autoantibodies. J Clin Endocrinol Metab, Vol.89, No.8,

the surgical management of initial and reoperative papillary thyroid cancer. *Arch* 

radioactive iodine remnant ablation achieves short-term clinical recurrence rates similar to those of traditional thyroid hormone withdrawal. *J Nucl Med*, Vol.49,

alterations in the phosphatidylinositol-3-kinase/akt pathway in thyroid tumors. *J.* 

with locally advanced or metastatic hereditary medullary thyroid cancer. *J Clin* 

accelerated radiotherapy for anaplastic thyroid carcinoma. *Radiotherapy and* 

prognosis for papillary thyroid cancer*. J. Clin. Endocrinol. Metab*, Vol.90, No.12,

survival improvement in young patients with localized disease treated by combination of surgery and radiotherapy. *Ann Surg Oncol*, Vol.15, No.9, pp.2500–


Mrozek, E.; Kloos, RT. & Shaha, MH. (2006). Phase II study of celecoxib in metastatic

Musholt. TJ.; Musholt, PB. & Klempnauer, J. (2000). Prognostic significance of RET and

Nikiforov, YE. (2002). RET/PTC rearrangement in thyroid tumors. *Endocr Pathol*, Vol.13,

Nikiforov, YE. (2006). RET/PTC Rearrangement – a link between Hashimoto's thyroiditis

Nikiforova, MN.; Kimura, ET. & Nikiforova, YE. (2003). BRAF mutations in thyroid tumors

Pal, T.; Vogl, ED. & Foulkes, WD. (2001). Increased risk for nonmedullary thyroid cancer in

Pasqualetti, G.; Ricci, S & Monzani, F. (2011). The emerging role of sunitinib in the treatment

Rago, T.; Santini, F. & Vitti, P. (2007). Elastography: new developments in ultrasound for

Robert, C.; Smallridge, SE. & Fatourechi, V. (2007). Monitoring thyreoglobulin in a sensitive

Roche, B.; Larroumets, G. & Tauveron, I. (2010). Epidemiology, clinical presentation,

Santoro, M.; Melillo, RM. & Fusco, A. (2006). RET/PTC activation in papillary thyroid

Schlumberger, M. & Pacini, F. (2006). Thyroid tumors. *Edition Nucleon*, Vol.18, pp. 313–340,

Schlumberger, M.; Berg, G. & Wiersinga, WM. (2004). Follow-up of low risk patients with

Schoenberger, J.; Grimm, D. & Eilles, C. (2004). Effects of PTK787/ZK222584, a tyrosine

animal study. *Endocrinology,* Vol.145, No.3, pp.1031–1038, ISSN.

irradiation. *Hell J Nucl Med.* Vol.12, No.3, pp.266-70, ISSN.

*Rev Med Chem,* Vol.11, No.9, pp.746-52, ISSN.

2204, ISSN.

No.6, pp.984-93, ISNN.

No.11, pp.5399–404, ISSN.

pp.2917–2922, ISSN

pp.38–45, ISSN.

ISSN.

Vol.92, No.1, pp.82-87, ISSN.

Vol.155, No.5, pp.645–53, ISSN.

No.2, pp.105–112, ISSN.

No.1, pp.3-16, ISSN.

differentiated thyroid carcinoma. *J Clin Endocrinol Metab*, Vol.91, No.6, pp. 2201–

NTRK1 rearrangements in sporadic papillary thyroid carcinoma. *Surgery*, Vol.128,

and thyroid cancer or not. *J. Clin. Endocrinol. Metab*, Vol. 91, No.6, pp.2040–2, ISNN.

are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. *J. Clin. Endocrinol. Metab,* Vol.88,

the first degree relatives of prevalent cases of nonmedullary thyroid cancer: a hospital-based study. *J. Clin. Endocrinol. Metab*, Vol.86, No.11, pp.5307–12, ISSN. Papadopoulou, F. & Efthimiou, E. (2009). Thyroid cancer after external or internal ionizing

of advanced epithelial thyroid cancer: our experience and review of literature. *Mini* 

predicting malignancy in thyroid nodules. *J Clin Endocrinol Metab*, Vol.92, No.8,

Immunoassay has comparable sensitivity to recombinant human TSH-stimulated thyroglobulin in follow-up of thyroid cancer patients. J Clin Endocrinol Metab,

treatment and prognosis of a regional series of 26 anaplastic thyroid carcinomas (ATC). Comparison with the literature. *Annales d'Endocrinologie*, Vol.71, No.1,

carcinoma: European Journal of Endocrinology Prize Lecture. *Eur. J. Endocrinol*,

differentiated thyroid carcinoma: a European perspective. *Eur J Endocrinol*, Vol.150,

kinase inhibitor, on the growth of a poorly differentiated thyroid carcinoma: an


**5** 

*Kraków Poland* 

**Thyroid Growth Factors** 

*Jagiellonian University College of Medicine* 

*Department of Endocrine Surgery* 

Aleksander Konturek and Marcin Barczynski

The effect of exogenous and endogenous factors on the thyroid is manifested as stimulation or inhibition of the gland's excretory activity and growth regulation of the thyroid tissue itself. External factors affecting thyroid enlargement were known as early as approximately 2000 years B.C. in ancient China, where marine products were administered to inhabitants of the central part of the country. This specific supplementation of iodine-rich products prevented goiter development. [1,2] In modern times, the first country to introduce iodine prophylaxis was Switzerland, followed by the United States. Poland has been implementing a program of common salt iodization since 1986, albeit with an interruption of less than a score of years. We presently know than apart from iodine, there are numerous factors that affect regulation of thyroid secretion and growth. Thyroid homeostasis is thus controlled by several different substances acting on various levels: directly and indirectly by thyrotropin TSH (thyroid stimulating hormone); locally by other growth stimulators, such as the epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), insulin growth factors (IGFs), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-β), as well as through programmed cell death in the mechanism of apoptosis. Hence, changes in thyroid size and hormone demand occur mostly through TSH and complex interactions of local growth factors with expression of specific receptors. It has been also observed recently that para- and autocrine effects of growth factors are also associated with expression of particular oncogenes. Except the embryonal and adolescent periods, the volume of a normal thyroid gland does not increase. Each thyroid follicular cell is programmed to undergo five mitotic cycles during adult life and the final population of thyrocytes demonstrates specific differentiation, manifested by hormone secretion in response to thyrotropin via the mechanism of negative feedback. Thus, participation of the thyroid gland in hemostasis is regulated via hormonal, neural and immune pathways. The first level of thyroid growth and function control occurs via the effect of thyrotropin (TSH). The second level of tissue hemostasis is controlled by local factors. The third level consists of interactions between thyroid cells and connective tissue stroma, while the fourth level

includes genetic factors with programmed death cell (apoptosis).

**1. Introduction** 


### **Thyroid Growth Factors**

Aleksander Konturek and Marcin Barczynski

*Department of Endocrine Surgery Jagiellonian University College of Medicine Kraków Poland* 

#### **1. Introduction**

76 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

Ying, H.; Suzuki, H. & Cheng, SE. (2003). Mutant thyroid hormone receptor beta represses

Zaydfudim, V.; Feurer, ID. & Phay, JE. (2008). The impact of lymph node involvement on

5280, ISSN.

Vol.144, No.6, pp.1070–1077, ISSN.

the expression and transcriptional activity of peroxisome proliferator- activated receptor gamma during thyroid carcinogenesis. *Cancer Res*, Vol.63, No.17, pp.5274-

survival in patients with papillary and follicular thyroid carcinoma. *Surgery*,

The effect of exogenous and endogenous factors on the thyroid is manifested as stimulation or inhibition of the gland's excretory activity and growth regulation of the thyroid tissue itself. External factors affecting thyroid enlargement were known as early as approximately 2000 years B.C. in ancient China, where marine products were administered to inhabitants of the central part of the country. This specific supplementation of iodine-rich products prevented goiter development. [1,2] In modern times, the first country to introduce iodine prophylaxis was Switzerland, followed by the United States. Poland has been implementing a program of common salt iodization since 1986, albeit with an interruption of less than a score of years. We presently know than apart from iodine, there are numerous factors that affect regulation of thyroid secretion and growth. Thyroid homeostasis is thus controlled by several different substances acting on various levels: directly and indirectly by thyrotropin TSH (thyroid stimulating hormone); locally by other growth stimulators, such as the epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), insulin growth factors (IGFs), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-β), as well as through programmed cell death in the mechanism of apoptosis. Hence, changes in thyroid size and hormone demand occur mostly through TSH and complex interactions of local growth factors with expression of specific receptors. It has been also observed recently that para- and autocrine effects of growth factors are also associated with expression of particular oncogenes. Except the embryonal and adolescent periods, the volume of a normal thyroid gland does not increase. Each thyroid follicular cell is programmed to undergo five mitotic cycles during adult life and the final population of thyrocytes demonstrates specific differentiation, manifested by hormone secretion in response to thyrotropin via the mechanism of negative feedback. Thus, participation of the thyroid gland in hemostasis is regulated via hormonal, neural and immune pathways. The first level of thyroid growth and function control occurs via the effect of thyrotropin (TSH). The second level of tissue hemostasis is controlled by local factors. The third level consists of interactions between thyroid cells and connective tissue stroma, while the fourth level includes genetic factors with programmed death cell (apoptosis).

Thyroid Growth Factors 79

is much shorter. The TSH receptor, in contrast to other receptors from this family, is modified post-translationally. Approximately 75% of the monomeric, membrane receptor is proteolized. In consequence of proteolysis, the so-called peptide C is released, while the generated subunits A and B are linked by disulfide bridges. Both forms of the receptor monomeric and dimeric - actively bind TSH. In the cell membrane of thyrocytes, the TSH receptor is found in two forms: active, which binds Gs protein, and inactive, which is prevalent. Both TSH and stimulating autoantibodies bind to the active form of the receptor and stabilize it. In turn, inhibiting antibodies stabilize the inactive ("closed") form of the receptor and thus, the genuine receptor agonist seems to be the active ("open") form of the

receptor rather than the thyrotropin (TSH) molecule itself [8,9,10,11].

**(EGFs) and transforming growth factor alpha (TGF-α)** 

**3. Vascular endothelial growth factor (VEGF), epidermal growth factors** 

Neoangiogenesis is a process consisting of numerous paracrine and endocrine interactions between cancer cells and vascular endothelial cells, connective tissue stromal cells and some morphotic blood elements, such as macrophages or mastocytes. The result of such interactions is a change in the microenvironment of a tumor that allows for its further uncontrollable growth and progression. A prerequisite for initiation of angiogenic phenomena is disturbance of balance between the system of pro- and anti-angiogenic factors. Not each of the proangiogenic factors that have been described to date (VEGF; bFGF; aFGF; PDGF; TGF-α; TGF-β; EGF; IGF-1) meets all the three characteristic criteria: exerting a specific effect on endothelium, possessing a system of specific cell receptors and manifesting fluctuations that inhibit or induce angiogenesis. Many of such factors, acting in conjunction with mediators secreted by other cells (e.g. macrophages - TNF), induce and promote development of cancer. In keeping with the presently accepted assumptions, angiogenesis is initiated via hypoxia of cancer cells that are situated at the highest distance from the lumen of a blood vessel, as well as via a defect of the genetic apparatus, the consequence of which is formation of the so-called angiogenic phenotype. This term denotes a state of permanent, constitutive activation of growth factors-encoding genes. An additional loss of function of suppressor genes (e.g. the p53 gene) favors the process of neoangiogenesis. One of the relatively well-understood epithelial growth factors is VEGF. This specific protein, defined and named in late eighties, is assumed to play a key role in vascularization of solid tumors, including thyroid tumors. At present, the VEGF group is believed to include six proteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and PIGF (Placenta Growth Factor). They act through binding to receptors: VEGFR1, VEGFR2 – on vascular endothelial cells, and VGFR3 – on lymphatic endothelium cells (Figure 3). The necessary cofactors for the VEGF receptor are neuropilins 1 and 2 (Nrp-1,2), which are indispensable for proper activation of the receptor by a ligand. Synthesis of the vascular growth factor is induced by numerous other substances, such as nitrogen oxide, insulin, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), tumor necrotic factor (TNF-α), transforming growth factor (TGF-β), IL-1,2. In keeping with the theory of Folkman, which states that tumor growth is limited by its vascularization, attempts have been made to demonstrated higher VEGF expression in cancer tissues as compared to normal cell populations. Similarly as in the case of cancer of the stomach, colon, uterus, breast and ovary, also in thyroid tumors a key role in neoangiogenesis is played by VEGF. As it has been already mentioned, not only thyroid

#### **2. Regulation of growth and hormonal secretion – Mitogenic pathways**

#### **2.1 Role of TSH**

#### **Mechanism of activation via a receptor (types of receptors)**

Thyrotropin (TSH) is traditionally believed to be the principal stimulator of growth, differentiation and maturation of thyroid follicular cells and connective tissue stroma. It is a 28 kD glycoprotein. Its level is among major parameters describing thyroid function and playing a decisive role in the clinical status of a patient. As early as in mid-thirties (1935 Kippen; Loeb; 1937 Dunhill), observations were made on the effect of TSH on thyroid follicular cells. Both a stimulatory and inhibitory effect of thyrotropin on thyroid follicular cells was described in cells cultured *in vitro*; attempts were undertaken at explaining the role of suppression therapy in preventing thyroid cancer development. For decades, particular research teams obtained contrary results of investigations on the proliferogenic effect on growth and differentiation of thyroid follicular cells. The effect on thyroid function and hormonal secretion has remained unquestioned [3,4]. In the last decade, numerous reports were published that discussed the complex regulation system of thyroid cell growth and proliferation, where thyrotropin alone may play an important role, but is not a prerequisite. Thus, TSH administration to rats resulted in thyroid enlargement both via cell hypertrophy and hyperplasia [5]. TSH-induced thyreocyte proliferation was triggered by human thyroid tissue implantation to mice (thymus-deficient nu/nu mice). In another study, thyrotropin was not necessary for compensatory thyroid growth following hemithyroidectomies, what suggested the effect of other factors that also play a role in the gland's growth [6,7]. Recently, attention has been also focused on the role of cAMP in thyroid growth-associated processes. Numerous reports on the proliferogenic effect of cAMP on thyroid follicular cells point to three pathways of activating growth and proliferation of thyroid cells:


An increase of cAMP level in the majority of differentiated thyroid tumors (in contrast to normal tissue collected from the same patient) was interpreted as a result of an increased response to TSH stimulation. In numerous differentiated thyroid cancers, the functional TSH-cAMP-thyroid follicular cell growth system was noted; nevertheless, the question on intercorrelations of the above factors continue to remain open (Table 1.). Numerous authors have also pointed to a double effect of TSH depending on activation of other transmitters. Thus, 1) thyroid follicular cell growth is stimulated via activation of phosphatidylinositol and protein kinase C, while 2) the function of thyroid follicular cells is regulated by cAMP and protein kinase A (Figure 1). The two alternative pathways may explain diversified effects of thyrotropin in cancerous thyroid tissue [2]. The turning point in explaining many growth-associated phenomena within the thyroid gland was determination of the receptor structure on the molecular level and demonstration of intracellular interactions via activation of other transmitters. The thyrotropin receptor itself belongs to the family of G protein-coupled receptors (similarly as FSH, LH, estrogens, hCG or other steroid receptors). The predominant property of all the above receptors is the presence of a transmembrane domain that crosses the lipid layer (Figure 2.). The N-terminal region of the receptors is the so-called ectodomain situated on cell surface. The C-terminal region, situated within the cell,

Thyrotropin (TSH) is traditionally believed to be the principal stimulator of growth, differentiation and maturation of thyroid follicular cells and connective tissue stroma. It is a 28 kD glycoprotein. Its level is among major parameters describing thyroid function and playing a decisive role in the clinical status of a patient. As early as in mid-thirties (1935 Kippen; Loeb; 1937 Dunhill), observations were made on the effect of TSH on thyroid follicular cells. Both a stimulatory and inhibitory effect of thyrotropin on thyroid follicular cells was described in cells cultured *in vitro*; attempts were undertaken at explaining the role of suppression therapy in preventing thyroid cancer development. For decades, particular research teams obtained contrary results of investigations on the proliferogenic effect on growth and differentiation of thyroid follicular cells. The effect on thyroid function and hormonal secretion has remained unquestioned [3,4]. In the last decade, numerous reports were published that discussed the complex regulation system of thyroid cell growth and proliferation, where thyrotropin alone may play an important role, but is not a prerequisite. Thus, TSH administration to rats resulted in thyroid enlargement both via cell hypertrophy and hyperplasia [5]. TSH-induced thyreocyte proliferation was triggered by human thyroid tissue implantation to mice (thymus-deficient nu/nu mice). In another study, thyrotropin was not necessary for compensatory thyroid growth following hemithyroidectomies, what suggested the effect of other factors that also play a role in the gland's growth [6,7]. Recently, attention has been also focused on the role of cAMP in thyroid growth-associated processes. Numerous reports on the proliferogenic effect of cAMP on thyroid follicular cells

**2. Regulation of growth and hormonal secretion – Mitogenic pathways** 

point to three pathways of activating growth and proliferation of thyroid cells: 1. first - activation of the adenyl cyclase-cAMP system, stimulated by TSH;

3. third - most likely independent of cAMP - protein phosphorylation of tyrosine.

An increase of cAMP level in the majority of differentiated thyroid tumors (in contrast to normal tissue collected from the same patient) was interpreted as a result of an increased response to TSH stimulation. In numerous differentiated thyroid cancers, the functional TSH-cAMP-thyroid follicular cell growth system was noted; nevertheless, the question on intercorrelations of the above factors continue to remain open (Table 1.). Numerous authors have also pointed to a double effect of TSH depending on activation of other transmitters. Thus, 1) thyroid follicular cell growth is stimulated via activation of phosphatidylinositol and protein kinase C, while 2) the function of thyroid follicular cells is regulated by cAMP and protein kinase A (Figure 1). The two alternative pathways may explain diversified effects of thyrotropin in cancerous thyroid tissue [2]. The turning point in explaining many growth-associated phenomena within the thyroid gland was determination of the receptor structure on the molecular level and demonstration of intracellular interactions via activation of other transmitters. The thyrotropin receptor itself belongs to the family of G protein-coupled receptors (similarly as FSH, LH, estrogens, hCG or other steroid receptors). The predominant property of all the above receptors is the presence of a transmembrane domain that crosses the lipid layer (Figure 2.). The N-terminal region of the receptors is the so-called ectodomain situated on cell surface. The C-terminal region, situated within the cell,

2. second - the phosphatidylinositol-Ca ion system,

**Mechanism of activation via a receptor (types of receptors)** 

**2.1 Role of TSH** 

is much shorter. The TSH receptor, in contrast to other receptors from this family, is modified post-translationally. Approximately 75% of the monomeric, membrane receptor is proteolized. In consequence of proteolysis, the so-called peptide C is released, while the generated subunits A and B are linked by disulfide bridges. Both forms of the receptor monomeric and dimeric - actively bind TSH. In the cell membrane of thyrocytes, the TSH receptor is found in two forms: active, which binds Gs protein, and inactive, which is prevalent. Both TSH and stimulating autoantibodies bind to the active form of the receptor and stabilize it. In turn, inhibiting antibodies stabilize the inactive ("closed") form of the receptor and thus, the genuine receptor agonist seems to be the active ("open") form of the receptor rather than the thyrotropin (TSH) molecule itself [8,9,10,11].

#### **3. Vascular endothelial growth factor (VEGF), epidermal growth factors (EGFs) and transforming growth factor alpha (TGF-α)**

Neoangiogenesis is a process consisting of numerous paracrine and endocrine interactions between cancer cells and vascular endothelial cells, connective tissue stromal cells and some morphotic blood elements, such as macrophages or mastocytes. The result of such interactions is a change in the microenvironment of a tumor that allows for its further uncontrollable growth and progression. A prerequisite for initiation of angiogenic phenomena is disturbance of balance between the system of pro- and anti-angiogenic factors. Not each of the proangiogenic factors that have been described to date (VEGF; bFGF; aFGF; PDGF; TGF-α; TGF-β; EGF; IGF-1) meets all the three characteristic criteria: exerting a specific effect on endothelium, possessing a system of specific cell receptors and manifesting fluctuations that inhibit or induce angiogenesis. Many of such factors, acting in conjunction with mediators secreted by other cells (e.g. macrophages - TNF), induce and promote development of cancer. In keeping with the presently accepted assumptions, angiogenesis is initiated via hypoxia of cancer cells that are situated at the highest distance from the lumen of a blood vessel, as well as via a defect of the genetic apparatus, the consequence of which is formation of the so-called angiogenic phenotype. This term denotes a state of permanent, constitutive activation of growth factors-encoding genes. An additional loss of function of suppressor genes (e.g. the p53 gene) favors the process of neoangiogenesis. One of the relatively well-understood epithelial growth factors is VEGF. This specific protein, defined and named in late eighties, is assumed to play a key role in vascularization of solid tumors, including thyroid tumors. At present, the VEGF group is believed to include six proteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and PIGF (Placenta Growth Factor). They act through binding to receptors: VEGFR1, VEGFR2 – on vascular endothelial cells, and VGFR3 – on lymphatic endothelium cells (Figure 3). The necessary cofactors for the VEGF receptor are neuropilins 1 and 2 (Nrp-1,2), which are indispensable for proper activation of the receptor by a ligand. Synthesis of the vascular growth factor is induced by numerous other substances, such as nitrogen oxide, insulin, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), tumor necrotic factor (TNF-α), transforming growth factor (TGF-β), IL-1,2. In keeping with the theory of Folkman, which states that tumor growth is limited by its vascularization, attempts have been made to demonstrated higher VEGF expression in cancer tissues as compared to normal cell populations. Similarly as in the case of cancer of the stomach, colon, uterus, breast and ovary, also in thyroid tumors a key role in neoangiogenesis is played by VEGF. As it has been already mentioned, not only thyroid

Thyroid Growth Factors 81

paracrine effect has been manifested in neovascularization seen in nodular goiter. Similarly as EGF, FGF has an inhibitory effect on thyroid function. The effect of FGF consists in inhibition of cAMP activation and weakening of TSH activity. FGF-1 administration to rats leads to development of colloid goiter, most likely through inhibition of TSH-dependent

The hepatocyte growth factor (HGF) is also a potent myogen for numerous diversified cell types in human body, especially these of epithelial origin. It operates through its receptor that is encoded by the c-met proto-oncogene and belongs to the family of tyrosine kinase receptors. HGF-R expression has been noted both in normal and cancer tissue, with the factor being present in papillary and follicular carcinoma, but absent in anaplastic carcinoma tissue. Some importance in neoplastic proliferation is also ascribed to the plateletderived growth factor PDGF), the presence of which has been noted in the papillary

**6. Interactions between stimulatory growth factors and their inhibitors** 

A group of factors that inhibit thyroid growth is the family of the thyroid growth inhibiting factor – β (TGF- β). Through their receptors TGF- β-R I to TGF- β-R III, the factors TGF- β1 to TGF- β3 affect inhibition of thyroid follicular cell growth in vitro; their role has also been implicated in development of benign lesions. A drop in TGF production has been observed

Stimulation of thyroid tissue growth occurs through an increased frequency of signals reaching the gland from the surrounding structures, resulting in sensitization of follicular cells to normal external stimuli. Such stimuli may be the afore-mentioned growth factors or antibodies or else dietary iodine deficit. A notion of the so-called "grey zone" has become popular in thyreology, to denote a population of cells with a high internal growth potential, which, in consequence of single or multiple genetic damage, change their growth potential (in hyperplastic tumors) towards cancer, yet without obvious neoplastic transformation. The effect on cell cycle, manifested as for example the shortened G0 phase or limitation of apoptosis may lead to an increase in the number of cell divisions prior to

Within the past decade, development of modern research methods, molecular biology and genetics has allowed for gradual understanding of molecular foundations of thyroid cancers. It is presently known that activation of certain oncogenes and inactivation of suppressor genes may lead to tumor development. Of 35 proto-oncogenes determined in neoplastic transformation of thyroid follicular cells, the following have their effect proven:

7. Trk encodes NGF (nerve growth factor) characteristic of papillary carcinoma and

colloid transport from the lumen of thyroid follicles (Figure 4.).

in thyroid follicular cells of patients with non-toxic goiter.

1. c-erb encodes the EGF (endothelial growth factor) receptor;

3. c-met encodes the HGF (hepatocyte growth factor) receptor; 4. c-sis encodes the PDGF (platelet-derived growth factor) receptor;

6. PTC/ret, TPC – characteristic of papillary carcinoma;

detected in nodular goiter.

2. flg, bek encodes the FGF receptor type 1 and 2 (fibroblast growth factor);

5. Ras is characteristic of follicular carcinoma, adenoma, anaplastic carcinoma;

carcinoma cell line.

programmed cell death.

epithelial cells, but also stromal cells are capable of producing and secreting VEGF. The above described results have allowed for formulating a hypothesis that VEGF participates in initiation of neoangiogenesis, yet further tumor development and progression most likely depend on the effect of other chemokines secreted both by tumor cells, its stroma and macrophages migrating to the neoplastic lesion [12.13,14]. In turn, epidermal growth factor EGF is one of the most potent stimulators of thyroid growth and its multiple effect is determined by its binding to specific EGF receptors. *In vitro*, it is a factor that stimulates proliferation of thyroid follicular cells. A factor that increases EGF binding to receptors is thyrotropin (TSH), which – stimulating the increase of the number of EGF receptors enhances its effect. Yet, in contrast to the above-mentioned TSH, to reveal its mitogenic effect, EGF does not require the presence of other chemokines. In subsequent studies, investigators attempted to determine the effect of positive EGF receptors expression in thyroid cancer tissue on the clinical course of the disease. A comparison was made between the presence of EGF receptors in various types of thyroid cancer, finding their highest expression in anaplastic and medullary carcinoma of the thyroid. Also adenomas demonstrated considerable expression of EGF receptors, but only in some limited areas within the tumor. EGF-R was also noted to bind not only to EGF -α l, but also to the transforming growth factor alpha (TGF-α). The autocrine mechanism of activating EGF receptors both by the epidermal growth factor molecule and by TGFα was seen in thyroid cancers (papillary carcinoma and its nodal metastases) [15].

#### **4. Insulin growth factors and their receptors (IGFs)**

Growth hormone (GH) affects growth processes in tissues and organs through specific substances called the insulin growth factors (IGF-I; IGF-II). IGF-I, termed somatomedin C, and IGF-II affect growth regulation of thyroid endothelial cells. In turn, thyroid follicular cells show high expression of specialized IGF-I and IGF-II receptors. The IGF-I receptor, as a member of the family of receptors that act via tyrosine kinase, is a mediator of the effect of IGF-I on stimulation and growth of thyroid follicular cells. IGF-I has been proven to strongly stimulate growth of the FRTL-5 line cells and to synergistically enhance the mitogenic effect of thyrotropin (TSH). In turn, the IGF-II receptor plays no significant role in thyroid growth stimulation. Autocrine secretion of IGF-I, IGF-II and expression of the IGF-I-Rs receptor were demonstrated in primary cultures of thyroid follicular cells, as well as in adenomas and thyroid papillary carcinoma cell lines [16,17,18].

#### **5. Fibroblast growth factor (FGFs), hepatocyte growth factor (HGF), plateletderived growth factor (PDGF)**

To date, nine subtypes of the fibroblast growth factor belonging to a single family (the "FGF family") have been determined. The FGF factor itself is known as a stimulator of proliferation, differentiation and functioning of various diverse cells of human body. FGF also plays a significant role in neoangiogenesis. The cell response to FGF effect is mediated by its four receptors (FGF-R 1-4) that belong to the family of tyrosine kinases. Thyroid endothelial cells show expression of FGF-R receptors, while the basement membrane of thyroid follicular cells is capable of producing the factor itself. Based on numerous observations in vitro and in vivo in rat thyroid follicular cells, an autocrine, stimulatory effect of FGF-2 ("basic FGF") on thyroid growth processes has been demonstrated. In turn, a

epithelial cells, but also stromal cells are capable of producing and secreting VEGF. The above described results have allowed for formulating a hypothesis that VEGF participates in initiation of neoangiogenesis, yet further tumor development and progression most likely depend on the effect of other chemokines secreted both by tumor cells, its stroma and macrophages migrating to the neoplastic lesion [12.13,14]. In turn, epidermal growth factor EGF is one of the most potent stimulators of thyroid growth and its multiple effect is determined by its binding to specific EGF receptors. *In vitro*, it is a factor that stimulates proliferation of thyroid follicular cells. A factor that increases EGF binding to receptors is thyrotropin (TSH), which – stimulating the increase of the number of EGF receptors enhances its effect. Yet, in contrast to the above-mentioned TSH, to reveal its mitogenic effect, EGF does not require the presence of other chemokines. In subsequent studies, investigators attempted to determine the effect of positive EGF receptors expression in thyroid cancer tissue on the clinical course of the disease. A comparison was made between the presence of EGF receptors in various types of thyroid cancer, finding their highest expression in anaplastic and medullary carcinoma of the thyroid. Also adenomas demonstrated considerable expression of EGF receptors, but only in some limited areas within the tumor. EGF-R was also noted to bind not only to EGF -α l, but also to the transforming growth factor alpha (TGF-α). The autocrine mechanism of activating EGF receptors both by the epidermal growth factor molecule and by TGFα was seen in thyroid

Growth hormone (GH) affects growth processes in tissues and organs through specific substances called the insulin growth factors (IGF-I; IGF-II). IGF-I, termed somatomedin C, and IGF-II affect growth regulation of thyroid endothelial cells. In turn, thyroid follicular cells show high expression of specialized IGF-I and IGF-II receptors. The IGF-I receptor, as a member of the family of receptors that act via tyrosine kinase, is a mediator of the effect of IGF-I on stimulation and growth of thyroid follicular cells. IGF-I has been proven to strongly stimulate growth of the FRTL-5 line cells and to synergistically enhance the mitogenic effect of thyrotropin (TSH). In turn, the IGF-II receptor plays no significant role in thyroid growth stimulation. Autocrine secretion of IGF-I, IGF-II and expression of the IGF-I-Rs receptor were demonstrated in primary cultures of thyroid follicular cells, as well as in adenomas

**5. Fibroblast growth factor (FGFs), hepatocyte growth factor (HGF), platelet-**

To date, nine subtypes of the fibroblast growth factor belonging to a single family (the "FGF family") have been determined. The FGF factor itself is known as a stimulator of proliferation, differentiation and functioning of various diverse cells of human body. FGF also plays a significant role in neoangiogenesis. The cell response to FGF effect is mediated by its four receptors (FGF-R 1-4) that belong to the family of tyrosine kinases. Thyroid endothelial cells show expression of FGF-R receptors, while the basement membrane of thyroid follicular cells is capable of producing the factor itself. Based on numerous observations in vitro and in vivo in rat thyroid follicular cells, an autocrine, stimulatory effect of FGF-2 ("basic FGF") on thyroid growth processes has been demonstrated. In turn, a

cancers (papillary carcinoma and its nodal metastases) [15].

**4. Insulin growth factors and their receptors (IGFs)** 

and thyroid papillary carcinoma cell lines [16,17,18].

**derived growth factor (PDGF)** 

paracrine effect has been manifested in neovascularization seen in nodular goiter. Similarly as EGF, FGF has an inhibitory effect on thyroid function. The effect of FGF consists in inhibition of cAMP activation and weakening of TSH activity. FGF-1 administration to rats leads to development of colloid goiter, most likely through inhibition of TSH-dependent colloid transport from the lumen of thyroid follicles (Figure 4.).

