Thyroid Cancer and Acromegaly

*Carla Souza Pereira Sobral, Marcelo Magalhães and Manuel dos Santos Faria*

#### **Abstract**

Acromegaly results from oversecretion of growth hormone and subsequent insulin growth factor-I. Some studies have described an association between acromegaly and increased risk of some cancers, including thyroid cancer, the most common endocrine malignancy. It is well known that follicular thyroid cells express IGF-I receptor and that GH and IGF-I have both proliferative and anti-apoptotic effects and their hypersecretion may theoretically induce tumor development and stimulate its growth, despite the fact that research data is conflicting and population-based data on thyroid cancer and acromegaly is rare. Some molecular alterations, including point mutations in *BRAF* and *RAS* genes and *RET/PTC* gene rearrangements, have been associated with oncogenesis of PTC. However, the implications of these genetic markers in the development of PTC in patients with acromegaly are not yet well known. In this chapter, we discuss epidemiology, pathogenesis, molecular biology aspects, and how to screen and to manage acromegalic patients with nodular thyroid disease and thyroid cancer.

**Keywords:** acromegaly and thyroid cancer, IGF-I and cancer, thyroid and acromegaly, GH and cancer, molecular markers and thyroid cancer

#### **1. Introduction**

Acromegaly is a rare disease that results from the oversecretion of growth hormone (GH) and subsequent insulin growth factor I (IGF-I) [1]. It is associated with important complications that may reduce life expectancy of these patients [2, 3].

Most acromegalic patients die from cardiovascular, cerebrovascular, or respiratory diseases [3, 4]. Nevertheless, in the past two decades, some studies have also described an association between acromegaly and an increased risk of some cancers such as colorectal and thyroid cancer (TC), which is the most common endocrine malignancy, among others [5].

Part of the difficulty in determining the true incidence of cancer in this population is due to the relative rarity of acromegaly [6]. On the other hand, with improvement in surgical and radiotherapeutic procedures as well as advances in medical treatment, an increase of the survival rate of patients with acromegaly has been shown. As a result, patients may have a longer exposure to high GH levels [7].

As the prevalence of thyroid cancer has been shown to increase among patients with acromegaly, this should draw attention for clinicians to investigate thyroid disease, particularly thyroid cancer.

#### **2. Epidemiology**

The association between acromegaly and TC is supported by preclinical data showing that GH-IGF system plays an important role in cancer development and progression [6]. However, clinical studies that addressed the association between acromegaly and cancer produced controversial results, partly due to the different methodological approaches used (case-control and population-based designs) [8].

A comprehensive meta-analysis showed an increased risk of both nodular thyroid disease (NTD) (OR = 6.9, RR = 2.1) and TC (OR = 7.5, RR = 7.2) in acromegaly. It showed a prevalence slightly below 60% of NTD and of around 4% of TC [8]. Within this context, a consistent Brazilian multicentric study with 124 acromegalic patients in a case-control design showed a higher prevalence of 7.2% for TC and 0.7% in the control group [9].

These findings may result from the fact of improving diagnostic and treatment of acromegaly extending the life duration which increases the prevalence of both benign and malignant neoplasms [3–11].

On the other hand, the co-occurrence of autoimmune thyroid diseases and acromegaly is not common. So far only a handful of cases of Graves-Basedow disease in acromegalic patients have been reported, while Hashimoto's disease occurs more frequently (4.6%) [12, 13].

#### **3. Molecular pathogenesis of TC in acromegalic patients**

#### **3.1 Molecular basis of acromegaly**

The pituitary gland integrates hormonal signs that control several homeostatic processes such as metabolism, growth, and reproduction. Cell clusters localized in the anterior pituitary, somatotrophs, secrete GH responsible for cellular proliferation through membrane-bound growth hormone receptor (GHR) present in various organs and systems [14]. The interaction between GH and GHR results in activation of intracellular protein Janus kinase 2 (JAK2). As shown in **Figure 1**, once phosphorylated JAK2 activates the signal transducers and activators of transcription (STAT) protein that is translocated to the nucleus and initiates transcription of genes in response to GH [15], the STAT is able to bind to IGF-I promoter regulating the transcription of this gene [16]. Thus, the presence of GH can induce the synthesis of IGF-I that occurs mainly in the liver and is composed of 70 amino acids and has mitotic and anti-apoptotic effects [1].

In the vast majority of cases, the excess of GH in acromegaly is originated from proliferating somatotrophs (somatotropinoma). The pituitary adenomas are of monoclonal origin, indicating that the tumor rises from a single cell that acquires proliferative advantage [17]. The primary defect that leads to development of somatotropinoma may result from genetic and epigenetic alterations inducing the activation of oncogenes or inactivation of tumor suppressor genes [1]. Mutations in the alpha subunit of transmembrane G protein is observed in 40% of GH-secreting tumors [1]. This abnormality may cause constitutive activation of cyclin AMP (cAMP) and consequent hypersecretion of GH. Loss of expression of proapoptotic molecules such as GADD45γ (growth arrest and DNA damage-inducible 45γ protein) and overexpression of oncoproteins, including PTTG (pituitary tumortransforming gene), are phenomena also observed in pituitary adenomas [17, 18].

Most cases of acromegaly occur sporadically; however, approximately 5% of cases may be related to inherited diseases such as multiple endocrine neoplasia type 1 (MEN1), Carney complex (CNC), and familial isolated pituitary adenoma

**61**

respectively [17].

**Figure 1.**

*this gene.*

**in acromegaly**

prognostic implications in thyroid cancer.

*Thyroid Cancer and Acromegaly*

*DOI: http://dx.doi.org/10.5772/intechopen.84541*

(FIPA) [17]. Germline mutations in aryl hydrocarbon receptor-interacting protein (*AIP*) gene seem to be the most frequent genetic alteration detected in sporadic and familial acromegaly patients [19]. The MEN1 and CNC are caused mainly by defects in genes *MEN1* (menin) and *PRKAR1A* (regulatory subunit type 1 alpha),

*Activation of JAK/STAT pathway mediated by GH (growth hormone). (a) JAK/STAT pathway components are inactive. (b) GH leads to dimerization of its receptor promoting phosphorylation of JAK and consequent activation of STAT proteins. (c) Once activated, STAT forms dimers that are translocated to the cell nucleus. (d) The STAT dimers in the nucleus are capable of binding to IGF-I promoter, initiating the transcription of* 

The serum GH excess may promote proliferation and suppress apoptosis in many tissues [15]. Thus, it is suggested that acromegaly is responsible for the increased risk for development of many malignancies. PTC is the most common thyroid cancer observed in acromegaly [7, 9]. This type of pituitary tumor can also be associated

The mechanism of thyroid carcinogenesis in acromegaly is attributed to an autocrine/paracrine loop for GH/IGF-I in tumor tissue [8]. As the thyroid follicular cells also produce IGF-I and express genes encoding IGF-IR, the long-term exposure of thyrocyte to high GH/IGF-I levels may work synergically with this loop in promot-

**3.3 Molecular mechanisms and potential biomarkers of thyroid carcinogenesis** 

As shown in **Figure 2**, the molecular oncogenesis of PTC is mainly related to deregulation of mitogen-activated protein kinase (MAPK) signaling pathway and involves point mutations in *BRAF* and *RAS* genes and *RET/PTC* gene rearrangements [21, 22]. Analysis of these molecular markers can have diagnostic and

**3.2 Cross talk between acromegaly and thyroid cancer**

ing goiter development and malignant transformation [20].

with benign thyroid conditions such as diffuse and nodular goiters [9].

#### **Figure 1.**

*Knowledges on Thyroid Cancer*

0.7% in the control group [9].

frequently (4.6%) [12, 13].

**3.1 Molecular basis of acromegaly**

has mitotic and anti-apoptotic effects [1].

benign and malignant neoplasms [3–11].

The association between acromegaly and TC is supported by preclinical data showing that GH-IGF system plays an important role in cancer development and progression [6]. However, clinical studies that addressed the association between acromegaly and cancer produced controversial results, partly due to the different methodological approaches used (case-control and population-based designs) [8]. A comprehensive meta-analysis showed an increased risk of both nodular thyroid disease (NTD) (OR = 6.9, RR = 2.1) and TC (OR = 7.5, RR = 7.2) in acromegaly. It showed a prevalence slightly below 60% of NTD and of around 4% of TC [8]. Within this context, a consistent Brazilian multicentric study with 124 acromegalic patients in a case-control design showed a higher prevalence of 7.2% for TC and

These findings may result from the fact of improving diagnostic and treatment of acromegaly extending the life duration which increases the prevalence of both

On the other hand, the co-occurrence of autoimmune thyroid diseases and acromegaly is not common. So far only a handful of cases of Graves-Basedow disease in acromegalic patients have been reported, while Hashimoto's disease occurs more

The pituitary gland integrates hormonal signs that control several homeostatic processes such as metabolism, growth, and reproduction. Cell clusters localized in the anterior pituitary, somatotrophs, secrete GH responsible for cellular proliferation through membrane-bound growth hormone receptor (GHR) present in various organs and systems [14]. The interaction between GH and GHR results in activation of intracellular protein Janus kinase 2 (JAK2). As shown in **Figure 1**, once phosphorylated JAK2 activates the signal transducers and activators of transcription (STAT) protein that is translocated to the nucleus and initiates transcription of genes in response to GH [15], the STAT is able to bind to IGF-I promoter regulating the transcription of this gene [16]. Thus, the presence of GH can induce the synthesis of IGF-I that occurs mainly in the liver and is composed of 70 amino acids and

In the vast majority of cases, the excess of GH in acromegaly is originated from proliferating somatotrophs (somatotropinoma). The pituitary adenomas are of monoclonal origin, indicating that the tumor rises from a single cell that acquires proliferative advantage [17]. The primary defect that leads to development of somatotropinoma may result from genetic and epigenetic alterations inducing the activation of oncogenes or inactivation of tumor suppressor genes [1]. Mutations in the alpha subunit of transmembrane G protein is observed in 40% of GH-secreting tumors [1]. This abnormality may cause constitutive activation of cyclin AMP (cAMP) and consequent hypersecretion of GH. Loss of expression of proapoptotic molecules such as GADD45γ (growth arrest and DNA damage-inducible 45γ protein) and overexpression of oncoproteins, including PTTG (pituitary tumortransforming gene), are phenomena also observed in pituitary adenomas [17, 18]. Most cases of acromegaly occur sporadically; however, approximately 5% of cases may be related to inherited diseases such as multiple endocrine neoplasia type 1 (MEN1), Carney complex (CNC), and familial isolated pituitary adenoma

**3. Molecular pathogenesis of TC in acromegalic patients**

**2. Epidemiology**

**60**

*Activation of JAK/STAT pathway mediated by GH (growth hormone). (a) JAK/STAT pathway components are inactive. (b) GH leads to dimerization of its receptor promoting phosphorylation of JAK and consequent activation of STAT proteins. (c) Once activated, STAT forms dimers that are translocated to the cell nucleus. (d) The STAT dimers in the nucleus are capable of binding to IGF-I promoter, initiating the transcription of this gene.*

(FIPA) [17]. Germline mutations in aryl hydrocarbon receptor-interacting protein (*AIP*) gene seem to be the most frequent genetic alteration detected in sporadic and familial acromegaly patients [19]. The MEN1 and CNC are caused mainly by defects in genes *MEN1* (menin) and *PRKAR1A* (regulatory subunit type 1 alpha), respectively [17].

#### **3.2 Cross talk between acromegaly and thyroid cancer**

The serum GH excess may promote proliferation and suppress apoptosis in many tissues [15]. Thus, it is suggested that acromegaly is responsible for the increased risk for development of many malignancies. PTC is the most common thyroid cancer observed in acromegaly [7, 9]. This type of pituitary tumor can also be associated with benign thyroid conditions such as diffuse and nodular goiters [9].

The mechanism of thyroid carcinogenesis in acromegaly is attributed to an autocrine/paracrine loop for GH/IGF-I in tumor tissue [8]. As the thyroid follicular cells also produce IGF-I and express genes encoding IGF-IR, the long-term exposure of thyrocyte to high GH/IGF-I levels may work synergically with this loop in promoting goiter development and malignant transformation [20].

#### **3.3 Molecular mechanisms and potential biomarkers of thyroid carcinogenesis in acromegaly**

As shown in **Figure 2**, the molecular oncogenesis of PTC is mainly related to deregulation of mitogen-activated protein kinase (MAPK) signaling pathway and involves point mutations in *BRAF* and *RAS* genes and *RET/PTC* gene rearrangements [21, 22]. Analysis of these molecular markers can have diagnostic and prognostic implications in thyroid cancer.

#### **Figure 2.**

*MAPK and PI3K pathways. (a) Growth factors bind to receptor tyrosine kinase and trigger the activation of (b) MAPK and/or (c) PI3K-AKT. (d) The signaling mediated to both pathways promotes the transcription of gene associated to different cellular processes such as proliferation and survival.*

#### *3.3.1 BRAF mutation*

BRAF (B-type RAF kinase) is a serine threonine kinase considered the most potent MAPK activator. This protein regulates important cellular processes such as proliferation, differentiation, and apoptosis [1].

In PTC, the main mechanism for activation of *BRAF* gene is a point mutation that promotes a substitution of nucleotide thymine by adenine at position 1799. This single nucleotide change promotes the replacement of valine by glutamate at protein residue 600 (V600E). The *BRAF* V600E mutation is the most frequent genetic abnormality reported in thyroid carcinomas in the general population, particularly in PTC [21].

In acromegalic patients, the importance of *BRAF* V600E mutation on PTC carcinogenesis is still not well defined. In an Italian cohort of acromegalic patients, the *BRAF* V600E mutation was detected in 70% of cases with PTC, suggesting that this mutation is the main genetic driver of neoplastic transformation of thyroid cells in acromegaly [23]. On the other hand, other studies have demonstrated that the *BRAF* V600E mutation is infrequent in patients PTC with and without acromegaly [20, 24]. In these reports lower prevalence of this genetic alteration in acromegalic patients with PTC than non-acromegalic cases with PTC was verified. These results suggest that *BRAF* V600E mutation may not be a main mechanism of malignant transformation of thyroid cells in patients with acromegaly.