The hepatocyte growth factor (HGF) is also a potent myogen for numerous diversified cell types in human body, especially these of epithelial origin. It operates through its receptor that is encoded by the c-met proto-oncogene and belongs to the family of tyrosine kinase receptors. HGF-R expression has been noted both in normal and cancer tissue, with the factor being present in papillary and follicular carcinoma, but absent in anaplastic carcinoma tissue. Some importance in neoplastic proliferation is also ascribed to the plateletderived growth factor PDGF), the presence of which has been noted in the papillary carcinoma cell line.

#### **6. Interactions between stimulatory growth factors and their inhibitors**

A group of factors that inhibit thyroid growth is the family of the thyroid growth inhibiting factor – β (TGF- β). Through their receptors TGF- β-R I to TGF- β-R III, the factors TGF- β1 to TGF- β3 affect inhibition of thyroid follicular cell growth in vitro; their role has also been implicated in development of benign lesions. A drop in TGF production has been observed in thyroid follicular cells of patients with non-toxic goiter.

Stimulation of thyroid tissue growth occurs through an increased frequency of signals reaching the gland from the surrounding structures, resulting in sensitization of follicular cells to normal external stimuli. Such stimuli may be the afore-mentioned growth factors or antibodies or else dietary iodine deficit. A notion of the so-called "grey zone" has become popular in thyreology, to denote a population of cells with a high internal growth potential, which, in consequence of single or multiple genetic damage, change their growth potential (in hyperplastic tumors) towards cancer, yet without obvious neoplastic transformation. The effect on cell cycle, manifested as for example the shortened G0 phase or limitation of apoptosis may lead to an increase in the number of cell divisions prior to programmed cell death.

Within the past decade, development of modern research methods, molecular biology and genetics has allowed for gradual understanding of molecular foundations of thyroid cancers. It is presently known that activation of certain oncogenes and inactivation of suppressor genes may lead to tumor development. Of 35 proto-oncogenes determined in neoplastic transformation of thyroid follicular cells, the following have their effect proven:


Thyroid Growth Factors 83

iodine (I-X) on various pathways. ATP = adenosine triphosphate.

**DNA <sup>D</sup><sup>E</sup>**

Development of small vessels angiogenesis arteriogenesis

Fig. 3. Vascular growth factors and the effects of their acting through receptors.

Mineralocorticoids;

Fig. 2. The family of steroid/thyroid receptors. The marked receptors have similar structure.

VEGF-A VEGF-B VEGF-C VEGF-D Ang-1 Ang-2

VEGFR-1 VEGFR-2 VEGFR-3 Tie - 2

Development of small lymph vessels

**NH 2 A/B <sup>C</sup>**

The C domain consists of DBD domains.

Migration of smooth muscle cells

a resultant increase of intracellular level of calcium (Ca2+) and PKC activity. Both PKA and PKC are serine-threonine kinases and they phosphorylate several different proteins. 3. The receptor tyrosine kinase pathway (RTK): Binding of a ligand to RTK leads to phosphorylation of tyrosine residues in the receptor molecule. The stimulated phosphorylated receptors connect to numerous different signaling pathways (the scheme does not show all the pathways) through direct binding of signaling proteins that contain a homology domain with Src proteins (SH2). Nevertheless, the main mitogenic pathway of numerous receptors tyrosine kinase (RTK) includes activation of a chain of events on the *ras* pathway (see the scheme). In brief, phosphorylated RTK interacts with Grb2 adaptor protein. Grb2 binds to a protein called Sos ("son of sevenless"), resulting in activation of the *ras* pathway. The activated form of *ras* GTP protein triggers increased activity of raf protein, with a resultant sequential activation of MAPK cascade proteins (mitogen-activated kinases), what ultimately leads to an increased transcriptional activity (MAPKK kinase, MAPK). The scheme also shows a negative effect of organic forms of

> Thyroid hormones Vitamin D Estrogens; Androgens; Progesterone; Glucocorticoids;

**HORMONES F COOH** 

Tie-2 arteriogenesis (Ang-1) angiogenesis (Ang-2 + VEGF) Angiogenesis inhibitor (Ang-2)

As it follows from the presented data, they play a key role in encoding growth factors and/or their receptors production

Summing up, it should be stressed that the theories presented in the chapter constitute only a very narrow fragment of the bulk of knowledge on the subject. An extensive presentation of the topic is possible only in a monograph, yet the above provided examples help in understanding the foundations of contemporary knowledge on the effect of external factors on cancer development.

Fig. 1. Mitogenic pathways in regulation of thyroid function and growth. The scheme present the main pathways that participate in regulation of thyroid function and growth.


As it follows from the presented data, they play a key role in encoding growth factors

Summing up, it should be stressed that the theories presented in the chapter constitute only a very narrow fragment of the bulk of knowledge on the subject. An extensive presentation of the topic is possible only in a monograph, yet the above provided examples help in understanding the foundations of contemporary knowledge on the effect of external factors

Fig. 1. Mitogenic pathways in regulation of thyroid function and growth. The scheme present the main pathways that participate in regulation of thyroid function and growth.

Gp protein, which stimulates phosphatidylinositol (PI) metabolism.

1. The AC/cAMP/PKA pathway: The main factor that stimulates the pathway in thyrocytes is thyrotropic hormone (TSH), which interacts with the TSH receptor (TSH-R). Stimulation of the TSH-R receptor leads to activations that bind guanosine triphosphate (GTP) of regulatory proteins; Ga protein in the plasma membrane activates adenyl cyclase (AC) that synthesizes cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA), and phospholipase C (PLC)-associated

2. The PI-PKC-Ca2+ pathway: In addition to TSH-R, the cascade of signal transmission is activated through numerous diversified receptors (marked as Rn in the scheme). Stimulation of TSH and other receptors leads to an increased PLC activity; in consequence, 1,4,5,-inositol triphosphate (IP3) and diacylglycerol (DAG) are formed with

and/or their receptors production

on cancer development.

a resultant increase of intracellular level of calcium (Ca2+) and PKC activity. Both PKA and PKC are serine-threonine kinases and they phosphorylate several different proteins.

3. The receptor tyrosine kinase pathway (RTK): Binding of a ligand to RTK leads to phosphorylation of tyrosine residues in the receptor molecule. The stimulated phosphorylated receptors connect to numerous different signaling pathways (the scheme does not show all the pathways) through direct binding of signaling proteins that contain a homology domain with Src proteins (SH2). Nevertheless, the main mitogenic pathway of numerous receptors tyrosine kinase (RTK) includes activation of a chain of events on the *ras* pathway (see the scheme). In brief, phosphorylated RTK interacts with Grb2 adaptor protein. Grb2 binds to a protein called Sos ("son of sevenless"), resulting in activation of the *ras* pathway. The activated form of *ras* GTP protein triggers increased activity of raf protein, with a resultant sequential activation of MAPK cascade proteins (mitogen-activated kinases), what ultimately leads to an increased transcriptional activity (MAPKK kinase, MAPK). The scheme also shows a negative effect of organic forms of iodine (I-X) on various pathways. ATP = adenosine triphosphate.

Fig. 2. The family of steroid/thyroid receptors. The marked receptors have similar structure. The C domain consists of DBD domains.

Fig. 3. Vascular growth factors and the effects of their acting through receptors.

Thyroid Growth Factors 85

[1] Cao X-Y., Jiang X-M., Kareem A.: Iodination of irrigation water as a method supplying

[2] Peter E. Goretzki, M.D., Dietmar Simon, M.D., Cornelia Dotzenrath, M.D., Klaus-Martin

[3] Werner SC 1991 History of the thyroid. In: Braverman LE, Utiger RD (eds) The Thyroid: A Fundamental and Clinical Text, ed 6. J, B. Lippincott, Philadelphia, pp 3-6 [4] Goretzki PE, Frilling A, Simon D, Roeher HD. Growth regulation of normal thyroids and

[5] Wynford Th,D, Stringer BMJ, Williams ED.: Goitrogen-induced thyroid growth in rat: A

[6] Smeds S, Boeryd B, Jortso E, Lennquist S.: normal and stimulated growth of different

[7] Lewiński A, Bartke A, Smith NKR.: Compensatory thyroid hyperplasia In hemithyroidectomized Snell dwarf mice. Endocrinology 1983;113:2317-2319. [8] Adler G.: Posttranslacyjne modyfikacje receptora tyreotropiny a choroby tarczycy

[9] Vassart G., Dumont JE.,: The Thyrotropin Receptor and the Regulation of Thyrocyte Function and Growth. Endocrine Reviews 1992 Vol. 13, No. 3, 596-613. [10] Bernard Rees Smith, Jadwiga Furmaniak, Jane Sanders TSH receptor blocking

[11] Lazar MA. Thyroid Hormone Receptors: Multiple Forms, Multiple Possibilities.

[12] Lin SY, Wang YY, Sheu WH (2003) Preoperative plasma concentrations of vascular

[14] Huang SM, Lee JC, Wu TJ, Chow NH (2001) Clinical revelance of vascular endothelial

[15] Konturek A, Barczyński M, Cichoń S, Pituch-Noworolska A, Jonkisz J, Cichoń W.

[16] Tode B, Serio M, Rotella CM et All: Insulin-like growth factor-I Autocrine secretion by

progression in papillary thyroid cancer. Clin Endocrinol 58:513-518. [13] Lennard CM, Patel A, Wilson J, Reinhardt B, Tuman C, Fenton C, Blair E, Francis GL,

growth factor for thyroid neoplasms. World J Surg 25:302-306.

endothelial growth factor and matrix metalloproteinase 9 are associated with stage

Tuttle RM (2001) Intensity of vascular endothelial growth factor expression associated with increased risk of recurrence and decreased disease-free survival in

Significance of vascular endothelial growth factor and epidermal growth factor in development of papillary thyroid cancer. Langenbecks Arch Surg. 2005

human thyroid follicular cells In primary culture. J Clin Endocrinol Metab

humman thyroid tissues in nude mice. In: Goretzki PE, Roeher HD . Growth regulation of thyroid gland and thyroid tumors Vol 18. Basel, Switzerland, Karger,

Thyroid Tumors in Humans. World J. Surg. 24, 913–922, 2000

thyroid tumors in man. Recent Results Cancer Res. 1990;118:48-63.

quantitive morphometric study. J Endocrinol. 1982,94,131-135

iodine to a severely iodine deficient population in Xinjiang, China. Lancet. 1994 Jul

Schulte, M.D., Hans-Dietrich Roeher, M.D.: Growth Regulation of Thyroid and

**7. References** 

9;344(8915):107-10].

1989, p98.

Jun;390(3):216-21

198969,621-626.

Endokrynol Pol 2005; 1(56): 72-77)

antibodies. Thyroid. 2008 Nov ;18 (11):1239

Endocrine Reviews, 1993; Vol. 14, N. 2; 184-193.)

papillary thyroid cancer. Surgery 129:552-558.

Fig. 4. Autocrine and paracrine regulation of growth and inhibition of thyrocytes via growth factors and their receptors. The factors may be also secreted by the surrounding stromal tissue, thus giving rise to proliferation of endothelial cells and fibroblasts. IGF-insulin growth factor; TGF – transforming growth factor; EGF – epidermal growth factor; FGF – fibroblast growth factor; PDGF – platelet-derived growth factor.


Table 1. Major factors with stimulatory and inhibitory effect on growth of thyroid follicular cells.

#### **7. References**

84 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

Fig. 4. Autocrine and paracrine regulation of growth and inhibition of thyrocytes via growth factors and their receptors. The factors may be also secreted by the surrounding stromal tissue, thus giving rise to proliferation of endothelial cells and fibroblasts. IGF-insulin growth factor; TGF – transforming growth factor; EGF – epidermal growth factor; FGF –

**(other transmitter) Factors Receptor Effect "Related"** 

TSH-R

of cAMP Stimulatory

TGF-a EGF-R Stimulatory

Insulin (ffi) IGF-I-R Stimulatory

IGF-I IGF-I-R Stimulatory IGF -n (IGF-n-R) IGF-I-R Stimulatory

TSH TSH-R Stimulatory Esters PKC Stimulatory TGF -β1 TGF--Rs (1-3) Inhibitory Iodides Inhibitory

Table 1. Major factors with stimulatory and inhibitory effect on growth of thyroid follicular

**proto-oncogene** 

I, FGF-R-2)

Stimulatory and inhibitory

Stimulatory

(via IGF-I-R)

(via IGF-I-R)

EGF EGF-R Stimulatory *c-erb* B (EGF-R)

FGFs FGF-Rs (1-4) Stimulatory *fIg, bek* (FGF-R-

HGF HGF-R Stimulatory c-met (HGF-R)

BB) PDGF-Rs *(a,* ) Stimulatory *c-sis* (PDGF-BB)

fibroblast growth factor; PDGF – platelet-derived growth factor.

TSH

Iodides (inhibition

Epinephrine (inhibition of cAMP)

PDGFs (AA, AB,

**Activation pathway**

**AC-cAMP-PKA** 

**Receptor tyrosine** 

**Phosphatidylinositol** 

**kinases** 

**cascade** 

cells.


**6**

*Poland* 

**Vascular Endothelial Growth Factor (VEGF)**

Early diagnosis and radical management of thyroid cancer do not always result in curing the patient. Long-term analyses of deaths due to thyroid cancer have allowed for establishing systems for distinguishing increased risk groups, such as AMES or AGES. These commonly employed prognostic systems recognize the role of four basic factors only (age, stage, extent of the tumor, its size [AGES] and metastases [AMES]), which constitute the foundation for establishing the low and high-risk groups. Despite the progress in oncology and oncological surgery, a major problem still lies in early cancer detection, when the tumor is still at a stage that allows for curing the patient, as well as in identifying individuals, in whom - despite radical treatment - the prognosis of a complete cure is poor and there is an increased risk of a local recurrence and death due to the

Papillary thyroid carcinoma accounts for the majority of thyroid cancers and is commonly believed to be the least malignant type. In Europe and the United States, it presently constitutes approximately 75-80% of all diagnosed thyroid cancers [1]. It is characterized by a mild clinical course and a slow growth rate. The tumor is detected in young individuals (usually before they turn 40 years of age) and is 2-3 times more common in females. As a rule, it is a multifocal disease (in 60% of patients) involving one thyroid lobe, although in 50% of cases microscopic neoplastic lesions are present in the contralateral lobe. One should be also aware of the possible presence of a small, 2-10 mm focus of papillary thyroid carcinoma termed "microcarcinoma", which is asymptomatic and detected by chance in the course of histopathology of the thyroid gland resected in a patient with goiter or in serial autopsies of the thyroid (such post-mortem examinations detected 35.6% microcarcinoma foci). Approximately 50% of patients demonstrate the presence of metastases in the lymph nodes. Papillary carcinoma of the thyroid is a hormonally dependent tumor (TSH). The 5-

**2. Epidemoilogy of papillary thyroid carcinoma** 

year survival rate is approximately 95% [2, 3]. [Fig. 1,2,3,4]

**1. Introduction** 

cancer.

**and Epidermal Growth Factor (EGF)**

**in Papillary Thyroid Cancer** 

*Department of Endocrine Surgery* 

Aleksander Konturek and Marcin Barczynski

*Jagiellonian University College of Medicine, Kraków* 


### **Vascular Endothelial Growth Factor (VEGF) and Epidermal Growth Factor (EGF) in Papillary Thyroid Cancer**

Aleksander Konturek and Marcin Barczynski *Department of Endocrine Surgery Jagiellonian University College of Medicine, Kraków Poland* 

#### **1. Introduction**

86 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

[17] Onoda N, Ohmura E, Tsushima T et All: Autocrine role of insulin-like growth factor

[18] Pouliaki V,Mitsiades CS, McMullan C et all: Regulation of Vascular Endothelial Growth

Factor Expression by Insulin-Like Growth Factor I in Thyroid Carcinomas. The

(IGF-I) in thyroid cancer cell Line. Eur J Cancer 1992,28A,1904-1908

Journal of Clinical Endocrinology & Metabolism 88(11):5392–5398

Early diagnosis and radical management of thyroid cancer do not always result in curing the patient. Long-term analyses of deaths due to thyroid cancer have allowed for establishing systems for distinguishing increased risk groups, such as AMES or AGES. These commonly employed prognostic systems recognize the role of four basic factors only (age, stage, extent of the tumor, its size [AGES] and metastases [AMES]), which constitute the foundation for establishing the low and high-risk groups. Despite the progress in oncology and oncological surgery, a major problem still lies in early cancer detection, when the tumor is still at a stage that allows for curing the patient, as well as in identifying individuals, in whom - despite radical treatment - the prognosis of a complete cure is poor and there is an increased risk of a local recurrence and death due to the cancer.

#### **2. Epidemoilogy of papillary thyroid carcinoma**

Papillary thyroid carcinoma accounts for the majority of thyroid cancers and is commonly believed to be the least malignant type. In Europe and the United States, it presently constitutes approximately 75-80% of all diagnosed thyroid cancers [1]. It is characterized by a mild clinical course and a slow growth rate. The tumor is detected in young individuals (usually before they turn 40 years of age) and is 2-3 times more common in females. As a rule, it is a multifocal disease (in 60% of patients) involving one thyroid lobe, although in 50% of cases microscopic neoplastic lesions are present in the contralateral lobe. One should be also aware of the possible presence of a small, 2-10 mm focus of papillary thyroid carcinoma termed "microcarcinoma", which is asymptomatic and detected by chance in the course of histopathology of the thyroid gland resected in a patient with goiter or in serial autopsies of the thyroid (such post-mortem examinations detected 35.6% microcarcinoma foci). Approximately 50% of patients demonstrate the presence of metastases in the lymph nodes. Papillary carcinoma of the thyroid is a hormonally dependent tumor (TSH). The 5 year survival rate is approximately 95% [2, 3]. [Fig. 1,2,3,4]

Vascular Endothelial Growth Factor (VEGF) and

arginine, show an angiogenic activity [6-10].

scleroderma, endometriosis) and in neoplastic diseases. [11-13].

factor (VEGF) in patients with papillary thyroid cancers.

thyroid gland [17].

**4. Angiogenesis** 

interstitial cells [18].

Epidermal Growth Factor (EGF) in Papillary Thyroid Cancer 89

extended by articles on the paracrine effect of connective tissue interstitial cells of the

The regulation of this process is complex and the contributing factors include both neoplastic cells capable of producing such factors as cytokines and chemokines, as well as immunocompetent cells situated in the vicinity of tumor cells or infiltrating the tumor itself; the latter also produce cytokines, chemokines and growth factors. The interrelation of such factor production, especially in the case of chemokines, significantly intensifies angiogenesis. Chemokines, which contain the repeated sequence of glutamine-leucine-

The basic process of the formation of new blood vessels originating from the previously existing structures is the branching off of capillary vessels and budding of new vascular limbs that takes place both in fetal life and in mature organisms. The process is short-lived (approximately 5 days on the average), subject to strict regulations, and its sudden termination results from the reduction of stimulatory factors and/or a decrease of inhibitor levels. [1-9]. Angiogenesis is a pathomechanism involved in lesions developing in autoimmune diseases (rheumatoid arthritis, lupus erythematosus, hemangiomas,

A good part of publications in world literature on the role of angiogenic cytokines and epithelial growth factors in the process of tumor growth concentrate on processes occurring in the gastrointestinal tract [14-16]. Nevertheless, their presence and possible effect on the development and growth of tumors of endocrine origin have been recently recognized [17, 18]. VEGF is among relatively well-known endothelial growth factors. This specific protein is believed to play a key role in vascularization of solid tumors, including thyroid cancers. In keeping with the theory adopted by Folkman that states that tumor growth is limited by its vascularization, attempts were made at demonstrating higher VEGF expression in neoplastic tissues as compared to the population of normal cells. These studies show such an association with respect to cancers involving the stomach, colon, uterus, mammary glands and ovaries [14-16]. Also in the case of thyroid tumors, the key role in neoangiogenesis is played by VEGF, especially in view of the fact that the ability to produce and release this factor is characteristic not only of epithelial thyroid cells, but also of

To date, infrequent reports have dealt with peripheral blood serum VEGF determinations in patients with highly differentiated thyroid cancers, what has prompted us to attempt assessing the clinical relevance of determining the level of vascular endothelial growth

EGF is among the most potent stimulators of thyroid gland growth and its multiple activity is determined by its binding with specific EGF receptors. In vitro, EGF is a factor that stimulates the proliferation of follicular thyroid cells. A factor that intensifies EGF binding with receptors is thyreotropin (TSH), which - stimulating an increase in the number of EGF receptors - potentiates its activity [24,25]. In contrast to TSH, however, to reveal its mitogenic activity, EGF does not require the presence of other chemokines. [28]. Subsequent

#### **3. Oncogenesis**

The presently prevalent opinion states that genetic factors play an ever-increasing role in the development of neoplastic lesions. In view of the present knowledge, a prerequisite for neoplastic transformation to occur is a mutation involving two basic groups of genes proto-oncogenes and suppressor genes, also called anti-oncogenes. Proto-oncogenes function as positive proliferation regulators. Under the effect of various external and internal factors, they may be converted into oncogenes. In turn, oncogene products may be divided into two groups of proteins, which are responsible for encoding the production of growth factors and affect the expression of surface receptors, either cytoplasmic or nuclear, thus indirectly participating in transcription inhibition or activation. Early neoplastic lesions usually involve a single cell line and appear as a consequence of a single or several serial mutations. Such mutations result in an increased capability of the cells to undergo mitotic divisions with a simultaneous decrease of their apoptotic capability as compared to the adjacent cells. Thus, a cell line develops that may give origin to for example hyperplasia of the thyroid tissue associated with neoplastic growth, since the borderline separating neoplastic transformation and hyperplastic proliferation is very thin [4,5].

In view of the high metabolism of cells undergoing division, the growth of a nonvascularized tumor is low. The clinical presentation of this growth phase is most commonly carcinoma in situ. The subsequent phase of tumor growth depends on the formation of new blood vessels (neoangiogenesis). Neoangiogenesis is a process composed of numerous interactions occurring in the paracrine and endocrine path between neoplastic cells and cells forming the vascular endothelium, connective tissue interstitium and some morphotic blood elements, such as macrophages or mastocytes. In consequence of these interactions, the microenvironment in the area surrounding the tumor changes, thus providing the neoplastic lesion with an opportunity for further uncontrollable growth and progression. A prerequisite for the initiation of angiogenic phenomena is a disturbed balance between the systems of pro- and anti-angiogenic factors. Of the identified to date proangiogenic factors (VEGF; bFGF; aFGF; PDGF; TGF-α; TGF-β; EGF; IGF-1), not all meet the three defining criteria: exerting a specific effect on the endothelium, possessing the system of specific cell receptors and inhibiting or inducing angiogenesis through changes in their levels. Some of these factors co-act with mediators secreted by other cells (e.g. macrophages - TNF) in triggering and promoting the development of neoplastic disease. In accordance with currently accepted theories, the initiation of angiogenesis occurs through hypoxia of neoplastic cells that are situated the most distally from the lumen of a blood vessel, as well as through a defect of the genetic apparatus, in consequence of which the so-called angiogenic phenotype emerges. The term denotes the condition characterized by a permanent, constitutional activation of genes that encode growth factors. An additional loss of function by suppressor genes (e.g. the p53 gene) facilitates neoangiogenesis [8,19,20]. [Figure 5]

Endocrine glands constitute typical, richly vascularized organs; the circulating blood is the basis of their normal functioning and provides a close control of the feedback systems. As early as more than 20 years ago, investigators demonstrated that an increase in thyroid vascularization in patients with hyperthyroid goiter was regulated by cytokines secreted by thyreocytes. In subsequent years (reports by Goodman), the concept was confirmed and extended by articles on the paracrine effect of connective tissue interstitial cells of the thyroid gland [17].

The regulation of this process is complex and the contributing factors include both neoplastic cells capable of producing such factors as cytokines and chemokines, as well as immunocompetent cells situated in the vicinity of tumor cells or infiltrating the tumor itself; the latter also produce cytokines, chemokines and growth factors. The interrelation of such factor production, especially in the case of chemokines, significantly intensifies angiogenesis. Chemokines, which contain the repeated sequence of glutamine-leucinearginine, show an angiogenic activity [6-10].

#### **4. Angiogenesis**

88 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

The presently prevalent opinion states that genetic factors play an ever-increasing role in the development of neoplastic lesions. In view of the present knowledge, a prerequisite for neoplastic transformation to occur is a mutation involving two basic groups of genes proto-oncogenes and suppressor genes, also called anti-oncogenes. Proto-oncogenes function as positive proliferation regulators. Under the effect of various external and internal factors, they may be converted into oncogenes. In turn, oncogene products may be divided into two groups of proteins, which are responsible for encoding the production of growth factors and affect the expression of surface receptors, either cytoplasmic or nuclear, thus indirectly participating in transcription inhibition or activation. Early neoplastic lesions usually involve a single cell line and appear as a consequence of a single or several serial mutations. Such mutations result in an increased capability of the cells to undergo mitotic divisions with a simultaneous decrease of their apoptotic capability as compared to the adjacent cells. Thus, a cell line develops that may give origin to for example hyperplasia of the thyroid tissue associated with neoplastic growth, since the borderline separating

In view of the high metabolism of cells undergoing division, the growth of a nonvascularized tumor is low. The clinical presentation of this growth phase is most commonly carcinoma in situ. The subsequent phase of tumor growth depends on the formation of new blood vessels (neoangiogenesis). Neoangiogenesis is a process composed of numerous interactions occurring in the paracrine and endocrine path between neoplastic cells and cells forming the vascular endothelium, connective tissue interstitium and some morphotic blood elements, such as macrophages or mastocytes. In consequence of these interactions, the microenvironment in the area surrounding the tumor changes, thus providing the neoplastic lesion with an opportunity for further uncontrollable growth and progression. A prerequisite for the initiation of angiogenic phenomena is a disturbed balance between the systems of pro- and anti-angiogenic factors. Of the identified to date proangiogenic factors (VEGF; bFGF; aFGF; PDGF; TGF-α; TGF-β; EGF; IGF-1), not all meet the three defining criteria: exerting a specific effect on the endothelium, possessing the system of specific cell receptors and inhibiting or inducing angiogenesis through changes in their levels. Some of these factors co-act with mediators secreted by other cells (e.g. macrophages - TNF) in triggering and promoting the development of neoplastic disease. In accordance with currently accepted theories, the initiation of angiogenesis occurs through hypoxia of neoplastic cells that are situated the most distally from the lumen of a blood vessel, as well as through a defect of the genetic apparatus, in consequence of which the so-called angiogenic phenotype emerges. The term denotes the condition characterized by a permanent, constitutional activation of genes that encode growth factors. An additional loss of function by suppressor genes (e.g. the p53 gene) facilitates

Endocrine glands constitute typical, richly vascularized organs; the circulating blood is the basis of their normal functioning and provides a close control of the feedback systems. As early as more than 20 years ago, investigators demonstrated that an increase in thyroid vascularization in patients with hyperthyroid goiter was regulated by cytokines secreted by thyreocytes. In subsequent years (reports by Goodman), the concept was confirmed and

neoplastic transformation and hyperplastic proliferation is very thin [4,5].

neoangiogenesis [8,19,20]. [Figure 5]

**3. Oncogenesis** 

The basic process of the formation of new blood vessels originating from the previously existing structures is the branching off of capillary vessels and budding of new vascular limbs that takes place both in fetal life and in mature organisms. The process is short-lived (approximately 5 days on the average), subject to strict regulations, and its sudden termination results from the reduction of stimulatory factors and/or a decrease of inhibitor levels. [1-9]. Angiogenesis is a pathomechanism involved in lesions developing in autoimmune diseases (rheumatoid arthritis, lupus erythematosus, hemangiomas, scleroderma, endometriosis) and in neoplastic diseases. [11-13].

A good part of publications in world literature on the role of angiogenic cytokines and epithelial growth factors in the process of tumor growth concentrate on processes occurring in the gastrointestinal tract [14-16]. Nevertheless, their presence and possible effect on the development and growth of tumors of endocrine origin have been recently recognized [17, 18].

VEGF is among relatively well-known endothelial growth factors. This specific protein is believed to play a key role in vascularization of solid tumors, including thyroid cancers. In keeping with the theory adopted by Folkman that states that tumor growth is limited by its vascularization, attempts were made at demonstrating higher VEGF expression in neoplastic tissues as compared to the population of normal cells. These studies show such an association with respect to cancers involving the stomach, colon, uterus, mammary glands and ovaries [14-16]. Also in the case of thyroid tumors, the key role in neoangiogenesis is played by VEGF, especially in view of the fact that the ability to produce and release this factor is characteristic not only of epithelial thyroid cells, but also of interstitial cells [18].

To date, infrequent reports have dealt with peripheral blood serum VEGF determinations in patients with highly differentiated thyroid cancers, what has prompted us to attempt assessing the clinical relevance of determining the level of vascular endothelial growth factor (VEGF) in patients with papillary thyroid cancers.

EGF is among the most potent stimulators of thyroid gland growth and its multiple activity is determined by its binding with specific EGF receptors. In vitro, EGF is a factor that stimulates the proliferation of follicular thyroid cells. A factor that intensifies EGF binding with receptors is thyreotropin (TSH), which - stimulating an increase in the number of EGF receptors - potentiates its activity [24,25]. In contrast to TSH, however, to reveal its mitogenic activity, EGF does not require the presence of other chemokines. [28]. Subsequent

Vascular Endothelial Growth Factor (VEGF) and

Fig. 2. Papillary thyroid cancer – a typically view

Fig. 3. Papillary thyroid cancer – a microcarcinoma variant

Epidermal Growth Factor (EGF) in Papillary Thyroid Cancer 91

investigators have attempted to determine the importance of positive EGF receptor expression in neoplastic thyroid tissue in the clinical course of the disease. EFG levels have been compared in various types of thyroid carcinomas and the highest expression has been found to be characteristic of anaplastic and medullary thyroid cancers. Also adenomas have been demonstrated to show marked expression of EGF receptors; however, this phenomenon involved solely certain regions of the tumor. The observation may weigh in favor of the possible neoplastic transformation of tumor tissues towards malignant processes [26]. The studies of Akslen et al. provided information on the importance of EGF receptor expression in the cytoplasm of papillary thyroid cancer cells, which was closely associated with extrathyroid growth of the tumor [27].

Summing up the results of studies on the serum concentration values of selected growth factors - VEGF and EGF - in patients with papillary thyroid cancer, one should state that they both participate in the induction and progression of neoplastic processes involving the thyroid gland, most likely acting, however, at various stages of tumor development - VEGF at the stage of neoangiogenesis induction, and EGF at the stage of invasion and possible remote metastases formation. [Figure 6]. To provide a firm and unambiguous confirmation of our observations it is necessary to conduct further investigations of the angiogenic activity, demonstrating a correlation between microvessel density (MVD) in the primary and metastatic tumors, as well as the presence and expression of receptors of these chemokines on the one hand, and the clinical stage of the tumor on the other; and showing whether these growth factors indeed have a prognostic value in identifying patients with a poor prognosis and expected shorter recurrence-free survival.

Fig. 1. Papillary thyroid cancer – psammoma body.

investigators have attempted to determine the importance of positive EGF receptor expression in neoplastic thyroid tissue in the clinical course of the disease. EFG levels have been compared in various types of thyroid carcinomas and the highest expression has been found to be characteristic of anaplastic and medullary thyroid cancers. Also adenomas have been demonstrated to show marked expression of EGF receptors; however, this phenomenon involved solely certain regions of the tumor. The observation may weigh in favor of the possible neoplastic transformation of tumor tissues towards malignant processes [26]. The studies of Akslen et al. provided information on the importance of EGF receptor expression in the cytoplasm of papillary thyroid cancer cells, which was closely

Summing up the results of studies on the serum concentration values of selected growth factors - VEGF and EGF - in patients with papillary thyroid cancer, one should state that they both participate in the induction and progression of neoplastic processes involving the thyroid gland, most likely acting, however, at various stages of tumor development - VEGF at the stage of neoangiogenesis induction, and EGF at the stage of invasion and possible remote metastases formation. [Figure 6]. To provide a firm and unambiguous confirmation of our observations it is necessary to conduct further investigations of the angiogenic activity, demonstrating a correlation between microvessel density (MVD) in the primary and metastatic tumors, as well as the presence and expression of receptors of these chemokines on the one hand, and the clinical stage of the tumor on the other; and showing whether these growth factors indeed have a prognostic value in identifying patients with a

associated with extrathyroid growth of the tumor [27].

poor prognosis and expected shorter recurrence-free survival.

Fig. 1. Papillary thyroid cancer – psammoma body.

Fig. 2. Papillary thyroid cancer – a typically view

Fig. 3. Papillary thyroid cancer – a microcarcinoma variant

Vascular Endothelial Growth Factor (VEGF) and

0

**5. Keywords** 

prognostic value

**6. References** 

New York.

6:389-395.

500

1000

1500

2000

**EGF [pg/ml]**

2500

3000

Epidermal Growth Factor (EGF) in Papillary Thyroid Cancer 93

r = 0.6104 p<0.05

r = -0.5168 p<0.05

Fig. 6. Correlation between VEGF, EGF and staging of papillary thyroid cancer in pTNM

Papillary thyroid cancer, vascular endothelial growth factor, epidermal growth factor,

[2] DeGroot LJ, Kaplan EL. McCormik M, Straus FH (1990) Natural history, treatment and course of papillary thyroid carcinoma. J Clin Endocrinol Metab 71: 414-424. [3] Schindler AM, van Melle G, Evequoz B, Scazziga B (1991) Prognostic factors in papillary

[4] Goretzki PE, Simon D, Dotzenrath C, Schulte KM, Röher HD (2000) *Growth Regulation of* 

[5] Falk SA (1997) Thyroid disease: endocrinology, surgery, nuclear medicine and

[7] Yancopoulos GD, Klagsburn M, Folkman J (1998) Vasculogenesis, angiogenesis and

[9] Carmeliet P (2000) Mechanism of angiogenesis and arteriogenesis. Nature Medicine

growth factors: ephrins enter the fray at the border. Cell 93:661-664.

radiotherapy. Lippincott, Williams & Wilkins and Raven Publishers, Philadelphia-

*Thyroid and Thyroid Tumors in Humans.* World J Surg 24:913-922.

0

100

200

EGF VEGF Linear (EGF) Linear (VEGF)

300

400

500

**VEGF [pg/ml]**

600

700

800

**pT1N0M0 pT2N0M0 pT3N1M0 pT4N1M0**

classification (r = Pearson's correlation coefficient). The hypothesis of influence.

[1] American Cancer Society (1991) Cancer Statistics 41: 28-29.

carcinoma of the thyroid. Cancer 68:324-330.

[6] Folkman J, Shing Y (1992) Angiogenesis. J Biol Chem 359:843-848.

[8] Risau W (1997) Mechanism of angiogenesis. Nature 386:671-674.

Fig. 4. Papillary thyroid cancer – high positive test of CK – 19

Fig. 5. Vascular growth factors and the effects of their acting.

Fig. 6. Correlation between VEGF, EGF and staging of papillary thyroid cancer in pTNM classification (r = Pearson's correlation coefficient). The hypothesis of influence.

#### **5. Keywords**

92 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

Fig. 4. Papillary thyroid cancer – high positive test of CK – 19

Fig. 5. Vascular growth factors and the effects of their acting.

Papillary thyroid cancer, vascular endothelial growth factor, epidermal growth factor, prognostic value

#### **6. References**


**7**

*Brazil* 

**Immune Profile and Signal Transduction**

*Endocrinology and Metabolism Unit of Santa Casa São Paulo, São Paulo* 

Autoimmune thyroiditis is of great importance because of its prevalence in global population and represents an organ-specific immune dysfunction whose pathophysiological stages have not yet been fully elucidated. It is well accepted that, as in other autoimmune diseases, there is loss of tolerance to auto-antigens (such as thyroperoxidase or thyroglobulin) with subsequent abnormal lymphocyte activation fostering aggression to

From the autoimmune dysfunction, especially in Hashimoto's thyroiditis (HT), the thyrocyte may undergo apoptosis by the FAS-mediated in CD4 + and CD8 + mechanism, or by downregulation of anti-apoptotic protein expression such as Bcl-2 (Mitsiades, Poulaki et al., 1998; Fountoulakis, Vartholomatos et al., 2008). In the HT, lymphocytic infiltrate is intense with formation of germinal centers and destruction of thyroid follicles by chronic inflammation through the natural killer cells (NK) and cytotoxicity induced by

In Hashimoto's thyroiditis, chronic inflammation and apoptosis are accepted as a mechanism of the disease and resultant hypothyroidism. It is noteworthy that presence of lymphocytic infiltrate alone does not necessarily induce hypothyroidism (Martin, Colonel et al., 2004) but induces immune dysfunction of T cells response due to genetic and

T cell and B cell immune dysfunction with production of autoantibodies, cytotoxic cell death, in addition to the previously mentioned apoptosis is the classical model of thyrocytes and thyroid follicle destruction. But recently described as non-classic mechanism and not related to cell death, but to the chronic inflammatory process by inhibiting the thyroid function mediated by inflammatory cytokines TNF-α and INF-γ based upon T cell dysfunction, even without a lymphocytic infiltrate, but by exposure *per se* to inflammatory

Differently, within the group of autoimmune thyroid diseases, Graves' disease (GD) occurs in a unique situation in autoimmunity, with dysfunction in T and B cells, however producing an autoantibody IgG, with great affinity to specific regions of the TSH (TSH-R)

environmental predisposition in different populations and ethnic groups.

cytokines (Caturegli, Hejazi et al., 2000; Kimura, Kimura et al., 2005).

**1. Introduction** 

thyroid tissue.

auto-antibodies.

**of T-Cell Receptor in Autoimmune** 

**Thyroid Diseases** 

Adriano Namo Cury


### **Immune Profile and Signal Transduction of T-Cell Receptor in Autoimmune Thyroid Diseases**

Adriano Namo Cury *Endocrinology and Metabolism Unit of Santa Casa São Paulo, São Paulo Brazil* 

#### **1. Introduction**

94 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

[12] Norrby W (1997) Angiogenesis: a new aspects relating to its initiation and control. Acta

[13] Eliseenko VI, Skobelkin OK, Chegin VM (1998) Microcirculation and angiogenesis

[14] Maeda K, Chung YS, Ogawa Y (1996) Prognostic value of vascular endothelial growth

[15] Okada F, Rak J, St.Croix B, Lieubeau B, Kaya M, Roncari L, Shirasawa S, Sasazuki T,

[16] Gasparini G, Toi M, Gion M, Verderio P, Dittadi R, Hanatani M, Matsubara I, Vinante

[17] Goodman AL, Rone JD (1987) Thyroid angiogenesis: endotheliotropic chemoattractant activity from rat thyroid cell in culture. Endocrinology 121:2131-2140. [18] Turner HE, Harris AL, Melmed SH, Wass JAH (2003) Angiogenesis in endocrine

[19] Rak J, Mrtsuhashi Y, Bayko L, Filmus J, Sasazuki T, Kerbel RS (1995) Mutant ras

[20] Kerbel RS, Vilona-Petit A, Okada F, Rak J (1998) Establishing a link between oncogenes

[21] Lin SY, Wang YY, Sheu WH (2003) Preoperative plasma concentrations of vascular

[23] Huang SM, Lee JC, Wu TJ, Chow NH (2001) Clinical revelance of vascular endothelial

[24] Westermark K, Westermark B (1982) Mitogenic effect of epidermal growth factor on

[25] Westermark K, Karlsson A, Westermark B (1985) Thyrotropin modulates EGF receptor functionin porcine thyroid follicle cells. Mol Cell Endocrinol 40:17-23. [26] Masuda H, Sugenoya A, Kobayashi S, Kasuga Y, Iida F (1988) Epidermal growth factor

[27] Alslen LA, Myking AO, Salvesen H, Varhaug JE (1993) Prognostic impact of EGF-

[28] Westermark K, Karlsson FA, Westermark B (1983) Epidermal growth factor modulates thyroid growth and function in culture. Endocrinology 112:1680-1686.

progression in papillary thyroid cancer. Clin Endocrinol 58:513-518. [22] Lennard CM, Patel A, Wilson J, Reinhardt B, Tuman C, Fenton C, Blair E, Francis GL,

growth factor for thyroid neoplasms. World J Surg 25:302-306.

receptor on human thyroid neoplasm. World J Surg 12:616-622.

receptor in papillary thyroid carcinoma. Br J Cancer 68:808-812.

sheep thyroid cells in culture. Exp Cell Res 138:47-55.

inhibition of tumor angiogenesis. Cancer Res 55:4575-4580.

and tumor angiogenesis. Molecular Medicine 4:286-295.

papillary thyroid cancer. Surgery 129:552-558.

human colorectal carcinoma cells. Proc Natl Acad Sci 95:3609-3614.

during wound healing by first and second intention. Bull Experiment Biol Med

Kerbel RS (1998) Impact of oncogenes on tumor angiogenesis: mutant K-ras upregulation of VEGF/VPF is necessary but not sufficient for tumorigenicyity of

O, Bonoldi E, Boracchi P, Gatti C, Suzuki H, Tominaga T (1997) Prognostic significance of vascular endothelial growth factor protein in node-negativ breast

oncogenes upregulate VEGF/VPF expression: implications for inducion or

endothelial growth factor and matrix metalloproteinase 9 are associated with stage

Tuttle RM (2001) Intensity of vascular endothelial growth factor expression associated with increased risk of recurrence and decreased disease-free survival in

[10] Balkwill F. (2003) :Chemokines biology in cancer. Seminars in Immunology 15: 49-55. [11] Reichlin M (1998) Systemic lupus erythematosus. In: Rose NR, Mackay IR (eds.) The

Autoimmune diseases. Academic Press, Philadelphia, pp 1-37.

factor expression in gastric carcinoma. Caner 77:858-863.

Pathol Microbiol Immunol Scand 105:417-437.

carcinoma. J Natl Cancer Inst 89:139-147.

tumors. Endocrine Rev 24:600-632.

105:289-292.

Autoimmune thyroiditis is of great importance because of its prevalence in global population and represents an organ-specific immune dysfunction whose pathophysiological stages have not yet been fully elucidated. It is well accepted that, as in other autoimmune diseases, there is loss of tolerance to auto-antigens (such as thyroperoxidase or thyroglobulin) with subsequent abnormal lymphocyte activation fostering aggression to thyroid tissue.

From the autoimmune dysfunction, especially in Hashimoto's thyroiditis (HT), the thyrocyte may undergo apoptosis by the FAS-mediated in CD4 + and CD8 + mechanism, or by downregulation of anti-apoptotic protein expression such as Bcl-2 (Mitsiades, Poulaki et al., 1998; Fountoulakis, Vartholomatos et al., 2008). In the HT, lymphocytic infiltrate is intense with formation of germinal centers and destruction of thyroid follicles by chronic inflammation through the natural killer cells (NK) and cytotoxicity induced by auto-antibodies.

In Hashimoto's thyroiditis, chronic inflammation and apoptosis are accepted as a mechanism of the disease and resultant hypothyroidism. It is noteworthy that presence of lymphocytic infiltrate alone does not necessarily induce hypothyroidism (Martin, Colonel et al., 2004) but induces immune dysfunction of T cells response due to genetic and environmental predisposition in different populations and ethnic groups.

T cell and B cell immune dysfunction with production of autoantibodies, cytotoxic cell death, in addition to the previously mentioned apoptosis is the classical model of thyrocytes and thyroid follicle destruction. But recently described as non-classic mechanism and not related to cell death, but to the chronic inflammatory process by inhibiting the thyroid function mediated by inflammatory cytokines TNF-α and INF-γ based upon T cell dysfunction, even without a lymphocytic infiltrate, but by exposure *per se* to inflammatory cytokines (Caturegli, Hejazi et al., 2000; Kimura, Kimura et al., 2005).

Differently, within the group of autoimmune thyroid diseases, Graves' disease (GD) occurs in a unique situation in autoimmunity, with dysfunction in T and B cells, however producing an autoantibody IgG, with great affinity to specific regions of the TSH (TSH-R)

Immune Profile and Tyrosine Phosphorylation

significant results in those with GD.

process (Green, Droin et al., 2003).

inflammation and cell destruction.

**2.2 Loss of tolerance and natural regulatory T cells** 

Hatton et al., 2007).

of T-Cell Receptor in Autoimmune Thyroid Diseases 97

and IL-22 (Wilson, Boniface et al., 2007 ) playing an important role in chronic inflammatory diseases such as asthma (Traves and Donnelly 2008) or systemic lupus erythematosus (Garrett-Sinha, John et al., 2008). IL-17 has potent proinflammatory action of chemotaxis, with chemokine synthesis and stimulus of cell proliferation (Weaver,

Proportion of Th17 lymphocytes in patients with GD was first described by Nanba *et. al.,* whose main finding was a higher rate of Th17, on the peripheral blood of patients with GD without treatment with anti-thyroid drugs when compared to patients with GD in remission (Nanba, Watanabe et al., 2009). Study of the profile of Th17 lymphocytes in Hashimoto's thyroiditis according to Figueroa-Vera *et al.,* (Figueroa-Vega, Alfonso-Perez et al., 2010) discloses a higher expression of the RORC2 gene responsible for differentiation of the Th17 phenotype, in addition to the sheer number of Th17 lymphocytes in peripheral blood and thyroid tissue of patients with HT, however without

Autoimmunity occurs mainly by loss of tolerance to auto-antigens from the perpetuation of autoreactive T cells and pathogenic for their cellular targets. Didactically, it can be understood that the stages of loss of tolerance occur in two moments: (1) failure of central tolerance and (2) peripheral. Initially, the correct reading of non-auto-antigens and no formation of autoreactive cells is known as clonal selection theory or negative selection when autoreactive lymphocytes are deleted at the initial stages of cell differentiation in the thymus (Burnet 1959). Evasion of cell clones from the negative central selection, autoreactive lymphocytes to the periphery may or may not be activated and trigger the process of autoimmune disease, however according to the genetic and environmental interaction. There is a break in the state of anergy or antigenic ignorance, with clonal activation and expansion that will then initiate the autoimmune and inflammatory

This cellular mechanism is known as intrinsic cellular mechanism of peripheral tolerance (Schwartz 2005). The extrinsic cellular mechanism of tolerance is carried out by regulatory T cells CD4 + CD25 + natural (Treg) that exist because of the expression of Forkhead Box Protein 3 (Foxp3), Treg are responsible for suppression of immune response of auto-reactive clones (Sakaguchi, Yamaguchi et al., 2008) and control amplification of the inflammatory response. Failure in negative central and peripheral selection promotes clonal expansion with differentiation of autoreactive cell subtypes, which according to the genetic and environmental triggers differentiate into T cells to produce inflammatory cytokines inducing

It is believed that failure of the negative selection process could not per *se* trigger autoimmunity. Perpetuation of pathogenic autoreactive cells associated with a lesser expression and differentiation of Tregs would be another condition for autoimmunity development. It was shown that CD4+ CD25 + cells may experimentally prevent development of autoimmune thyroiditis (Gangi, Vasu et al., 2005; Vergini, Li et al., 2005). As the proportion and function of Tregs seems altered in the ATD when analyzing peripheral and cells of the thyroid ambient itself without the ability to downmodulate the autoimmune

receptor that determines hyperfunction, hypertrophy of the thyroid follicle, abnormal dynamics of activation, or even blockade of TSH-R and hyperthyroidism itself (Hadj-Kacem, Rebuffat et al., 2009). Production of TRAb stimulator and sometimes blocker is an expression of the break of tolerance to TSH-R specific epitopes with biological effect similar to TSH, yet with longer lasting and slower signaling to thyrocytes.

Exposure of TSH-R subunit-A seems to be responsible for generating the TRAb stimulator, which generates an atypical biological signal to the thyroid cells which respond with activation of the intracellular machinery, hypertrophy, and hypersecretion (Rapoport and McLachlan 2007). In GD the inflammatory infiltrate is less intense when compared to HT, and the phenomenon of apoptosis is less pronounced due to probable protection by soluble FAS (sFas), interfering with the classic mechanism of apoptosis FAS-FASL (Feldkamp, Pascher et al., 2001; Fountoulakis, Vartholomatos et al., 2008) or by upregulation of anti-apoptotic proteins in thyrocytes such as Bcl-2, Bcl-xL and cFLIP (Stassi, Di Liberto et al., 2000).

Comprehension of cell phenotype, dynamics of lymphocyte activation in the break of immune tolerance and its correlation with immunoregulator genes is mandatory to understand the etiology and development mechanism of autoimmune thyroid diseases. Moreover, for the attempt to elucidate the pathways of cellular and humoral dysfunctions that determine the route leading to HT or GD.

#### **2. Immunity and T cells**

#### **2.1 Inflammatory response in autoimmune thyroid diseases**

Immune behavior of autoimmune thyroid diseases lies in the characterization of the concept of breaking the mechanism of tolerance to self antigens, activation and differentiation of cell clones in charge (T-cell subtypes) of the amplification and execution of inflammatory response in thyroid tissue and dynamics of lymphocyte receptors in HT or GD. Differentiation and amplification of the inflammatory response in different types of T cells plays an essential role in the pathogenesis of the disease.

The understanding of cell phenotype, dynamics of lymphocytes activation in the break of immune tolerance and its correlation with immunoregulator genes is mandatory for the etiology and development mechanism of autoimmune thyroid diseases. Or even for the attempt to elucidate the pathways of cellular and humoral dysfunctions that rule the path leading to HT or GD.

The CD4 + T helper lymphocytes (Th) can be classified into at least three subtypes, Th1, Th2 and Th17 in accordance with a profile of cytokine production (Abbas, Murphy et al., 1996; Mosmann and Sad 1996). In HT the Th1 cell response prevails with predominant production of IFN-γ, IL-2 and TNF-β (Fisfalen, Palmer et al., 1997; Fisfalen, Soltani et al., 1997; Watanabe, Yamamoto et al., 2002). The Th2 humoral response pertains to GD with production of cytokines IL-4, IL-5, IL-6, IL-10 and IL-13 and suppression of INF-γ (Yano, Sone et al., 1995; Abbas Murphy et al., 1996; Fisfalen, Palmer et al., 1997; Fisfalen, Soltani et al., 1997).

Th17 lymphocytes were recently described and specific studies on thyroid autoimmune diseases are scarce. T cells that differentiate to Th17 subtypes secrete IL-17, IL-17F, IL-21

receptor that determines hyperfunction, hypertrophy of the thyroid follicle, abnormal dynamics of activation, or even blockade of TSH-R and hyperthyroidism itself (Hadj-Kacem, Rebuffat et al., 2009). Production of TRAb stimulator and sometimes blocker is an expression of the break of tolerance to TSH-R specific epitopes with biological effect similar

Exposure of TSH-R subunit-A seems to be responsible for generating the TRAb stimulator, which generates an atypical biological signal to the thyroid cells which respond with activation of the intracellular machinery, hypertrophy, and hypersecretion (Rapoport and McLachlan 2007). In GD the inflammatory infiltrate is less intense when compared to HT, and the phenomenon of apoptosis is less pronounced due to probable protection by soluble FAS (sFas), interfering with the classic mechanism of apoptosis FAS-FASL (Feldkamp, Pascher et al., 2001; Fountoulakis, Vartholomatos et al., 2008) or by upregulation of anti-apoptotic proteins in thyrocytes such as Bcl-2, Bcl-xL and cFLIP

Comprehension of cell phenotype, dynamics of lymphocyte activation in the break of immune tolerance and its correlation with immunoregulator genes is mandatory to understand the etiology and development mechanism of autoimmune thyroid diseases. Moreover, for the attempt to elucidate the pathways of cellular and humoral dysfunctions

Immune behavior of autoimmune thyroid diseases lies in the characterization of the concept of breaking the mechanism of tolerance to self antigens, activation and differentiation of cell clones in charge (T-cell subtypes) of the amplification and execution of inflammatory response in thyroid tissue and dynamics of lymphocyte receptors in HT or GD. Differentiation and amplification of the inflammatory response in different types of T cells

The understanding of cell phenotype, dynamics of lymphocytes activation in the break of immune tolerance and its correlation with immunoregulator genes is mandatory for the etiology and development mechanism of autoimmune thyroid diseases. Or even for the attempt to elucidate the pathways of cellular and humoral dysfunctions that rule the path

The CD4 + T helper lymphocytes (Th) can be classified into at least three subtypes, Th1, Th2 and Th17 in accordance with a profile of cytokine production (Abbas, Murphy et al., 1996; Mosmann and Sad 1996). In HT the Th1 cell response prevails with predominant production of IFN-γ, IL-2 and TNF-β (Fisfalen, Palmer et al., 1997; Fisfalen, Soltani et al., 1997; Watanabe, Yamamoto et al., 2002). The Th2 humoral response pertains to GD with production of cytokines IL-4, IL-5, IL-6, IL-10 and IL-13 and suppression of INF-γ (Yano, Sone et al., 1995; Abbas Murphy et al., 1996; Fisfalen, Palmer et al., 1997; Fisfalen, Soltani

Th17 lymphocytes were recently described and specific studies on thyroid autoimmune diseases are scarce. T cells that differentiate to Th17 subtypes secrete IL-17, IL-17F, IL-21

to TSH, yet with longer lasting and slower signaling to thyrocytes.

(Stassi, Di Liberto et al., 2000).

**2. Immunity and T cells** 

leading to HT or GD.

et al., 1997).

that determine the route leading to HT or GD.

**2.1 Inflammatory response in autoimmune thyroid diseases** 

plays an essential role in the pathogenesis of the disease.

and IL-22 (Wilson, Boniface et al., 2007 ) playing an important role in chronic inflammatory diseases such as asthma (Traves and Donnelly 2008) or systemic lupus erythematosus (Garrett-Sinha, John et al., 2008). IL-17 has potent proinflammatory action of chemotaxis, with chemokine synthesis and stimulus of cell proliferation (Weaver, Hatton et al., 2007).

Proportion of Th17 lymphocytes in patients with GD was first described by Nanba *et. al.,* whose main finding was a higher rate of Th17, on the peripheral blood of patients with GD without treatment with anti-thyroid drugs when compared to patients with GD in remission (Nanba, Watanabe et al., 2009). Study of the profile of Th17 lymphocytes in Hashimoto's thyroiditis according to Figueroa-Vera *et al.,* (Figueroa-Vega, Alfonso-Perez et al., 2010) discloses a higher expression of the RORC2 gene responsible for differentiation of the Th17 phenotype, in addition to the sheer number of Th17 lymphocytes in peripheral blood and thyroid tissue of patients with HT, however without significant results in those with GD.

#### **2.2 Loss of tolerance and natural regulatory T cells**

Autoimmunity occurs mainly by loss of tolerance to auto-antigens from the perpetuation of autoreactive T cells and pathogenic for their cellular targets. Didactically, it can be understood that the stages of loss of tolerance occur in two moments: (1) failure of central tolerance and (2) peripheral. Initially, the correct reading of non-auto-antigens and no formation of autoreactive cells is known as clonal selection theory or negative selection when autoreactive lymphocytes are deleted at the initial stages of cell differentiation in the thymus (Burnet 1959). Evasion of cell clones from the negative central selection, autoreactive lymphocytes to the periphery may or may not be activated and trigger the process of autoimmune disease, however according to the genetic and environmental interaction. There is a break in the state of anergy or antigenic ignorance, with clonal activation and expansion that will then initiate the autoimmune and inflammatory process (Green, Droin et al., 2003).

This cellular mechanism is known as intrinsic cellular mechanism of peripheral tolerance (Schwartz 2005). The extrinsic cellular mechanism of tolerance is carried out by regulatory T cells CD4 + CD25 + natural (Treg) that exist because of the expression of Forkhead Box Protein 3 (Foxp3), Treg are responsible for suppression of immune response of auto-reactive clones (Sakaguchi, Yamaguchi et al., 2008) and control amplification of the inflammatory response. Failure in negative central and peripheral selection promotes clonal expansion with differentiation of autoreactive cell subtypes, which according to the genetic and environmental triggers differentiate into T cells to produce inflammatory cytokines inducing inflammation and cell destruction.

It is believed that failure of the negative selection process could not per *se* trigger autoimmunity. Perpetuation of pathogenic autoreactive cells associated with a lesser expression and differentiation of Tregs would be another condition for autoimmunity development. It was shown that CD4+ CD25 + cells may experimentally prevent development of autoimmune thyroiditis (Gangi, Vasu et al., 2005; Vergini, Li et al., 2005). As the proportion and function of Tregs seems altered in the ATD when analyzing peripheral and cells of the thyroid ambient itself without the ability to downmodulate the autoimmune

Immune Profile and Tyrosine Phosphorylation

al., 1999; Vaidya, Imrie et al., 1999).

quantities of inflammatory cytokines.

cells (Treg) (Atabani, Thio et al., 2005).

of T-Cell Receptor in Autoimmune Thyroid Diseases 99

and Vaidya 2004; Kavvoura, Akamizu et al., 2007). and in familial studies was associated with GD in Caucasian, Japanese, Chinese and Korean populations (Heward, Allahabadia et

Proteins CD28 and CTLA4 are costimulatory molecules found on the surface of T cells that bind to the family of B7 receptors expressed on antigen presenting cells (APC) (Reiser and Stadecker 1996). Immune response relies on the generation of two signals: the first, from the interaction of antigenic peptides with receptors on T cells in the context of MHC and the second signal (costimulatory) that activates, enhances and promotes T cell proliferation by production of cytokines (such as IL-2), where complex CD28/B7 functions as a positive regulator of T cells, and the CTLA4/B7 expressed exclusively in activated T lymphocytes, provides an inhibitory signal, required to limit proliferation of T cells and regulates the autoimmune response (Oosterwegel, Greenwald et al., 1999; Sharpe and Abbas 2006).

**4. Polymorphisms, functional impact on the T cell and transduction of TCR**  The polymorphism +49 A> G (rs231775) of gene CTLA-4, which promotes the exchange of amino acid threonine for alanine at position 17, has emerged as the natural candidate, among polymorphisms of CTLA-4 gene, because of the ability to promote functional changes of protein CTLA- 4. Kouki *et al.,* (Kouki, Sawai et al., 2000) showed a higher frequency of genotype GG or AG at position 49 of the CTLA-4 gene in GD patients, and lesser control over proliferation and clonal expansion of T cells. Whereas Maurer *et al (Maurer, Loserth et al., 2002)* found differences in the pool of intracellular CTLA-4 protein, prompting imbalance in the expression and competition between CTLA-4 and CD28 on the surface of T cells, possibly modifying the suppression of T cells and generating larger

The CT60 polymorphism (rs30807243) seems to determine a distinct expression of mRNA isoforms through alternative splicing, with a lesser expression of soluble CTLA-4 (sCTLA-4) in relation to the total length isoform of CTLA-4 (*fl*CTLA-4) (Ueda, Howson et al., 2003). The correlation between immune-cell genotype and phenotype was demonstrated by presence of susceptibility allele G or allele A of protection, to specific subtypes of T cells in healthy controls, and the quantitative variations of the type CD4 + CD25 + cells called regulatory T

Other studies have shown an association between the SNP at exon 2 of CTLA-4 gene in mice, and a new variant of the protein called ligand independent of CTLA-4 (liCTLA-4) (Wicker, Chamberlain et al., 2004), that also has a significant inhibitor function on T cell

Expression of the isoform liCTLA-4 seems to be greater in regulatory and memory T cells (Vijayakrishnan, Slavik et al., 2004), a possible association between the gene CTLA-4 (its isoforms) and immune response after antigen presentation by APC and T cell activation as from the T cell receptor. As such, genetic variations in the CTLA-4 gene region play an important role in T cell signaling and therefore in its function and proliferation of T cells. Different genotypes may determine different phenotypes and probable predisposition to autoimmunity by loss of negative selection mechanisms (central and peripheral immune

response when binding and dephosphorilating the T cell receptor (TCR).

system dysfunction) and a distinct pattern of CD4 + T cells.

response in the thyroid environment (Marazuela, Garcia-Lopez et al., 2006), or even suffer increased apoptosis in the thyroid environment (Nakano, Watanabe et al., 2007) a significant event considering that regulatory action of Treg cells modulates and inhibits inflammatory immune response Th1, Th2 and Th17.

Therefore, the evasion of autoreactive cells, the reduced presence of Tregs, the non- control of inflammatory response, in the genetic context and environmental factor, the predominance of phenotypes Th1, Th2 or even Th17 characterizes autoimmune thyroid disease. Hashimoto's thyroiditis with typical Th1 response and cell infiltrate by thyroid tissue and thyrocyte apoptosis.

In GD, product of a more humoral response Th2, lesser lymphocytic infiltrate and specific failures for TSHR that generate, presumably the only autoimmune condition that promotes hyperplasia, with a lesser degree of apoptosis and IgG by affinity for THSR. The Th17 response might possibly be involved in HT or GD. However studies encompass a limited number of patients and primarily use peripheral blood to isolate lymphocytes, while study of lymphocytes from the thyroid is scarce, since indications for surgery for patients with GD or HT are less frequent nowadays.

#### **3. Immunoregulator genes**

The autoimmune thyroid diseases (ATD), such as Hashimoto's thyroiditis and Graves' disease are found in the general population and have an estimated prevalence of 5% (Ban, Davies et al., 2003). Pathogenesis of ATD especially that of GD is brought about by complex interaction between environmental and genetic factors. Genetics of predisposition for ATD involve HLA (human leukocyte antigen) system genes and specific genes that affect any step of the immune response regulation, i.e. activation and suppression of T cells and consequent modulation of B-cells.

Besides genes of the MHC class II system such as HLA-DR3 and DQA1 \* 0501 on chromosome 6p21 (Yanagawa, Mangklabruks et al., 1993; Zamani, Spaepen et al., 2000; Maciel, Rodrigues et al., 2001), on chromosome 2q33, we found the *loci* of genes involved in the regulation of T lymphocytes: CD28, CTLA4 and ICOS in which, specifically polymorphisms of the cytotoxic T lymphocyte antigen-4 (CTLA-4) are associated to various autoimmune diseases (Chistiakov and Turakulov 2003; Vaidya and Pearce 2004).

Allelic variants of the CTLA-4 gene with potential effect on functional modulation of the T cell were shown as single-nucleotide polymorphism (SNP) + 49 A> G in exon 1, which seems to modify both the structure and protein expression of CTLA-4 [7]. In 2003, Ueda *et al.,* identified the SNP +6230 G> A in the stop codon of gene CTLA-4 (CT60) associating it to higher risk for GD, HT and type 1 diabetes (DM1) due to expression of different isoforms of mRNA gene CTLA-4 (Ueda, Howson et al., 2003).