#### *3.3.2 RAS mutations*

The *HRAS*, *KRAS*, and *NRAS* are homologous gene members of the *RAS* (retrovirus-associated DNA sequences) family. These genes encode GTP-binding

**63**

*Thyroid Cancer and Acromegaly*

to be clarified.

*3.3.3 RET/PTC rearrangements*

*3.3.4 Other molecular alterations*

of this protein on thyroid carcinogenesis.

*DOI: http://dx.doi.org/10.5772/intechopen.84541*

cases and 40–50% of follicular carcinomas [22].

proteins localized at the inner superficial of the cell membrane involved in signaling MAPK and PI3K-AKT pathways [1]. Together, *RAS* mutations are the second most frequent molecular alteration found in thyroid cancer, occurring in 10–20% of PTC

Point mutations are commonly restricted at codon 61 of the *HRAS* and *NRAS* genes and at codons 12 and 13 in the *KRAS* gene. *RAS* mutations in thyroid cancer have been associated to favorable prognosis such as tumor encapsulation and absence of metastases but also may represent a poor prognostic factor predisposing to cellular dedifferentiation and anaplastic transformation [22]. *NRAS* codon 61 mutation has been referred as the most frequent genetic alteration in PTC patients with acromegaly. Aydin et al. pointed out that patients with *NRAS* codon 61 mutation have aggressive histologic features such as vascular and capsular invasion [24]. However, another study revealed no case in a cohort of acromegalic patients with PTC-harbored *RAS* mutations [23]. These contradictory findings indicate that the importance of *RAS* mutational status in thyroid oncogenesis in acromegaly remains

The *RET* is a proto-oncogene that encodes a receptor-type tyrosine kinase with three domains: extracellular, transmembrane, and intracellular tyrosine kinase. The activation of this gene can contribute to the development of several neoplasms [25]. Rearrangements of *RET* that originated from fusion with unrelated genes (RET/PTC rearrangements) have been reported in thyroid follicular cells [26]. This genomic alteration can produce a chimeric oncoprotein with inappropriate tyrosine kinase activity able to continually stimulate the MAPK and PI3K-AKT pathways [26]. Among the fusion variants of *RET*, the rearrangements RET/PTC1 and RET/ PTC3 are the most frequent in thyroid cancer. Whereas in RET/PTC1 the *RET* gene is fused to *CCDC6* (known as *H4*), in RET/PTC3 the rearrangement occurs with *NCOA4* (known as *ELE1* or *RFG*) [25]. RET/PTC rearrangement appears to be an important mechanism of thyroid carcinogenesis, but its frequency has oscillated in different reports. This genetic abnormality was not detected in PTC patients with acromegaly [24], although studies with this approach are rare in acromegaly.

Besides the potential classic marker, other molecules have been evaluated in relation to their implication on PTC development in acromegaly, among them are

The analysis of immunohistochemical staining for IGF-IRβ revealed a high expression of this receptor in PTC samples [20]. Although differences in IGF-IRβ tumoral staining between PTC patients with and without acromegaly have not been observed, this marker had significantly less expression in adjacent normal tissue of patients with acromegaly. These data suggest that high GH levels may trigger autocrine and paracrine effects of IGF-I in thyroid follicular cells resulting in overexpression of IGF-IRβ in tumor tissue of acromegalic patients. In line with these results, it was observed that PTC patients with acromegaly have higher expression of IGF-I than PTC cases without acromegaly [27]. Additionally, an intense expression was verified of Gal-3 in PTC with acromegaly, speculating a possible influence

As previously mentioned, inactivation of *AIP* gene is frequently reported in pituitary tumors. However, this genetic abnormality seems not to be determinant to thyroid carcinogenesis in acromegalic patients [23]. Furthermore, there are no

IGF-I, IGF-IRβ, AIP, AHR, and galectin-3 (Gal-3) [20, 23–24, 27].

#### *Thyroid Cancer and Acromegaly DOI: http://dx.doi.org/10.5772/intechopen.84541*

*Knowledges on Thyroid Cancer*

*3.3.1 BRAF mutation*

**Figure 2.**

proliferation, differentiation, and apoptosis [1].

*gene associated to different cellular processes such as proliferation and survival.*

transformation of thyroid cells in patients with acromegaly.

The *HRAS*, *KRAS*, and *NRAS* are homologous gene members of the *RAS* (retrovirus-associated DNA sequences) family. These genes encode GTP-binding

BRAF (B-type RAF kinase) is a serine threonine kinase considered the most potent MAPK activator. This protein regulates important cellular processes such as

*MAPK and PI3K pathways. (a) Growth factors bind to receptor tyrosine kinase and trigger the activation of (b) MAPK and/or (c) PI3K-AKT. (d) The signaling mediated to both pathways promotes the transcription of* 

In PTC, the main mechanism for activation of *BRAF* gene is a point mutation that promotes a substitution of nucleotide thymine by adenine at position 1799. This single nucleotide change promotes the replacement of valine by glutamate at protein residue 600 (V600E). The *BRAF* V600E mutation is the most frequent genetic abnormality reported in thyroid carcinomas in the general population, particularly in PTC [21]. In acromegalic patients, the importance of *BRAF* V600E mutation on PTC carcinogenesis is still not well defined. In an Italian cohort of acromegalic patients, the *BRAF* V600E mutation was detected in 70% of cases with PTC, suggesting that this mutation is the main genetic driver of neoplastic transformation of thyroid cells in acromegaly [23]. On the other hand, other studies have demonstrated that the *BRAF* V600E mutation is infrequent in patients PTC with and without acromegaly [20, 24]. In these reports lower prevalence of this genetic alteration in acromegalic patients with PTC than non-acromegalic cases with PTC was verified. These results suggest that *BRAF* V600E mutation may not be a main mechanism of malignant

**62**

*3.3.2 RAS mutations*

proteins localized at the inner superficial of the cell membrane involved in signaling MAPK and PI3K-AKT pathways [1]. Together, *RAS* mutations are the second most frequent molecular alteration found in thyroid cancer, occurring in 10–20% of PTC cases and 40–50% of follicular carcinomas [22].

Point mutations are commonly restricted at codon 61 of the *HRAS* and *NRAS* genes and at codons 12 and 13 in the *KRAS* gene. *RAS* mutations in thyroid cancer have been associated to favorable prognosis such as tumor encapsulation and absence of metastases but also may represent a poor prognostic factor predisposing to cellular dedifferentiation and anaplastic transformation [22]. *NRAS* codon 61 mutation has been referred as the most frequent genetic alteration in PTC patients with acromegaly. Aydin et al. pointed out that patients with *NRAS* codon 61 mutation have aggressive histologic features such as vascular and capsular invasion [24]. However, another study revealed no case in a cohort of acromegalic patients with PTC-harbored *RAS* mutations [23]. These contradictory findings indicate that the importance of *RAS* mutational status in thyroid oncogenesis in acromegaly remains to be clarified.

#### *3.3.3 RET/PTC rearrangements*

The *RET* is a proto-oncogene that encodes a receptor-type tyrosine kinase with three domains: extracellular, transmembrane, and intracellular tyrosine kinase. The activation of this gene can contribute to the development of several neoplasms [25]. Rearrangements of *RET* that originated from fusion with unrelated genes (RET/PTC rearrangements) have been reported in thyroid follicular cells [26]. This genomic alteration can produce a chimeric oncoprotein with inappropriate tyrosine kinase activity able to continually stimulate the MAPK and PI3K-AKT pathways [26]. Among the fusion variants of *RET*, the rearrangements RET/PTC1 and RET/ PTC3 are the most frequent in thyroid cancer. Whereas in RET/PTC1 the *RET* gene is fused to *CCDC6* (known as *H4*), in RET/PTC3 the rearrangement occurs with *NCOA4* (known as *ELE1* or *RFG*) [25]. RET/PTC rearrangement appears to be an important mechanism of thyroid carcinogenesis, but its frequency has oscillated in different reports. This genetic abnormality was not detected in PTC patients with acromegaly [24], although studies with this approach are rare in acromegaly.

#### *3.3.4 Other molecular alterations*

Besides the potential classic marker, other molecules have been evaluated in relation to their implication on PTC development in acromegaly, among them are IGF-I, IGF-IRβ, AIP, AHR, and galectin-3 (Gal-3) [20, 23–24, 27].

The analysis of immunohistochemical staining for IGF-IRβ revealed a high expression of this receptor in PTC samples [20]. Although differences in IGF-IRβ tumoral staining between PTC patients with and without acromegaly have not been observed, this marker had significantly less expression in adjacent normal tissue of patients with acromegaly. These data suggest that high GH levels may trigger autocrine and paracrine effects of IGF-I in thyroid follicular cells resulting in overexpression of IGF-IRβ in tumor tissue of acromegalic patients. In line with these results, it was observed that PTC patients with acromegaly have higher expression of IGF-I than PTC cases without acromegaly [27]. Additionally, an intense expression was verified of Gal-3 in PTC with acromegaly, speculating a possible influence of this protein on thyroid carcinogenesis.

As previously mentioned, inactivation of *AIP* gene is frequently reported in pituitary tumors. However, this genetic abnormality seems not to be determinant to thyroid carcinogenesis in acromegalic patients [23]. Furthermore, there are no

differences in AIP protein expression between PTC in patients with and without acromegaly. Although immunohistochemical analysis for AIP receptor (AHR) has shown strong staining of PTC samples carrying *BRAF* V600E compared with wild type, differences were not found in AHR staining between PTC in acromegalic and non-acromegalic patients [23]. Thus, molecular alterations in AIP and AHR cannot be related to PTC carcinogenesis in acromegaly.

#### **4. How to screen NTD in acromegalic patients**

NTD seems to be significantly more frequent in patients with acromegaly. Even palpable thyroid nodules occur significantly more often in these patients [9, 13].

Periodic thyroid ultrasound (US) and careful evaluation of detected lesions are important parts in the follow-up of acromegalic patients. The sonographic characteristics considered to be suspicious of TC, such as microcalcifications, irregular margins (infiltrative and microlobulated), taller than wide shape, and rim calcifications with small extrusive soft tissue component (evidence of extrathyroidal extension), are the same of the general population with NTD [5, 9].

Fine-needle aspiration (FNA) is the procedure of choice in the evaluation on NTD, and it should be performed when clinically indicated according to nodule's size and US appearance. The FNA cytology result must be reported using the Bethesda System for Reporting Thyroid Cytopathology [9, 28].

In summary, as the risk of malignancy in thyroid nodules in these patients is about 8%, which is in the range considered for the general population, the management of NTD should follow the current guidelines [9, 28].

#### **5. How to treat TC in acromegalic patients**

Although there is a risk of TC in acromegalic patients, its clinical behavior does not seem to be different [5]. Therefore, acromegalic patients with TC may be treated with total thyroidectomy or hemithyroidectomy according to its FNA result and size and the presence of clinically apparent metastatic lymph nodes [28].

Before surgery, we suggest that all acromegalic patients should do a preoperative voice assessment (preoperative laryngeal exam—laryngoscopy) because they frequently have soft tissue thickening and edema of the tongue, pharynx, and upper airways [3]. Also, they must have a careful evaluation of comorbidities as hypertension, diabetes mellitus, and cardiovascular disease [3].

After surgery, these patients may or may not receive radioiodine depending, if it is a differentiated TC, on its risk of recurrence [28]. Studies about the relationship between medullary thyroid cancer (MTC) and acromegaly are lacking.

The frequency of US and laboratory tests during TC follow-up should follow the current guidelines.

#### **6. Conclusion**

NTC and TC are more frequent in acromegalic patients. On the other hand, the studies about potential mechanisms involved in this association between TC and acromegaly are still scarce, and besides they include small sample sizes. Furthermore, in these few reports, there is no marker clearly implicated on diagnosis or prognosis of PTC. Thus, further studies with this approach are needed.

**65**

**Author details**

(HUUFMA), Brazil

and Manuel dos Santos Faria1,2,3\*

(ENDOCLIM—HUUFMA), Brazil

provided the original work is properly cited.

Carla Souza Pereira Sobral1,3, Marcelo Magalhães1,2,3

Maranhão, Brazil (CEPEC—HUUFMA), Brazil

\*Address all correspondence to: mfaria@inlabmail.com

*Thyroid Cancer and Acromegaly*

and treatment of TC.

**Acknowledgements**

**Conflict of interest**

There is no conflict of interest.

manuscript.

*DOI: http://dx.doi.org/10.5772/intechopen.84541*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Endocrinology Unit, University Hospital of the Federal University of Maranhão

We suggest that acromegalic patients should be routinely screened by thyroid ultrasound and during their follow-up as necessary. Its management should follow the current guidelines. This is very important because it may allow early diagnosis

We would like to thank Doctor Gilvan Nascimento Cortês for the review of the

2 Clinical Research Center of the University Hospital of the Federal University of

3 Research Group in Clinical and Molecular Endocrinology and Metabology

*Thyroid Cancer and Acromegaly DOI: http://dx.doi.org/10.5772/intechopen.84541*

We suggest that acromegalic patients should be routinely screened by thyroid ultrasound and during their follow-up as necessary. Its management should follow the current guidelines. This is very important because it may allow early diagnosis and treatment of TC.

#### **Acknowledgements**

*Knowledges on Thyroid Cancer*

be related to PTC carcinogenesis in acromegaly.

**4. How to screen NTD in acromegalic patients**

extension), are the same of the general population with NTD [5, 9].

and the presence of clinically apparent metastatic lymph nodes [28].

between medullary thyroid cancer (MTC) and acromegaly are lacking.

Bethesda System for Reporting Thyroid Cytopathology [9, 28].

ment of NTD should follow the current guidelines [9, 28].

**5. How to treat TC in acromegalic patients**

sion, diabetes mellitus, and cardiovascular disease [3].

differences in AIP protein expression between PTC in patients with and without acromegaly. Although immunohistochemical analysis for AIP receptor (AHR) has shown strong staining of PTC samples carrying *BRAF* V600E compared with wild type, differences were not found in AHR staining between PTC in acromegalic and non-acromegalic patients [23]. Thus, molecular alterations in AIP and AHR cannot

NTD seems to be significantly more frequent in patients with acromegaly. Even palpable thyroid nodules occur significantly more often in these patients [9, 13]. Periodic thyroid ultrasound (US) and careful evaluation of detected lesions are important parts in the follow-up of acromegalic patients. The sonographic characteristics considered to be suspicious of TC, such as microcalcifications, irregular margins (infiltrative and microlobulated), taller than wide shape, and rim calcifications with small extrusive soft tissue component (evidence of extrathyroidal

Fine-needle aspiration (FNA) is the procedure of choice in the evaluation on NTD, and it should be performed when clinically indicated according to nodule's size and US appearance. The FNA cytology result must be reported using the

In summary, as the risk of malignancy in thyroid nodules in these patients is about 8%, which is in the range considered for the general population, the manage-

Although there is a risk of TC in acromegalic patients, its clinical behavior does not seem to be different [5]. Therefore, acromegalic patients with TC may be treated with total thyroidectomy or hemithyroidectomy according to its FNA result and size

Before surgery, we suggest that all acromegalic patients should do a preoperative voice assessment (preoperative laryngeal exam—laryngoscopy) because they frequently have soft tissue thickening and edema of the tongue, pharynx, and upper airways [3]. Also, they must have a careful evaluation of comorbidities as hyperten-

After surgery, these patients may or may not receive radioiodine depending, if it is a differentiated TC, on its risk of recurrence [28]. Studies about the relationship

The frequency of US and laboratory tests during TC follow-up should follow the

NTC and TC are more frequent in acromegalic patients. On the other hand, the studies about potential mechanisms involved in this association between TC and acromegaly are still scarce, and besides they include small sample sizes. Furthermore, in these few reports, there is no marker clearly implicated on diagnosis or prognosis of PTC. Thus, further studies with this approach are

**64**

needed.

current guidelines.

**6. Conclusion**

We would like to thank Doctor Gilvan Nascimento Cortês for the review of the manuscript.

#### **Conflict of interest**

There is no conflict of interest.

### **Author details**

Carla Souza Pereira Sobral1,3, Marcelo Magalhães1,2,3 and Manuel dos Santos Faria1,2,3\*

1 Endocrinology Unit, University Hospital of the Federal University of Maranhão (HUUFMA), Brazil

2 Clinical Research Center of the University Hospital of the Federal University of Maranhão, Brazil (CEPEC—HUUFMA), Brazil

3 Research Group in Clinical and Molecular Endocrinology and Metabology (ENDOCLIM—HUUFMA), Brazil

\*Address all correspondence to: mfaria@inlabmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Melmed S. Acromegaly pathogenesis and treatment. Journal of Clinical Investigation. 2009;**119**:3189-3202. DOI: 10.1172/JCI39375

[2] Holdaway IM, Rajasoorya RC, Gamble GD. Factors influencing mortality in acromegaly. Journal of Clinical Endocrinology and Metabolism. 2004;**89**:667-674. DOI: 10.1210/ jc.2003-031199

[3] Katznelson L, Laws ER Jr, Melmed S. Acromegaly: An endocrine society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism. 2014;**99**:3933-3951. DOI: 10.1210/ jc.2014-2700

[4] Arosio M, Reimondo G, Malchiodi E. Predictors of morbidity and mortality in acromegaly: An Italian survey. European Journal of Endocrinology. 2012;**167**:189-198. DOI: 10.1530/ EJE-12-0084

[5] Terzolo M, Reimondo G, Berchialla P. Acromegaly is associated with increased cancer risk: A survey in Italy. Endocrine-Related Cancer. 2017;**24**:495- 504. DOI: 10.1530/ERC-16-0553

[6] Wolinski K, Czarnywojtek A, Ruchala M. Risk of thyroid nodular disease and thyroid cancer in patients with acromegaly—Meta-analysis and systematic review. PLoS One. 2014;**9**:e8878. DOI: 10.1371/journal. pone.0088787

[7] Reverter JL, Fajardo C, Resmini E. Benign and malignant nodular thyroid disease in acromegaly. Is a routine thyroid ultrasound evaluation advisable? PLoS One. 2014;**9**:e104174. DOI: 10.1371/journal.pone.0104174

[8] Loeper S, Ezzat S. Acromegaly: Re-thinking the cancer risk. Reviews in Endocrine and Metabolic Disorders. 2008;**9**:41-58. DOI: 10.1007/ s11154-007-9063-z

[9] Santos MC, Nascimento GC, Nascimento AG. Thyroid cancer in patients with acromegaly: A case– control study. Pituitary. 2013;**16**:109- 114. DOI: 10.1007/s11102-012-0383-y

[10] Boguszewski CL, Ayuk J. Management of endocrine disease: Acromegaly and cancer risk: An old debate revisited. European Journal of Endocrinology. 2016;**175**:147-156. DOI: 10.1530/EJE-16-0178

[11] Maione L, Brue T, Beckers A. Changes in the management and comorbidities of acromegaly over three decades: The French Acromegaly Registry. European Journal of Endocrinology. 2017;**176**:645-655. DOI: 10.1530/EJE-16-1064

[12] Xia W, Gao L, Guo X. GH, IGF-1, and age are important contributors to thyroid abnormalities in patients with acromegaly. International Journal of Endocrinology. 2018;**2018**:6546832. DOI: 10.1155/2018/6546832

[13] Wüster C, Steger G, Schmelzle A.Increased incidence of euthyroid and hyper-thyroid GO iters in dependently of thyrotropin in patients with acromegaly. Hormone and Metabolic Research. 1991;**23**:131-134. DOI: 10.1055/s-2007-1003632

[14] Nathan J, Lanning CC. Recent advances in growth hormone signaling. Reviews in Endocrine & Metabolic Disorders. 2006;**7**:225-223. DOI: 10.1007/s11154-007-9025-5

[15] Loeper S, Ezzat S. Acromegaly: Re-thinking the cancer risk. Reviews in Endocrine & Metabolic Disorders. 2008;**9**:41-58. DOI: 10.1007/ s11154-007-9063-z

**67**

*Thyroid Cancer and Acromegaly*

[16] Chia DJ, Ono M, Woelfle J. Characterization of distinct Stat5b binding sites that mediate growth hormone-stimulated IGF-I gene transcription. Journal of Biological Chemistry. 2006;**6**:3190-3197. DOI:

10.1074/jbc.M510204200

s11154-007-9066-9

10.1126/science.1126100

pone.0110241

[20] Kim HK, Lee JS, Park MH. Tumorigenesis of papillary thyroid cancer is not BRAF-dependent in patients with acromegaly. PLoS One. 2014;**10**:e110241. DOI: 10.1371/journal.

[21] Kimura ET, Nikiforova MN, Zhaowen Z. High prevalence of BRAF mutations in thyroid cancer: Genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Research. 2003;**63**:1454-1457

[22] Koster EJ, Geus-Oei LF, Dekkers OM. Diagnostic utility of molecular and imaging biomarkers in cytological indeterminate thyroid nodules. Endocrine Reviews. 2018;**39**:154-191.

[23] Mian C, Ceccato F, Barollo S. AHR over-expression in papillary thyroid

DOI: 10.1210/er.2017-00133

[17] Horvath A, Constantine A. Stratakis: Clinical and molecular genetics of acromegaly: MEN1, Carney complex, McCune-Albright syndrome, familial acromegaly and genetic defects in sporadic tumors. Reviews in Endocrine & Metabolic Disorders. 2008;**9**:1-11. DOI: 10.1007/

[18] Melmed S. Acromegaly: Review article. The New England Journal of Medicine. 2006;**355**:2558-2573

[19] Vierimaa O, Georgitsi M, Lehtonen R. Pituitary adenoma predisposition caused by germline mutations in the AIP gene. Science. 2006;**312**:1228. DOI:

*DOI: http://dx.doi.org/10.5772/intechopen.84541*

carcinoma: Clinical and molecular assessments in a series of italian acromegalic patients with a long-term follow-up. PLoS One. 2014;**7**:e101560. DOI: 10.1371/journal.pone.0101560

[24] Aydin K, Aydin C, Dagdelen S. Genetic alterations in differentiated

[25] Mulligan LM. RET revisited: Expanding the oncogenic portfolio. Nature Reviews Cancer. 2014;**14**: 173-186. DOI: 10.1038/nrc3680

[26] Nikiforov YE, Nikiforova MN. Molecular genetics and diagnosis of thyroid cancer. Focus on Thyroid Cancer. 2011;**7**:569-580. DOI: 10.1038/

[27] Keskin FE, Ozkaya HM, Ferahman S. The role of different molecular markers in papillary thyroid cancer patients with acromegaly. Experimental and Clinical Endocrinology & Diabetes.

2018. DOI: 10.1055/a-0629-9223

[28] Bruchim I, Attias Z, Werner H. Targeting the IGF1 axis in cancer proliferation. Expert Opinion on

Therapeutic Targets. 2009;**13**:1179-1181. DOI: 10.1517/14728220903201702

thyroid cancer patients with acromegaly. Experimental and Clinical Endocrinology & Diabetes. 2015;**124**:198-202. DOI:

10.1055/s-0035-1565061

nrendo.2011.142

*Thyroid Cancer and Acromegaly DOI: http://dx.doi.org/10.5772/intechopen.84541*

[16] Chia DJ, Ono M, Woelfle J. Characterization of distinct Stat5b binding sites that mediate growth hormone-stimulated IGF-I gene transcription. Journal of Biological Chemistry. 2006;**6**:3190-3197. DOI: 10.1074/jbc.M510204200

[17] Horvath A, Constantine A. Stratakis: Clinical and molecular genetics of acromegaly: MEN1, Carney complex, McCune-Albright syndrome, familial acromegaly and genetic defects in sporadic tumors. Reviews in Endocrine & Metabolic Disorders. 2008;**9**:1-11. DOI: 10.1007/ s11154-007-9066-9

[18] Melmed S. Acromegaly: Review article. The New England Journal of Medicine. 2006;**355**:2558-2573

[19] Vierimaa O, Georgitsi M, Lehtonen R. Pituitary adenoma predisposition caused by germline mutations in the AIP gene. Science. 2006;**312**:1228. DOI: 10.1126/science.1126100

[20] Kim HK, Lee JS, Park MH. Tumorigenesis of papillary thyroid cancer is not BRAF-dependent in patients with acromegaly. PLoS One. 2014;**10**:e110241. DOI: 10.1371/journal. pone.0110241

[21] Kimura ET, Nikiforova MN, Zhaowen Z. High prevalence of BRAF mutations in thyroid cancer: Genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Research. 2003;**63**:1454-1457

[22] Koster EJ, Geus-Oei LF, Dekkers OM. Diagnostic utility of molecular and imaging biomarkers in cytological indeterminate thyroid nodules. Endocrine Reviews. 2018;**39**:154-191. DOI: 10.1210/er.2017-00133

[23] Mian C, Ceccato F, Barollo S. AHR over-expression in papillary thyroid

carcinoma: Clinical and molecular assessments in a series of italian acromegalic patients with a long-term follow-up. PLoS One. 2014;**7**:e101560. DOI: 10.1371/journal.pone.0101560

[24] Aydin K, Aydin C, Dagdelen S. Genetic alterations in differentiated thyroid cancer patients with acromegaly. Experimental and Clinical Endocrinology & Diabetes. 2015;**124**:198-202. DOI: 10.1055/s-0035-1565061

[25] Mulligan LM. RET revisited: Expanding the oncogenic portfolio. Nature Reviews Cancer. 2014;**14**: 173-186. DOI: 10.1038/nrc3680

[26] Nikiforov YE, Nikiforova MN. Molecular genetics and diagnosis of thyroid cancer. Focus on Thyroid Cancer. 2011;**7**:569-580. DOI: 10.1038/ nrendo.2011.142

[27] Keskin FE, Ozkaya HM, Ferahman S. The role of different molecular markers in papillary thyroid cancer patients with acromegaly. Experimental and Clinical Endocrinology & Diabetes. 2018. DOI: 10.1055/a-0629-9223

[28] Bruchim I, Attias Z, Werner H. Targeting the IGF1 axis in cancer proliferation. Expert Opinion on Therapeutic Targets. 2009;**13**:1179-1181. DOI: 10.1517/14728220903201702

**66**

*Knowledges on Thyroid Cancer*

[1] Melmed S. Acromegaly pathogenesis and treatment. Journal of Clinical Investigation. 2009;**119**:3189-3202. DOI: 2008;**9**:41-58. DOI: 10.1007/

[9] Santos MC, Nascimento GC, Nascimento AG. Thyroid cancer in patients with acromegaly: A case– control study. Pituitary. 2013;**16**:109- 114. DOI: 10.1007/s11102-012-0383-y

[10] Boguszewski CL, Ayuk J. Management of endocrine disease: Acromegaly and cancer risk: An old debate revisited. European Journal of Endocrinology. 2016;**175**:147-156. DOI:

[11] Maione L, Brue T, Beckers A. Changes in the management and comorbidities of acromegaly over three decades: The French Acromegaly

Registry. European Journal of

DOI: 10.1155/2018/6546832

[13] Wüster C, Steger G, Schmelzle A.Increased incidence of euthyroid and hyper-thyroid GO iters in dependently

of thyrotropin in patients with acromegaly. Hormone and Metabolic Research. 1991;**23**:131-134. DOI:

[14] Nathan J, Lanning CC. Recent advances in growth hormone signaling. Reviews in Endocrine & Metabolic Disorders. 2006;**7**:225-223. DOI: 10.1007/s11154-007-9025-5

[15] Loeper S, Ezzat S. Acromegaly: Re-thinking the cancer risk. Reviews in Endocrine & Metabolic Disorders.

2008;**9**:41-58. DOI: 10.1007/

s11154-007-9063-z

10.1055/s-2007-1003632

Endocrinology. 2017;**176**:645-655. DOI:

[12] Xia W, Gao L, Guo X. GH, IGF-1, and age are important contributors to thyroid abnormalities in patients with acromegaly. International Journal of Endocrinology. 2018;**2018**:6546832.

10.1530/EJE-16-0178

10.1530/EJE-16-1064

s11154-007-9063-z

[2] Holdaway IM, Rajasoorya RC, Gamble GD. Factors influencing mortality in acromegaly. Journal of Clinical Endocrinology and Metabolism.