In case-control studies during the last decade, polymorphism +49 A> G of CTLA4 gene exon 1 was associated with autoimmune diseases, such as GD, DM1, HT, rheumatoid arthritis, autoimmune Addison's disease, multiple sclerosis (Yanagawa, Hidaka et al., 1995; Nistico, Buzzetti et al., 1996; Kotsa, Watson et al., 1997; Yanagawa, Taniyama et al., 1997; Awata, Kurihara et al., 1998; Fukazawa, Yanagawa et al., 1999; Heward, Allahabadia et al., 1999; Vaidya, Imrie et al., 2000; Ueda, Howson et al., 2003; Blomhoff, Lie et al., 2004; Young-Min

response in the thyroid environment (Marazuela, Garcia-Lopez et al., 2006), or even suffer increased apoptosis in the thyroid environment (Nakano, Watanabe et al., 2007) a significant event considering that regulatory action of Treg cells modulates and inhibits inflammatory

Therefore, the evasion of autoreactive cells, the reduced presence of Tregs, the non- control of inflammatory response, in the genetic context and environmental factor, the predominance of phenotypes Th1, Th2 or even Th17 characterizes autoimmune thyroid disease. Hashimoto's thyroiditis with typical Th1 response and cell infiltrate by thyroid

In GD, product of a more humoral response Th2, lesser lymphocytic infiltrate and specific failures for TSHR that generate, presumably the only autoimmune condition that promotes hyperplasia, with a lesser degree of apoptosis and IgG by affinity for THSR. The Th17 response might possibly be involved in HT or GD. However studies encompass a limited number of patients and primarily use peripheral blood to isolate lymphocytes, while study of lymphocytes from the thyroid is scarce, since indications for surgery for patients with GD

The autoimmune thyroid diseases (ATD), such as Hashimoto's thyroiditis and Graves' disease are found in the general population and have an estimated prevalence of 5% (Ban, Davies et al., 2003). Pathogenesis of ATD especially that of GD is brought about by complex interaction between environmental and genetic factors. Genetics of predisposition for ATD involve HLA (human leukocyte antigen) system genes and specific genes that affect any step of the immune response regulation, i.e. activation and suppression of T cells and

Besides genes of the MHC class II system such as HLA-DR3 and DQA1 \* 0501 on chromosome 6p21 (Yanagawa, Mangklabruks et al., 1993; Zamani, Spaepen et al., 2000; Maciel, Rodrigues et al., 2001), on chromosome 2q33, we found the *loci* of genes involved in the regulation of T lymphocytes: CD28, CTLA4 and ICOS in which, specifically polymorphisms of the cytotoxic T lymphocyte antigen-4 (CTLA-4) are associated to various

Allelic variants of the CTLA-4 gene with potential effect on functional modulation of the T cell were shown as single-nucleotide polymorphism (SNP) + 49 A> G in exon 1, which seems to modify both the structure and protein expression of CTLA-4 [7]. In 2003, Ueda *et al.,* identified the SNP +6230 G> A in the stop codon of gene CTLA-4 (CT60) associating it to higher risk for GD, HT and type 1 diabetes (DM1) due to expression of different isoforms of

In case-control studies during the last decade, polymorphism +49 A> G of CTLA4 gene exon 1 was associated with autoimmune diseases, such as GD, DM1, HT, rheumatoid arthritis, autoimmune Addison's disease, multiple sclerosis (Yanagawa, Hidaka et al., 1995; Nistico, Buzzetti et al., 1996; Kotsa, Watson et al., 1997; Yanagawa, Taniyama et al., 1997; Awata, Kurihara et al., 1998; Fukazawa, Yanagawa et al., 1999; Heward, Allahabadia et al., 1999; Vaidya, Imrie et al., 2000; Ueda, Howson et al., 2003; Blomhoff, Lie et al., 2004; Young-Min

autoimmune diseases (Chistiakov and Turakulov 2003; Vaidya and Pearce 2004).

immune response Th1, Th2 and Th17.

tissue and thyrocyte apoptosis.

or HT are less frequent nowadays.

**3. Immunoregulator genes** 

consequent modulation of B-cells.

mRNA gene CTLA-4 (Ueda, Howson et al., 2003).

and Vaidya 2004; Kavvoura, Akamizu et al., 2007). and in familial studies was associated with GD in Caucasian, Japanese, Chinese and Korean populations (Heward, Allahabadia et al., 1999; Vaidya, Imrie et al., 1999).

Proteins CD28 and CTLA4 are costimulatory molecules found on the surface of T cells that bind to the family of B7 receptors expressed on antigen presenting cells (APC) (Reiser and Stadecker 1996). Immune response relies on the generation of two signals: the first, from the interaction of antigenic peptides with receptors on T cells in the context of MHC and the second signal (costimulatory) that activates, enhances and promotes T cell proliferation by production of cytokines (such as IL-2), where complex CD28/B7 functions as a positive regulator of T cells, and the CTLA4/B7 expressed exclusively in activated T lymphocytes, provides an inhibitory signal, required to limit proliferation of T cells and regulates the autoimmune response (Oosterwegel, Greenwald et al., 1999; Sharpe and Abbas 2006).

### **4. Polymorphisms, functional impact on the T cell and transduction of TCR**

The polymorphism +49 A> G (rs231775) of gene CTLA-4, which promotes the exchange of amino acid threonine for alanine at position 17, has emerged as the natural candidate, among polymorphisms of CTLA-4 gene, because of the ability to promote functional changes of protein CTLA- 4. Kouki *et al.,* (Kouki, Sawai et al., 2000) showed a higher frequency of genotype GG or AG at position 49 of the CTLA-4 gene in GD patients, and lesser control over proliferation and clonal expansion of T cells. Whereas Maurer *et al (Maurer, Loserth et al., 2002)* found differences in the pool of intracellular CTLA-4 protein, prompting imbalance in the expression and competition between CTLA-4 and CD28 on the surface of T cells, possibly modifying the suppression of T cells and generating larger quantities of inflammatory cytokines.

The CT60 polymorphism (rs30807243) seems to determine a distinct expression of mRNA isoforms through alternative splicing, with a lesser expression of soluble CTLA-4 (sCTLA-4) in relation to the total length isoform of CTLA-4 (*fl*CTLA-4) (Ueda, Howson et al., 2003). The correlation between immune-cell genotype and phenotype was demonstrated by presence of susceptibility allele G or allele A of protection, to specific subtypes of T cells in healthy controls, and the quantitative variations of the type CD4 + CD25 + cells called regulatory T cells (Treg) (Atabani, Thio et al., 2005).

Other studies have shown an association between the SNP at exon 2 of CTLA-4 gene in mice, and a new variant of the protein called ligand independent of CTLA-4 (liCTLA-4) (Wicker, Chamberlain et al., 2004), that also has a significant inhibitor function on T cell response when binding and dephosphorilating the T cell receptor (TCR).

Expression of the isoform liCTLA-4 seems to be greater in regulatory and memory T cells (Vijayakrishnan, Slavik et al., 2004), a possible association between the gene CTLA-4 (its isoforms) and immune response after antigen presentation by APC and T cell activation as from the T cell receptor. As such, genetic variations in the CTLA-4 gene region play an important role in T cell signaling and therefore in its function and proliferation of T cells. Different genotypes may determine different phenotypes and probable predisposition to autoimmunity by loss of negative selection mechanisms (central and peripheral immune system dysfunction) and a distinct pattern of CD4 + T cells.

Immune Profile and Tyrosine Phosphorylation

protein LYP (LYP\*W620) (Bottini, Musumeci et al., 2004).

of T-Cell Receptor in Autoimmune Thyroid Diseases 101

(C1859T) causes at codon 620, the exchange of amino acid arginine by tryptophan and possible changes in signaling and dephosphorylation post- TCR of family kinases through

Fig. 1. Representation of T/CD3 cell receptor and signaling pathways and phosphorylation of effector molecules according to the subtype of T lymphocytes (Hussain *et al.*, 2002).

The main function of LYP protein would be to downregulate T cells through TCR signaling, by direct effect on dephosphorylation of the protein kinases Lck and Fyn, from complex TCRζ/CD3 and ZAP70 protein among others (Figure 2) (Cloutier and Veillette 1999; Gjorloff-Wingren, Saxena et al., 1999; Wu, Katrekar et al., 2006). It is noteworthy that, according to the genotype of PTPN22 and the two alleles of LYP (LYP \* W620 or LYP \*

The LYP \* W620 of "predisposition" (CT or TT genotype of PTPN22) leads to a gain of function, proteins Lck dephosphorylation, TCRζ much more efficiently than LYP \* R620 (CC genotype of PTPN22), with less mobilization of intracellular calcium or transactivation of the IL-2 gene (Vang, Congia et al., 2005). That is, predisposition alleles (LYP \* W620) activate phosphatases leading to suppression of TCR better than CC homozygotes (LYP \* R620 / \* R620 LYP), possibly by binding to a larger number of intracellular proteins in TCR signaling

As such LYP \* W620 shows gain of function based upon the allelic variation of gene PTPN22 C1859T and possibly predisposes to autoimmune diseases by suppressing the TCR signaling in a much more potent way during thymic development, resulting in loss of negative selection and survival of a greater number of self-reactive cells (Vang, Miletic et al., 2007). Whether they can or not also jeopardize selection of T cells CD4 + CD25 + (Treg), if lineages of T cells are committed in accordance to expression of protein LYP, nevertheless remain

The ATD are a group of autoimmune diseases of poorly understood pathophysiology considering the determinant type of inflammatory response (Th1 or Th2) in the target organ,

R620), a distinct TCR signaling takes place (Vang, Congia et al., 2005).

than LYP \* R620 (Vang, Miletic et al., 2007).

scarcely known.

The possibility of analyzing the actions of protein CTLA-4 (and its isoforms) and its correlation with the type of activated T cell (either the memory/effector or naive cell) as well as the profile of tyrosine residues phosphorylation and activity of protein kinases in the intracellular environment (Maier, Anderson et al., 2007) may explain how the genetic profile influences immune response and promotes autoimmune thyroid disease for different clinical or subclinical poles as in HT and GD.

The expression of surface molecules, phenotype, discloses the history of antigenic exposure (Appay, Dunbar et al., 2002) or indicates the functional capacity of each cell subtype (Rufer, Zippelius et al., 2003). Naive or memory / effector cells are pointed out by presence or absence of CD45RA, naive cells are mainly CD4 + CCR7 + CD45RAhigh, memory cells CD4+ CCR7 + CD45RAlow and effector cells CD4 + CCR7-CD45RAlow (Amyes, McMichael et al., 2005).

During the antigen presentation process, costimulation and clonal expansion of the different populations of T lymphocytes, memory, effector or naive cells use the same pathway on the cell surface by means of the TCR/CD3 complex (Farber 2000), however with different properties in activation of the immune response. Naive cells are hyper-responsive to antigenic and non- antigenic stimuli, with increased susceptibility to apoptosis, while memory cells activated by slower kinetics, and are hyporesponsive to stimulation of the TCR and less susceptible to apoptosis (Hussain, Anderson et al., 2002).

In all T cell lines and their cellular clones, the same mechanism of signal transduction linked to the TCR was identified (Germain and Stefanova 1999), exhibiting phosphorylation of tyrosine residues in the subunits linked to the TCR/CD3 (ζ, ε, δ, γ) by the family proteins tyrosine kinase p56 lck (Iwashima, Irving et al., 1994).

Phosphorylation of the CD3ζ subunit brings about activation and recruitment of other tyrosine kinases such as ZAP-70 that phosphorylate multiple molecules like SLP-76 (Bubeck Wardenburg, Fu et al., 1996) and ligand for activation of the T cell (LAT) (Zhang, Sloan-Lancaster et al., 1998) that associated with Grb2 and GADS (Liu, Fang et al., 1999) activate according to messengers Ras/Erk MAP kinases and activation or suppression of intracytoplasmic events, such as activation of enzymes, modulation of transcription genes, synthesis of inflammatory cytokines, mobilization of intracellular calcium or induction of cell proliferation or cell apoptosis (Wange and Samelson 1996) (figure 1).

Therefore, the antigen-specific response can be well characterized by specific intracellular markers of phosphorylation and development of antibodies for specific epitopes of cytoplasmic proteins (Rosette, Werlen et al., 2001). The phenotypic correlation of T cell subtypes and pattern of TCR/CD3ζ phosphorylation with allelic variants of CTLA-4 (CT60 SNP) gene was recently demonstrated by Maier *et al.*,*.*(Maier, Anderson et al., 2007), with a different signaling pattern of CD4 + T cells according to presence of allele G.

Polymorphism (rs2476601) of gene PTPN22 (protein tyrosine phosphatase nonreceptor 22) on chromosome 1p13 responsible for the expression of protein tyrosine-specific phosphatase (LYP), with a suppressive and regulatory function of post- TCR phosphorylation are associated with autoimmune diseases such as GD, DM1 and to polyglandular autoimmune conditions (Bottini, Musumeci et al., 2004; Velaga, Wilson et al., 2004; Skorka, Bednarczuk et al., 2005; Dultz, Matheis et al., 2009). The exchange of nucleotide C by T at position 1858

The possibility of analyzing the actions of protein CTLA-4 (and its isoforms) and its correlation with the type of activated T cell (either the memory/effector or naive cell) as well as the profile of tyrosine residues phosphorylation and activity of protein kinases in the intracellular environment (Maier, Anderson et al., 2007) may explain how the genetic profile influences immune response and promotes autoimmune thyroid disease for different

The expression of surface molecules, phenotype, discloses the history of antigenic exposure (Appay, Dunbar et al., 2002) or indicates the functional capacity of each cell subtype (Rufer, Zippelius et al., 2003). Naive or memory / effector cells are pointed out by presence or absence of CD45RA, naive cells are mainly CD4 + CCR7 + CD45RAhigh, memory cells CD4+ CCR7 + CD45RAlow and effector cells CD4 + CCR7-CD45RAlow (Amyes,

During the antigen presentation process, costimulation and clonal expansion of the different populations of T lymphocytes, memory, effector or naive cells use the same pathway on the cell surface by means of the TCR/CD3 complex (Farber 2000), however with different properties in activation of the immune response. Naive cells are hyper-responsive to antigenic and non- antigenic stimuli, with increased susceptibility to apoptosis, while memory cells activated by slower kinetics, and are hyporesponsive to stimulation of the

In all T cell lines and their cellular clones, the same mechanism of signal transduction linked to the TCR was identified (Germain and Stefanova 1999), exhibiting phosphorylation of tyrosine residues in the subunits linked to the TCR/CD3 (ζ, ε, δ, γ) by the family proteins

Phosphorylation of the CD3ζ subunit brings about activation and recruitment of other tyrosine kinases such as ZAP-70 that phosphorylate multiple molecules like SLP-76 (Bubeck Wardenburg, Fu et al., 1996) and ligand for activation of the T cell (LAT) (Zhang, Sloan-Lancaster et al., 1998) that associated with Grb2 and GADS (Liu, Fang et al., 1999) activate according to messengers Ras/Erk MAP kinases and activation or suppression of intracytoplasmic events, such as activation of enzymes, modulation of transcription genes, synthesis of inflammatory cytokines, mobilization of intracellular calcium or induction of

Therefore, the antigen-specific response can be well characterized by specific intracellular markers of phosphorylation and development of antibodies for specific epitopes of cytoplasmic proteins (Rosette, Werlen et al., 2001). The phenotypic correlation of T cell subtypes and pattern of TCR/CD3ζ phosphorylation with allelic variants of CTLA-4 (CT60 SNP) gene was recently demonstrated by Maier *et al.*,*.*(Maier, Anderson et al., 2007), with a

Polymorphism (rs2476601) of gene PTPN22 (protein tyrosine phosphatase nonreceptor 22) on chromosome 1p13 responsible for the expression of protein tyrosine-specific phosphatase (LYP), with a suppressive and regulatory function of post- TCR phosphorylation are associated with autoimmune diseases such as GD, DM1 and to polyglandular autoimmune conditions (Bottini, Musumeci et al., 2004; Velaga, Wilson et al., 2004; Skorka, Bednarczuk et al., 2005; Dultz, Matheis et al., 2009). The exchange of nucleotide C by T at position 1858

TCR and less susceptible to apoptosis (Hussain, Anderson et al., 2002).

cell proliferation or cell apoptosis (Wange and Samelson 1996) (figure 1).

different signaling pattern of CD4 + T cells according to presence of allele G.

tyrosine kinase p56 lck (Iwashima, Irving et al., 1994).

clinical or subclinical poles as in HT and GD.

McMichael et al., 2005).

(C1859T) causes at codon 620, the exchange of amino acid arginine by tryptophan and possible changes in signaling and dephosphorylation post- TCR of family kinases through protein LYP (LYP\*W620) (Bottini, Musumeci et al., 2004).

Fig. 1. Representation of T/CD3 cell receptor and signaling pathways and phosphorylation of effector molecules according to the subtype of T lymphocytes (Hussain *et al.*, 2002).

The main function of LYP protein would be to downregulate T cells through TCR signaling, by direct effect on dephosphorylation of the protein kinases Lck and Fyn, from complex TCRζ/CD3 and ZAP70 protein among others (Figure 2) (Cloutier and Veillette 1999; Gjorloff-Wingren, Saxena et al., 1999; Wu, Katrekar et al., 2006). It is noteworthy that, according to the genotype of PTPN22 and the two alleles of LYP (LYP \* W620 or LYP \* R620), a distinct TCR signaling takes place (Vang, Congia et al., 2005).

The LYP \* W620 of "predisposition" (CT or TT genotype of PTPN22) leads to a gain of function, proteins Lck dephosphorylation, TCRζ much more efficiently than LYP \* R620 (CC genotype of PTPN22), with less mobilization of intracellular calcium or transactivation of the IL-2 gene (Vang, Congia et al., 2005). That is, predisposition alleles (LYP \* W620) activate phosphatases leading to suppression of TCR better than CC homozygotes (LYP \* R620 / \* R620 LYP), possibly by binding to a larger number of intracellular proteins in TCR signaling than LYP \* R620 (Vang, Miletic et al., 2007).

As such LYP \* W620 shows gain of function based upon the allelic variation of gene PTPN22 C1859T and possibly predisposes to autoimmune diseases by suppressing the TCR signaling in a much more potent way during thymic development, resulting in loss of negative selection and survival of a greater number of self-reactive cells (Vang, Miletic et al., 2007). Whether they can or not also jeopardize selection of T cells CD4 + CD25 + (Treg), if lineages of T cells are committed in accordance to expression of protein LYP, nevertheless remain scarcely known.

The ATD are a group of autoimmune diseases of poorly understood pathophysiology considering the determinant type of inflammatory response (Th1 or Th2) in the target organ,

Immune Profile and Tyrosine Phosphorylation

Nature 383(6603): 787-793.

175(9): 5765-5773.

586-593.

89(7): 3474-3476.

271(33): 19641-19644.

medical journal 2(5153): 645-650.

follicular lymphoma." Blood 108(9): 2957-2964.

United States of America 97(4): 1719-1724.

disease." J Mol Endocrinol 31(1): 21-36.

**6. References** 

polyglandular syndrome or frequency in different ethnic groups.

Japanese population." Diabetes 47(1): 128-129.

of T-Cell Receptor in Autoimmune Thyroid Diseases 103

clinical endocrinology and the main genetic and biochemical markers of autoimmune thyroid diseases. As well as the actual evolution of subclinical and clinical phases, therapeutic response in GD and new predictors of remission or relapse, or even the varying presentation of HT, duration of progression to hypothyroidism, and variations on antibodies and different epitopes, glandular volume and texture at ultrasound as well as association with autoimmune

Abbas, A. K., K. M. Murphy, et al., (1996). "Functional diversity of helper T lymphocytes."

Amyes, E., A. J. McMichael, et al., (2005). "Human CD4+ T cells are predominantly

Appay, V., P. R. Dunbar, et al., (2002). "Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections." Nat Med 8(4): 379-385. Atabani, S. F., C. L. Thio, et al., (2005). "Association of CTLA4 polymorphism with

Awata, T., S. Kurihara, et al., (1998). "Association of CTLA-4 gene A-G polymorphism

Ban, Y., T. F. Davies, et al., (2003). "Analysis of the CTLA-4, CD28, and inducible

Blomhoff, A., B. A. Lie, et al., (2004). "Polymorphisms in the cytotoxic T lymphocyte antigen-

Bottini, N., L. Musumeci, et al., (2004). "A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes." Nat Genet 36(4): 337-338. Bubeck Wardenburg, J., C. Fu, et al., (1996). "Phosphorylation of SLP-76 by the ZAP-70

Burnet, M. (1959). "Auto-immune disease. I. Modern immunological concepts." British

Carreras, J., A. Lopez-Guillermo, et al., (2006). "High numbers of tumor-infiltrating FOXP3-

Caturegli, P., M. Hejazi, et al., (2000). "Hypothyroidism in transgenic mice expressing IFN-

Chistiakov, D. A. and R. I. Turakulov (2003). "CTLA-4 and its role in autoimmune thyroid

regulatory T cell frequency." Eur J Immunol 35(7): 2157-2162.

distributed among six phenotypically and functionally distinct subsets." J Immunol

(IDDM12 locus) with acute-onset and insulin-depleted IDDM as well as autoimmune thyroid disease (Graves' disease and Hashimoto's thyroiditis) in the

costimulator (ICOS) genes in autoimmune thyroid disease." Genes Immun 4(8):

4 gene region confer susceptibility to Addison's disease." J Clin Endocrinol Metab

protein-tyrosine kinase is required for T-cell receptor function." J Biol Chem

positive regulatory T cells are associated with improved overall survival in

gamma in the thyroid." Proceedings of the National Academy of Sciences of the

inducing apoptosis or hyperstimulation and a heterogeneous clinical condition. Thus, different cell phenotypes and immune response may take place when patients with ATD are compared to the population with no autoimmune diseases. There are few studies of autoimmune diseases that have studied the pattern of intracellular signaling of T lymphocytes and possible dysfunctions in T cell activation and immune response, except in rheumatic diseases such as systemic lupus erythematosus (Pang, Setoyama et al., 2002).

Fig. 2. Possible *loci* of action of protein LYP in signaling of T cell receptor (Vang *et al.,* 2007)

#### **5. Conclusions**

Therefore, the main conclusions of this chapter, that is to say correlation of CT60 polymorphism +49 A> G and of the PTPN22 gene with differences in T cell subtypes and intracellular signaling in memory and naive T cells has been established for patients with ATD. But the transduction o TCR activation need to be elucidated in GD a HT. Comparison of immunophenotyping and phosphorylation of TCR, cells in the periphery and of the thyroid environment may answer some questions about genetic profile and predominant phenotype in HT and GD.

Study of CD4 + cell subtypes has extended their association with other thyroid diseases are important, such as the association of Treg and aggressiveness of papillary thyroid carcinoma (French, Weber et al., 2010) was recently published, involving ATD in a different way and impact. Considering that frequency and existence of CD4+CD25+ has already been associated to a worse prognosis in breast adenocarcinoma and lymphomas (Carreras, Lopez-Guillermo et al., 2006; Gobert, Treilleux et al., 2009).

Therefore, study of the microenvironment as well as dynamics of TCR activation and association with specific genotypes might also contribute to a better association between clinical endocrinology and the main genetic and biochemical markers of autoimmune thyroid diseases. As well as the actual evolution of subclinical and clinical phases, therapeutic response in GD and new predictors of remission or relapse, or even the varying presentation of HT, duration of progression to hypothyroidism, and variations on antibodies and different epitopes, glandular volume and texture at ultrasound as well as association with autoimmune polyglandular syndrome or frequency in different ethnic groups.

#### **6. References**

102 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

inducing apoptosis or hyperstimulation and a heterogeneous clinical condition. Thus, different cell phenotypes and immune response may take place when patients with ATD are compared to the population with no autoimmune diseases. There are few studies of autoimmune diseases that have studied the pattern of intracellular signaling of T lymphocytes and possible dysfunctions in T cell activation and immune response, except in rheumatic diseases such as systemic lupus erythematosus (Pang, Setoyama et al., 2002).

Fig. 2. Possible *loci* of action of protein LYP in signaling of T cell receptor (Vang *et al.,* 2007)

Therefore, the main conclusions of this chapter, that is to say correlation of CT60 polymorphism +49 A> G and of the PTPN22 gene with differences in T cell subtypes and intracellular signaling in memory and naive T cells has been established for patients with ATD. But the transduction o TCR activation need to be elucidated in GD a HT. Comparison of immunophenotyping and phosphorylation of TCR, cells in the periphery and of the thyroid environment may answer some questions about genetic profile and predominant

Study of CD4 + cell subtypes has extended their association with other thyroid diseases are important, such as the association of Treg and aggressiveness of papillary thyroid carcinoma (French, Weber et al., 2010) was recently published, involving ATD in a different way and impact. Considering that frequency and existence of CD4+CD25+ has already been associated to a worse prognosis in breast adenocarcinoma and lymphomas (Carreras,

Therefore, study of the microenvironment as well as dynamics of TCR activation and association with specific genotypes might also contribute to a better association between

Lopez-Guillermo et al., 2006; Gobert, Treilleux et al., 2009).

**5. Conclusions** 

phenotype in HT and GD.


Immune Profile and Tyrosine Phosphorylation

1565.

86(2): 97-106.

551-554.

9(2): 67-75.

metabolism 91(9): 3639-3646.

Immunogenetics 54(1): 1-8.

Journal of immunology 173(8): 4791-4798.

Immunological reviews 193: 70-81.

Endocrinol Metab 92(8): 3162-3170.

Graves' disease." J Immunol 165(11): 6606-6611.

International journal of immunogenetics 36(2): 85-96.

of T-Cell Receptor in Autoimmune Thyroid Diseases 105

Gobert, M., I. Treilleux, et al., (2009). "Regulatory T cells recruited through CCL22/CCR4 are

and lead to an adverse clinical outcome." Cancer research 69(5): 2000-2009. Green, D. R., N. Droin, et al., (2003). "Activation-induced cell death in T cells."

Hadj-Kacem, H., S. Rebuffat, et al., (2009). "Autoimmune thyroid diseases: genetic

Heward, J. M., A. Allahabadia, et al., (1999). "The development of Graves' disease and the CTLA-4 gene on chromosome 2q33." J Clin Endocrinol Metab 84(7): 2398-2401. Hussain, S. F., C. F. Anderson, et al., (2002). "Differential SLP-76 expression and TCR-

Iwashima, M., B. A. Irving, et al., (1994). "Sequential interactions of the TCR with two

Kavvoura, F. K., T. Akamizu, et al., (2007). "Cytotoxic T-lymphocyte associated antigen 4

Kimura, H., M. Kimura, et al., (2005). "Increased thyroidal fat and goitrous hypothyroidism

Kotsa, K., P. F. Watson, et al., (1997). "A CTLA-4 gene polymorphism is associated with both

Kouki, T., Y. Sawai, et al., (2000). "CTLA-4 gene polymorphism at position 49 in exon 1

Liu, S. K., N. Fang, et al., (1999). "The hematopoietic-specific adaptor protein gads functions

Maciel, L. M., S. S. Rodrigues, et al., (2001). "Association of the HLA-DRB1\*0301 and HLA-

Martin, A. P., E. C. Coronel, et al., (2004). "A novel model for lymphocytic infiltration of the

Maurer, M., S. Loserth, et al., (2002). "A polymorphism in the human cytotoxic T-

contribution from several ethnic backgrounds." Thyroid 11(1): 31-35. Maier, L. M., D. E. Anderson, et al., (2007). "Allelic variant in CTLA4 alters T cell phosphorylation patterns." Proc Natl Acad Sci U S A 104(47): 18607-18612. Marazuela, M., M. A. Garcia-Lopez, et al., (2006). "Regulatory T cells in human

distinct cytoplasmic tyrosine kinases." Science 263(5150): 1136-1139.

selectively activated in lymphoid infiltrates surrounding primary breast tumors

susceptibility of thyroid-specific genes and thyroid autoantigens contributions."

mediated signaling in effector and memory CD4 T cells." J Immunol 168(4): 1557-

gene polymorphisms and autoimmune thyroid disease: a meta-analysis." J Clin

induced by interferon-gamma." International journal of experimental pathology

Graves disease and autoimmune hypothyroidism." Clin Endocrinol (Oxf) 46(5):

reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of

in T-cell signaling via interactions with the SLP-76 and LAT adaptors." Curr Biol

DQA1\*0501 alleles with Graves' disease in a population representing the gene

autoimmune thyroid disease." The Journal of clinical endocrinology and

thyroid gland generated by transgenic expression of the CC chemokine CCL21."

lymphocyte antigen 4 ( CTLA4) gene (exon 1 +49) alters T-cell activation."


Cloutier, J. F. and A. Veillette (1999). "Cooperative inhibition of T-cell antigen receptor

Dultz, G., N. Matheis, et al., (2009). "The protein tyrosine phosphatase non-receptor type 22

Farber, D. L. (2000). "T cell memory: heterogeneity and mechanisms." Clin Immunol 95(3):

Feldkamp, J., E. Pascher, et al., (2001). "Soluble Fas is increased in hyperthyroidism

Figueroa-Vega, N., M. Alfonso-Perez, et al., (2010). "Increased circulating pro-inflammatory

Fisfalen, M. E., E. M. Palmer, et al., (1997). "Thyrotropin-receptor and thyroid peroxidase-

Fountoulakis, S., G. Vartholomatos, et al., (2008). "Differential expression of Fas system

French, J. D., Z. J. Weber, et al., (2010). "Tumor-associated lymphocytes and increased

Fukazawa, T., T. Yanagawa, et al., (1999). "CTLA-4 gene polymorphism may modulate disease in Japanese multiple sclerosis patients." J Neurol Sci 171(1): 49-55. Gangi, E., C. Vasu, et al., (2005). "IL-10-producing CD4+CD25+ regulatory T cells play a

Garrett-Sinha, L. A., S. John, et al., (2008). "IL-17 and the Th17 lineage in systemic lupus

Germain, R. N. and I. Stefanova (1999). "The dynamics of T cell receptor signaling:

Gjorloff-Wingren, A., M. Saxena, et al., (1999). "Characterization of TCR-induced receptor-

erythematosus." Current opinion in rheumatology 20(5): 519-525.

The Journal of clinical endocrinology and metabolism 82(11): 3655-3663. Fisfalen, M. E., K. Soltani, et al., (1997). "Evaluating the role of Th0 and Th1 clones in

autoimmune diabetes." Thyroid 19(2): 143-148.

endocrinology and metabolism 86(9): 4250-4253.

endocrinology and metabolism 95(2): 953-962.

Federation of Endocrine Societies 158(6): 853-859.

and immunopathology 85(3): 253-264.

111-121.

173-181.

2325-2333.

174(11): 7006-7013.

Immunol 17: 467-522.