2004;**89**:667-674. DOI: 10.1210/

[3] Katznelson L, Laws ER Jr, Melmed S. Acromegaly: An endocrine society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism.

2014;**99**:3933-3951. DOI: 10.1210/

in acromegaly: An Italian survey. European Journal of Endocrinology. 2012;**167**:189-198. DOI: 10.1530/

[4] Arosio M, Reimondo G, Malchiodi E. Predictors of morbidity and mortality

[5] Terzolo M, Reimondo G, Berchialla P. Acromegaly is associated with increased cancer risk: A survey in Italy. Endocrine-Related Cancer. 2017;**24**:495-

504. DOI: 10.1530/ERC-16-0553

[6] Wolinski K, Czarnywojtek A, Ruchala M. Risk of thyroid nodular disease and thyroid cancer in patients with acromegaly—Meta-analysis and systematic review. PLoS One. 2014;**9**:e8878. DOI: 10.1371/journal.

[7] Reverter JL, Fajardo C, Resmini E. Benign and malignant nodular thyroid disease in acromegaly. Is a routine thyroid ultrasound evaluation advisable? PLoS One. 2014;**9**:e104174. DOI: 10.1371/journal.pone.0104174

[8] Loeper S, Ezzat S. Acromegaly: Re-thinking the cancer risk. Reviews in Endocrine and Metabolic Disorders.

**References**

10.1172/JCI39375

jc.2003-031199

jc.2014-2700

EJE-12-0084

pone.0088787

**69**

**Chapter 6**

**Abstract**

*and Pietro Giorgio*

Intraoperative Neuromonitoring

Recurrent laryngeal nerve (RLN) injury is the most feared complication in thyroid surgery, resulting in a worse patients' quality of life, and is the most common cause of medical claim. Visualization of RLN before proceeding with dissection of the gland is considered the gold standard. In the last decade, intraoperative neuromonitoring (IONM) of RLN has progressively gained acceptance; nowadays, this method is widely spread, being routinely used in large workflow centers. IONM is helpful in the identification of RLN and allows to asses nerve functionality during and at the end of surgical procedure. In this chapter, IONM features, its advantages

**Keywords:** thyroid surgery, thyroid carcinoma, intraoperative neuromonitoring,

Postoperative recurrent laryngeal nerve (RLN) injury is one of the most feared complications during thyroid surgery. Even in experienced hands, transient RLN palsy occurs in 0.4–12% of cases [1–3], while permanent palsy is reported in up to 5–6% [2, 4]; its frequency is lower (0.2–0.8%) in hospitals with a large workflow [5]. RLN palsy can significantly deteriorate patients' quality of life, causing hoarseness of voice, dysphonia and dysphagia. Bilateral RLN palsy is an uncommon but life-threatening complication, even if transient, since it is associated to airway obstruction, which is potentially lethal for the patient. Intraoperative RLN injuries are due to transection and traction of the nerve, electrical and thermal injuries, suture entrapment and excessive skeletization. The risk of nerve palsy increases during surgery for thyroid cancer, especially in case of large or locally advanced tumors that can dislocate or infiltrate the nerve, during central compartment lymph node dissection (CLND) or in case of revision surgery. Over the years, surgical approach to RLN has consistently changed: the first approach, consisting in nonvisualization and avoidance of the nerve, has been replaced with routine identification of RLN, which has been reported to be associated with a lower incidence of

Intraoperative neuromonitoring (IONM) of RNL during thyroid surgery has widely spread during the last decade as an adjunct to the gold standard of direct visualization of the nerve; nowadays, it has become standard practice in thyroid surgery for many surgeons [11]. The most common method currently in use for

in Thyroid Surgery

and limits, and its usefulness will be discussed.

recurrent laryngeal nerve, nerve injury

**1. Introduction**

RLN palsy [6–10].

*Fabio Medas, Gian Luigi Canu, Enrico Erdas*

#### **Chapter 6**

## Intraoperative Neuromonitoring in Thyroid Surgery

*Fabio Medas, Gian Luigi Canu, Enrico Erdas and Pietro Giorgio*

#### **Abstract**

Recurrent laryngeal nerve (RLN) injury is the most feared complication in thyroid surgery, resulting in a worse patients' quality of life, and is the most common cause of medical claim. Visualization of RLN before proceeding with dissection of the gland is considered the gold standard. In the last decade, intraoperative neuromonitoring (IONM) of RLN has progressively gained acceptance; nowadays, this method is widely spread, being routinely used in large workflow centers. IONM is helpful in the identification of RLN and allows to asses nerve functionality during and at the end of surgical procedure. In this chapter, IONM features, its advantages and limits, and its usefulness will be discussed.

**Keywords:** thyroid surgery, thyroid carcinoma, intraoperative neuromonitoring, recurrent laryngeal nerve, nerve injury

#### **1. Introduction**

Postoperative recurrent laryngeal nerve (RLN) injury is one of the most feared complications during thyroid surgery. Even in experienced hands, transient RLN palsy occurs in 0.4–12% of cases [1–3], while permanent palsy is reported in up to 5–6% [2, 4]; its frequency is lower (0.2–0.8%) in hospitals with a large workflow [5]. RLN palsy can significantly deteriorate patients' quality of life, causing hoarseness of voice, dysphonia and dysphagia. Bilateral RLN palsy is an uncommon but life-threatening complication, even if transient, since it is associated to airway obstruction, which is potentially lethal for the patient. Intraoperative RLN injuries are due to transection and traction of the nerve, electrical and thermal injuries, suture entrapment and excessive skeletization. The risk of nerve palsy increases during surgery for thyroid cancer, especially in case of large or locally advanced tumors that can dislocate or infiltrate the nerve, during central compartment lymph node dissection (CLND) or in case of revision surgery. Over the years, surgical approach to RLN has consistently changed: the first approach, consisting in nonvisualization and avoidance of the nerve, has been replaced with routine identification of RLN, which has been reported to be associated with a lower incidence of RLN palsy [6–10].

Intraoperative neuromonitoring (IONM) of RNL during thyroid surgery has widely spread during the last decade as an adjunct to the gold standard of direct visualization of the nerve; nowadays, it has become standard practice in thyroid surgery for many surgeons [11]. The most common method currently in use for

RLN monitoring is an endotracheal tube containing electrodes embedded on it, placed in close proximity to vocal cords, that register effects of stimulation of RLN. IONM is able to detect anatomic variations of the nerve and to clarify the mechanism and the site of the injury, and to predict vocal cord function after surgery. During surgical procedure, repeated tissue stimulation is helpful to correctly identify the nerve. Furthermore, IONM can detect non-function nerves that appear anatomically intact.

#### **2. Intraoperative neuromonitoring in thyroid surgery**

Intraoperative neuromonitoring has been introduced in thyroid surgery as an adjunct to standard visual identification of the recurrent laryngeal nerve (RLN) to prevent nerve lesion. The use of IONM, besides helping to identify the RLN, gives an objective evaluation of its function during the whole dissection [12–15]. IONM was introduced about 50 years ago, and various neuromonitoring methods (glottic pressure method, glottic monitoring method, insertion of needle electrodes in vocal cords endoscopically or through cricothyroid membrane, laryngeal palpation method, and monitoring via endotracheal tube with surface electrodes) have been utilized [16]. For several reasons, such as simplicity, non-invasiveness, and safety, IONM via endotracheal (ET) tube with surface electrodes has become the standard method [12]. It consists in an electromyography (EMG) that evaluates the vocal cord adductor function by using surface electrodes on the ET tube. NIM-Response 3.0 System (Medtronic Xomed, Jacksonville, Florida, USA) is the most widely used device for RLN monitoring (**Figure 1**). It transforms laryngeal muscle activity into audible and visual EMG signals whenever the RLN or vagus nerve is stimulated intraoperatively. This system basically consists of the combination of two electrical circuits: stimulation and recording sides. The stimulation side consists of a stimulator probe (nerve stimulator probe, continuous vagus nerve stimulator probe), which transmits electric current to the nerve, and a grounding electrode. The nerve stimulation probe can be monopolar or bipolar, while the continuous monitoring probes applied to the vagus nerve can be monopolar, bipolar, or tripolar. The

#### **Figure 1.**

*NIM-Response 3.0 System (Medtronic Xomed, Jacksonville, Florida, USA) (Italian configuration). In thyroid surgery, two channels for the right and left vocal cords are sufficient and 2 EMG screens appear on the monitor.*

**71**

**Figure 3.**

*monitor.*

**Figure 2.**

recording side, instead, consists of the ET tube with surface electrodes, which are placed at the level of the vocal cord, and their ground electrode (**Figure 2**). These two main systems combine on the interconnection box (**Figure 3**), through which

*Stimulation and recording sides are combined on the interconnect box, through which they connect to the* 

*ET tube with surface electrodes and grounding electrodes (white: stimulation side, green: recording side).*

*Intraoperative Neuromonitoring in Thyroid Surgery DOI: http://dx.doi.org/10.5772/intechopen.83840*

*Intraoperative Neuromonitoring in Thyroid Surgery DOI: http://dx.doi.org/10.5772/intechopen.83840*

*Knowledges on Thyroid Cancer*

anatomically intact.

RLN monitoring is an endotracheal tube containing electrodes embedded on it, placed in close proximity to vocal cords, that register effects of stimulation of RLN. IONM is able to detect anatomic variations of the nerve and to clarify the mechanism and the site of the injury, and to predict vocal cord function after surgery. During surgical procedure, repeated tissue stimulation is helpful to correctly identify the nerve. Furthermore, IONM can detect non-function nerves that appear

Intraoperative neuromonitoring has been introduced in thyroid surgery as an adjunct to standard visual identification of the recurrent laryngeal nerve (RLN) to prevent nerve lesion. The use of IONM, besides helping to identify the RLN, gives an objective evaluation of its function during the whole dissection [12–15]. IONM was introduced about 50 years ago, and various neuromonitoring methods (glottic pressure method, glottic monitoring method, insertion of needle electrodes in vocal cords endoscopically or through cricothyroid membrane, laryngeal palpation method, and monitoring via endotracheal tube with surface electrodes) have been utilized [16]. For several reasons, such as simplicity, non-invasiveness, and safety, IONM via endotracheal (ET) tube with surface electrodes has become the standard method [12]. It consists in an electromyography (EMG) that evaluates the vocal cord adductor function by using surface electrodes on the ET tube. NIM-Response 3.0 System (Medtronic Xomed, Jacksonville, Florida, USA) is the most widely used device for RLN monitoring (**Figure 1**). It transforms laryngeal muscle activity into audible and visual EMG signals whenever the RLN or vagus nerve is stimulated intraoperatively. This system basically consists of the combination of two electrical circuits: stimulation and recording sides. The stimulation side consists of a stimulator probe (nerve stimulator probe, continuous vagus nerve stimulator probe), which transmits electric current to the nerve, and a grounding electrode. The nerve stimulation probe can be monopolar or bipolar, while the continuous monitoring probes applied to the vagus nerve can be monopolar, bipolar, or tripolar. The

*NIM-Response 3.0 System (Medtronic Xomed, Jacksonville, Florida, USA) (Italian configuration). In thyroid surgery, two channels for the right and left vocal cords are sufficient and 2 EMG screens appear on the monitor.*

**2. Intraoperative neuromonitoring in thyroid surgery**

**70**

**Figure 1.**

**Figure 2.** *ET tube with surface electrodes and grounding electrodes (white: stimulation side, green: recording side).*

*Stimulation and recording sides are combined on the interconnect box, through which they connect to the monitor.*

recording side, instead, consists of the ET tube with surface electrodes, which are placed at the level of the vocal cord, and their ground electrode (**Figure 2**). These two main systems combine on the interconnection box (**Figure 3**), through which they connect to the monitor. The monitoring systems can be with 2, 4, 8, or 16 channels. The number of channels indicates the number of nerves that can be monitored. A separate EMG screen appears for each nerve on the monitor. In thyroid surgery, two channels for the right and left vocal cords are sufficient and two EMG screens appear on the monitor (**Figure 1**). In case of continuous vagus nerve stimulation, instead, two EMG screens for intermittent monitoring appear on the left side of the monitor, and another EMG screen for continuous vagus nerve stimulation appears on the right side.

#### **3. Standardization of intraoperative neuromonitoring**

Adequate knowledge of the neuromonitoring system and standardization of the procedure are required for proper use of IONM [12–17]. In this context, both surgeons and anaesthesiologists are involved. In order to reach an adequate experience, it has been stated that the learning curve is approximately of 50–100 cases [18–20].

The anesthetist plays a key role in IONM procedure, particularly with regard to the type of drugs used to induce anaesthesia and to the positioning of the ET tube. After these steps, anesthesia can be obtained by inhaler or intravenous anesthetics: these agents do not have significant effects on EMG signal, providing an adequate depth of anesthesia. Differently, neuromuscular blocking agents (NMBAs) interfere with monitoring, reducing EMG amplitude and the optimal laryngeal response, thus making neuromonitoring less effective. For this reason, after induction, NMBA should be avoided for the rest of the operation. Small doses of a non-depolarizing muscle relaxant (usually rocuronium and atracurium) are used at intubation, as these agents allow the restoration of basic physiological functions, such as spontaneous respiration and normal muscle twitch activity, within a few minutes.

Endotracheal tubes are available in sizes 6.0, 6.5, 7.0, 7.5, and 8.0. The largest tube that can be passed between the patient's vocal cords has to be used. The ET tube is placed under direct laryngoscopy with the middle of the blue marked region (the exposed electrodes) in contact with the true vocal cords. The tube has to be placed in the right position to obtain adequate functioning of the system. Endotracheal tube positioning errors include not only depth errors but also rotational errors. The malposition of the ET can lead to misleading information and is a potential cause of loss of signal during surgery. After the tube is inserted, the patient is given the right operation position, with hyperextended neck, by applying a pillow under the shoulders. During positioning, the anesthetist must protect the ET tube to keep its position unchanged. If the tube is fixed to the rim of the patient's mouth before the patient is correctly positioned, the position of the tube in the airway can change. This can lead to a disruption of the relationship between the surface electrodes of the ET tube and the vocal cords. Thus, the tube has to be secured to the rim of the lip after the patient is correctly positioned.

Once the patient is positioned, the grounding electrode of the recording side and the grounding electrode of the stimulation side are subdermally applied to the presternal region or to the shoulder at the side of the monitor. The second one should be placed 1–2 cm below the first one.

After all connections are made, the correct positioning of the ET tube must be checked. This can be done from the monitor by verifying the impedance value of the electrodes (**Figure 4**). For each electrode, it has to be less than 5 kΩ. Moreover, the impedance difference between positive and negative electrodes of each channel should be less than 1 kΩ. Values above these thresholds indicate that the contact between the patient's vocal cords and the ET tube electrodes is not adequate. Other

**73**

**Figure 4.**

RLN and vagus nerve is performed.

*Intraoperative Neuromonitoring in Thyroid Surgery DOI: http://dx.doi.org/10.5772/intechopen.83840*

tests to verify the correct location of the ET tube include the evaluation of respiratory changes or a further laryngoscopy. About the first method, in the short-term window period between the loss of the effect of short-acting NMBA and the deepening of anesthesia following intubation, spontaneous respiratory movements should result in waveforms with an amplitude of 30–70 μV on the monitor. These

At this point, the monitor should be set as follows: a threshold value of 100 μV, an excitation electrode stimulation level of 0.5–2 mA (mean: 1 mA), a stimulation

At the beginning of the operation, the stimulator probe should be tested directly

on the infrahyoid or sternocleidomastoid muscle to confirm the presence of an appropriate muscle twitching. This confirms that the nerve stimulation probe is working properly and the absence of ongoing paralytic agent. Moreover, to confirm the overall system function, before the identification of RLN, an EMG signal should initially be obtained from the vagus nerve. This step is crucial to assess that IONM system is functioning correctly and that the normal pathway of RLN signal is elicited. The vagus nerve can be directly stimulated after dissection of the carotid sheath, or its stimulation can be performed simply by increasing the stimulation level up to 2–3 mA with the probe on the carotid sheath without dissecting it. The RLN is situated at the tracheoesophageal groove in proximity to the inferior thyroid artery. It can be initially searched with a stimulation level of 2 mA and fully mapped out; then, it can be isolated and visually confirmed. Once the nerve is visualized, the stimulation level can be turned down to 1 mA. It is important to keep in mind that RLN extralaryngeal branching can be found in about 30–40% of patients, particularly at the level of Berry's ligament. Thus, it is necessary to dissect the RLN from the lower neck up to the nerve entrance into the larynx. In case of branched RLN, each branch should be stimulated separately by using a stimulation current of 0.4–0.5 mA. EMG signal of these individual branches should be assessed to allow a reliable evaluation of the distribution of the motor and sensory fibers. After removing the surgical specimen and ensuring a complete hemostasis, the final testing of

respiratory changes should be detected for both vocal cords.

*Check of the impedance value of the electrodes from the monitor.*

period of 100 μs, and a stimulation frequency of 4 stimuli per second.

*Intraoperative Neuromonitoring in Thyroid Surgery DOI: http://dx.doi.org/10.5772/intechopen.83840*

*Knowledges on Thyroid Cancer*

on the right side.

they connect to the monitor. The monitoring systems can be with 2, 4, 8, or 16 channels. The number of channels indicates the number of nerves that can be monitored. A separate EMG screen appears for each nerve on the monitor. In thyroid surgery, two channels for the right and left vocal cords are sufficient and two EMG screens appear on the monitor (**Figure 1**). In case of continuous vagus nerve stimulation, instead, two EMG screens for intermittent monitoring appear on the left side of the monitor, and another EMG screen for continuous vagus nerve stimulation appears

Adequate knowledge of the neuromonitoring system and standardization of the procedure are required for proper use of IONM [12–17]. In this context, both surgeons and anaesthesiologists are involved. In order to reach an adequate experience, it has been stated that the learning curve is approximately of 50–100 cases [18–20]. The anesthetist plays a key role in IONM procedure, particularly with regard to the type of drugs used to induce anaesthesia and to the positioning of the ET tube. After these steps, anesthesia can be obtained by inhaler or intravenous anesthetics: these agents do not have significant effects on EMG signal, providing an adequate depth of anesthesia. Differently, neuromuscular blocking agents (NMBAs) interfere with monitoring, reducing EMG amplitude and the optimal laryngeal response, thus making neuromonitoring less effective. For this reason, after induction, NMBA should be avoided for the rest of the operation. Small doses of a non-depolarizing muscle relaxant (usually rocuronium and atracurium) are used at intubation, as these agents allow the restoration of basic physiological functions, such as spontane-

**3. Standardization of intraoperative neuromonitoring**

ous respiration and normal muscle twitch activity, within a few minutes.

secured to the rim of the lip after the patient is correctly positioned.

should be placed 1–2 cm below the first one.

Once the patient is positioned, the grounding electrode of the recording side and the grounding electrode of the stimulation side are subdermally applied to the presternal region or to the shoulder at the side of the monitor. The second one

After all connections are made, the correct positioning of the ET tube must be checked. This can be done from the monitor by verifying the impedance value of the electrodes (**Figure 4**). For each electrode, it has to be less than 5 kΩ. Moreover, the impedance difference between positive and negative electrodes of each channel should be less than 1 kΩ. Values above these thresholds indicate that the contact between the patient's vocal cords and the ET tube electrodes is not adequate. Other

Endotracheal tubes are available in sizes 6.0, 6.5, 7.0, 7.5, and 8.0. The largest tube that can be passed between the patient's vocal cords has to be used. The ET tube is placed under direct laryngoscopy with the middle of the blue marked region (the exposed electrodes) in contact with the true vocal cords. The tube has to be placed in the right position to obtain adequate functioning of the system. Endotracheal tube positioning errors include not only depth errors but also rotational errors. The malposition of the ET can lead to misleading information and is a potential cause of loss of signal during surgery. After the tube is inserted, the patient is given the right operation position, with hyperextended neck, by applying a pillow under the shoulders. During positioning, the anesthetist must protect the ET tube to keep its position unchanged. If the tube is fixed to the rim of the patient's mouth before the patient is correctly positioned, the position of the tube in the airway can change. This can lead to a disruption of the relationship between the surface electrodes of the ET tube and the vocal cords. Thus, the tube has to be

**72**

#### **Figure 4.** *Check of the impedance value of the electrodes from the monitor.*

tests to verify the correct location of the ET tube include the evaluation of respiratory changes or a further laryngoscopy. About the first method, in the short-term window period between the loss of the effect of short-acting NMBA and the deepening of anesthesia following intubation, spontaneous respiratory movements should result in waveforms with an amplitude of 30–70 μV on the monitor. These respiratory changes should be detected for both vocal cords.

At this point, the monitor should be set as follows: a threshold value of 100 μV, an excitation electrode stimulation level of 0.5–2 mA (mean: 1 mA), a stimulation period of 100 μs, and a stimulation frequency of 4 stimuli per second.

At the beginning of the operation, the stimulator probe should be tested directly on the infrahyoid or sternocleidomastoid muscle to confirm the presence of an appropriate muscle twitching. This confirms that the nerve stimulation probe is working properly and the absence of ongoing paralytic agent. Moreover, to confirm the overall system function, before the identification of RLN, an EMG signal should initially be obtained from the vagus nerve. This step is crucial to assess that IONM system is functioning correctly and that the normal pathway of RLN signal is elicited. The vagus nerve can be directly stimulated after dissection of the carotid sheath, or its stimulation can be performed simply by increasing the stimulation level up to 2–3 mA with the probe on the carotid sheath without dissecting it. The RLN is situated at the tracheoesophageal groove in proximity to the inferior thyroid artery. It can be initially searched with a stimulation level of 2 mA and fully mapped out; then, it can be isolated and visually confirmed. Once the nerve is visualized, the stimulation level can be turned down to 1 mA. It is important to keep in mind that RLN extralaryngeal branching can be found in about 30–40% of patients, particularly at the level of Berry's ligament. Thus, it is necessary to dissect the RLN from the lower neck up to the nerve entrance into the larynx. In case of branched RLN, each branch should be stimulated separately by using a stimulation current of 0.4–0.5 mA. EMG signal of these individual branches should be assessed to allow a reliable evaluation of the distribution of the motor and sensory fibers. After removing the surgical specimen and ensuring a complete hemostasis, the final testing of RLN and vagus nerve is performed.

**Figure 5.** *RLN stimulation at the end of thyroid dissection and complete hemostasis (R2).*

In 2011, the International Neural Monitoring Study Group (INMSG) defined the standard stages of intraoperative neuromonitoring in thyroid surgery by adding preoperative and postoperative vocal cord examinations to the four-step method previously proposed by Chiang et al. [12, 21]. The six stages can be summarized as follows:


As well as being useful during traditional open thyroidectomy, IONM can be very useful for RLN preservation also in case of endoscopic thyroid surgery [22, 23]. The fundamental steps of IONM during video-assisted thyroidectomy (VAT) are the same as those used during traditional thyroidectomy (L1, V1, R1, R2, V2, L2) [22]. IONM has proven to be very helpful also in the case of other endoscopic techniques, such as transaxillary or transoral thyroidectomy [23], even if a standardized technique is still lacking with these approaches.

#### **4. Types of intraoperative neuromonitoring (I-IONM and C-IONM)**

Currently, two types of IONM are available: the intermittent IONM (I-IONM) and the continuous IONM (C-IONM). With I-IONM, the functional integrity of the

**75**

*Intraoperative Neuromonitoring in Thyroid Surgery DOI: http://dx.doi.org/10.5772/intechopen.83840*

occurs [24].

**5. Loss of signal**

(Type 2).

nerve.

proximally to this site.

**5.1 Definition of loss of signal**

RLN is limited to the site of direct nerve stimulation. For this reason, in proximal lesions of the RLN, distal stimulation near the larynx may produce a false negative IONM signal. Moreover, with this system, the RLN is at risk for damage during the time gap between two nerve stimulations. Ultimately, I-IONM allows the evaluation of the RLN only at the time of stimulation, and detects RLN lesion merely after it

To overcome these limits, a C-IONM technology has been introduced [25–28]. C-IONM consists in a probe that is applied directly to the vagus nerve, allowing the surgeon to constantly test the RLN function while dissecting the thyroid gland. However, the intermittent method is not a separate process from the continuous IONM technique. The intermittent IONM probe, in fact, is an integral and complementary part of the C-IONM [15]. The goal of continuous vagus nerve probing is to inform the surgeon immediately of any critical insult of the RLN, like traction, thus avoiding signal loss and vocal cord paralysis. A 50% decrease in amplitude and a 10% increase in latency time have been defined as critical changes [25, 28]. The device alarms when these thresholds are exceeded. According to recent observation, the most common cause of RLN lesion is tractional trauma [25–29]; C-IONM has proved to be useful in preventing traction injury by promptly detecting progressive decreases in EMG amplitude combined with progressive increases in latency. The surgeon can so avoid eventual RLN injury by changing his strategy. However, this system is not effective in case of acute injury of the nerve (section or thermal injury) [25–28].

Loss of signal (LOS) occurs when the original EMG signal obtained from the vagus nerve and/or RLN nerve can no longer be elicited [12]. It is classified as true positive if vocal cord palsy is confirmed on postoperative laryngoscopy and false

There are two types of LOS: the segmental type (Type 1) and the global type

Type 1 LOS consists in the loss of signal at a certain point in the nerve; signal is obtained distally to the point where the nerve is injured, but no signal is detected

In case of Type 2 LOS, instead, no specific damage point is recognizable, and no signal is acquired stimulating the RLN all along its course or stimulating the vagus

About this argument, it is important to introduce another concept: the intraoperative signal recovery. Especially with the introduction of the C-IONM with continuous vagus nerve stimulation, it has been noted that, in some patients with

When LOS occurs, a troubleshooting protocol should be followed to check the

In this case, the first procedure to be performed is to palpate the larynx with a finger behind the posterior plate of the cricoid to feel the posterior cricoarytenoid

If digital detection of the laryngeal twitch is present in response to nerve stimulation, the stimulation side of the system is working properly, and a malfunction of the recording side should be considered. The most frequent causes of malfunction

positive if no vocal cord palsy is present on postoperative laryngoscopy.

signal loss, the signal can improve in the course of the operation.

**5.2 Troubleshooting algorithm for loss of signal**

IONM system for technical problems [13, 30, 31].

muscle contraction in response to RLN stimulation.

*Intraoperative Neuromonitoring in Thyroid Surgery DOI: http://dx.doi.org/10.5772/intechopen.83840*

*Knowledges on Thyroid Cancer*

a. preoperative laryngoscopy (L1);

f. postoperative laryngoscopy (L2).

lacking with these approaches.

(**Figure 5**);

**Figure 5.**

b. vagus nerve stimulation before thyroidectomy (V1);

*RLN stimulation at the end of thyroid dissection and complete hemostasis (R2).*

c. RLN stimulation upon initial identification (R1);

In 2011, the International Neural Monitoring Study Group (INMSG) defined the standard stages of intraoperative neuromonitoring in thyroid surgery by adding preoperative and postoperative vocal cord examinations to the four-step method previously proposed by Chiang et al. [12, 21]. The six stages can be summarized as follows:

d. RLN stimulation at the end of thyroid dissection and complete hemostasis (R2)

e. vagus nerve stimulation after complete thyroidectomy and hemostasis (V2);

As well as being useful during traditional open thyroidectomy, IONM can be very useful for RLN preservation also in case of endoscopic thyroid surgery [22, 23]. The fundamental steps of IONM during video-assisted thyroidectomy (VAT) are the same as those used during traditional thyroidectomy (L1, V1, R1, R2, V2, L2) [22]. IONM has proven to be very helpful also in the case of other endoscopic techniques, such as transaxillary or transoral thyroidectomy [23], even if a standardized technique is still

**4. Types of intraoperative neuromonitoring (I-IONM and C-IONM)**

Currently, two types of IONM are available: the intermittent IONM (I-IONM) and the continuous IONM (C-IONM). With I-IONM, the functional integrity of the

**74**

RLN is limited to the site of direct nerve stimulation. For this reason, in proximal lesions of the RLN, distal stimulation near the larynx may produce a false negative IONM signal. Moreover, with this system, the RLN is at risk for damage during the time gap between two nerve stimulations. Ultimately, I-IONM allows the evaluation of the RLN only at the time of stimulation, and detects RLN lesion merely after it occurs [24].