PEP." Eur J Immunol 29(12): 3845-3854.

signaling by a complex between a kinase and a phosphatase." J Exp Med 189(1):

C1858T polymorphism is a joint susceptibility locus for immunthyroiditis and

independent of the underlying thyroid disease." The Journal of clinical

cytokines and Th17 lymphocytes in Hashimoto's thyroiditis." The Journal of clinical

specific T cell clones and their cytokine profile in autoimmune thyroid disease."

autoimmune thyroid disease by use of Hu-SCID chimeras." Clinical immunology

apoptotic molecules in peripheral lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis." European journal of endocrinology / European

FoxP3+ regulatory T cell frequency correlate with more aggressive papillary thyroid cancer." The Journal of clinical endocrinology and metabolism 95(5):

critical role in granulocyte-macrophage colony-stimulating factor-induced suppression of experimental autoimmune thyroiditis." Journal of immunology

complex orchestration and the key roles of tempo and cooperation." Annu Rev

proximal signaling events negatively regulated by the protein tyrosine phosphatase


Immune Profile and Tyrosine Phosphorylation

of T-Cell Receptor in Autoimmune Thyroid Diseases 107

Ueda, H., J. M. Howson, et al., (2003). "Association of the T-cell regulatory gene CTLA4 with

Vaidya, B., H. Imrie, et al., (2000). "Association analysis of the cytotoxic T lymphocyte

Vaidya, B. and S. Pearce (2004). "The emerging role of the CTLA-4 gene in autoimmune

Vang, T., M. Congia, et al., (2005). "Autoimmune-associated lymphoid tyrosine phosphatase

Vang, T., A. V. Miletic, et al., (2007). "Protein tyrosine phosphatase PTPN22 in human

Velaga, M. R., V. Wilson, et al., (2004). "The codon 620 tryptophan allele of the lymphoid

Verginis, P., H. S. Li, et al., (2005). "Tolerogenic semimature dendritic cells suppress

Vijayakrishnan, L., J. M. Slavik, et al., (2004). "An autoimmune disease-associated CTLA-4

Wange, R. L. and L. E. Samelson (1996). "Complex complexes: signaling at the TCR."

Watanabe, M., N. Yamamoto, et al., (2002). "Independent involvement of CD8+ CD25+ cells

Weaver, C. T., R. D. Hatton, et al., (2007). "IL-17 family cytokines and the expanding diversity of effector T cell lineages." Annual review of immunology 25: 821-852. Wicker, L. S., G. Chamberlain, et al., (2004). "Fine mapping, gene content, comparative

Wu, J., A. Katrekar, et al., (2006). "Identification of substrates of human protein-tyrosine

Yanagawa, T., Y. Hidaka, et al., (1995). "CTLA-4 gene polymorphism associated with Graves' disease in a Caucasian population." J Clin Endocrinol Metab 80(1): 41-45. Yanagawa, T., A. Mangklabruks, et al., (1993). "Human histocompatibility leukocyte

in a Caucasian population." J Clin Endocrinol Metab 76(6): 1569-1574. Yanagawa, T., M. Taniyama, et al., (1997). "CTLA4 gene polymorphism confers

susceptibility to Graves' disease in Japanese." Thyroid 7(6): 843-846.

phosphatase PTPN22." J Biol Chem 281(16): 11002-11010.

official journal of the American Thyroid Association 12(9): 801-808.

CD4+CD25+ T cells." Journal of immunology 174(11): 7433-7439.

tyrosine phosphatase (LYP) gene is a major determinant of Graves' disease." J Clin

experimental autoimmune thyroiditis by activation of thyroglobulin-specific

splice variant lacking the B7 binding domain signals negatively in T cells."

and thyroid autoantibodies in disease severity of Hashimoto's disease." Thyroid :

sequencing, and expression analyses support Ctla4 and Nramp1 as candidates for Idd5.1 and Idd5.2 in the nonobese diabetic mouse." J Immunol 173(1): 164-173. Wilson, N. J., K. Boniface, et al., (2007). "Development, cytokine profile and function of

human interleukin 17-producing helper T cells." Nature immunology 8(9): 950-

antigen-DQA1\*0501 allele associated with genetic susceptibility to Graves' disease

autoimmune Addison's disease." J Clin Endocrinol Metab 85(2): 688-691. Vaidya, B., H. Imrie, et al., (1999). "The cytotoxic T lymphocyte antigen-4 is a major Graves'

antigen-4 (CTLA-4) and autoimmune regulator-1 (AIRE-1) genes in sporadic

susceptibility to autoimmune disease." Nature 423(6939): 506-511.

disease locus." Hum Mol Genet 8(7): 1195-1199.

autoimmunity." Autoimmunity 40(6): 453-461.

Endocrinol Metab 89(11): 5862-5865.

Immunity 20(5): 563-575.

Immunity 5(3): 197-205.

957.

endocrinopathies." Eur J Endocrinol 150(5): 619-626.

is a gain-of-function variant." Nat Genet 37(12): 1317-1319.


Mitsiades, N., V. Poulaki, et al., (1998). "Fas/Fas ligand up-regulation and Bcl-2 down-

Mosmann, T. R. and S. Sad (1996). "The expanding universe of T-cell subsets: Th1, Th2 and

Nakano, A., M. Watanabe, et al., (2007). "Apoptosis-induced decrease of intrathyroidal

Nanba, T., M. Watanabe, et al., (2009). "Increases of the Th1/Th2 cell ratio in severe

Nistico, L., R. Buzzetti, et al., (1996). "The CTLA-4 gene region of chromosome 2q33 is linked

Oosterwegel, M. A., R. J. Greenwald, et al., (1999). "CTLA-4 and T cell activation." Curr Opin

Pang, M., Y. Setoyama, et al., (2002). "Defective expression and tyrosine phosphorylation of

Rapoport, B. and S. M. McLachlan (2007). "The thyrotropin receptor in Graves' disease." Thyroid: official journal of the American Thyroid Association 17(10): 911-922. Reiser, H. and M. J. Stadecker (1996). "Costimulatory B7 molecules in the pathogenesis of infectious and autoimmune diseases." N Engl J Med 335(18): 1369-1377. Rosette, C., G. Werlen, et al., (2001). "The impact of duration versus extent of TCR

Rufer, N., A. Zippelius, et al., (2003). "Ex vivo characterization of human CD8+ T subsets

Sakaguchi, S., T. Yamaguchi, et al., (2008). "Regulatory T cells and immune tolerance." Cell

Schwartz, R. H. (2005). "Natural regulatory T cells and self-tolerance." Nature immunology

Sharpe, A. H. and A. K. Abbas (2006). "T-cell costimulation--biology, therapeutic potential,

Skorka, A., T. Bednarczuk, et al., (2005). "Lymphoid tyrosine phosphatase (PTPN22/LYP)

Traves, S. L. and L. E. Donnelly (2008). "Th17 cells in airway diseases." Current molecular

variant and Graves' disease in a Polish population: association and gene dosedependent correlation with age of onset." Clin Endocrinol (Oxf) 62(6): 679-682. Stassi, G., D. Di Liberto, et al., (2000). "Control of target cell survival in thyroid

autoimmunity by T helper cytokines via regulation of apoptotic proteins." Nature

and challenges." N Engl J Med 355(10): 973-975.

erythematosus patients." Clin Exp Immunol 129(1): 160-168.

Journal of clinical endocrinology and metabolism 83(6): 2199-2203.

official journal of the American Thyroid Association 17(1): 25-31.

more." Immunology today 17(3): 138-146.

501.

Genet 5(7): 1075-1080.

Immunol 11(3): 294-300.

Immunity 15(1): 59-70.

immunology 1(6): 483-488.

medicine 8(5): 416-426.

1779-1787.

133(5): 775-787.

6(4): 327-330.

regulation may be significant in the pathogenesis of Hashimoto's thyroiditis." The

CD4(+)CD25(+) regulatory T cells in autoimmune thyroid diseases." Thyroid :

Hashimoto's disease and in the proportion of Th17 cells in intractable Graves' disease." Thyroid: official journal of the American Thyroid Association 19(5): 495-

to, and associated with, type 1 diabetes. Belgian Diabetes Registry." Hum Mol

the T cell receptor zeta chain in peripheral blood T cells from systemic lupus

occupancy on T cell activation: a revision of the kinetic proofreading model."

with distinct replicative history and partial effector functions." Blood 102(5):


**8**

*USA* 

**Estrogen Signaling and Thyrocyte Proliferation** 

The development of thyroid cancer is a multifactorial and multistep process. Several factors are thought to predispose people to thyroid cancer, including genetics, environment, and sex hormones. The incidence of thyroid cancer is three to four times higher in women than in men (Libutti, 2005; Machens et al., 2006). This difference in incidence between genders suggests that the growth and outcome of thyroid tumors may be influenced by female sex hormones, particularly E2, which has been widely implicated in the development and progression of several cancers, such as breast, ovarian and prostate cancer (Arnold et al., 2007; Stender et al., 2007). Animal studies support these epidemiological data, and suggest that exogenous estrogen (17β-estradiol, E2) can promote thyroid tumors (Mori et al., 1990;

Several studies have been carried out to address the role of estrogens in the pathogenesis of proliferative and neoplastic disorders. Although the precise mechanism still remains illdefined, a range of plausible mechanisms explaining their carcinogenic effects has been proposed. On one hand, estrogens may promote cellular proliferation through their receptor-mediated activity (Arnold et al., 2007; Lee et al., 2005). In addition, the natural estrogen E2 or its metabolites 2- hydroxy, 4-hydroxy, and 16-α-hydroxy-estradiol (2-OH-E2, 4-OH-E2, and 16-α-OH E2) can cause neoplastic transformation through a direct genotoxic effect, increasing the spontaneous mutation rate of normal cells (Cavalieri et al., 1997).

In this review, we will analyze the role of estrogen signaling in the proliferation and transformation of the thyroid gland, with a special emphasis on the cross-talk between

Thyroid carcinoma is the most common and prevalent of all endocrine malignancies, accounting for more than 95% of all endocrine-related cancers (Hodgson et al., 2004; Jemal et al*.*, 2009). Papillary and follicular carcinomas (PTC and FTC respectively) are differentiated tumors arising from thyroid epithelial cells (thyrocytes), while medullary carcinoma originates from parafollicular cells. PTC is by far the most common type of thyroid cancer, representing up to 80% of all thyroid malignancies. Anaplastic carcinomas are undifferentiated tumors deriving from thyroid epithelial cells. They are usually lethal with

**1. Introduction** 

Thiruvengadam et al., 2003).

estrogen signaling and the PI3K pathway.

**2. Thyroid cancer** 

Valeria Gabriela Antico Arciuch and Antonio Di Cristofano

*Department of Developmental and Molecular Biology* 

*Albert Einstein College of Medicine, Bronx* 


### **8**

### **Estrogen Signaling and Thyrocyte Proliferation**

Valeria Gabriela Antico Arciuch and Antonio Di Cristofano

*Department of Developmental and Molecular Biology Albert Einstein College of Medicine, Bronx USA* 

#### **1. Introduction**

108 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

Yano, S., S. Sone, et al., (1995). "Differential effects of anti-inflammatory cytokines (IL-4, IL-

Young-Min, S. and B. Vaidya (2004). "CTLA4 exon 1 polymorphism, rheumatoid arthritis

Zamani, M., M. Spaepen, et al., (2000). "Primary role of the HLA class II DRB1\*0301 allele in

Zhang, W., J. Sloan-Lancaster, et al., (1998). "LAT: the ZAP-70 tyrosine kinase substrate that

and autoimmune endocrinopathy." Clin Rheumatol 23(6): 568-569.

links T cell receptor to cellular activation." Cell 92(1): 83-92.

Graves disease." Am J Med Genet 95(5): 432-437.

biology 57(2): 303-309.

10 and IL-13) on tumoricidal and chemotactic properties of human monocytes induced by monocyte chemotactic and activating factor." Journal of leukocyte

> The development of thyroid cancer is a multifactorial and multistep process. Several factors are thought to predispose people to thyroid cancer, including genetics, environment, and sex hormones. The incidence of thyroid cancer is three to four times higher in women than in men (Libutti, 2005; Machens et al., 2006). This difference in incidence between genders suggests that the growth and outcome of thyroid tumors may be influenced by female sex hormones, particularly E2, which has been widely implicated in the development and progression of several cancers, such as breast, ovarian and prostate cancer (Arnold et al., 2007; Stender et al., 2007). Animal studies support these epidemiological data, and suggest that exogenous estrogen (17β-estradiol, E2) can promote thyroid tumors (Mori et al., 1990; Thiruvengadam et al., 2003).

> Several studies have been carried out to address the role of estrogens in the pathogenesis of proliferative and neoplastic disorders. Although the precise mechanism still remains illdefined, a range of plausible mechanisms explaining their carcinogenic effects has been proposed. On one hand, estrogens may promote cellular proliferation through their receptor-mediated activity (Arnold et al., 2007; Lee et al., 2005). In addition, the natural estrogen E2 or its metabolites 2- hydroxy, 4-hydroxy, and 16-α-hydroxy-estradiol (2-OH-E2, 4-OH-E2, and 16-α-OH E2) can cause neoplastic transformation through a direct genotoxic effect, increasing the spontaneous mutation rate of normal cells (Cavalieri et al., 1997).

> In this review, we will analyze the role of estrogen signaling in the proliferation and transformation of the thyroid gland, with a special emphasis on the cross-talk between estrogen signaling and the PI3K pathway.

#### **2. Thyroid cancer**

Thyroid carcinoma is the most common and prevalent of all endocrine malignancies, accounting for more than 95% of all endocrine-related cancers (Hodgson et al., 2004; Jemal et al*.*, 2009). Papillary and follicular carcinomas (PTC and FTC respectively) are differentiated tumors arising from thyroid epithelial cells (thyrocytes), while medullary carcinoma originates from parafollicular cells. PTC is by far the most common type of thyroid cancer, representing up to 80% of all thyroid malignancies. Anaplastic carcinomas are undifferentiated tumors deriving from thyroid epithelial cells. They are usually lethal with

Estrogen Signaling and Thyrocyte Proliferation 111

not only in the reproductive tract but also in other tissues such as bone, brain, liver,

Although estrogens are present in both men and women, their levels are significantly higher in women of reproductive age. They are mainly produced by the adrenal cortex and ovary. The three major naturally occurring estrogens in women are: estrone, estradiol and estriol (Speroff et al., 1999). In premenopausal women, 17β-estradiol (E2), produced by the ovary, is the estrogen formed in the largest quantity and is the most potent since it has the highest affinity for estrogen receptors. In premenopausal women, the level of circulating E2 varies from 40 to 400 pg/mL during the menstrual cycle (Ruggiero et al., 2002). After menopause, the level of E2 drops to less than 20 pg/mL (Jones, 1992). The second endogenous estrogen is estrone (E1), a less potent metabolite of E2. Estrone is produced from androstenedione in adipose tissue. In postmenopausal women, the ovary ceases to produce E2 while the adrenal gland continues to produce androstenedione, with the result that the level of estrone remains unchanged while the level of E2 falls significantly. The third endogenous estrogen is estriol (E3), also a metabolite of E2. E3 is the main estrogen produced by the placenta during pregnancy, and is found in smaller quantities than E2 and E1 in nonpregnant women

The actions of estrogens occur through activation of estrogen receptors (ERα, ERβ and GPR30). ERα was initially described in 1973 (Jensen and De Sombre, 1973) while ERβ was identified much later (Kuiper et al., 1996). ERα and ERβ are encoded by separate genes, *ESR1* and *ESR2*, respectively, which share similarities in the DNA-binding domain (97% amino acid similarity) and ligand-binding domain (60% amino acid similarity) (Hall et al., 2001). These two ERs differ in their tissue distributions (Kuiper et al., 1997; Dechering et al., 2000), suggesting that ERα and ERβ might have different physiological functions. It has also been demonstrated that in many systems the activity of ERβ is opposed to that of ERα. For example, in breast cancer cells, ERα is the receptor responsible for E2-induced proliferation, whereas activation of ERβ inhibits this effect (Strom et al., 2004). In the uterus, E2 induces proliferation of both epithelial and stromal cells through ERα, which is the predominant ER in the mature organ, while in the immature uterus, ERα and ERβ are found at similar expression levels in both epithelium and stroma, and ERβ mediates the action of E2 as a

suppressor of cell proliferation against activation of ERα by E2 (Weihua et al., 2000).

G protein-coupled receptor 30 (GPR30), a novel transmembrane ER, was identified in different cells by four laboratories between 1996 and 1998 (Takada et al., 1997; Owman et al., 1996; Carmeci et al., 1997; O'Dowd et al., 1998). Since its ligand was unknown at that time, it was named based on its homology to the G protein-coupled receptor (GPCR) super-family. In addition, this receptor was found to be associated with ER expression in breast cancer cell lines (Carmeci et al., 1997). Later in 2000, Filardo et al. demonstrated that estrogen promptly activated ERK1/2 in two breast cancer cell lines, MCF-7 and SKBR3, with the cell line SKBR3 non-expressing ERs. These results demonstrated that estrogen might be a potential ligand for GPR30 (Filardo et al., 2000). This fact was further confirmed by the observation that estrogen did not activate ERK1/2 in the breast cancer cell line MDA-MB-231 without GPR30 expression, whereas ERK1/2 was activated by estrogen after GPR30 transfection into the cells (Filardo et al., 2000). Therefore, GPR30 is necessary for the activation of ERK1/2 by

cardiovascular system, and endocrine glands.

(Jones, 1992; Ruggiero et al., 2002).

**3.2 Estrogen receptors and their ligands** 

no effective system therapy. The factors leading to thyroid carcinoma development are not fully understood despite some well-established associations, such as between ionizing radiation and papillary carcinoma, and between iodine deficiency and follicular carcinoma.

From the molecular point of view, papillary and follicular thyroid cancers are completely different diseases. This notion is supported by dissimilar molecular initiating events leading to neoplastic transformation and by differences in DNA ploidy level (PTCs are generally diploid, FTCs aneuploid) (Handkiewicz-Junak et al., 2010).


Table 1. Most frequent genetic alterations in thyroid cancer

The genetic alterations found in PTC primarily affect two central signalling pathways in thyroid cells: TSH receptor (TSHR)-mediated signalling and mitogen-activated protein kinase (MAPK) pathways (Kim and Zhu, 2009; Lemoine et al., 1998; Nikiforov, 2008). Three important initiating events, *RET/PTC* (rearranged during transfection/ papillary thyroid cancer), *RAS* (resistance to audiogenic seizures) and *BRAF* mutations, are considered mutually exclusive (Fagin, 2004). BRAF mutation and RET/PTC rearrangements differ to some extent in their effects on the shared oncogenic pathway, resulting more frequently in the classic or the solid variant of PTC, respectively, while RAS mutations are more likely to induce the follicular variant of PTC (Xing, 2005).

Follicular carcinomas are often characterized by *RAS* mutations (up to 50%) and *PAX8- PPARγ* rearrangements (20–35%), which lead to a mutant protein incapable of transactivating a PPARγ signal (Gilfillan, 2010). Phosphatidylinositol 3-kinase (PI3K)/AKT alterations are frequently found in FTC and, even more distinctly, in ATC. In FTC, phosphorylation of AKT, the key player in this pathway, is by far more frequent than that of ERK (Liu et al., 2008).

Anaplastic thyroid carcinomas (ATCs) comprise 2% of thyroid malignancies, and are usually lethal, with no effective therapy (Are and Shaha, 2006). Dedifferentiation, a common hallmark of ATC, is manifested by a loss of specific thyroid cell characteristics and functions, including expression of thyroglobulin, thyroid peroxidase, thyroid stimulating hormone receptor and the Na/I symporter (Neff et al., 2008; Smallridge et al., 2009). Molecular signature events that characterize ATC involve either *BRAF* activation or sustained hyperactivation of the PI3K/AKT cascade, together with *TP53* loss or inactivation (Kouniavsky and Zeiger, 2010).

#### **3. Physiological functions of estrogen and estrogen receptors**

#### **3.1 Estrogen production**

Estrogens are a group of steroid compounds acting as the primary female sex hormones. Estrogens regulate several physiological processes, including cell growth and development,

no effective system therapy. The factors leading to thyroid carcinoma development are not fully understood despite some well-established associations, such as between ionizing radiation and papillary carcinoma, and between iodine deficiency and follicular carcinoma. From the molecular point of view, papillary and follicular thyroid cancers are completely different diseases. This notion is supported by dissimilar molecular initiating events leading to neoplastic transformation and by differences in DNA ploidy level (PTCs are generally

Follicular Carcinoma Papillary Carcinoma Anaplastic Carcinoma *RAS*: 20-50% *BRAF*: 40-45% *TP53*: 50-80% *PAX8-PPARγ*: 20-35% *RAS*: 10-20% *BRAF*: 20-40% PI3K pathway: 20% *RET-PTC*: 10-30% *RAS*: 20-40%

The genetic alterations found in PTC primarily affect two central signalling pathways in thyroid cells: TSH receptor (TSHR)-mediated signalling and mitogen-activated protein kinase (MAPK) pathways (Kim and Zhu, 2009; Lemoine et al., 1998; Nikiforov, 2008). Three important initiating events, *RET/PTC* (rearranged during transfection/ papillary thyroid cancer), *RAS* (resistance to audiogenic seizures) and *BRAF* mutations, are considered mutually exclusive (Fagin, 2004). BRAF mutation and RET/PTC rearrangements differ to some extent in their effects on the shared oncogenic pathway, resulting more frequently in the classic or the solid variant of PTC, respectively, while RAS mutations are more likely to

Follicular carcinomas are often characterized by *RAS* mutations (up to 50%) and *PAX8- PPARγ* rearrangements (20–35%), which lead to a mutant protein incapable of transactivating a PPARγ signal (Gilfillan, 2010). Phosphatidylinositol 3-kinase (PI3K)/AKT alterations are frequently found in FTC and, even more distinctly, in ATC. In FTC, phosphorylation of AKT, the key player in this pathway, is by far more frequent than that of

Anaplastic thyroid carcinomas (ATCs) comprise 2% of thyroid malignancies, and are usually lethal, with no effective therapy (Are and Shaha, 2006). Dedifferentiation, a common hallmark of ATC, is manifested by a loss of specific thyroid cell characteristics and functions, including expression of thyroglobulin, thyroid peroxidase, thyroid stimulating hormone receptor and the Na/I symporter (Neff et al., 2008; Smallridge et al., 2009). Molecular signature events that characterize ATC involve either *BRAF* activation or sustained hyperactivation of the PI3K/AKT cascade, together with *TP53* loss or inactivation

Estrogens are a group of steroid compounds acting as the primary female sex hormones. Estrogens regulate several physiological processes, including cell growth and development,

**3. Physiological functions of estrogen and estrogen receptors** 

PI3K pathway: 20-50%

diploid, FTCs aneuploid) (Handkiewicz-Junak et al., 2010).

Table 1. Most frequent genetic alterations in thyroid cancer

induce the follicular variant of PTC (Xing, 2005).

ERK (Liu et al., 2008).

(Kouniavsky and Zeiger, 2010).

**3.1 Estrogen production** 

not only in the reproductive tract but also in other tissues such as bone, brain, liver, cardiovascular system, and endocrine glands.

Although estrogens are present in both men and women, their levels are significantly higher in women of reproductive age. They are mainly produced by the adrenal cortex and ovary. The three major naturally occurring estrogens in women are: estrone, estradiol and estriol (Speroff et al., 1999). In premenopausal women, 17β-estradiol (E2), produced by the ovary, is the estrogen formed in the largest quantity and is the most potent since it has the highest affinity for estrogen receptors. In premenopausal women, the level of circulating E2 varies from 40 to 400 pg/mL during the menstrual cycle (Ruggiero et al., 2002). After menopause, the level of E2 drops to less than 20 pg/mL (Jones, 1992). The second endogenous estrogen is estrone (E1), a less potent metabolite of E2. Estrone is produced from androstenedione in adipose tissue. In postmenopausal women, the ovary ceases to produce E2 while the adrenal gland continues to produce androstenedione, with the result that the level of estrone remains unchanged while the level of E2 falls significantly. The third endogenous estrogen is estriol (E3), also a metabolite of E2. E3 is the main estrogen produced by the placenta during pregnancy, and is found in smaller quantities than E2 and E1 in nonpregnant women (Jones, 1992; Ruggiero et al., 2002).

#### **3.2 Estrogen receptors and their ligands**

The actions of estrogens occur through activation of estrogen receptors (ERα, ERβ and GPR30). ERα was initially described in 1973 (Jensen and De Sombre, 1973) while ERβ was identified much later (Kuiper et al., 1996). ERα and ERβ are encoded by separate genes, *ESR1* and *ESR2*, respectively, which share similarities in the DNA-binding domain (97% amino acid similarity) and ligand-binding domain (60% amino acid similarity) (Hall et al., 2001). These two ERs differ in their tissue distributions (Kuiper et al., 1997; Dechering et al., 2000), suggesting that ERα and ERβ might have different physiological functions. It has also been demonstrated that in many systems the activity of ERβ is opposed to that of ERα. For example, in breast cancer cells, ERα is the receptor responsible for E2-induced proliferation, whereas activation of ERβ inhibits this effect (Strom et al., 2004). In the uterus, E2 induces proliferation of both epithelial and stromal cells through ERα, which is the predominant ER in the mature organ, while in the immature uterus, ERα and ERβ are found at similar expression levels in both epithelium and stroma, and ERβ mediates the action of E2 as a suppressor of cell proliferation against activation of ERα by E2 (Weihua et al., 2000).

G protein-coupled receptor 30 (GPR30), a novel transmembrane ER, was identified in different cells by four laboratories between 1996 and 1998 (Takada et al., 1997; Owman et al., 1996; Carmeci et al., 1997; O'Dowd et al., 1998). Since its ligand was unknown at that time, it was named based on its homology to the G protein-coupled receptor (GPCR) super-family. In addition, this receptor was found to be associated with ER expression in breast cancer cell lines (Carmeci et al., 1997). Later in 2000, Filardo et al. demonstrated that estrogen promptly activated ERK1/2 in two breast cancer cell lines, MCF-7 and SKBR3, with the cell line SKBR3 non-expressing ERs. These results demonstrated that estrogen might be a potential ligand for GPR30 (Filardo et al., 2000). This fact was further confirmed by the observation that estrogen did not activate ERK1/2 in the breast cancer cell line MDA-MB-231 without GPR30 expression, whereas ERK1/2 was activated by estrogen after GPR30 transfection into the cells (Filardo et al., 2000). Therefore, GPR30 is necessary for the activation of ERK1/2 by

Estrogen Signaling and Thyrocyte Proliferation 113

Glucocorticoid and thyroid hormones have been shown to modify the levels of mtDNAencoded gene transcripts. These effects are mediated through direct interactions of their receptors with mtDNA. It has also been established that thyroid hormone can cause the direct stimulation of mitochondrial RNA synthesis (Casas et al., 1999; Enriquez et al., 1999) and that a variant form of the thyroid hormone receptor is imported in and localized within

These findings suggest that mitochondria could also be a target site for the action of estrogens. Monje and colleagues (Monje and Boland 2001; Monje et al., 2001) demonstrated the presence of both ERα and ERβ in mitochondria of rabbit uterine and ovarian tissue, and ER translocation into mitochondria suggests the presence of E2 effects on mitochondrial function and protein expression (Chen et al*.*, 2004). The mitochondrial genome contains estrogen response elements (ERE)-like sequences (Demonacos et al., 1996; Sekeris et al., 1990). Furthermore, several studies have detected the presence of estrogen-binding proteins (EBPs) in the organelle (Grossman et al., 1989; Moats and Ramirez 2000). Estrogen treatment increases the transcript levels of several mitochondrial DNA (mtDNA)-encoded genes in rat

Estrogen response elements have been found in the D-loop, in the master regulatory region, and within the structural genes of the mtDNA (Demonacos et al*.*, 1996). As a consequence, E2 may exert coordinated effects on both nuclear and mitochondrial gene expression. E2 can increase mtDNA transcripts for cytochrome oxidase IV subunits I and II in cultured cancer cells (Chen et al*.*, 2004). E2 profoundly affects mitochondrial function in cerebral blood vessels, enhancing efficiency of energy production and suppressing mitochondrial oxidative stress by increasing protein levels of Mn-SOD and aconitase, and stabilizing

The mechanisms of ER translocation into mitochondria are still quite elusive but recent data in MCF7 cells demonstrated that human ERβ posses a putative internal mitochondrial targeting peptide signal to the organelle (Chen et al., 2004). These authors observed that around 12% of total cellular ERα and 18% of ERβ is present in the mitochondrial fraction in E2-treated MCF7 cells. Furthermore, the localization of both ERα and ERβ to mitochondria in response to E2-treatment is accompanied by a concomitant time- and concentrationdependent increase in the transcript levels of the mtDNA-encoded genes (Chen et al., 2004).

Besides the adrenal cortex and ovary, also the human thyroid gland has the ability to synthesize estrogens and such ability seems to be higher in women than men (Dalla Valle et al., 1998). In the thyroid gland, E2 provokes a considerable increase in the thyroid weight, stimulates thyroid iodide uptake, enhances thyroperoxidase activity, and increases the level

ERK1/2 regulate various cellular activities, such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis (Roberts and Der, 2007; Dunn et al., 2005). Zeng and colleagues have demonstrated that E2 can activate ERK1/2 in the thyroid by inducing its phosphorylation (Zeng et al., 2007). ERK1/2 activation by E2 depends on the interaction

**3.4 Estrogen receptors in the mitochondria** 

mitochondrial membrane (Stirone et al*.*, 2005).

between estradiol and ERα (Zeng et al., 2007).

of T3 (Lima et al., 2006).

liver mitochondria (Casas et al., 1999; Wrutniak et al., 1995).

hepatocytes and human Hep G2 cells (Chen et al., 1996; Chen et al., 1998).

**3.5 Target molecules of estrogen receptors in the thyroid gland** 

estrogen. So far, GPR30 has been detected in numerous human tissues such as heart, liver, lung, intestine, ovary, brain, breast, uterus, placenta and prostate (He et al., 2009; Filardo et al., 2006; Zhang et al., 2008; Haas et al., 2007; Hugo et al., 2008).

#### **3.3 Genomic and non-genomic actions of estrogen receptors**

In the classical, genomic estrogen-signaling pathway, estradiol (E2)-activated ERα translocates to the nucleus, dimerizes, and binds to the 15-bp palindromic estrogen response element (ERE) or interacts with other transcription factors on target genes, recruits coactivators, and stimulates gene transcription thereby promoting cell proliferation (Klinge, 2000). ERα interacts with a number of coactivators and corepressors in a ligand-dependent manner (Klinge, 2000). ERα may also function in a non-traditional manner, interacting with other DNA-binding transcription factors such as activator protein 1 (AP-1) or Sp-1, that in turn bind their cognate DNA elements, leading to remodeling of chromatin, and interactions with components of the basal transcription machinery complex (Ascenzi et al., 2006; Deroo and Korach, 2006).

Another more rapid mechanism of estrogen action is termed 'non-genomic' or 'membraneinitiated' because it involves E2 activation of plasma membrane-associated ERα or ERβ and leads to rapid activation of intracellular signaling pathways, e.g., ERK1/2 and PI3K/AKT (Wong et al*.*, 2002; Watson et al*.*, 2007; He et al*.,* 2009). It can also result in an increase of Ca2+ or nitric oxide and the promotion of cell cycle progression. The ERs may be targeted to the plasma membrane by adaptor proteins such as caveolin-1 or Shc (Kim et al., 2008). GPR30 also activates ERK1/2 and PI3K/AKT signaling, although its exact role in estrogen action remains controversial (Pedram et al*.*, 2006). GPR30 ligands, for example, estrogen (Muller et al., 1979), tamoxifen (Dick et al., 2002) and ICI 182780 (Hermenegildo and Cano, 2000) bind to GPR30, and activate heterotrimeric G proteins, which then activate Src and adenylyl cyclase (AC) resulting in intracellular cAMP production. Src is involved in matrix metalloproteinases (MMP) activation, which cleave pro-heparan-bound epidermal growth factor (pro-HB-EGF) and release free HB-EGF. The latter activates EGF receptor (EGFR), leading to multiple downstream events such as activation of phospholipase C (PLC), PI3K, and MAPK. Activated PLC produces inositol triphosphate (IP3), which further binds to IP3 receptor and leads to intracellular calcium mobilization. The activation of MAPK and PI3K results in activation of numerous cytosolic pathways and nuclear proteins, which further regulate transcription factors such as serum response factor and members of the E26 transformation specific (ETS) family by direct phosphorylation (Posern and Treisman, 2006; Gutierrez-Hartmann et al., 2007).

The non-genomic pathway may cross-talk with the genomic pathway, since ERα can be translocated from the membrane into the nucleus both in a E2-dependent or independent manner (Lu et al., 2002). It has also been demonstrated that E2-induced ERK activation stimulates the expression of AP-1-mediated genes via both serum response factor ELK-1 (ER activated in the membrane) and the recruitment of coactivators to AP-1 sites on gene promoters by the nuclear ER (Ascenzi et al., 2006). The intricate relationship between membrane and nuclear effects induced by estrogens has also been observed in the regulation of many other genes including PI3K (Ascenzi et al., 2006).

Therefore, integrative signaling by E2 from several places in the cell can lead to both rapid and sustained actions, which synergize to provide plasticity for cell response.

estrogen. So far, GPR30 has been detected in numerous human tissues such as heart, liver, lung, intestine, ovary, brain, breast, uterus, placenta and prostate (He et al., 2009; Filardo et

In the classical, genomic estrogen-signaling pathway, estradiol (E2)-activated ERα translocates to the nucleus, dimerizes, and binds to the 15-bp palindromic estrogen response element (ERE) or interacts with other transcription factors on target genes, recruits coactivators, and stimulates gene transcription thereby promoting cell proliferation (Klinge, 2000). ERα interacts with a number of coactivators and corepressors in a ligand-dependent manner (Klinge, 2000). ERα may also function in a non-traditional manner, interacting with other DNA-binding transcription factors such as activator protein 1 (AP-1) or Sp-1, that in turn bind their cognate DNA elements, leading to remodeling of chromatin, and interactions with components of the

basal transcription machinery complex (Ascenzi et al., 2006; Deroo and Korach, 2006).

Another more rapid mechanism of estrogen action is termed 'non-genomic' or 'membraneinitiated' because it involves E2 activation of plasma membrane-associated ERα or ERβ and leads to rapid activation of intracellular signaling pathways, e.g., ERK1/2 and PI3K/AKT (Wong et al*.*, 2002; Watson et al*.*, 2007; He et al*.,* 2009). It can also result in an increase of Ca2+ or nitric oxide and the promotion of cell cycle progression. The ERs may be targeted to the plasma membrane by adaptor proteins such as caveolin-1 or Shc (Kim et al., 2008). GPR30 also activates ERK1/2 and PI3K/AKT signaling, although its exact role in estrogen action remains controversial (Pedram et al*.*, 2006). GPR30 ligands, for example, estrogen (Muller et al., 1979), tamoxifen (Dick et al., 2002) and ICI 182780 (Hermenegildo and Cano, 2000) bind to GPR30, and activate heterotrimeric G proteins, which then activate Src and adenylyl cyclase (AC) resulting in intracellular cAMP production. Src is involved in matrix metalloproteinases (MMP) activation, which cleave pro-heparan-bound epidermal growth factor (pro-HB-EGF) and release free HB-EGF. The latter activates EGF receptor (EGFR), leading to multiple downstream events such as activation of phospholipase C (PLC), PI3K, and MAPK. Activated PLC produces inositol triphosphate (IP3), which further binds to IP3 receptor and leads to intracellular calcium mobilization. The activation of MAPK and PI3K results in activation of numerous cytosolic pathways and nuclear proteins, which further regulate transcription factors such as serum response factor and members of the E26 transformation specific (ETS) family by direct phosphorylation (Posern and Treisman, 2006;

The non-genomic pathway may cross-talk with the genomic pathway, since ERα can be translocated from the membrane into the nucleus both in a E2-dependent or independent manner (Lu et al., 2002). It has also been demonstrated that E2-induced ERK activation stimulates the expression of AP-1-mediated genes via both serum response factor ELK-1 (ER activated in the membrane) and the recruitment of coactivators to AP-1 sites on gene promoters by the nuclear ER (Ascenzi et al., 2006). The intricate relationship between membrane and nuclear effects induced by estrogens has also been observed in the

Therefore, integrative signaling by E2 from several places in the cell can lead to both rapid

regulation of many other genes including PI3K (Ascenzi et al., 2006).

and sustained actions, which synergize to provide plasticity for cell response.

al., 2006; Zhang et al., 2008; Haas et al., 2007; Hugo et al., 2008).

**3.3 Genomic and non-genomic actions of estrogen receptors** 

Gutierrez-Hartmann et al., 2007).

#### **3.4 Estrogen receptors in the mitochondria**

Glucocorticoid and thyroid hormones have been shown to modify the levels of mtDNAencoded gene transcripts. These effects are mediated through direct interactions of their receptors with mtDNA. It has also been established that thyroid hormone can cause the direct stimulation of mitochondrial RNA synthesis (Casas et al., 1999; Enriquez et al., 1999) and that a variant form of the thyroid hormone receptor is imported in and localized within liver mitochondria (Casas et al., 1999; Wrutniak et al., 1995).

These findings suggest that mitochondria could also be a target site for the action of estrogens. Monje and colleagues (Monje and Boland 2001; Monje et al., 2001) demonstrated the presence of both ERα and ERβ in mitochondria of rabbit uterine and ovarian tissue, and ER translocation into mitochondria suggests the presence of E2 effects on mitochondrial function and protein expression (Chen et al*.*, 2004). The mitochondrial genome contains estrogen response elements (ERE)-like sequences (Demonacos et al., 1996; Sekeris et al., 1990). Furthermore, several studies have detected the presence of estrogen-binding proteins (EBPs) in the organelle (Grossman et al., 1989; Moats and Ramirez 2000). Estrogen treatment increases the transcript levels of several mitochondrial DNA (mtDNA)-encoded genes in rat hepatocytes and human Hep G2 cells (Chen et al., 1996; Chen et al., 1998).

Estrogen response elements have been found in the D-loop, in the master regulatory region, and within the structural genes of the mtDNA (Demonacos et al*.*, 1996). As a consequence, E2 may exert coordinated effects on both nuclear and mitochondrial gene expression. E2 can increase mtDNA transcripts for cytochrome oxidase IV subunits I and II in cultured cancer cells (Chen et al*.*, 2004). E2 profoundly affects mitochondrial function in cerebral blood vessels, enhancing efficiency of energy production and suppressing mitochondrial oxidative stress by increasing protein levels of Mn-SOD and aconitase, and stabilizing mitochondrial membrane (Stirone et al*.*, 2005).

The mechanisms of ER translocation into mitochondria are still quite elusive but recent data in MCF7 cells demonstrated that human ERβ posses a putative internal mitochondrial targeting peptide signal to the organelle (Chen et al., 2004). These authors observed that around 12% of total cellular ERα and 18% of ERβ is present in the mitochondrial fraction in E2-treated MCF7 cells. Furthermore, the localization of both ERα and ERβ to mitochondria in response to E2-treatment is accompanied by a concomitant time- and concentrationdependent increase in the transcript levels of the mtDNA-encoded genes (Chen et al., 2004).

#### **3.5 Target molecules of estrogen receptors in the thyroid gland**

Besides the adrenal cortex and ovary, also the human thyroid gland has the ability to synthesize estrogens and such ability seems to be higher in women than men (Dalla Valle et al., 1998). In the thyroid gland, E2 provokes a considerable increase in the thyroid weight, stimulates thyroid iodide uptake, enhances thyroperoxidase activity, and increases the level of T3 (Lima et al., 2006).

ERK1/2 regulate various cellular activities, such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis (Roberts and Der, 2007; Dunn et al., 2005). Zeng and colleagues have demonstrated that E2 can activate ERK1/2 in the thyroid by inducing its phosphorylation (Zeng et al., 2007). ERK1/2 activation by E2 depends on the interaction between estradiol and ERα (Zeng et al., 2007).

Estrogen Signaling and Thyrocyte Proliferation 115

also been shown that cells derived from *Akt1* deficient mouse embryos are also more susceptible to pro-apoptotic stimuli (Chen et al., 2001). On the other hand, deficiency of *AKT2* alone is sufficient to cause a diabetic phenotype in mice (Withers et al., 1998; Cho et al., 2001) and a loss-of-function mutation in AKT2 is associated with diabetes in one human

AKT kinases are typically activated by engagement of receptor tyrosine kinases by growth factors and cytokines, as well as oxidative stress and heat shock. AKT activation relies on phosphatidylinositol 3,4,5-triphosphate (PtdIns-3,4,5-P3) which is produced from phosphatidylinositol 4,5-biphosphate (PtdIns-4,5-P2) by phosphatidylinositol 3-kinase (PI3K) (Franke et al., 1995). The interaction between the Pleckstrin homology (PH) domain of AKT with PtdIns-3,4,5-P3 favors its phosphorylation at two residues, one in the Cterminal tail (Ser473) and the other in the activation loop (Thr308). Phosphorylation at Ser473 appears to precede and facilitate phosphorylation at Thr308 (Sarbassov et al., 2005). AKT is phosphorylated in Ser473 by mTORC2 (Ikenoue et al., 2008), while PI-3K-dependent kinase 1

The proliferative effects of AKT result from phosphorylation of several substrates. For example, GSK3*β* once phosphorylated is inactivated and this prevents degradation of cyclin D1 (Diehl et al., 1998). Furthermore, AKT activation leads to increased translation of cyclin D1 and D3 transcripts via mTOR (Muise-Helmericks et al., 1998). AKT phosphorylates the cell cycle inhibitors p21WAF1 and p27Kip1 inducing their cytoplasmic retention (Testa and

AKT activity prevents apoptosis through the phosphorylation and inhibition of proapoptotic mediators such as Bad, FOXO family members, and IκB kinase-β (IKK-β) (Datta et al., 1999). AKT activity also attenuates the response of cells to the release of cytochrome *c*

AKT can also antagonize p53-mediated cell cycle checkpoints by modulating the subcellular localization of Mdm2. Phosphorylation of Mdm2 by AKT triggers its localization to the nucleus, where Mdm2 can complex with p53 to promote its ubiquitin/proteasome-mediated

The crucial role of the PI3K signaling cascade in the pathogenesis of thyroid neoplastic disorders has been recently confirmed by the development and study of a relevant mouse model (Yeager et al., 2007, 2008; Miller et al., 2009), as well as by solid clinicopathological data (Garcia-Rostan et al., 2005; Hou et al., 2007, 2008; Vasko and Saji, 2007; Wang et al., 2007). Thyrocyte-specific deletion of the *Pten* tumor suppressor constitutively activates the PI3K signaling cascade, leading to hyperplastic thyroid glands at birth, and to the development of thyroid nodules and follicular adenomas by 6-8 months of age (Yeager et

The *Pten* mouse model of thyroid disease displays a unique and remarkable characteristic: the higher proliferative index of female mutant thyrocytes, compared with males. This difference leads to increased cellularity in the thyroids of female mutants at a young age, to an increased incidence of thyroid adenomas in mutant females at 8 months of age (Yeager et

al., 2007) and thyroid carcinomas by one year of age (Antico Arciuch et al., 2010).

**5. PI3K-estrogen cooperation during proliferation** 

(PDK1) accounts for the phosphorylation in Thr308 (Chan et al., 1999).

family (George et al., 2004).

Bellacosa, 2001).

into the cytoplasm (Kennedy et al., 1999).

degradation (Mayo and Donner, 2001).

Bcl-2 family proteins play a central role in controlling mitochondrial-mediated apoptosis. They include proteins that suppress apoptosis such as Bcl-2 and Bcl-XL, and proteins that promote apoptosis such as Bax, Bad and Bcl-XS (Antonsson and Martinou, 2000). Bcl-2 proteins localize or translocate to the mitochondrial membrane and modulate apoptosis by permeabilization of the inner and/or outer membrane, leading to the release of citochrome *c* or stabilization of the barrier function. Bcl-2 family members are altered in thyroid cancer (Kossmehl et al., 2003) and their levels are regulated by estrogen in some cell systems (Song and Santen, 2003). The antiapoptotic member Bcl-2 is up-regulated by E2 and by the ERα agonist PPT, but downregulated by the ERβ agonist DPN in thyroid cancer cells, suggesting that ERα induces Bcl-2 expression whereas ERβ reduces it (Zeng et al., 2007). In addition, it has been shown that ERβ but not ERα promotes the expression of Bax (Lee et al., 2005; Zeng et al., 2007).

Recent work on the WRO thyroid cancer cells revealed that E2 increases cathepsin D transcription and that cathepsin D expression is inhibited upon siRNA-mediated knockdown of ERα and ERβ (Kumar et al., 2010). Cathepsin D is a classical E2 target gene regulated by Sp1-ERα promoter binding (Wang et al., 1997). It is well established that cathepsin D expression is elevated in thyroid tumors and correlates with disease aggressiveness (Leto et al., 2004).

The expression of another classical E2 target gene, cyclin D1 (Pestell et al., 1999), is stimulated by E2 in thyroid cancer cell lines, and co-treatment with siERα and siERβ shows roles for ERα and ERβ in regulating cyclin D1 transcription. E2 regulation of cyclin D1 transcription involves ERα-Sp1 (Castro-Rivera et al., 2001) and AP-1-ERα (Liu et al., 2002) interactions.

In Nthy-ori3-1 and BCPAP cells (derived from thyroid carcinoma), ERα was found to be complexed with Hsp90 and AKT (Rajoria et al., 2010). The complex of Hsp90 and AKT with ERα has major implications for its non-genomic signaling. In the presence of E2, Hsp90 dissociates, allowing ERα to dimerize and induce gene expression. At the same time, AKT is also rendered free to participate in the signal transduction cascade.

Rajoria and colleagues observed that E2 dramatically increases the ability of thyroid cells to adhere (137-140%) and migrate (27-75%). They also found downregulation of β-catenin in the thyroid cells treated with E2 (Rajoria et al., 2010).

### **4. PI3K-AKT pathway**

In 1991, three independent research groups identified the gene that encodes for the serin/threonin kinase AKT/PKB (Jones et al., 1991; Bellacosa et al., 1991; Coffer and Woodgent, 1991). AKT plays a major role in cell proliferation, survival, adhesion, migration, metabolism and tumorigenesis. The effects of AKT activation are determined by the phosphorylation of its downstream effectors located in the cytoplasm, nucleus and mitochondria (Manning and Cantley, 2007; Bijur and Jope, 2003; Antico Arciuch et al., 2009). Mammals have three closely related PKB genes, encoding the isoforms AKT1/PKBα, AKT2/PKBβ and AKT3/PKBγ. Although the AKT isoforms are ubiquitously expressed, evidence suggests that the relative isoform expression levels differ between tissues. AKT1 is the mainly expressed isoform in most tissues, while AKT2 is highly enriched in insulin target tissues. *Akt1* deficient mice show normal glucose tolerance and insulin-stimulated glucose clearance from blood, but display severe growth retardation (Cho et al., 2001). It has

Bcl-2 family proteins play a central role in controlling mitochondrial-mediated apoptosis. They include proteins that suppress apoptosis such as Bcl-2 and Bcl-XL, and proteins that promote apoptosis such as Bax, Bad and Bcl-XS (Antonsson and Martinou, 2000). Bcl-2 proteins localize or translocate to the mitochondrial membrane and modulate apoptosis by permeabilization of the inner and/or outer membrane, leading to the release of citochrome *c* or stabilization of the barrier function. Bcl-2 family members are altered in thyroid cancer (Kossmehl et al., 2003) and their levels are regulated by estrogen in some cell systems (Song and Santen, 2003). The antiapoptotic member Bcl-2 is up-regulated by E2 and by the ERα agonist PPT, but downregulated by the ERβ agonist DPN in thyroid cancer cells, suggesting that ERα induces Bcl-2 expression whereas ERβ reduces it (Zeng et al., 2007). In addition, it has been shown that ERβ

Recent work on the WRO thyroid cancer cells revealed that E2 increases cathepsin D transcription and that cathepsin D expression is inhibited upon siRNA-mediated knockdown of ERα and ERβ (Kumar et al., 2010). Cathepsin D is a classical E2 target gene regulated by Sp1-ERα promoter binding (Wang et al., 1997). It is well established that cathepsin D expression is elevated in thyroid tumors and correlates with disease

The expression of another classical E2 target gene, cyclin D1 (Pestell et al., 1999), is stimulated by E2 in thyroid cancer cell lines, and co-treatment with siERα and siERβ shows roles for ERα and ERβ in regulating cyclin D1 transcription. E2 regulation of cyclin D1 transcription involves

In Nthy-ori3-1 and BCPAP cells (derived from thyroid carcinoma), ERα was found to be complexed with Hsp90 and AKT (Rajoria et al., 2010). The complex of Hsp90 and AKT with ERα has major implications for its non-genomic signaling. In the presence of E2, Hsp90 dissociates, allowing ERα to dimerize and induce gene expression. At the same time, AKT is

Rajoria and colleagues observed that E2 dramatically increases the ability of thyroid cells to adhere (137-140%) and migrate (27-75%). They also found downregulation of β-catenin in

In 1991, three independent research groups identified the gene that encodes for the serin/threonin kinase AKT/PKB (Jones et al., 1991; Bellacosa et al., 1991; Coffer and Woodgent, 1991). AKT plays a major role in cell proliferation, survival, adhesion, migration, metabolism and tumorigenesis. The effects of AKT activation are determined by the phosphorylation of its downstream effectors located in the cytoplasm, nucleus and mitochondria (Manning and Cantley, 2007; Bijur and Jope, 2003; Antico Arciuch et al., 2009). Mammals have three closely related PKB genes, encoding the isoforms AKT1/PKBα, AKT2/PKBβ and AKT3/PKBγ. Although the AKT isoforms are ubiquitously expressed, evidence suggests that the relative isoform expression levels differ between tissues. AKT1 is the mainly expressed isoform in most tissues, while AKT2 is highly enriched in insulin target tissues. *Akt1* deficient mice show normal glucose tolerance and insulin-stimulated glucose clearance from blood, but display severe growth retardation (Cho et al., 2001). It has

but not ERα promotes the expression of Bax (Lee et al., 2005; Zeng et al., 2007).

ERα-Sp1 (Castro-Rivera et al., 2001) and AP-1-ERα (Liu et al., 2002) interactions.

also rendered free to participate in the signal transduction cascade.

the thyroid cells treated with E2 (Rajoria et al., 2010).

aggressiveness (Leto et al., 2004).

**4. PI3K-AKT pathway** 

also been shown that cells derived from *Akt1* deficient mouse embryos are also more susceptible to pro-apoptotic stimuli (Chen et al., 2001). On the other hand, deficiency of *AKT2* alone is sufficient to cause a diabetic phenotype in mice (Withers et al., 1998; Cho et al., 2001) and a loss-of-function mutation in AKT2 is associated with diabetes in one human family (George et al., 2004).

AKT kinases are typically activated by engagement of receptor tyrosine kinases by growth factors and cytokines, as well as oxidative stress and heat shock. AKT activation relies on phosphatidylinositol 3,4,5-triphosphate (PtdIns-3,4,5-P3) which is produced from phosphatidylinositol 4,5-biphosphate (PtdIns-4,5-P2) by phosphatidylinositol 3-kinase (PI3K) (Franke et al., 1995). The interaction between the Pleckstrin homology (PH) domain of AKT with PtdIns-3,4,5-P3 favors its phosphorylation at two residues, one in the Cterminal tail (Ser473) and the other in the activation loop (Thr308). Phosphorylation at Ser473 appears to precede and facilitate phosphorylation at Thr308 (Sarbassov et al., 2005). AKT is phosphorylated in Ser473 by mTORC2 (Ikenoue et al., 2008), while PI-3K-dependent kinase 1 (PDK1) accounts for the phosphorylation in Thr308 (Chan et al., 1999).

The proliferative effects of AKT result from phosphorylation of several substrates. For example, GSK3*β* once phosphorylated is inactivated and this prevents degradation of cyclin D1 (Diehl et al., 1998). Furthermore, AKT activation leads to increased translation of cyclin D1 and D3 transcripts via mTOR (Muise-Helmericks et al., 1998). AKT phosphorylates the cell cycle inhibitors p21WAF1 and p27Kip1 inducing their cytoplasmic retention (Testa and Bellacosa, 2001).

AKT activity prevents apoptosis through the phosphorylation and inhibition of proapoptotic mediators such as Bad, FOXO family members, and IκB kinase-β (IKK-β) (Datta et al., 1999). AKT activity also attenuates the response of cells to the release of cytochrome *c* into the cytoplasm (Kennedy et al., 1999).

AKT can also antagonize p53-mediated cell cycle checkpoints by modulating the subcellular localization of Mdm2. Phosphorylation of Mdm2 by AKT triggers its localization to the nucleus, where Mdm2 can complex with p53 to promote its ubiquitin/proteasome-mediated degradation (Mayo and Donner, 2001).

The crucial role of the PI3K signaling cascade in the pathogenesis of thyroid neoplastic disorders has been recently confirmed by the development and study of a relevant mouse model (Yeager et al., 2007, 2008; Miller et al., 2009), as well as by solid clinicopathological data (Garcia-Rostan et al., 2005; Hou et al., 2007, 2008; Vasko and Saji, 2007; Wang et al., 2007). Thyrocyte-specific deletion of the *Pten* tumor suppressor constitutively activates the PI3K signaling cascade, leading to hyperplastic thyroid glands at birth, and to the development of thyroid nodules and follicular adenomas by 6-8 months of age (Yeager et al., 2007) and thyroid carcinomas by one year of age (Antico Arciuch et al., 2010).

#### **5. PI3K-estrogen cooperation during proliferation**

The *Pten* mouse model of thyroid disease displays a unique and remarkable characteristic: the higher proliferative index of female mutant thyrocytes, compared with males. This difference leads to increased cellularity in the thyroids of female mutants at a young age, to an increased incidence of thyroid adenomas in mutant females at 8 months of age (Yeager et

Estrogen Signaling and Thyrocyte Proliferation 117

and AP-1 are strongly responsive to redox regulation (Droge, 2002). Recent data have suggested that physiological concentrations of E2 trigger a rapid production of intracellular reactive oxygen species (ROS) in endothelial and epithelial cells, and that E2-induced DNA synthesis is at least in part mediated by ROS signaling in these cells (Felty et al., 2005; Felty, 2006). This notion is particularly intriguing, since E2-mediated ROS production in thyroid follicular cells would have two effects: an immediate stimulation of cell proliferation, and a long-term accumulation of oxidative DNA damage. Furthermore, these effects would be further enhanced if PI3K activation resulted in an alteration of the thyrocyte antioxidant and detoxification system. Strikingly, in an ongoing proteomic effort (manuscript in preparation), we have recently identified Glutathione S-transferase Mu 1 (GSTM1), an enzyme important for the reduction (detoxification) of hydrogen peroxide, as one of the most significantly downregulated proteins in mutant thyroids, suggesting that, indeed, PI3K-mediated GSTM1

Finally, the increased expression level of *Tpo*, *Duox1* and *Slc5a5* genes in female mice, irrespective of their genotype, strongly suggests that estrogen has a significant role in their transcriptional regulation, providing additional targets for future studies on the role of

A role for estrogen in thyroid proliferation has been proposed for several years, based on the analysis of the effects of estrogen on thyroid cells in culture. Now, for the first time, our hormone manipulation experiments in a relevant mouse model of thyroid proliferative disorders and neoplastic transformation have provided *in vivo* evidence that circulating estrogens increase thyroid follicular cells proliferation. It is tempting to suggest that the relatively mild effect of estrogens on thyroid cells is uncovered and amplified by oncogenic events lowering the thyrocyte proliferation threshold. Further studies will validate this

Antico Arciuch VG, Dima M, Liao XH, Refetoff S and Di Cristofano A. (2010) Cross-talk

Antonsson B and Martinou JC. (2000) The Bcl-2 protein family. Exp Cell Res. 256:50-7. Are C and Shaha AR. (2006) Anaplastic thyroid carcinoma: biology, pathogenesis, prognostic factors, and treatment approaches. *Ann Surg Oncol*. 13:453-64. Arnold JT, Liu X, Allen JD, Le H, McFann KK and Blackman MR. (2007) Androgen receptor or

and PSA production in human prostate cancer cells. *Prostate.* 67:1152-62. Ascenzi P, Bocedi A, Marino M. (2006) Structure-function relationship of estrogen receptor alpha and beta: impact on human health. *Mol Aspects Med*. 27:299-402.

between PI3K and estrogen in the mouse thyroid predisposes to the development of follicular carcinomas with a higher incidence in females. *Oncogene*. 29:5678-86. Antico Arciuch VG, Galli S, Franco MC, Lam PY, Cadenas E, Carreras MC, and Poderoso JJ.

(2009) AKT1 intramitochondrial cycling is a crucial step in the redox modulation of

estrogen receptor-beta blockade alters DHEA-, DHT-, and E(2)-induced proliferation

reduction might indeed further amplify the effects of ROS in the thyroid.

estrogen in the pathophysiology of the thyroid gland.

hypothesis in the context of different oncogenic mutations.

cell cycle progression. *PLoS One* 4: e7523.

**6. Conclusion** 

**7. References** 

al., 2007), and to an increased incidence of thyroid carcinomas in mutant females at one year of age (Antico Arciuch et al., 2010). The direct role of estrogen signaling in determining this difference in proliferative response to PI3K activation is underlined by the fact that these effects could be completely reversed by estrogen depletion in the females, and by slowrelease estrogen pellet implantation in the males.

Several groups had anticipated a role for estrogen in thyroid proliferation, based on the effects of estradiol on thyroid carcinoma cells in culture (Manole et al., 2001; Vivacqua et al., 2006; Chen et al., 2008; Kumar et al., 2010; Rajoria et al., 2010). The *Pten* mouse model represents the first *in vivo* validation of the direct role played by estrogen in establishing the increased prevalence of thyroid disorders in the female.

Fig. 1. Schematic model of the cooperation between estrogen signaling and PI3K activation.

The analysis of *Pten* mutant mice also shed some light on the molecular basis of the differential thyrocyte proliferative index and risk of adenoma and carcinoma development between male and female mutant mice. Genetic approaches, by crossing *Pten* mutant mice and *p27* mutant mice, and cell culture-based experiments have provided evidence that these gender-based differences in this mouse model are due, at least in part, to the ability of estrogens to down-regulate p27 levels through mechanisms that include transcriptional regulation, in addition to the known effects on p27 protein degradation through regulation of Skp2 (Antico Arciuch et al., 2010; Foster et al., 2003).

Thus it is conceivable that, in thyroids harboring mutations that confer elevated proliferative signals and thus a low cell cycle progression threshold, E2-mediated p27 depletion further increases the thyrocyte proliferative index (Figure 1).

Additional mechanisms, including E2-mediated mitochondrial effects, are also likely to contribute to this phenotype. Maintenance of a normal intracellular redox status plays an important role in such processes as DNA synthesis, gene expression, enzymatic activity, and others. Signaling cascades involving protein tyrosine kinases can be enhanced by oxidative inhibition of protein tyrosine phosphatases, and pathways involving NF-kB, JNK, p38 MAPK, and AP-1 are strongly responsive to redox regulation (Droge, 2002). Recent data have suggested that physiological concentrations of E2 trigger a rapid production of intracellular reactive oxygen species (ROS) in endothelial and epithelial cells, and that E2-induced DNA synthesis is at least in part mediated by ROS signaling in these cells (Felty et al., 2005; Felty, 2006). This notion is particularly intriguing, since E2-mediated ROS production in thyroid follicular cells would have two effects: an immediate stimulation of cell proliferation, and a long-term accumulation of oxidative DNA damage. Furthermore, these effects would be further enhanced if PI3K activation resulted in an alteration of the thyrocyte antioxidant and detoxification system. Strikingly, in an ongoing proteomic effort (manuscript in preparation), we have recently identified Glutathione S-transferase Mu 1 (GSTM1), an enzyme important for the reduction (detoxification) of hydrogen peroxide, as one of the most significantly downregulated proteins in mutant thyroids, suggesting that, indeed, PI3K-mediated GSTM1 reduction might indeed further amplify the effects of ROS in the thyroid.

Finally, the increased expression level of *Tpo*, *Duox1* and *Slc5a5* genes in female mice, irrespective of their genotype, strongly suggests that estrogen has a significant role in their transcriptional regulation, providing additional targets for future studies on the role of estrogen in the pathophysiology of the thyroid gland.

#### **6. Conclusion**

116 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

al., 2007), and to an increased incidence of thyroid carcinomas in mutant females at one year of age (Antico Arciuch et al., 2010). The direct role of estrogen signaling in determining this difference in proliferative response to PI3K activation is underlined by the fact that these effects could be completely reversed by estrogen depletion in the females, and by slow-

Several groups had anticipated a role for estrogen in thyroid proliferation, based on the effects of estradiol on thyroid carcinoma cells in culture (Manole et al., 2001; Vivacqua et al., 2006; Chen et al., 2008; Kumar et al., 2010; Rajoria et al., 2010). The *Pten* mouse model represents the first *in vivo* validation of the direct role played by estrogen in establishing the

Fig. 1. Schematic model of the cooperation between estrogen signaling and PI3K activation.

The analysis of *Pten* mutant mice also shed some light on the molecular basis of the differential thyrocyte proliferative index and risk of adenoma and carcinoma development between male and female mutant mice. Genetic approaches, by crossing *Pten* mutant mice and *p27* mutant mice, and cell culture-based experiments have provided evidence that these gender-based differences in this mouse model are due, at least in part, to the ability of estrogens to down-regulate p27 levels through mechanisms that include transcriptional regulation, in addition to the known effects on p27 protein degradation through regulation

Thus it is conceivable that, in thyroids harboring mutations that confer elevated proliferative signals and thus a low cell cycle progression threshold, E2-mediated p27 depletion further

Additional mechanisms, including E2-mediated mitochondrial effects, are also likely to contribute to this phenotype. Maintenance of a normal intracellular redox status plays an important role in such processes as DNA synthesis, gene expression, enzymatic activity, and others. Signaling cascades involving protein tyrosine kinases can be enhanced by oxidative inhibition of protein tyrosine phosphatases, and pathways involving NF-kB, JNK, p38 MAPK,

release estrogen pellet implantation in the males.

increased prevalence of thyroid disorders in the female.

of Skp2 (Antico Arciuch et al., 2010; Foster et al., 2003).

increases the thyrocyte proliferative index (Figure 1).

A role for estrogen in thyroid proliferation has been proposed for several years, based on the analysis of the effects of estrogen on thyroid cells in culture. Now, for the first time, our hormone manipulation experiments in a relevant mouse model of thyroid proliferative disorders and neoplastic transformation have provided *in vivo* evidence that circulating estrogens increase thyroid follicular cells proliferation. It is tempting to suggest that the relatively mild effect of estrogens on thyroid cells is uncovered and amplified by oncogenic events lowering the thyrocyte proliferation threshold. Further studies will validate this hypothesis in the context of different oncogenic mutations.

#### **7. References**


Estrogen Signaling and Thyrocyte Proliferation 119

Cho H, Thorvaldsen JL, Chu Q, Feng F and Birnbaum MJ. (2001) AKT1/pkbalpha is

Coffer PJ and Woodgett JR. (1991) Molecular cloning and characterisation of a novel

Dalla Valle L, Ramina A, Vianello S, Fassina A, Belvedere P and Colombo L. (1998) Potential

Datta SR, Brunet A and Greenberg ME. Cellular survival: a play in three AKTs. (1999) *Genes* 

Dechering K, Boersma and Mosselman S. (2000) Estrogen receptors alpha and beta: two

Demonacos CV, Karayanni N, Hatzoglou E, Tsiriyiotis C, Spandidos DA and Sekeris CE.