To overcome these limits, a C-IONM technology has been introduced [25–28]. C-IONM consists in a probe that is applied directly to the vagus nerve, allowing the surgeon to constantly test the RLN function while dissecting the thyroid gland. However, the intermittent method is not a separate process from the continuous IONM technique. The intermittent IONM probe, in fact, is an integral and complementary part of the C-IONM [15]. The goal of continuous vagus nerve probing is to inform the surgeon immediately of any critical insult of the RLN, like traction, thus avoiding signal loss and vocal cord paralysis. A 50% decrease in amplitude and a 10% increase in latency time have been defined as critical changes [25, 28]. The device alarms when these thresholds are exceeded. According to recent observation, the most common cause of RLN lesion is tractional trauma [25–29]; C-IONM has proved to be useful in preventing traction injury by promptly detecting progressive decreases in EMG amplitude combined with progressive increases in latency. The surgeon can so avoid eventual RLN injury by changing his strategy. However, this system is not effective in case of acute injury of the nerve (section or thermal injury) [25–28].

#### **5. Loss of signal**

#### **5.1 Definition of loss of signal**

Loss of signal (LOS) occurs when the original EMG signal obtained from the vagus nerve and/or RLN nerve can no longer be elicited [12]. It is classified as true positive if vocal cord palsy is confirmed on postoperative laryngoscopy and false positive if no vocal cord palsy is present on postoperative laryngoscopy.

There are two types of LOS: the segmental type (Type 1) and the global type (Type 2).

Type 1 LOS consists in the loss of signal at a certain point in the nerve; signal is obtained distally to the point where the nerve is injured, but no signal is detected proximally to this site.

In case of Type 2 LOS, instead, no specific damage point is recognizable, and no signal is acquired stimulating the RLN all along its course or stimulating the vagus nerve.

About this argument, it is important to introduce another concept: the intraoperative signal recovery. Especially with the introduction of the C-IONM with continuous vagus nerve stimulation, it has been noted that, in some patients with signal loss, the signal can improve in the course of the operation.

#### **5.2 Troubleshooting algorithm for loss of signal**

When LOS occurs, a troubleshooting protocol should be followed to check the IONM system for technical problems [13, 30, 31].

In this case, the first procedure to be performed is to palpate the larynx with a finger behind the posterior plate of the cricoid to feel the posterior cricoarytenoid muscle contraction in response to RLN stimulation.

If digital detection of the laryngeal twitch is present in response to nerve stimulation, the stimulation side of the system is working properly, and a malfunction of the recording side should be considered. The most frequent causes of malfunction

of the recording side are ET tube malposition, displacement of grounding electrodes, or malfunction of the ET tube electrodes.

In addition to laryngeal palpation, contralateral vagal assessment also represents a useful option for troubleshooting. If contralateral vagus nerve stimulation does not elicit an adequate EMG signal, a malfunction of the recording side should be investigated as first option. Differently, if the contralateral vagus nerve is properly functioning, a possible nerve lesion must be considered.

If laryngeal twitch is absent when the nerve is stimulated, a malfunction of the stimulation side should be considered, thus the nerve stimulator probe and monitor have to be checked. Nerve stimulator probe function should be checked by applying its tip directly on a muscle to confirm a muscle twitching. Moreover, the whole system, with special attention focused on the monitor screen, must be fully reviewed. Again, with regard to stimulation side error, if C-IONM is used, LOS may be due to a dislocation of the vagal nerve electrode.

Finally, in case of LOS, it is of fundamental importance to rule any administration of NMBA during the operation.

#### **6. Advantages and limits of IONM**

#### **6.1 Advantages of IONM**

Visualization of RLNs is considered the gold standard in thyroid surgery to reduce the incidence of nerve palsy. Nevertheless, visualization of the nerve can only suggest an anatomic integrity, which does not ensure functionality. In fact, some studies have demonstrated that the most common mechanism of nerve injury is traction [29, 32], resulting in a palsy with complete anatomic integrity. Furthermore, direct visualization of the nerve can be difficult, especially in case of revision neck surgery, because of the scar tissue [1, 32–34], in case of anatomic variations of the nerve, during central compartment lymph node dissection, or in course of surgery for advanced thyroid cancer. For these reasons, in the last decades, the use of IONM has widely spread among endocrine surgeons, in order to facilitate identification and dissection of the nerve and to evaluate its functionality, predicting vocal cord function outcome.

However, to date, studies have failed to demonstrate a statistically significant reduction of incidence of nerve injury using IONM [2, 3]; this lack of data may be related to the very low rate of nerve palsy. It has been estimated that in prospective, randomized trials, the calculated sample size needed to demonstrate that incidence of palsy is lower with IONM use is about 9000 nerves at risk [1, 7]. Other authors have reported that at least 39,000 nerves at risk per arm should be necessary to achieve statistical power that could demonstrate a significant difference in RLN palsy rate [3]. To date, only one randomized controlled trial [35] has demonstrated a significant reduction of transient RLN palsy from 5 to 2.7% (p = 0.007).

Furthermore, it is important to underline that voice impairment isn't always due to RLN palsy; other causes can be vocal cord damage due to orotracheal intubation and damage of the strap muscles. In these cases, the good surgical practice and the integrity and functionality of the nerve are assessed by IONM, preserving from medical claim.

Advantages of IONM of RLNs in thyroid surgery are:

• Early identification of RLN and aid in dissection: Stimulation of paratracheal area allows to identify the course of the nerve before it is visualized; the identified area is then carefully dissected until the nerve is satisfactorily exposed.

**77**

*Intraoperative Neuromonitoring in Thyroid Surgery DOI: http://dx.doi.org/10.5772/intechopen.83840*

of anatomic variants.

nerve.

palsy.

**6.2 Limits of IONM**

Once the nerve is identified, stimulation of the adjacent structures can help to distinguish the nerve from other non-neural elements, like vessels. IONM is also helpful in identifying neural branches and to clarify nerve course in case

• Intraoperative diagnosis of RLN injury and postoperative prognosis: The most common causes of RLN injuries are transection of the nerve, suture entrapment, traction, compression, contusion, pressure, ischemia by excessive skeletization, thermal trauma caused by dissection or hemostatic instruments used too close to the nerve. In most of the cases of nerve injury, the nerve is anatomically intact, thus visualization only is not predictive of a vocal cord palsy; on the contrary, IONM allows to predict most of the nerve injuries, improving the accuracy of prognostic evaluation of nerve functionality. Once a loss of signal is detected, IONM allows to identify with high accuracy the site of the lesion by stimulating the nerve all long its course and the vagus

• Reduction of bilateral RLN palsy: Once RLN palsy has been diagnosed with IONM on the first side of resection, the surgeon can decide to modify the initially scheduled bilateral surgery and to perform a delayed completion thyroidectomy (two-staged thyroidectomy), in order to avoid a bilateral RLN

• Difficult cases: IONM has beneficial effects especially in difficult cases, like reoperative surgery, locally advanced thyroid cancer and cervico-mediastinal goiter, or in case of lymphadenectomy of central neck compartment, facilitat-

• Improvement of radicality in total thyroidectomy: Most of RLN injuries are produced during dissection of thyroid tissue from Berry ligament, within the last 2 cm of the nerve. The two main reasons of lesion are a ramification of RLN, which occurs usually less than 5 mm of its entry in larynx, or an intracapsular course of RLN, which is reported in 15–38% of cases. Thus, in the absence of IONM, surgeons often tend to leave a small amount of thyroid tissue to avoid lesion of RLN in the last part of its course. On the contrary, IONM allows to completely resect the thyroid gland, reducing the risk of nerve palsy. Furthermore, hemostatic maneuvers can be safely conducted with IONM, ensuring that surgical sutures do not exert a traction on the nerve and

The first evidence is that, despite IONM usage, RLN injury still remains one of the most common complications in thyroid surgery. Multiple studies have failed to demonstrate a reduction of incidence of RLN injury when IONM is used in thyroid surgery; as already discussed, this may be due to the very low incidence of RLN palsy. Surgeon should be conscious that IONM does not prevent at all RLN injury. In fact, stimulation of the nerve can assure integrity of the nerve only after dissection, while nerve palsy can be identified only after the injury has been produced. Thus, IONM should be considered an adjunct, but direct visualization of the nerve and

Although the specificity of IONM in detecting nerve injury is very high (94–99%), a small number of patients will have a vocal cord dysfunction despite

ing identification and dissection of the nerve.

that cauterization is far enough from the nerve.

careful dissection of the tissues are needed.

*Knowledges on Thyroid Cancer*

trodes, or malfunction of the ET tube electrodes.

a dislocation of the vagal nerve electrode.

tion of NMBA during the operation.

**6. Advantages and limits of IONM**

predicting vocal cord function outcome.

**6.1 Advantages of IONM**

functioning, a possible nerve lesion must be considered.

of the recording side are ET tube malposition, displacement of grounding elec-

In addition to laryngeal palpation, contralateral vagal assessment also represents a useful option for troubleshooting. If contralateral vagus nerve stimulation does not elicit an adequate EMG signal, a malfunction of the recording side should be investigated as first option. Differently, if the contralateral vagus nerve is properly

If laryngeal twitch is absent when the nerve is stimulated, a malfunction of the stimulation side should be considered, thus the nerve stimulator probe and monitor have to be checked. Nerve stimulator probe function should be checked by applying its tip directly on a muscle to confirm a muscle twitching. Moreover, the whole system, with special attention focused on the monitor screen, must be fully reviewed. Again, with regard to stimulation side error, if C-IONM is used, LOS may be due to

Finally, in case of LOS, it is of fundamental importance to rule any administra-

Visualization of RLNs is considered the gold standard in thyroid surgery to reduce the incidence of nerve palsy. Nevertheless, visualization of the nerve can only suggest an anatomic integrity, which does not ensure functionality. In fact, some studies have demonstrated that the most common mechanism of nerve injury is traction [29, 32], resulting in a palsy with complete anatomic integrity. Furthermore, direct visualization of the nerve can be difficult, especially in case of revision neck surgery, because of the scar tissue [1, 32–34], in case of anatomic variations of the nerve, during central compartment lymph node dissection, or in course of surgery for advanced thyroid cancer. For these reasons, in the last decades, the use of IONM has widely spread among endocrine surgeons, in order to facilitate identification and dissection of the nerve and to evaluate its functionality,

However, to date, studies have failed to demonstrate a statistically significant reduction of incidence of nerve injury using IONM [2, 3]; this lack of data may be related to the very low rate of nerve palsy. It has been estimated that in prospective, randomized trials, the calculated sample size needed to demonstrate that incidence of palsy is lower with IONM use is about 9000 nerves at risk [1, 7]. Other authors have reported that at least 39,000 nerves at risk per arm should be necessary to achieve statistical power that could demonstrate a significant difference in RLN palsy rate [3]. To date, only one randomized controlled trial [35] has demonstrated

Furthermore, it is important to underline that voice impairment isn't always due to RLN palsy; other causes can be vocal cord damage due to orotracheal intubation and damage of the strap muscles. In these cases, the good surgical practice and the integrity and functionality of the nerve are assessed by IONM, preserving from

• Early identification of RLN and aid in dissection: Stimulation of paratracheal area allows to identify the course of the nerve before it is visualized; the identified area is then carefully dissected until the nerve is satisfactorily exposed.

a significant reduction of transient RLN palsy from 5 to 2.7% (p = 0.007).

Advantages of IONM of RLNs in thyroid surgery are:

**76**

medical claim.

Once the nerve is identified, stimulation of the adjacent structures can help to distinguish the nerve from other non-neural elements, like vessels. IONM is also helpful in identifying neural branches and to clarify nerve course in case of anatomic variants.


#### **6.2 Limits of IONM**

The first evidence is that, despite IONM usage, RLN injury still remains one of the most common complications in thyroid surgery. Multiple studies have failed to demonstrate a reduction of incidence of RLN injury when IONM is used in thyroid surgery; as already discussed, this may be due to the very low incidence of RLN palsy. Surgeon should be conscious that IONM does not prevent at all RLN injury. In fact, stimulation of the nerve can assure integrity of the nerve only after dissection, while nerve palsy can be identified only after the injury has been produced. Thus, IONM should be considered an adjunct, but direct visualization of the nerve and careful dissection of the tissues are needed.

Although the specificity of IONM in detecting nerve injury is very high (94–99%), a small number of patients will have a vocal cord dysfunction despite regular neuromonitoring signal (false-negative IONM). By the other side, a loss of signal (LOS) at the end of procedure is predictive of a postoperative vocal cord paralysis, but positive predictive value ranges widely from 33 to 90%.; this is probably related to the poor uniformity in application of IONM across different centers. It has been reported that preoperative and postoperative laryngoscopy is performed routinely only in 15% of centers who use IONM [36], and that vagal stimulation is not routinely performed in most of the centers [37]. Furthermore, usage of IONM needs a learning curve, a precise knowledge of the components and of the issues that may occur: insufficient experience in managing IONM may result in misleading information that can increase the risk of RLN injury.

The most frequent causes of false negative results are malposition of endotracheal tube, technical problems related to stimulation or registration devices, neuromuscular blocking due to anaesthetic drugs; another cause of false positive result is thought to be transient neuropraxia with rapid recovery before end of surgery.

Thus, low positive predictive value is the main limit of IONM. A low positive predictive value means that, in the worst-case scenario, two out of three patients with LOS will not suffer any alteration of vocal cord motility after surgery. In this regard, a standardization of IONM methods and reporting has been undertaken to provide uniformity and to minimize variations in application of IONM. As already discussed, standardization of IONM should include pre- and postoperative laryngoscopy, stimulation of vagus nerve before dissection and at the end of surgery, and stimulation of RLN when identified and at the end of lobectomy.