Deroo BJ, Korach KS. (2006) Estrogen receptors and human disease. *J Clin Invest.* 116:561-70. Dick GM, Hunter AC and Sanders KM. (2002) Ethylbromide tamoxifen, a membrane-

Diehl JA, Cheng M, Roussel MF and Sherr CJ. (1998) Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. *Genes Dev.* 12:3499-3511. Droge W. (2002) Free radicals in the physiological control of cell function. *Physiol Rev* 82, 47-95. Dunn KL, Espino PS, Drobic B, He S and Davie JR. (2005) The Ras-MAPK signal

Enriquez JA, Fernandez-Silva P, Garrido-Perez N, Lopez-Perez MJ, Perez-Martos A, and

Fagin JA. (2004) How thyroid tumors start and why it matters: kinase mutants as targets for

Felty Q. (2006) Estrogen-induced DNA synthesis in vascular endothelial cells is mediated by

Felty Q., Singh K.P. and Roy, D. (2005) Estrogen-induced G1/S transition of G0-arrested

Filardo EJ, Quinn JA, Bland KI, Frackelton AR Jr. (2000) Estrogen-induced activation of Erk-

Filardo EJ, Graeber CT, Quinn JA, Resnick MB, Giri D, DeLellis RA, Steinhoff MM and Sabo

solid cancer pharmacotherapy. *J Endocrinol.* 183:249-56.

ROS signaling. *BMC Cardiovasc Disord* 6, 16-22.

tumor progression. *Clin. Cancer Res.* 12:6359-6366.

homeostasis in mice. *J Biol Chem* 276:38349-52.

receptors of a kind? *Curr. Med. Chem.* 7:561-576.

families. *Eur J Biochem.* 201:475-481.

*J. Clin. Endocrinol. Metab.* 83:3702-3709.

hormone. *Mol Cell Biol.* 19:657-670.

signaling. *Oncogene* 24, 4883-93.

EGF. *Mol. Endocrinol.* 14:1649-1660.

*Dev*. 13:2905-27.

61:226-232.

61(5):1105-13.

required for normal growth but dispensable for maintenance of glucose

putative protein-serine kinase related to the cAMP-dependent and protein kinase C

for estrogen synthesis and action in human normal and neoplastic thyroid tissues.

(1996) Mitochondrial genes as sites of primary action of steroid hormones. *Steroids.*

impermeant antiestrogen, activates smooth muscle calcium-activated largeconductance potassium channels from the extracellular side. *Mol Pharmacol*.

transduction pathway, cancer and chromatin remodeling. *Biochem Cell Biol.* 83:1-14.

Montoya J. (1999) Direct regulation of mitochondrial RNA synthesis by thyroid

estrogen-dependent breast cancer cells is regulated by mitochondrial oxidant

1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-

E. (2006) Distribution of GPR30, a seven membrane-spanning estrogen receptor, in primary breast cancer and its association with clinicopathologic determinants of


Bellacosa A, Testa JR, Staal SP and Tsichlis PN. (1991) A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. *Science.* 254:274-277. Bijur GN, Jope RS. Rapid accumulation of AKT in mitochondria following phosphatidylinositol 3-kinase activation. (2003) *J Neurochem* 87:1427-35. Carmeci C, Thompson DA, Ring HZ, Francke U and Weigel RJ. (1997) Identification of a

Casas F, Rochard P, Rodier A, Cassar-Malek I, Marchal-Victorion S, Wiesner RJ, Cabello G,

Castro-Rivera E, Samudio I and Safe S. (2001) Estrogen regulation of cyclin D1 gene

Cavalieri EL and Rogan EG. (2001) Evidence that a burst of DNA depurination in SENCAR

Cavalieri EL, Stack DE, Devanesan PD, Todorovic R, Dwivedy I, Higginbotham S,

Chan TO, Rittenhouse SE and Tsichlis PN. AKT/PKB and other D3

Chen GG, Liu ZM, Vlantis AC, Tse GM, Leung BC and van Hasselt CA. (2004) Heme

Chen J, Gokhale M, Li Y, Trush MA, and Yager JD. (1998) Enhanced levels of several

Chen J, Schwartz DA, Young TA, Norris JS, and Yager JD. (1996) Identification of genes

Chen JQ, Delannoy M, Cooke C and Yager JD. (2004) Mitochondrial localization of ERalpha

Chen WS, Xu PZ, Gottlob K, Chen ML, Sokol K, Shiyanova T, Roninson I, Weng W, Suzuki

and ERbeta in human MCF7 cells. *Am J Physiol.* 286:E1011-E1022.

endogenous tumor initiators. *Proc Natl Acad Sci U S A.* 94:10937-42.

dependent phosphorylation. (1999) *Annu Rev Biochem* 68: 965-1014.

*Cell Biol.* 19:7913-7924.

*Chem.* 276:30853-30861.

*Oncogene* 20:7945-7953.

8:367-377.

292:1728-1731.

HepG2 cells. *Carcinogenesis.* 19:2187-2193.

treated female rats. *Carcinogenesis.* 17:2783-2786.

gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer. *Genomics*. 45:607-617.

and Wrutniak C. (1999) A variant form of the nuclear triiodothyronine receptor c-ErbAalpha1 plays a direct role in regulation of mitochondrial RNA synthesis. *Mol* 

expression in ZR-75 breast cancer cells involves multiple enhancer elements. *J Biol* 

mouse skin induces error-prone repair and forms mutations in the H-ras gene.

Johansson SL, Patil KD, Gross ML, Gooden JK, Ramanathan R, Cerny RL and Rogan EG. (1997) Molecular origin of cancer: catechol estrogen-3,4-quinones as

phosphoinositideregulated kinases: kinase activation by phosphoinositide-

oxygenase-1 protects against apoptosis induced by tumor necrosis factor-alpha and cycloheximide in papillary thyroid carcinoma cells. *J Cell Biochem*. 92:1246-56. Chen GG, Vlantis AC, Zeng Q and van Hasselt CA. (2008) Regulation of cell growth by

estrogen signaling and potential targets in thyroid cancer. *Curr Cancer Drug Targets*.

mitochondrial mRNA transcripts and mitochondrial superoxide production during ethinyl estradiol-induced hepatocarcinogenesis and after estrogen treatment of

whose expression is altered during mitosuppression in livers of ethinyl estradiol-

R, Tobe K, Kadowaki T and Hay N. Growth retardation and increased apoptosis in mice with homozygous disruption of the AKT1 gene. (2001) *Genes Dev* 15:2203-8. Cho H, Mu J, Kim JK, Thorvaldsen JL, Chu Q, Crenshaw EB 3rd, Kaestner KH, Bartolomei

MS, Shulman GI and Birnbaum MJ*.* Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase AKT2 (PKB beta). (2001) *Science*


Estrogen Signaling and Thyrocyte Proliferation 121

Hou P, Liu D, Shan Y, Hu S, Studeman K, Condouris S et al. (2007) Genetic alterations and

Hugo ER, Brandebourg TD, Woo JG Loftus J, Alexander JW, Ben-Jonathan N. (2008) Bisphenol

Jemal A, Siegel R, Ward E, Hao Y, Xu J and Thun MJ. (2009) Cancer statistics, 2009. *CA* 

Jones KP (1992). Estrogens and progestins: what to use and how to use it. *Clin. Obstet.* 

Jones PF, Jakubowicz T, Pitossi FJ, Maurer F and Hemmings BA. (1991) Molecular cloning

Kennedy SG, Kandel ES, Cross TK and Hay N. (1999) AKT/Protein kinase B inhibits cell

Kim CS and Zhu X. (2009) Lessons from mouse models of thyroid cancer. *Thyroid*. 19:1317-31. Kim KH, Moriarty K and Bender JR. (2008) Vascular cell signaling by membrane estrogen

Klinge CM. (2000) Estrogen receptor interaction with co-activators and co-repressors.

Kossmehl P, Shakibaei M, Cogoli A, Infanger M, Curcio F, Schönberger J, Eilles C, Bauer J,

Kouniavsky G and Zeiger MA. (2010) Thyroid tumorigenesis and molecular markers in

Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S and Gustafsson JA. (1996) Cloning of a

Kuiper GG, Carlsson B, Grandien K, Enmark E, Häggblad J, Nilsson S and Gustafsson JA.

Lee ML, Chen GG, Vlantis A C, Tse GM, Leung BC and van Hasselt CA. (2005) Induction of

Lemoine NR, Mayall ES, Wyllie FS, Farr CJ, Hughes D, Padua RA, Thurston V, Williams ED

extrinsic and intrinsic pathways. *Endocrinology*. 144:4172-9.

Jensen EV, DeSombre ER. (1973) Estrogen-receptor interaction. *Science*. 182:126-134.

subfamily. *Proc Natl Acad Sci U S A.* 88:4171-4175.

receptors. *Steroids.* 73(9-10):864-9.

thyroid cancer. *Curr Opin Oncol.* 22:23-9.

altered expression of Bcl-xL. *Cancer J.* 11:113-121.

cancer. *Clin Cancer Res.* 13:1161-1170.

23:1919-31.

*Cancer J Clin.* 59:225-49.

*Gynecol.* 32:871-883.

19:5800-5810.

*Steroids* 65: 227-25.

*Int J Oncol.* 36:1067-1080.

*Cancer Res*. 48:4459-4463.

5930.

their relationship in the phosphatidylinositol 3-kinase/AKT pathway in thyroid

A at environmentally relevant doses inhibits adiponectin release from human adipose tissue explants and adipocytes. *Environ. Health Perspect*. 116:1642-1647. Ikenoue T, Inoki K, Yang Q, Zhou X and Guan KL. Essential function of TORC2 in PKC and

AKT turn motif phosphorylation, maturation and signaling. (2008) *EMBO J* 

and identification of a serine/threonine protein kinase of the second-messenger

death by preventing the release of cytochrome c from mitochondria. *Mol Cell Biol*

Pickenhahn H, Schulze-Tanzil G, Paul M and Grimm D. (2003) Weightlessness induced apoptosis in normal thyroid cells and papillary thyroid carcinoma cells via

novel receptor expressed in rat prostate and ovary. *Proc Natl Acad Sci USA*. 93:5925-

(1997) Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. *Endocrinology.* 138:863-870. Kumar A, Klinge CM and Goldstein RE. (2010) Estradiol-induced proliferation of papillary

and follicular thyroid cancer cells is mediated by estrogen receptors alpha and beta.

thyroid papillary carcinoma cell proliferation by estrogen is associated with an

and Wynford-Thomas D. (1988) Activated ras oncogenes in human thyroid cancers.


Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR and Tsichlis

Garcia-Rostan G, Costa AM, Pereira-Castro I, Salvatore G, Hernandez R, Hermsem MJ et al.

George S, Rochford JJ, Wolfrum C, Gray SL, Schinner S, Wilson JC, Soos MA, Murgatroyd

Gilfillan, CP. (2010) Review of the genetics of thyroid tumours: diagnostic and prognostic

Grossman A, Oppenheim J, Grondin G, St. Jean P and Beaudoin AR. (1989)

Gutierrez-Hartmann A, Duval DL and Bradford AP. (2007) ETS transcription factors in

Haas E, Meyer MR, Schurr U, Bhattacharya I, Minotti R, Nguyen HH, Heigl A, Lachat M,

Handkiewicz-Junak D, Czarniecka A and Jarzab B. (2010) Molecular prognostic markers in

He YY, Cai B, Yang YX, Liu XL and Wan XP. (2009) Estrogenic G protein-coupled receptor

Hodgson NC, Button J and Solorzano CC. (2004) Thyroid cancer: is the incidence still

Hou P, Ji M and Xing M. (2008) Association of PTEN gene methylation with genetic

mitogen-activated protein kinase pathway. *Cancer Sci.* 100:1051-1061. Hermenegildo C and Cano A. (2000) Pure anti-oestrogens. *Hum Reprod Update*. 6(3):237-43. Herrmann BL, Saller B, Janssen OE, Gocke P, Bockisch A. Sperling H, Mann K and Broecker

PDGF-activated phosphatidylinositol 3-kinase. (1995) *Cell* 2:727-36. Foster JS, Fernando RI, Ishida N, Nakayama KI and Wimalasena J. (2003) Estrogens down-

mediated by the ERK pathway. *J Biol Chem*. 278:41355-41366.

implications. *ANZ Journal of Surgery*. 80:33-40.-

pancreatic acinar cells. *Endocrinology.* 124:2857-2866.

endocrine systems. *Trends Endocrinol Metab*. 18(4):150-8.

estrogen receptor signaling. *J. Biol. Chem*. 276:36869-36872.

65:10199-10207.

*Cell Endocrinol.* 322:8-28.

*Endocrinol. Metab.* 8:5476-5484.

tumors. *Cancer*. 113:2440-2447.

increasing? *Ann Surg Oncol*. 11:1093-7.

1328.

PN. The protein kinase encoded by the AKT proto-oncogene is a target of the

regulate p27Kip1 in breast cancer cells through Skp2 and through nuclear export

(2005) Mutation of the PIK3CA gene in anaplastic thyroid cancer. *Cancer Res.*

PR, Williams RM, Acerini CL, Dunger DB, Barford D, Umpleby AM, Wareham NJ, Davies HA, Schafer AJ, Stoffel M, O'Rahilly S and Barroso I*.* A family with severe insulin resistance and diabetes due to a mutation in AKT2. (2004) *Science* 304:1325-

Immunocytochemical localization of the [3H]estradiol-binding protein in rat

Genoni M and Barton M. (2007) Differential effects of 17beta-estradiol on function and expression of estrogen receptor alpha, estrogen receptor beta, and GPR30 in arteries and veins of patients with atherosclerosis. *Hypertension*. 49:1358-1363. Hall JM, Couse JF and Korach KS. (2001) The multifaceted mechanisms of estradiol and

papillary and follicular thyroid cancer: Current status and future directions. *Mol* 

30 signaling is involved in regulation of endometrial carcinoma by promoting proliferation, invasion potential, and interleukin-6 secretion via the MEK/ERK

M. (2002) Impact of estrogen replacement therapy in a male with congenital aromatase deficiency caused by a novel mutation in the CYP19 gene. *J. Clin.* 

alterations in the phosphatidylinositol 3-kinase/AKT signaling pathway in thyroid


Estrogen Signaling and Thyrocyte Proliferation 123

Neff RL, Farrar WB, Kloos RT and Burman KD. (2008) Anaplastic thyroid cancer. *Endocrinol* 

Nikiforov YE. (2008) Thyroid carcinoma: molecular pathways and therapeutic targets. *Mod* 

O'Dowd BF, Nguyen T, Marchese A, Cheng R, Lynch KR, Heng HH, Kolakowski LF Jr and

Owman C, Blay P, Nilsson C and Lolait SJ. (1996) Cloning of human cDNA encoding a

in brain and peripheral tissues. *Biochem. Biophys. Res. Commun*. 228:285-292. Pedram A, Razandi M and Levin ER. (2006) Nature of functional estrogen receptors at the

Pestell RG, Albanese C, Reutens AT, Segall JE, Lee RJ and Arnold A. (1999) The cyclins and

Posern G and Treisman R. (2006) Actin' together: serum response factor, its cofactors and the

Rajoria S, Suriano R, Shanmugam A, Wilson YL, Schantz SP, Geliebter J and Tiwari RK. (2010) Metastatic phenotype is regulated by estrogen in thyroid cells. *Thyroid*. 20:33-41. Roberts PJ and Der CJ. (2007) Targeting the Raf-MEK-ERK mitogen-activated protein kinase

Ruggiero RJ and Likis FE. (2002) Estrogen: physiology, pharmacology, and formulations for

Sarbassov DD, Guertin DA, Ali SM and Sabatini DM. Phosphorylation and regulation of AKT/PKB by the rictor-mTOR complex. (2005) *Science* 18:1098-1101. Sekeris CE. (1990) The mitochondrial genome: a possible primary site of action of steroid

Smallridge RC, Marlow LA and Copland JA. (2009) Anaplastic thyroid cancer: molecular

Speroff L, Glass RH and Kase NG. (1999) *Clin. Gynecol. Endocrinol. Infert. (6th ed)*. Lippincott

Stender JD, Frasor J, Komm B, Chang KC, Kraus WL and Katzenellenbogen BS. (2007)

Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J and Gustafsson JA. (2004) Estrogen

Takada Y, Kato C, Kondo S, Korenaga R and Ando J. (1997) Cloning of cDNAs encoding G

Testa JR and Bellacosa A. AKT plays a central role in tumorigenesis. (2001) *Proc. Natl. Acad.* 

E2F1 in the regulation of cell proliferation*. Mol Endocrinol.* 21:2112-23. Stirone C, Duckles SP, Krause DN and Procaccio V. (2005) Estrogen increases mitochondrial

cell line T47D. *Proc. Natl. Acad. Sci. USA*. 101:1566-1571.

shear stress. *Biochem. Biophys. Res. Commun*. 240:737-741.

Estrogen-regulated gene networks in human breast cancer cells: involvement of

efficiency and reduces oxidative stress in cerebral blood vessels. *Mol Pharmacol*.

receptor beta inhibits 17beta-estradiol-stimulated proliferation of the breast cancer

protein-coupled receptor expressed in human endothelial cells exposed to fluid

pathogenesis and emerging therapies. *Endocr Relat Cancer.* 16:17-44. Song RX and Santen RJ. (2003) Apoptotic action of estrogen. *Apoptosis*. 8:55-60.

George SR. (1998) Discovery of three novel G-protein-coupled receptor genes.

novel heptahelix receptor expressed in Burkitt's lymphoma and widely distributed

cyclin-dependent kinase inhibitors in hormonal regulation of proliferation and

*Metab Clin North Am.* 37:525-38.

plasma membrane. *Mol Endocrinol* 20:1996-2009.

link to signal transduction. *Trends Cell Biol*. 16(11):588-96.

cascade for the treatment of cancer. *Oncogene.* 26:3291-310.

replacement therapy. *J. Midwifery Womens Health.* 47:130-138.

differentiation. *Endocr Rev.* 20:501-534.

hormones. *In Vivo.* 4:317-320.

Williams & Wilkins: Philadelphia.

68:959-965.

*Sci. USA* 98:10983-10985.

*Pathol.* 2:S37-S43.

*Genomics.* 47:310-313.


Leto G, Tumminello FM, Crescimanno M, Flandina C and Gebbia N. (2004) Cathepsin D

Libutti SK. (2005) Understanding the role of gender in the incidence of thyroid cancer.

Lima LP, Barros IA, Lisbôa PC, Araújo RL, Silva AC, Rosenthal D, Ferreira AC and Carvalho

Liu MM, Albanese C, Anderson CM, Hilty K, Webb P, Uht RM, Price RH Jr, Pestell RG and

Liu Z, Hou P, Ji M, Guan H, Studeman K, Jensen K, Vasko V, El-Naggar AK and Xing M.

Machens A, Hauptmann S and Dralle H. (2006) Disparities between male and female

Manole D, Schildknecht B, Gosnell B, Adams E and Derwahl M. (2001) Estrogen promotes

Mayo LD and Donner DB. (2001) A phosphatidylinositol 3-kinase/AKT pathway promotes

Miller KA, Yeager N, Baker K, Liao XH, Refetoff S and Di Cristofano A. (2009) Oncogenic

Moats RK II and Ramirez VD. (2000) Electron microscopic visualization of membrane-

Monje P and Boland R. (2001) Subcellular distribution of native estrogen receptor alpha and

Monje P, Zanello S, Holick M and Boland R. (2001) Differential cellular localization of estrogen receptor alpha in uterine and mammary cells. *Mol Cell Endocrinol.* 181:117-129. Mori M, Naito M, Watanabe H, Takeichi N, Dohi K and Ito A. (1990) Effects of sex

Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE, Tsichlis PN and Rosen N. (1998)

Müller RE, Johnston TC and Wotiz HH. (1979) Binding of estradiol to purified uterine

beta isoforms in rabbit uterus and ovary. *J Cell Biochem.* 82:467-479.

kinase/AKT-dependent pathway. *J. Biol. Chem.* 273:29864-29872.

plasma membranes. *J Biol Chem*. 254(16):7895-900.

thyroid epithelial cells in vivo. *Cancer Res.* 69:3689-3694.

implications. *Clin Exp Metastasis.* 21:91-106.

in normal and ovariectomized rats. *Steroids.* 71:653-9.

D1 gene expression. *J Biol Chem.* 277:24353-24360.

*Cancer J.* 11:104-105.

downstream. *Cell* 129:1261-74.

*Endocrinol Metab.* 86:1072-1077.

*(Oxf).* 65:500-5.

98:11598-603.

*Endocrinol.* 166:631-647.

thyroid tumors. *Cancer Res.* 50:7662-7.

expression levels in non-gynecological solid tumors: clinical and therapeutic

DP. (2006) Estrogen effects on thyroid iodide uptake and thyroperoxidase activity

Kushner PJ. (2002) Opposing action of estrogen receptors alpha and beta on cyclin

(2008) Highly prevalent genetic alterations in receptor tyrosine kinases and phosphatidylinositol 3-kinase/akt and mitogen-activated protein kinase pathways in anaplastic and follicular thyroid cancers. *J. Clin. Endocrinol. Metab*. 93:3106-3116. Lu Q, Ebling H, Mittler J, Baur WE and Karas RH. (2002) MAP kinase mediates growth

factor-induced nuclear translocation of estrogen receptor alpha. *FEBS Lett*. 516(1- 3):1-8.Manning BD, and Cantley LC. (2007) AKT/PKB signaling: navigating

patients with thyroid cancers: sex difference or gender divide? *Clin Endocrinol* 

growth of human thyroid tumor cells by different molecular mechanisms. *J Clin* 

translocation of Mdm2 from the cytoplasm to the nucleus. *Proc Natl Acad Sci U S A.*

Kras requires simultaneous PI3K signaling to induce ERK activation and transform

mediated uptake and translocation of estrogen-BSA:colloidal gold by hep G2 cells. *J* 

difference, gonadectomy, and estrogen on N-methyl-N-nitrosourea induced rat

Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-


**9**

*Brazil* 

**Suspicious Thyroid Fine Needle Aspiration** 

Renata Boldrin de Araujo, Célia Regina Nogueira, Jose Vicente Tagliarini, Emanuel Celice Castilho, Mariângela de Alencar Marques, Yoshio Kiy,

Thyroid nodules are common, affecting from 5 to 15% of the population (Tunbridge et al., 1977; Vander et al., 1968). Thyroid cancer, on the other hand, is uncommon and represents only 5% of all nodules, with an incidence in the United States of 1/10,000 inhabitants (Davies & Welch, 2006). Among thyroid neoplasms, the differentiated carcinomas (DTC) are the most frequent, and are responsible for about 75% of the malignant nodules of this gland

Despite the fact that the great majority of thyroid lesions are benign and the mortality rate due to thyroid cancer is low (Schlumberger & Pacini, 1997), the incidence of thyroid cancer is increasing at a rate of greater than 5% per year (Davies & Welch, 2006). Thus, it is important to identify the nodules which are malignant and require surgical treatment.

The method of choice for the diagnostic evaluation of thyroid nodules is fine needle aspiration biopsy (FNAB) (Bennedbaek et al., 1999; Bennedbaek & Hegedus, 2000; Cooper et al., 2006; Schlumberger, 1998). FNAB is both highly sensitive (65 – 98%) and highly specific (72 – 100%) (Gharib & Goellner, 1993; Mazzaferri, 1993; Sherman, 2003), and provides satisfactory diagnostic results in 80% of cases, with an increase of this percentage after a new FNAB (Gharib & Goellner, 1993). In cases of DTC, FNAB has been shown to be particularly useful in the diagnosis of papillary carcinoma (PC). However, in cases of follicular (FC) and Hürthle carcinoma (HC), as in the case of the follicular variant of PC (FVPC), FNAB is useful only as a screening test. In these cases FNAB indicates the corresponding cytological pattern (follicular or Hürthle), but is not able to differentiate benign tumors from the malignant tumors. In these cases, the patients undergo surgery for histological analysis and definitive

Faced with the uncertainty of the diagnostic evaluation of thyroid nodules, several clinical risk factors (Kimura et al., 2009; Tuttle et al., 1998), imaging tests (Wiest, 1998; Frates, 2006) and molecular markers (Melck & Yip, 2011) have been proposed as malignancy indicators. Recently, some studies have reported an association between increased serum levels of

**1. Introduction**

(Schlumberger & Pacini, 1997).

diagnosis (Faquin & Baloch, 2010; Tuttle et al., 1998).

**Aspiration Biopsy: TSH as a**

Lidia R. Carvalho and Gláucia M. F. S. Mazeto *Botucatu Medical School, Sao Paulo State University, Unesp* 

**Malignancy Marker?** 


### **Suspicious Thyroid Fine Needle Aspiration Aspiration Biopsy: TSH as a Malignancy Marker?**

Renata Boldrin de Araujo, Célia Regina Nogueira, Jose Vicente Tagliarini, Emanuel Celice Castilho, Mariângela de Alencar Marques, Yoshio Kiy, Lidia R. Carvalho and Gláucia M. F. S. Mazeto *Botucatu Medical School, Sao Paulo State University, Unesp Brazil* 

#### **1. Introduction**

124 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

Thiruvengadam A, Govindarajulu P and Aruldhas MM. (2003) Modulatory effect of

Vasko VV and Saji M. (2007) Molecular mechanisms involved in differentiated thyroid

Vivacqua A, Bonofiglio D, Albanito L, Madeo A, Rago V, Carpino A et al. (2006) 17beta-

Wang F, Porter W, Xing W, Archer TK and Safe S. (1997) Identification of a functional

Wang Y, Hou P, Yu H, Wang W, Ji M, Zhao S et al. (2007) High prevalence and mutual

Watson CS, Alyea RA, Jeng YJ and Kochukov MY. (2007) Nongenomic actions of low

Weihua Z, Saji S, Makinen S, Cheng G, Jensen EV, Warner M and Gustafsson JA. Estrogen

Withers DJ, Gutierrez JS, Towery H, Burks DJ, Ren JM, Previs S, Zhang Y, Bernal D, Pons S,

Wong CW, McNally C, Nickbarg E, Komm BS and Cheskis BJ. (2002) Estrogen receptor-

Wrutniak C, Cassar-Malek I, Marchal S, Rascle A, Heusser S, Keller JM, Flechon J, Dauca M,

Yeager N, Brewer C, Cai KQ, Xu XX and Di Cristofano A. (2008) mTOR is the key effector of

Yeager N, Klein-Szanto A, Kimura S and Di Cristofano A. (2007) Pten loss in the mouse

Zeng Q, Chen GG, Vlantis AC and van Hasselt CA. (2007) Oestrogen mediates the growth of

Zhang Z, Duan L, Du X, Ma H, Park I, Lee C, Zhang J and Shi J. (2008) The proliferative

phosphorylation cascade. *Proc Natl Acad Sci U S A*. 99:14783-14788.

Xing M. (2005) BRAF mutation in thyroid cancer*. Endocr. Relat. Cancer.* 12:245-262.

Cowden disease pathogenesis. *Cancer Res.* 67:959-66.

induced thyroid tumors in female rats. *Endocr Res.* 29:43-51.

cancer invasion and metastasis. *Curr Opin Oncol.* 19:11-17.

in thyroid tumors. *J Clin Endocrinol Metab.* 92:2387-2390.

cathepsin D gene. *Biochemistry.* 36:7793-7801.

diabetes in mice. *Nature* 391:900-904.

1423.

274:1-7.

16354.

68:444-449.

40:921-35.

of ERK. *Prostate.* 68:508-516.

97:5936-5941.

estradiol and testosterone on the development of N-nitrosodiisopropanolamine

estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the G protein-coupled receptor GPR30. *Mol Pharmacol*. 70:1414-

imperfect estrogen-responsive element in the 5'- promoter region of the human

exclusivity of genetic alterations in the phosphatidylinositol-3-kinase/akt pathway

concentration estrogens and xenoestrogens on multiple tissues. *Mol Cell Endocrinol*

receptor (ER) beta, a modulator of ERalpha in the uterus. *Proc. Natl. Acad. Sci. USA*.

Shulman GI, Bonner-Weir S and White MF. (1998) Disruption of IRS-2 causes type 2

interacting protein that modulates its nongenomic activity-crosstalk with Src/Erk

Samarut J, Ghysdael J and Cabello G. (1995) A 43-kDa protein related to c-Erb A alpha 1 is located in the mitochondrial matrix of rat liver. *J Biol Chem.* 270:16347-

PI3K-initiated proliferative signals in the thyroid follicular epithelium. *Cancer Res.*

thyroid causes goiter and follicular adenomas: insights into thyroid function and

human thyroid carcinoma cells via an oestrogen receptor-ERK pathway. *Cell Prolif*.

effect of estradiol on human prostate stromal cells is mediated through activation

Thyroid nodules are common, affecting from 5 to 15% of the population (Tunbridge et al., 1977; Vander et al., 1968). Thyroid cancer, on the other hand, is uncommon and represents only 5% of all nodules, with an incidence in the United States of 1/10,000 inhabitants (Davies & Welch, 2006). Among thyroid neoplasms, the differentiated carcinomas (DTC) are the most frequent, and are responsible for about 75% of the malignant nodules of this gland (Schlumberger & Pacini, 1997).

Despite the fact that the great majority of thyroid lesions are benign and the mortality rate due to thyroid cancer is low (Schlumberger & Pacini, 1997), the incidence of thyroid cancer is increasing at a rate of greater than 5% per year (Davies & Welch, 2006). Thus, it is important to identify the nodules which are malignant and require surgical treatment.

The method of choice for the diagnostic evaluation of thyroid nodules is fine needle aspiration biopsy (FNAB) (Bennedbaek et al., 1999; Bennedbaek & Hegedus, 2000; Cooper et al., 2006; Schlumberger, 1998). FNAB is both highly sensitive (65 – 98%) and highly specific (72 – 100%) (Gharib & Goellner, 1993; Mazzaferri, 1993; Sherman, 2003), and provides satisfactory diagnostic results in 80% of cases, with an increase of this percentage after a new FNAB (Gharib & Goellner, 1993). In cases of DTC, FNAB has been shown to be particularly useful in the diagnosis of papillary carcinoma (PC). However, in cases of follicular (FC) and Hürthle carcinoma (HC), as in the case of the follicular variant of PC (FVPC), FNAB is useful only as a screening test. In these cases FNAB indicates the corresponding cytological pattern (follicular or Hürthle), but is not able to differentiate benign tumors from the malignant tumors. In these cases, the patients undergo surgery for histological analysis and definitive diagnosis (Faquin & Baloch, 2010; Tuttle et al., 1998).