#### **7. Two-staged thyroidectomy**

Routine use of IONM in thyroid surgery has led to two-stage operations to prevent bilateral RLN palsy. This approach is defined as removal of the thyroid gland in two different procedures: in the first one, surgery is limited to the main lobe, while the remnant gland is excised in a second intervention. In fact, in case of LOS after excision of the first lobe, the surgeon can evaluate the opportunity to delay removal of the second lobe. Thus, a LOS during first lobectomy should induce to consider timing for contralateral lobectomy. This decision should take into account several elements, including especially thyroid pathology. Over the years, oncologic radicality in case of two-staged thyroidectomy has been a matter of debate; in this regard, we should consider that differentiated thyroid tumors have a good prognosis even in case of local or distant metastases, and that radioablative therapy with iodine-131 can be delayed safely. Thus twostaged thyroidectomy seems to be adequate also in case of thyroid carcinoma [38]; in this case, the endocrinologist can prescribe a TSH-suppressive therapy to reduce the risk of tumor progression of eventually unresected foci of tumor. Alternatively, a near-total lobectomy could be performed on the second side to preserve contralateral RLN. In this scenario, it is necessary to underline once again the importance of a correct standardization of IONM to reduce false positive results; that may lead to an unnecessary two-staged thyroidectomy. In case of thyroid cancer, thyroidectomy should always begin from tumoral side, or, in case of bilateral carcinoma, from the side where the nodule has more aggressive features. In case of two-staged thyroidectomy, contralateral lobectomy should be carefully planned after recovery of vocal cord motility, typically 6–8 weeks after surgery, or, in case of permanent palsy, after demonstration of enough respiratory space.

**79**

**Author details**

provided the original work is properly cited.

*Intraoperative Neuromonitoring in Thyroid Surgery DOI: http://dx.doi.org/10.5772/intechopen.83840*

IONM is a valuable instrument in thyroid surgery. A correct standardization of the method is necessary to reduce the number of false positive results. In case of loss of signal, two-staged thyroidectomy can be safely performed even in case of

The authors declare that there is no conflict of interest.

**8. Conclusions**

malignancy.

**Conflict of interest**

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Fabio Medas\*, Gian Luigi Canu, Enrico Erdas and Pietro Giorgio Department of Surgical Sciences, University of Cagliari, Italy

\*Address all correspondence to: fabiomedas@gmail.com

### **8. Conclusions**

*Knowledges on Thyroid Cancer*

surgery.

lobectomy.

**7. Two-staged thyroidectomy**

regular neuromonitoring signal (false-negative IONM). By the other side, a loss of signal (LOS) at the end of procedure is predictive of a postoperative vocal cord paralysis, but positive predictive value ranges widely from 33 to 90%.; this is probably related to the poor uniformity in application of IONM across different centers. It has been reported that preoperative and postoperative laryngoscopy is performed routinely only in 15% of centers who use IONM [36], and that vagal stimulation is not routinely performed in most of the centers [37]. Furthermore, usage of IONM needs a learning curve, a precise knowledge of the components and of the issues that may occur: insufficient experience in managing IONM may result in mislead-

The most frequent causes of false negative results are malposition of endotracheal tube, technical problems related to stimulation or registration devices, neuromuscular blocking due to anaesthetic drugs; another cause of false positive result is thought to be transient neuropraxia with rapid recovery before end of

Thus, low positive predictive value is the main limit of IONM. A low positive predictive value means that, in the worst-case scenario, two out of three patients with LOS will not suffer any alteration of vocal cord motility after surgery. In this regard, a standardization of IONM methods and reporting has been undertaken to provide uniformity and to minimize variations in application of IONM. As already discussed, standardization of IONM should include pre- and postoperative laryngoscopy, stimulation of vagus nerve before dissection and at the end of surgery, and stimulation of RLN when identified and at the end of

Routine use of IONM in thyroid surgery has led to two-stage operations to prevent bilateral RLN palsy. This approach is defined as removal of the thyroid gland in two different procedures: in the first one, surgery is limited to the main lobe, while the remnant gland is excised in a second intervention. In fact, in case of LOS after excision of the first lobe, the surgeon can evaluate the opportunity to delay removal of the second lobe. Thus, a LOS during first lobectomy should induce to consider timing for contralateral lobectomy. This decision should take into account several elements, including especially thyroid pathology. Over the years, oncologic radicality in case of two-staged thyroidectomy has been a matter of debate; in this regard, we should consider that differentiated thyroid tumors have a good prognosis even in case of local or distant metastases, and that radioablative therapy with iodine-131 can be delayed safely. Thus twostaged thyroidectomy seems to be adequate also in case of thyroid carcinoma [38]; in this case, the endocrinologist can prescribe a TSH-suppressive therapy to reduce the risk of tumor progression of eventually unresected foci of tumor. Alternatively, a near-total lobectomy could be performed on the second side to preserve contralateral RLN. In this scenario, it is necessary to underline once again the importance of a correct standardization of IONM to reduce false positive results; that may lead to an unnecessary two-staged thyroidectomy. In case of thyroid cancer, thyroidectomy should always begin from tumoral side, or, in case of bilateral carcinoma, from the side where the nodule has more aggressive features. In case of two-staged thyroidectomy, contralateral lobectomy should be carefully planned after recovery of vocal cord motility, typically 6–8 weeks after surgery, or, in case of permanent palsy, after demonstration of enough

ing information that can increase the risk of RLN injury.

**78**

respiratory space.

IONM is a valuable instrument in thyroid surgery. A correct standardization of the method is necessary to reduce the number of false positive results. In case of loss of signal, two-staged thyroidectomy can be safely performed even in case of malignancy.

### **Conflict of interest**

The authors declare that there is no conflict of interest.

### **Author details**

Fabio Medas\*, Gian Luigi Canu, Enrico Erdas and Pietro Giorgio Department of Surgical Sciences, University of Cagliari, Italy

\*Address all correspondence to: fabiomedas@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

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[2] Pisanu A, Porceddu G, Podda M, Cois A, Uccheddu A. Systematic review with metaanalysis of studies comparing intraoperative neuromonitoring of recurrent laryngeal nerves versus visualization alone during thyroidectomy. The Journal of Surgical Research. 2014;**188**:152-161. DOI: 10.1016/j.jss.2013.12.022

[3] Deniwar A, Bhatia P, Kandil E. Electrophysiological neuromonitoring of the laryngeal nerves in thyroid and parathyroid surgery: A review. World Journal of Experimental Medicine. 2015;**5**:120-123. DOI: 10.5493/wjem.v5.i2.120

[4] Gurrado A, Bellantone R, Cavallaro G, Citton M, Constantinides V, Conzo G, et al. Can total thyroidectomy be safely performed by residents?: A comparative retrospective multicenter study. Medicine (Baltimore). 2016;**95**:e3241. DOI: 10.1097/ MD.0000000000003241

[5] Goretzki PE, Schwarz K, Brinkmann J, Wirowski D, Lammers BJ. The impact of intraoperative neuromonitoring (IONM) on surgical strategy in bilateral thyroid diseases: Is it worth the effort? World Journal of Surgery. 2010;**34**: 1274-1284. DOI: 10.1007/ s00268-009-0353-3

[6] Jeannon JP, Orabi AA, Bruch GA, Abdalsalm HA, Simo R. Diagnosis of recurrent laryngeal nerve palsy after thyroidectomy: A systematic review. International Journal of Clinical Practice. 2009;**63**:624-629. DOI: 10.1111/j.1742-1241.2008.01875.x

[7] Dralle H, Lorenz K, Machens A. Verdicts on malpractice claims after thyroid surgery: Emerging trends and future directions. Head & Neck. 2012;**34**:1591-1596. DOI: 10.1002/ hed.21970

[8] Rosato L, Avenia N, Bernante P, De Palma M, Gulino G, Nasi PG, et al. Complications of thyroid surgery: Analysis of a multicentric study on 14,934 patients operated on in Italy over 5 years. World Journal of Surgery. 2004;**28**:271-276. DOI: 10.1007/ s00268-003-6903-1

[9] Bergenfelz A, Jansson S, Kristoffersson A, Mårtensson H, Reihnér E, Wallin G, et al. Complications to thyroid surgery: Results as reported in a database from a multicentre audit comprising 3660 patients. Langenbeck's Archives of Surgery. 2008;**393**:667-673. DOI: 10.1007/s00423-008-0366-7

[10] Snyder SK, Lairmore TC, Hendricks JC, Roberts JW. Elucidating mechanisms of recurrent laryngeal nerve injury during thyroidectomy and parathyroidectomy. Journal of the American College of Surgeons. 2008;**206**:123130. DOI: 10.1016/j. jamcollsurg.2007.07.017

[11] Melin M, Schwarz K, Pearson MD, Lammers BJ, Goretzki PE. Postoperative vocal cord dysfunction despite normal intraoeprative neuromonitoring: An unexpected complication with the risk of bilateral palsy. World Journal of Surgery. 2014;**38**:2597-2602. DOI: 10.1007/s00268-014-2591-2

[12] Randolph GW, Dralle H, International Intraoperative Monitoring Study Group, Abdullah H, Barczynski M, Bellantone R, et al. Electrophysiologic recurrent laryngeal nerve monitoring during thyroid and parathyroid surgery: International

**81**

ijsu.2008.12.023

*Intraoperative Neuromonitoring in Thyroid Surgery DOI: http://dx.doi.org/10.5772/intechopen.83840*

> [19] Jonas J, Bähr R. Intraoperative neuromonitoring of the recurrent laryngeal nerve—Results and learning curve. Zentralblatt für Chirurgie. 2006;**131**:443-448. DOI:

10.1055/s-2006-955453

pjs-2015-0005

[20] Pragacz K, Barczyński M. Evaluation of the learning curve for intraoperative neural monitoring of the recurrent laryngeal nerves in thyroid surgery. Polski Przeglad Chirurgiczny.

2015;**86**:584-593. DOI: 10.1515/

[21] Chiang FY, Lee KW, Chen HC, Chen HY, Lu IC, Kuo WR, et al. Standardization of intraoperative neuromonitoring of recurrent laryngeal nerve in thyroid operation. World Journal of Surgery. 2010;**34**:223-229. DOI: 10.1007/s00268-009-0316-8

[22] Dionigi G, Boni L, Rovera F,

[23] Lang BH, Wong KP. Feasibility on the use of intraoperative vagal nerve stimulation in gasless,

transaxillary endoscopic, and roboticassisted thyroidectomy. Journal of Laparoendoscopic & Advanced Surgical Techniques. Part A. 2011;**21**:911-917.

DOI: 10.1089/lap.2011.0204

[24] Dionigi G, Van Slycke S, Boni L, Rausei S, Mangano A. Limits of neuromonitoring in thyroid surgery. Annals of Surgery. 2013;**258**:e1-e2. DOI:

10.1097/SLA.0b013e318294559d

[25] Schneider R, Przybyl J, Pliquett U, Hermann M, Wehner M, Pietsch UC, et al. A new vagal anchor electrode for real-time monitoring of the recurrent laryngeal nerve. American Journal of Surgery. 2010;**199**:507514. DOI: 10.1016/j.amjsurg.2009.04.036

s00464-008-0098-3

Bacuzzi A, Dionigi R. Neuromonitoring and video-assisted thyroidectomy: A prospective, randomized case-control evaluation. Surgical Endoscopy. 2009;**23**:996-1003. DOI: 10.1007/

standards guideline statement. The Laryngoscope. 2011;**121**(S1):Suppl 1-Suppl16. DOI: 10.1002/lary.21119

[13] Durán Poveda M, Dionigi G, Sitges-Serra A, Barczynski M, Angelos P, Dralle H, et al. Intraoperative monitoring of the recurrent laryngeal nerve during thyroidectomy: A standardized approach part 2. World Journal of Endocrine Surgery.

2012;**4**:33-40. DOI: 10.5005/ jp-journals-10002-1091

SEMB.20170216084444

s40136-013-0033-6

1966;**163**:47-50

[15] Dionigi G, Wu CW, Lombardi D, Accorona R, Bozzola A, Kim HY, et al. The current state of recurrent laryngeal nerve monitoring for thyroid surgery. Current Otorhinolaryngology Reports. 2014;**2**:44-54. DOI: 10.1007/

[16] Shedd DP, Durham C. Electrical identification of the recurrent laryngeal nerve. I. Response of the canine larynx to electrical stimulation of the recurrent laryngeal nerve. Annals of Surgery.

[17] Calò PG, Medas F, Gordini L, Podda F, Erdas E, Pisano G, et al. Interpretation of intraoperative recurrent laryngeal nerve monitoring signals: The importance of a correct standardization. International Journal of Surgery. 2016;**28**(Suppl 1):S54-S58.

DOI:10.1016/j.ijsu.2015.12.039

[18] Dionigi G, Bacuzzi A, Boni L, Rovera F, Dionigi R. What is the learning curve for intraoperative neuromonitoring in thyroid surgery? International Journal of Surgery. 2008;**6**(Suppl 1):S7-S12. DOI: 10.1016/j.