Faced with the uncertainty of the diagnostic evaluation of thyroid nodules, several clinical risk factors (Kimura et al., 2009; Tuttle et al., 1998), imaging tests (Wiest, 1998; Frates, 2006) and molecular markers (Melck & Yip, 2011) have been proposed as malignancy indicators. Recently, some studies have reported an association between increased serum levels of

Suspicious Thyroid Fine Needle Aspiration Aspiration Biopsy: TSH as a Malignancy Marker? 127

Of the 31 cases submitted to surgery, 14 showed malignancy upon histopathological analysis (group M). The malignancies included nine PCs, three FCs (one case with both PC and FC), one HC, one Hürthle tumor and one follicular tumor of uncertain malignant meaning. Thus, the concordance of suspicious or inconclusive FNAB for FC or HC with malignancy was 45.2% and the concordance with FC or HC was 12.9%. 17 patients (54.8%)

The M and WM groups did not differ significantly as to serum levels of TSH and FT4 (p>0.05). There were no significant differences in age or gender distribution (p>0.05). There were two smokers in the WM group and no smokers in the M group. In addition one patient

Seven patients (22.6%) presented hypo- or hyperthyroidism and were under treatment with thyroid medication. These included two (28.6%) patients from the M group and five (71.4%) patients from the WM group. After excluding such cases, the M and WM groups still did not

Female Gender n (%)\*\* 12 (85.7) 16 (94.1) 0.43 Age (years)\* 48.8 ± 12.5 54.8 ± 13.9 0.21 Smoking n (%)\*\*\* 0 (0.0) 2 (100.0) - Previous exporsure to radiation \*\*\* 0 (0.0) 1 (100.0) - TSH (mUI/L)\* 2.04 ± 1.74 3.08 ± 2.67 0.22 FT4 (ng/dL)\* 1.56 ± 0.59 1.35 ± 0.19 0.17 \* Average ± standard deviation (Student's T test); \*\* Chi-square test; \*\*\*not submitted to statistical analysis due to small sample number. M: presence of malignancy; WM: absence of malignancy; TSH:

Table 1. General data from 31 patients, with fine needle aspiration biopsies (FNAB) with suspicious or inconclusive cytological diagnosis for FC or HC, submitted to thyroidectomy, according to the final histological diagnosis of presence (group M) or absence (group WM)

Female Gender n (%)\*\* 10 (83.3) 11 (91.7) 0.54 Age (years)\* 50.4 ± 11.3 52.3 ± 15.8 0.74 Smoking n (%)\*\*\* 0 (0.0) 2 (100.0) - TSH (mUI/L)\* 1.79 ± 1.03 1.81 ± 1.16 0.96 FT4 (ng/dL)\* 1.59 ± 0.64 1.33 ± 0.18 0.19 \* Average ± standard deviation (Student's T test); \*\* Chi-square test; \*\*\*not submitted to statistical analysis due to small sample number. M: presence of malignancy; WM: absence of malignancy; TSH:

Table 2. General data from 24 patients, without hypo- or hyperthyroidism, with fine needle aspiration biopsies (FNAB) with suspicious or inconclusive cytological diagnosis for FC or HC, submitted to thyroidectomy, according to the final histological diagnosis of presence

**Data Group P M WM** 

**Data Group P M WM**

**3. Results**

had benign histological reports (group WM).

in the WM group had previous exposure to radiation (Table 1).

differ in regards to the analyzed parameters (Table 2).

thyroid stimulating hormone; FT4: free thyroxine.

thyroid stimulating hormone; FT4: free thyroxine.

(group M) or absence (group WM) of malignancy.

of malignancy.

thyroid stimulating hormone (TSH) and thyroid cancer (Boelaert et al., 2006; Gul et al., 2010; Jonklaas et al., 2008). However, this relationship has not yet been established for the more doubtful cases, such as those with an inconclusive cytological diagnosis for FC or HC.

The objective of this study was to evaluate whether the TSH serum levels can help to differentiate benign cases from the malignant cases in patients with an FNAB that shows a follicular or Hürthle pattern.

### **2. Material and methods**

This retrospective study was approved by the Committee of Ethics in Research of Botucatu Medical School (FMB) of the Sao Paulo State University - UNESP (protocol number 3626-2010).

We analyzed the cytological reports from patients carrying thyroid nodules that were submitted for thyroid FNAB at the Clinics Hospital FMB–Unesp between the years of 2003 and 2008. Of these, 59 cases with suspicious or inconclusive cytological diagnosis for FC or HC were selected. We included those nodules that presented a follicular or Hürthle pattern with the following reported descriptions: "follicular lesion," "follicular tumor," "follicular neoplasia," "Hürthle follicular lesion," "Hürthle tumor" or "Hürthle neoplasia." The medical data of these patients were evaluated, and we found 31 cases that were submitted to surgery and with histological diagnosis. The effective study sample consisted of 28 women and three men, with an average age of 52.1 years.

The patients were divided into two groups according to the presence (group M) or absence (group WM) of a histological diagnosis of malignancy. Pre-operative TSH serum levels were compared between group M and group WM. The two groups were also compared in regards to gender, age, smoking history, previous exposure to radiation and free thyroxine (FT4) serum levels. These same comparisons were performed after the exclusion of the cases which presented hypo- or hyperthyroidism.

The histological diagnosis of malignancy was based on the criteria set by the World Health Organization (WHO). Reports of PC, FC, HC and FVPC were considered to be malignant, and reports of follicular adenoma, Hürthle adenoma, colloid goiter and Hashimoto's thyroiditis were considered to be benign (DeLellis et al., 2004).

The serum levels of TSH and FT4 were obtained from the medical records. The average preoperative hormone levels of each patient were determined by the average of three separate test results for these hormones, which were collected at different times up to one year prior to surgery. TSH and FT4 were measured by chemiluminescence, with a normal range of 0.8- 1.9 ng/dL and 0.4-4.0 mUI/mL, respectively (DPC, Los Angeles, CA). Thyroid function was considered normal when TSH and FT4 were within normal reference ranges; hypothyroid, when TSH was elevated; and hyperthyroid, when TSH was suppressed.

#### **2.1 Statistical analysis**

The collected data were charted in a Microsoft Excel® worksheet (Microsoft Corporation, EUA) and submitted to statistical analysis through the computer program SPSS/Windows (version 10.0.7). To study the association between the qualitative variables, we used the Chisquare test. For the quantitative variables, we used the Student's T test. The significance level was of 5% (Zar, 1999).

### **3. Results**

126 Thyroid and Parathyroid Diseases – New Insights into Some Old and Some New Issues

thyroid stimulating hormone (TSH) and thyroid cancer (Boelaert et al., 2006; Gul et al., 2010; Jonklaas et al., 2008). However, this relationship has not yet been established for the more doubtful cases, such as those with an inconclusive cytological diagnosis for FC or HC.

The objective of this study was to evaluate whether the TSH serum levels can help to differentiate benign cases from the malignant cases in patients with an FNAB that shows a

This retrospective study was approved by the Committee of Ethics in Research of Botucatu Medical School (FMB) of the Sao Paulo State University - UNESP (protocol number 3626-2010). We analyzed the cytological reports from patients carrying thyroid nodules that were submitted for thyroid FNAB at the Clinics Hospital FMB–Unesp between the years of 2003 and 2008. Of these, 59 cases with suspicious or inconclusive cytological diagnosis for FC or HC were selected. We included those nodules that presented a follicular or Hürthle pattern with the following reported descriptions: "follicular lesion," "follicular tumor," "follicular neoplasia," "Hürthle follicular lesion," "Hürthle tumor" or "Hürthle neoplasia." The medical data of these patients were evaluated, and we found 31 cases that were submitted to surgery and with histological diagnosis. The effective study sample consisted of 28 women

The patients were divided into two groups according to the presence (group M) or absence (group WM) of a histological diagnosis of malignancy. Pre-operative TSH serum levels were compared between group M and group WM. The two groups were also compared in regards to gender, age, smoking history, previous exposure to radiation and free thyroxine (FT4) serum levels. These same comparisons were performed after the exclusion of the cases

The histological diagnosis of malignancy was based on the criteria set by the World Health Organization (WHO). Reports of PC, FC, HC and FVPC were considered to be malignant, and reports of follicular adenoma, Hürthle adenoma, colloid goiter and Hashimoto's

The serum levels of TSH and FT4 were obtained from the medical records. The average preoperative hormone levels of each patient were determined by the average of three separate test results for these hormones, which were collected at different times up to one year prior to surgery. TSH and FT4 were measured by chemiluminescence, with a normal range of 0.8- 1.9 ng/dL and 0.4-4.0 mUI/mL, respectively (DPC, Los Angeles, CA). Thyroid function was considered normal when TSH and FT4 were within normal reference ranges; hypothyroid,

The collected data were charted in a Microsoft Excel® worksheet (Microsoft Corporation, EUA) and submitted to statistical analysis through the computer program SPSS/Windows (version 10.0.7). To study the association between the qualitative variables, we used the Chisquare test. For the quantitative variables, we used the Student's T test. The significance

follicular or Hürthle pattern.

**2. Material and methods**

and three men, with an average age of 52.1 years.

which presented hypo- or hyperthyroidism.

**2.1 Statistical analysis**

level was of 5% (Zar, 1999).

thyroiditis were considered to be benign (DeLellis et al., 2004).

when TSH was elevated; and hyperthyroid, when TSH was suppressed.

Of the 31 cases submitted to surgery, 14 showed malignancy upon histopathological analysis (group M). The malignancies included nine PCs, three FCs (one case with both PC and FC), one HC, one Hürthle tumor and one follicular tumor of uncertain malignant meaning. Thus, the concordance of suspicious or inconclusive FNAB for FC or HC with malignancy was 45.2% and the concordance with FC or HC was 12.9%. 17 patients (54.8%) had benign histological reports (group WM).

The M and WM groups did not differ significantly as to serum levels of TSH and FT4 (p>0.05). There were no significant differences in age or gender distribution (p>0.05). There were two smokers in the WM group and no smokers in the M group. In addition one patient in the WM group had previous exposure to radiation (Table 1).

Seven patients (22.6%) presented hypo- or hyperthyroidism and were under treatment with thyroid medication. These included two (28.6%) patients from the M group and five (71.4%) patients from the WM group. After excluding such cases, the M and WM groups still did not differ in regards to the analyzed parameters (Table 2).


\* Average ± standard deviation (Student's T test); \*\* Chi-square test; \*\*\*not submitted to statistical analysis due to small sample number. M: presence of malignancy; WM: absence of malignancy; TSH: thyroid stimulating hormone; FT4: free thyroxine.

Table 1. General data from 31 patients, with fine needle aspiration biopsies (FNAB) with suspicious or inconclusive cytological diagnosis for FC or HC, submitted to thyroidectomy, according to the final histological diagnosis of presence (group M) or absence (group WM) of malignancy.


\* Average ± standard deviation (Student's T test); \*\* Chi-square test; \*\*\*not submitted to statistical analysis due to small sample number. M: presence of malignancy; WM: absence of malignancy; TSH: thyroid stimulating hormone; FT4: free thyroxine.

Table 2. General data from 24 patients, without hypo- or hyperthyroidism, with fine needle aspiration biopsies (FNAB) with suspicious or inconclusive cytological diagnosis for FC or HC, submitted to thyroidectomy, according to the final histological diagnosis of presence (group M) or absence (group WM) of malignancy.

Suspicious Thyroid Fine Needle Aspiration Aspiration Biopsy: TSH as a Malignancy Marker? 129

In regards to the remaining clinical aspects examined in this study, there was also divergence between the present study and previous reports mentioned. We did not find any significant differences regarding age and gender that might predict nodular malignancy. In fact, the influence of such aspects is controversial and, similar to TSH evaluation, many of the previous studies did not restrict their evaluation only to cases with suspicion of FC or HC. Boelaert et al. showed an association of the male sex or the age extremes with a greater malignancy risk (Boelaert et al., 2006). Haymart et al. has also reported an association of greater risk of malignancy with the male sex and younger age groups, although they found no association with older age (Haymart et al., 2008). On the other hand, Gul et al. associated age of greater than 60 years to a greater risk of malignancy, but did not find an association with gender (Gul et al., 2010). Considering the lesions of follicular pattern, Raber et al., when evaluating cases with diagnostic FNAB for follicular neoplasia, also did not associate the age extremes or the male gender to a greater risk of malignancy (Raber et al., 2000). Tuttle et al. did not find an association between age and the occurrence of malignant tumors, although they did associate the male gender to a greater risk of malignancy (Tuttle et al., 1998). Schlinkert et al. also studied cases with diagnostic FNAB for follicular neoplasia and associated younger ages, but not older ages, with a greater risk of malignancy (Schlinkert et al., 1997). However, other authors have found an association between older ages and greater probability of malignant tumor (Cooper et al., 2006; Tuttle et al., 1998). Thus, there is a great divergence even among findings of studies that are restricted to the cytological diagnosis of follicular neoplasia. Moreover, the criteria for surgery submission were different in each

Another characteristic examined in this study was smoking history, which also could not be associated with a greater risk of malignancy. This finding is in agreement with other casecontrol studies (Kreiger & Parkes, 2000; Mack et al., 2003). In contrast to these studies, Sokic et al. found an association between smoking and a greater risk of thyroid cancer. However, the Sokic study was carried out in a population of hospitalized patients (Sokic et al., 1994),

We must highlight that the present study presents important limitations. One of the most relevant limitations is the small number of cases evaluated (31 patients). The retrospective nature of the study contributed to that small number by presenting problems such as irregular follow-up, non-submission to thyroidectomy and therefore absence of a histopathological report, and the absence of TSH measurements close in time to the surgical procedure. However, independent of the limitations of this and similar studies, it is a fact that the reports investigating nodular malignancy criteria in follicular tumors cases are not

Therefore, there is still much controversy surrounding the pre-operative diagnosis of thyroid nodules with FNAB compatible with a follicular or Hürthle pattern. Recently, the *National Cancer Institute Thyroid Fine-needle Aspiration State of the Science Conference* (Bethesda, Maryland, USA) attempted to minimize such divergences by reclassifying many of these lesions as benign, follicular lesions of uncertain meaning or follicular neoplasia, presenting a malignancy risk lower than 1%, between 5 and 10% and between 20 and 30%, respectively (Baloch et al., 2008). However, many services have not yet adhered to this new cytological classification and, even when this classification is used, there is still a significant percentage of thyroid nodules for which diagnostic doubt will only be clarified after surgical approach.

study, which complicates the comparison between studies.

in unanimous agreement.

for whom tobacco exposure was modified due to the hospital condition.

#### **4. Discussion**

Considering that in DTC the follicular cell physiologic characteristic of TSH responsiveness is preserved (Biondi et al., 2005; Carayon et al., 1980; Ichikawa et al., 1976), it is presumable that a greater stimulation provided by this hormone might be a contributing factor to the tumor genesis. In fact, some studies have reported an association between increased TSH serum levels and thyroid malignancy. Boelaert et al. have observed that the malignancy risk of a thyroid nodule rises along with the TSH serum levels, indicating that these levels are an independent prognostic factor for malignancy and may be used in conjunction with FNAB in detecting such tumors (Boelaert et al., 2006). Gul et al. have reported that low serum levels of FT4, associated with high levels of TSH (still within the normal patterns), are associated with a greater probability of thyroid cancer, independent of gender and goiter type (Gul et al., 2010). This study also suggests that hormone levels may be used in conjunction with FNAB in diagnosing thyroid cancer, as do gender, age and goiter type (Boelaert et al., 2006; Kumar et al., 1999; Tuttle et al., 1998).

Although FNAB is the method of choice for the evaluation of thyroid nodules, the technique presents limitations in the investigation of lesions of follicular or Hürthle patterns (Faquin & Baloch, 2010). Thus, the TSH serum levels might be used as a malignancy marker in such lesions. However, few studies have evaluated the relationship between thyroid malignancy and levels of this hormone specifically in follicular or Hürthle lesions (Tuttle et al., 1998). In the present study we have evaluated such relationship, and observed no association between TSH serum levels and malignancy in follicular or Hürthle lesions: the average serum levels of TSH and FT4 were not significantly different between the benign and the malignant cases.

One possible reason for such discordance in comparison with the majority of previous studies may be that, in this study, only the cases with suspicious or inconclusive aspiration for FC or HC were analyzed. Others have evaluated the presence of malignancy by studying DTC in general, including PC (Gul et al., 2010; Haymart et al., 2008; Jonklaas et al., 2008), or several types of carcinomas, including those independent from TSH (Boelaert et al., 2006; Polyzos et al., 2008). When evaluating only the FNABs with a diagnosis of follicular neoplasia, Tuttle et al. also did not find any differences in the results of thyroid function tests between benign and malignant cases (Tuttle et al., 1998).

Another reason for the distinct findings in this study may be that we have evaluated only the cases with histological confirmation of the diagnosis. Others have included nonthyroidectomized patients, who had "diagnostic confirmation" only through the evolutionary evaluation during a two year follow-up period (Boelaert et al., 2006; Polyzos et al., 2008; Fiore et al., 2009), a time that might be considered insufficient in the DTC cases.

Another divergence between this study and others (Gul et al., 2010; Jonklaas et al., 2008) is that this study excluded the cases with cytological diagnosis of malignancy and those without recent TSH level measurements. At first, the cases with radiation exposure were not excluded. However, only one patient had been submitted to previous external radiotherapy, presenting a final histological diagnosis of benignity. Although those with thyroid dysfunction were also not excluded at first, when these patients were withdrawn from the analysis, there was still no statistically significant difference between the hormone levels in benign and malignant cases.

Considering that in DTC the follicular cell physiologic characteristic of TSH responsiveness is preserved (Biondi et al., 2005; Carayon et al., 1980; Ichikawa et al., 1976), it is presumable that a greater stimulation provided by this hormone might be a contributing factor to the tumor genesis. In fact, some studies have reported an association between increased TSH serum levels and thyroid malignancy. Boelaert et al. have observed that the malignancy risk of a thyroid nodule rises along with the TSH serum levels, indicating that these levels are an independent prognostic factor for malignancy and may be used in conjunction with FNAB in detecting such tumors (Boelaert et al., 2006). Gul et al. have reported that low serum levels of FT4, associated with high levels of TSH (still within the normal patterns), are associated with a greater probability of thyroid cancer, independent of gender and goiter type (Gul et al., 2010). This study also suggests that hormone levels may be used in conjunction with FNAB in diagnosing thyroid cancer, as do gender, age and goiter type

Although FNAB is the method of choice for the evaluation of thyroid nodules, the technique presents limitations in the investigation of lesions of follicular or Hürthle patterns (Faquin & Baloch, 2010). Thus, the TSH serum levels might be used as a malignancy marker in such lesions. However, few studies have evaluated the relationship between thyroid malignancy and levels of this hormone specifically in follicular or Hürthle lesions (Tuttle et al., 1998). In the present study we have evaluated such relationship, and observed no association between TSH serum levels and malignancy in follicular or Hürthle lesions: the average serum levels of TSH and FT4 were not significantly different between the benign and the

One possible reason for such discordance in comparison with the majority of previous studies may be that, in this study, only the cases with suspicious or inconclusive aspiration for FC or HC were analyzed. Others have evaluated the presence of malignancy by studying DTC in general, including PC (Gul et al., 2010; Haymart et al., 2008; Jonklaas et al., 2008), or several types of carcinomas, including those independent from TSH (Boelaert et al., 2006; Polyzos et al., 2008). When evaluating only the FNABs with a diagnosis of follicular neoplasia, Tuttle et al. also did not find any differences in the results of thyroid function

Another reason for the distinct findings in this study may be that we have evaluated only the cases with histological confirmation of the diagnosis. Others have included nonthyroidectomized patients, who had "diagnostic confirmation" only through the evolutionary evaluation during a two year follow-up period (Boelaert et al., 2006; Polyzos et al., 2008; Fiore et al., 2009), a time that might be considered insufficient in the DTC cases.

Another divergence between this study and others (Gul et al., 2010; Jonklaas et al., 2008) is that this study excluded the cases with cytological diagnosis of malignancy and those without recent TSH level measurements. At first, the cases with radiation exposure were not excluded. However, only one patient had been submitted to previous external radiotherapy, presenting a final histological diagnosis of benignity. Although those with thyroid dysfunction were also not excluded at first, when these patients were withdrawn from the analysis, there was still no statistically significant difference between the hormone levels in

(Boelaert et al., 2006; Kumar et al., 1999; Tuttle et al., 1998).

tests between benign and malignant cases (Tuttle et al., 1998).

**4. Discussion**

malignant cases.

benign and malignant cases.

In regards to the remaining clinical aspects examined in this study, there was also divergence between the present study and previous reports mentioned. We did not find any significant differences regarding age and gender that might predict nodular malignancy. In fact, the influence of such aspects is controversial and, similar to TSH evaluation, many of the previous studies did not restrict their evaluation only to cases with suspicion of FC or HC. Boelaert et al. showed an association of the male sex or the age extremes with a greater malignancy risk (Boelaert et al., 2006). Haymart et al. has also reported an association of greater risk of malignancy with the male sex and younger age groups, although they found no association with older age (Haymart et al., 2008). On the other hand, Gul et al. associated age of greater than 60 years to a greater risk of malignancy, but did not find an association with gender (Gul et al., 2010). Considering the lesions of follicular pattern, Raber et al., when evaluating cases with diagnostic FNAB for follicular neoplasia, also did not associate the age extremes or the male gender to a greater risk of malignancy (Raber et al., 2000). Tuttle et al. did not find an association between age and the occurrence of malignant tumors, although they did associate the male gender to a greater risk of malignancy (Tuttle et al., 1998). Schlinkert et al. also studied cases with diagnostic FNAB for follicular neoplasia and associated younger ages, but not older ages, with a greater risk of malignancy (Schlinkert et al., 1997). However, other authors have found an association between older ages and greater probability of malignant tumor (Cooper et al., 2006; Tuttle et al., 1998). Thus, there is a great divergence even among findings of studies that are restricted to the cytological diagnosis of follicular neoplasia. Moreover, the criteria for surgery submission were different in each study, which complicates the comparison between studies.

Another characteristic examined in this study was smoking history, which also could not be associated with a greater risk of malignancy. This finding is in agreement with other casecontrol studies (Kreiger & Parkes, 2000; Mack et al., 2003). In contrast to these studies, Sokic et al. found an association between smoking and a greater risk of thyroid cancer. However, the Sokic study was carried out in a population of hospitalized patients (Sokic et al., 1994), for whom tobacco exposure was modified due to the hospital condition.

We must highlight that the present study presents important limitations. One of the most relevant limitations is the small number of cases evaluated (31 patients). The retrospective nature of the study contributed to that small number by presenting problems such as irregular follow-up, non-submission to thyroidectomy and therefore absence of a histopathological report, and the absence of TSH measurements close in time to the surgical procedure. However, independent of the limitations of this and similar studies, it is a fact that the reports investigating nodular malignancy criteria in follicular tumors cases are not in unanimous agreement.

Therefore, there is still much controversy surrounding the pre-operative diagnosis of thyroid nodules with FNAB compatible with a follicular or Hürthle pattern. Recently, the *National Cancer Institute Thyroid Fine-needle Aspiration State of the Science Conference* (Bethesda, Maryland, USA) attempted to minimize such divergences by reclassifying many of these lesions as benign, follicular lesions of uncertain meaning or follicular neoplasia, presenting a malignancy risk lower than 1%, between 5 and 10% and between 20 and 30%, respectively (Baloch et al., 2008). However, many services have not yet adhered to this new cytological classification and, even when this classification is used, there is still a significant percentage of thyroid nodules for which diagnostic doubt will only be clarified after surgical approach.

Suspicious Thyroid Fine Needle Aspiration Aspiration Biopsy: TSH as a Malignancy Marker? 131

Gul K., Ozdemir D., Dirikoc A., Oguz A., Tuzun D., Baser H., Ersoy R., & Cakir B. (2010).

Ichikawa Y., Saito E., Abe Y., Homma M., & Muraki T. (1976). Presence of TSH receptor in

Jonklaas J, Nsouli-Maktabi H, & Soldin SJ. (2008). Endogenous thyrotropin and

Kimura ET, Tincani AJ, Ward LS, Nogueira CR, Carvalho GA, Maia AL, Tavares MR,

Kreiger N. & Parkes R. (2000). Cigarrete smoking and the risk of thyroid cancer. *European* 

Kumar H., Daykin J., Holder R., Watkinson J. C., Sheppard M. C., & Franklin J. A. (1999)

Investigated by Fine-Needle Aspiration Cytology. *Thyroid,* 9(11):1105-1109. Mack, W.J., Martin, S.P., Dal Maso, L., Galanti, R., Xiang M., Franceschi S., Hallquist A., Jin

Mazzaferri E.L. (1993). Management of a solitary thyroid nodule. *N Engl J Med,* 328:553–559. Melck AL, Yip L. (2011). Predicting malignancy in thyroid nodules: Molecular advances.

Polyzos S.A., Kita M., Efstathiadou Z., Poulakos P., Slavakis A., Sofianou D., Flaris N.,

Raber W., Kaserer K., Niederle B., & Vierhapper H. (2000). Risk factors for malignancy of

Schlinkert, R.T., van Heerden, J.A., Goellner, J.R., Gharib, H., Smith, S.L., Rosales, R.F., &

Schlumberger M. & Pacini F. (1997). *Tumeurs de la thyroïde*, Nucléon. Sherman S.I. (2003).

*Head Neck.* Aug 4. doi: 10.1002/hed.21818. [Epub ahead of print].

Gender, Clinical Findings, and Serum Thyrotropin Measurements in the Prediction of Thyroid Neoplasia in 1005 Patients Presenting with Thyroid Enlargement and

F., Kolonel L., La Vecchia C., Levi F., Linos A., Lund E., McTiernan A., Mabuchi K., Negri E., Wingren G., & Ron E. (2003). A pooled analysis of case–control studies of thyroid cancer: cigarette smoking and consumption of alcohol, coffee, and tea.

Leontsini M., Kourtis A., & Avramidis A. (2008). *J. Cancer Res. Clin. Oncol.,* 134,

thyroid nodules initially identified as follicular neoplasia by fine-needle aspiration: results of a prospective study of one hundred twenty patients. *Thyroid*,

Weaver, A.L. (1997). Factors that predict malignant thyroid lesions when Fine-Needle Aspiration is "Suspicious for Follicular Neoplasm". *Mayo Clin Proc,* 72:913-916. Schlumberger M.J. (1998). Papillary and follicular thyroid carcinoma. *N Engl J Med,* 338:297–

(2008). *J. Clin. Endocrinol. Metab.,* 93, 809–814.

Saúde Suplementar, retrieved from

*Cancer Causes and Control,* 14: 773–785.

*Thyroid carcinoma,* Lancet, 361:501–511.

*Journal of Cancer,* 36, 1969-1973.

18(9):943-52.

953–960.

306.

Aug;10(8):709-712.

thyroid neoplasms. *J Clin Endocrinol Metab,* 42:395–398.

http://www.projetodiretrizes.org.br/ans/diretrizes/29.pdf.

Are endogenously lower serum thyroid hormones new predictors for thyroid malignancy in addition to higher serum thyrotropin? *Endocrine*. 37(2):253-260. Haymart M.R., Repplinger D.J., Leverson G.E, Elson D.F, Sippel R.S., Jaume J.C., & Chen H.

triiodothyronine concentrations in individuals with thyroid cancer. *Thyroid*,

Teixeira G, Kulcsar MAV, Biscolla RPM, Cavalcanti CEO, Correa LAC, del Negro A, Friguglieti CUM, Hojaij F, Abrahão M, & Andrada NC. (2009). Doença nodular da tireóide: Diagnóstico, In: *Diretrizes Clínicas na Saúde Suplementar,* Sociedade Brasileira de Endocrinologia e Metabolismo, Sociedade Brasileira de Cirurgia de Cabeça e Pescoço, pp. 1-14, Associação Médica Brasileira e Agência Nacional de

#### **5. Conclusion**

In conclusion, in this study the serum levels of TSH and FT4, in addition to gender, age and smoking habits, were not useful in differentiating FC or HC from benign lesions of similar cytological patterns. Considering the controversy surrounding this area and the absence of significant evidence, there is a need for more studies examining the correlation between the cytological diagnoses in cases of follicular tumors with the pre-operative TSH serum levels. Future studies should have an adequate study design and a greater study population, in order to improve the diagnosis of these lesions.

#### **6. References**


In conclusion, in this study the serum levels of TSH and FT4, in addition to gender, age and smoking habits, were not useful in differentiating FC or HC from benign lesions of similar cytological patterns. Considering the controversy surrounding this area and the absence of significant evidence, there is a need for more studies examining the correlation between the cytological diagnoses in cases of follicular tumors with the pre-operative TSH serum levels. Future studies should have an adequate study design and a greater study population, in

Baloch Z.W., LiVolsi V.A., Asa S.L., Rosai J., Merino M.J., Randolph G., Vielh P., DeMay

Bennedbaek F.N., Perrild H., & Hegedus L. (1999). Diagnosis and treatment of the solitary thyroid nodule. Results of a European survey. *Clin Endocrinol* (Oxf), 50:357–363 Bennedbaek F.N. & Hegedus L. (2000). Management of the solitary thyroid nodule: results

Biondi B., Filetti S. & Schlumberger M. (2005). Thyroidhormone herapy and thyroid cancer: a reassessment. Nature Clinical Practice. *Endocrinology & Metabolism,* 1(1): 32–40. Boelaert K., Horacek J., Holder R. L., Watkinson J. C., Sheppard M. C., & Franklyn J. A.

Carayon P., Thomas-Morvan C., Castanas E., & Tubiana M. (1980). Human thyroid cancer:

Cooper D.S., Doherty G.M., Haugen B.R., Kloos R.T., Lee S.L., Mandel S.J., Mazzaferri E.L.,

Faquin W.C., Baloch Z.W. (2010). Fine-Needle Aspiration of Follicular Patterned Lesions of

Frates M.C., Benson C.B., Doubilet P.M., Kunreuther E., Contreras M., & Cibas E.S. (2006).

Gharib H. & Goellner J.R. (1993). Fine-needle aspiration biopsy of the thyroid: an appraisal.

thyroid nodules on sonography. *J Clin Endocrinol Metab,* 91:3411-7.

with thyroid nodules and differentiated thyroid cancer. *Thyroid,* 16:1–33 Davies L. & Welch H.G. (2006). Increasing incidence of thyroid cancer in the United States, 1973–2002. *Journal of the American Medical Association,* 295 2164–2167. DeLellis R.A., Lloyd R.D., Heitz P.U., Eng C. (editors). (2004) WHO: Pathology and Genetics,

In: *Tumours of Endocrine Organs,* IARC Press, Lyon, France.

(2006). Serum Thyrotropin Concentration as a Novel Predictor of Malignancy in Thyroid Nodules Investigated by Fine-Needle Aspiration. *J Clin Endocrinol Metab*,

membrane thyrotropin binding and adenylate cyclase activity. *J Clin Endocrinol* 

McIver B., Sherman S.I., & Tuttle R.M. (2006). Management guidelines for patients

the Thyroid: Diagnosis, Management, and Follow-Up According to National Cancer Institute (NCI) Recommendations. Diagn Cytopathol, 38(10):731-739. Fiore E., Rago T., Provenzale M.A., Scutari M., Ugolini C., Basolo F., Di Coscio G., Berti P.,

Grasso L., Elisei R., Pinchera A., Vitti P. (2009). *Endocr. Relat. Cancer*, 16, 1251–1260.

Prevalence and distribution of carcinoma in patients with solitary and multiple

of a North American survey. *J Clin Endocrinol Metab* 85:2493–2498.

R.M., Sidawy M.K., & Frable W.J. (2008). Diagnostic Terminology and Morphologic Criteria for Cytologic Diagnosis of Thyroid Lesions: A Synopsis of the National Cancer Institute Thyroid Fine-Needle Aspiration State of the Science Conference.

**5. Conclusion**

**6. References**

order to improve the diagnosis of these lesions.

Diagn Cytopathol., 36:425–437.

91(11):4295-4301.

*Metab,* 51:915–920.

*Ann Intern Med,* 118:282–289


http://www.projetodiretrizes.org.br/ans/diretrizes/29.pdf.


**Part 2** 

**Treatment of Thyroid and Parathyroid Diseases** 

Sherman SI. (2003). Thyroid carcinoma. *Lancet* 361:501–511.