[14] Uludag M, Aygun N, Kaya C, Tanal M, Oba S, Isgor A. Basic principles and standardization of intraoperative nerve monitoring in thyroid surgery. The Medical Bulletin of Sisli Etfal Hospital. 2017;**51**:13-25. DOI: 10.5350/

*Intraoperative Neuromonitoring in Thyroid Surgery DOI: http://dx.doi.org/10.5772/intechopen.83840*

standards guideline statement. The Laryngoscope. 2011;**121**(S1):Suppl 1-Suppl16. DOI: 10.1002/lary.21119

[13] Durán Poveda M, Dionigi G, Sitges-Serra A, Barczynski M, Angelos P, Dralle H, et al. Intraoperative monitoring of the recurrent laryngeal nerve during thyroidectomy: A standardized approach part 2. World Journal of Endocrine Surgery. 2012;**4**:33-40. DOI: 10.5005/ jp-journals-10002-1091

[14] Uludag M, Aygun N, Kaya C, Tanal M, Oba S, Isgor A. Basic principles and standardization of intraoperative nerve monitoring in thyroid surgery. The Medical Bulletin of Sisli Etfal Hospital. 2017;**51**:13-25. DOI: 10.5350/ SEMB.20170216084444

[15] Dionigi G, Wu CW, Lombardi D, Accorona R, Bozzola A, Kim HY, et al. The current state of recurrent laryngeal nerve monitoring for thyroid surgery. Current Otorhinolaryngology Reports. 2014;**2**:44-54. DOI: 10.1007/ s40136-013-0033-6

[16] Shedd DP, Durham C. Electrical identification of the recurrent laryngeal nerve. I. Response of the canine larynx to electrical stimulation of the recurrent laryngeal nerve. Annals of Surgery. 1966;**163**:47-50

[17] Calò PG, Medas F, Gordini L, Podda F, Erdas E, Pisano G, et al. Interpretation of intraoperative recurrent laryngeal nerve monitoring signals: The importance of a correct standardization. International Journal of Surgery. 2016;**28**(Suppl 1):S54-S58. DOI:10.1016/j.ijsu.2015.12.039

[18] Dionigi G, Bacuzzi A, Boni L, Rovera F, Dionigi R. What is the learning curve for intraoperative neuromonitoring in thyroid surgery? International Journal of Surgery. 2008;**6**(Suppl 1):S7-S12. DOI: 10.1016/j. ijsu.2008.12.023

[19] Jonas J, Bähr R. Intraoperative neuromonitoring of the recurrent laryngeal nerve—Results and learning curve. Zentralblatt für Chirurgie. 2006;**131**:443-448. DOI: 10.1055/s-2006-955453

[20] Pragacz K, Barczyński M. Evaluation of the learning curve for intraoperative neural monitoring of the recurrent laryngeal nerves in thyroid surgery. Polski Przeglad Chirurgiczny. 2015;**86**:584-593. DOI: 10.1515/ pjs-2015-0005

[21] Chiang FY, Lee KW, Chen HC, Chen HY, Lu IC, Kuo WR, et al. Standardization of intraoperative neuromonitoring of recurrent laryngeal nerve in thyroid operation. World Journal of Surgery. 2010;**34**:223-229. DOI: 10.1007/s00268-009-0316-8

[22] Dionigi G, Boni L, Rovera F, Bacuzzi A, Dionigi R. Neuromonitoring and video-assisted thyroidectomy: A prospective, randomized case-control evaluation. Surgical Endoscopy. 2009;**23**:996-1003. DOI: 10.1007/ s00464-008-0098-3

[23] Lang BH, Wong KP. Feasibility on the use of intraoperative vagal nerve stimulation in gasless, transaxillary endoscopic, and roboticassisted thyroidectomy. Journal of Laparoendoscopic & Advanced Surgical Techniques. Part A. 2011;**21**:911-917. DOI: 10.1089/lap.2011.0204

[24] Dionigi G, Van Slycke S, Boni L, Rausei S, Mangano A. Limits of neuromonitoring in thyroid surgery. Annals of Surgery. 2013;**258**:e1-e2. DOI: 10.1097/SLA.0b013e318294559d

[25] Schneider R, Przybyl J, Pliquett U, Hermann M, Wehner M, Pietsch UC, et al. A new vagal anchor electrode for real-time monitoring of the recurrent laryngeal nerve. American Journal of Surgery. 2010;**199**:507514. DOI: 10.1016/j.amjsurg.2009.04.036

**80**

*Knowledges on Thyroid Cancer*

**References**

Electrophysiological neural monitoring of the laryngeal nerves in thyroid surgery: Review of the current literature. Gland Surgery. 2015;**4**:368-375. DOI: 10.3978/j. issn.2227-684X.2015.04.04

[1] Deniwar A, Kandil E, Randolph G.

[7] Dralle H, Lorenz K, Machens A. Verdicts on malpractice claims after thyroid surgery: Emerging trends and future directions. Head & Neck. 2012;**34**:1591-1596. DOI: 10.1002/

[8] Rosato L, Avenia N, Bernante P, De Palma M, Gulino G, Nasi PG, et al. Complications of thyroid surgery: Analysis of a multicentric study on 14,934 patients operated on in Italy over 5 years. World Journal of Surgery.

2004;**28**:271-276. DOI: 10.1007/

[9] Bergenfelz A, Jansson S, Kristoffersson A, Mårtensson H, Reihnér E, Wallin G, et al. Complications to thyroid surgery: Results as reported in a database from a multicentre audit comprising 3660 patients. Langenbeck's Archives of Surgery. 2008;**393**:667-673. DOI: 10.1007/s00423-008-0366-7

[10] Snyder SK, Lairmore TC,

jamcollsurg.2007.07.017

10.1007/s00268-014-2591-2

[12] Randolph GW, Dralle H, International Intraoperative Monitoring Study Group, Abdullah H, Barczynski M, Bellantone R, et al. Electrophysiologic recurrent laryngeal nerve monitoring during thyroid and parathyroid surgery: International

Hendricks JC, Roberts JW. Elucidating mechanisms of recurrent laryngeal nerve injury during thyroidectomy and parathyroidectomy. Journal of the American College of Surgeons. 2008;**206**:123130. DOI: 10.1016/j.

[11] Melin M, Schwarz K, Pearson MD, Lammers BJ, Goretzki PE. Postoperative vocal cord dysfunction despite normal intraoeprative neuromonitoring: An unexpected complication with the risk of bilateral palsy. World Journal of Surgery. 2014;**38**:2597-2602. DOI:

s00268-003-6903-1

hed.21970

[2] Pisanu A, Porceddu G, Podda M, Cois A, Uccheddu A. Systematic review with metaanalysis of studies comparing

intraoperative neuromonitoring of recurrent laryngeal nerves versus visualization alone during thyroidectomy. The Journal of Surgical Research. 2014;**188**:152-161. DOI:

10.1016/j.jss.2013.12.022

[3] Deniwar A, Bhatia P, Kandil E. Electrophysiological neuromonitoring of the laryngeal nerves in thyroid and parathyroid surgery: A review. World Journal of Experimental Medicine. 2015;**5**:120-123.

DOI: 10.5493/wjem.v5.i2.120

study. Medicine (Baltimore). 2016;**95**:e3241. DOI: 10.1097/ MD.0000000000003241

1274-1284. DOI: 10.1007/ s00268-009-0353-3

[4] Gurrado A, Bellantone R, Cavallaro G, Citton M, Constantinides V, Conzo G, et al. Can total thyroidectomy be safely performed by residents?: A comparative retrospective multicenter

[5] Goretzki PE, Schwarz K, Brinkmann J, Wirowski D, Lammers BJ. The impact of intraoperative neuromonitoring (IONM) on surgical strategy in bilateral thyroid diseases: Is it worth the effort? World Journal of Surgery. 2010;**34**:

[6] Jeannon JP, Orabi AA, Bruch GA, Abdalsalm HA, Simo R. Diagnosis of recurrent laryngeal nerve palsy after thyroidectomy: A systematic review. International Journal of Clinical Practice. 2009;**63**:624-629. DOI: 10.1111/j.1742-1241.2008.01875.x

[26] Schneider R, Bures C, Lorenz K, Dralle H, Freissmuth M, Hermann M. Evolution of nerve injury with unexpected EMG signal recovery in thyroid surgery using continuous intraoperative neuromonitoring. World Journal of Surgery. 2013;**37**:364-368. DOI: 10.1007/s00268-012-1853-0

[27] Lamadé W, Ulmer C, Friedrich C, Rieber F, Schymik K, Gemkow HM, et al. Signal stability as key requirement for continuous intraoperative neuromonitoring. Der Chirurg. 2011;**82**:913-920. DOI: 10.1007/ s00104-011-2080-1

[28] Schneider R, Randolph GW, Sekulla C, Phelan E, Thanh PN, Bucher M, et al. Continuous intraoperative vagus nerve stimulation for identification of imminent recurrent laryngeal nerve injury. Head & Neck. 2013;**35**: 1591-1598. DOI: 10.1002/hed.23187

[29] Dionigi G, Wu CW, Kim HY, Rausei S, Boni L, Chiang FY. Severity of recurrent laryngeal nerve injuries in thyroid surgery. World Journal of Surgery. 2016;**40**:1373-1381. DOI: 10.1007/s00268-016-3415-3

[30] Randolph GW, Kobler JB, Wilkins J. Recurrent laryngeal nerve identification and assessment during thyroid surgery: Laryngeal palpation. World Journal of Surgery. 2004;**28**:755-760. DOI: 10.1007/ s00268-004-7348-x

[31] Tomoda C, Hirokawa Y, Uruno T, Takamura Y, Ito Y, Miya A, et al. Sensitivity and specificity of intraoperative recurrent laryngeal nerve stimulation test for predicting vocal cord palsy after thyroid surgery. World Journal of Surgery. 2006;**30**:1230-1233. DOI: 10.1007/s00268-005-0351-z

[32] Calò PG, Pisano G, Medas F, Marcialis J, Gordini L, Erdas E, et al. Total thyroidectomy without prophylactic central neck dissection in clinically node-negative papillary

thyroid cancer: Is it an adequate treatment? World Journal of Surgical Oncology. 2014;**12**:152. DOI: 10.1186/1477-7819-12-152

Chapter 7

Abstract

Genetic Alterations of RET:

Syed Mudassar, Mosin S. Khan, Shariq R. Masoodi,

Mahboob Ul Hussain and Khurshid I. Andrabi

penetrance alleles for predisposition of thyroid cancer.

papillary thyroid cancer, follicular thyroid cancer

1. Introduction

83

Correlations in Thyroid

Carcinogenesis

Possible Implications and Clinical

Thyroid cancers are malignant tumors in the thyroid gland. DNA polymorphisms are playing a decisive role in unscrambling the genomic basis of tumor formation and development in cancer. Thyroid cancer is influenced in a polygenic and low-penetrance manner by RET gene polymorphisms and this part of the world (North India) has not recorded any study regarding RET alterations in this very cancer. We assessed RET G691S (rs1799939), L769L (rs1800861) and S904S (rs1800863) polymorphisms by restriction fragment length polymorphism (RFLP) in order to explain their potential role in the diagnosis and prognosis of Papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). In RET G691S polymorphism, the total dissemination of variant alleles (GA + AA) was 62.9% in cases as related to 44.5% in controls (P < 0.05). RET L769L variant alleles (TG + GG) was 70% in cases versus 88% in controls (P < 0.05). In RET S904S, occurrence of variant alleles (CG + GG) was 56% in cases versus 44% in controls (P < 0.05). G691S and L769L polymorphism advocate a "Dominant mode of inheritance". The S904S polymorphism approves an "Additive mode of inheritance". In conclusion, there was an over-representation of RET G691S/S904S polymorphisms and underrepresentation of L769L polymorphism in PTC and FTC patients. Additionally, our data suggest that some haplotypes (A T G, G T G and A T C) of RET may act as low

Keywords: thyroid cancer, rearranged during transfection, RET, polymorphism,

Cancer is a large group of diseases that vary in their age of onset, rate of growth, state of cellular differentiation, diagnostic detectability, invasiveness, metastatic potential, response to treatment, and prognosis. However, cancer may be a relatively small number of diseases caused by similar molecular defects in cell function

[33] Calò PG, Pisano G, Medas F, Tatti A, Tuveri M, Nicolosi A. Risk factors in reoperative thyroid surgery for recurrent goiter: Our experience. Il Giornale di Chirurgia. 2012;**33**:335-338

[34] Pironi D, Pontone S, Vendettuoli M, Podzemny V, Mascagni D, Arcieri S, et al. Prevention of complications during reoperative thyroid surgery. La Clinica Terapeutica. 2014;**165**:e285-e290. DOI: 10.7417/ CT.2014.1744

[35] Barczynski M, Konturek A, Cichon S. Randomized clinical trial of visualization versus neuromonitoring of recurrent laryngeal nerves during thyroidectomy. The British Journal of Surgery. 2009;**96**:240-246. DOI: 10.1002/bjs.6417

[36] Dionigi G, Tanda ML, Piantanida E, Boni L, Rovera F, Dionigi R, et al. Time interval in diagnosis and treatment of papillary thyroid cancer: A descriptive, retrospective study. American Journal of Surgery. 2009;**197**:434-438. DOI: 10.1016/j.amjsurg.2008.01.031

[37] Dionigi G, Bacuzzi A, Barczynski M, Biondi A, Boni L, Chiang FY, et al. Implementation of systematic neuromonitoring training for thyroid surgery. Updates in Surgery. 2011;**63**:201-207. DOI: 10.1007/ s13304-011-0098-z

[38] Calò PG, Medas F, Conzo G, Podda F, Canu GL, Gambardella C, et al. Intraoperative neuromonitoring in thyroid surgery: Is the two-staged thyroidectomy justified? International Journal of Surgery. 2017;**41**(Suppl 1): S13-S20. DOI: 10.1016/j.ijsu.2017.02.001

#### Chapter 7

*Knowledges on Thyroid Cancer*

[26] Schneider R, Bures C, Lorenz K, Dralle H, Freissmuth M, Hermann M. Evolution of nerve injury with unexpected EMG signal recovery in thyroid surgery using continuous intraoperative neuromonitoring. World Journal of Surgery. 2013;**37**:364-368. DOI: 10.1007/s00268-012-1853-0

thyroid cancer: Is it an adequate treatment? World Journal of Surgical

[33] Calò PG, Pisano G, Medas F, Tatti A, Tuveri M, Nicolosi A. Risk factors in reoperative thyroid surgery for recurrent goiter: Our experience. Il Giornale di Chirurgia. 2012;**33**:335-338

[34] Pironi D, Pontone S, Vendettuoli M, Podzemny V, Mascagni D, Arcieri S, et al. Prevention of complications

during reoperative thyroid surgery. La Clinica Terapeutica. 2014;**165**:e285-e290. DOI: 10.7417/

[35] Barczynski M, Konturek A, Cichon S. Randomized clinical trial of visualization versus neuromonitoring of recurrent laryngeal nerves during thyroidectomy. The British Journal of Surgery. 2009;**96**:240-246. DOI:

[36] Dionigi G, Tanda ML, Piantanida E, Boni L, Rovera F, Dionigi R, et al. Time interval in diagnosis and treatment of papillary thyroid cancer: A descriptive, retrospective study. American Journal of Surgery. 2009;**197**:434-438. DOI: 10.1016/j.amjsurg.2008.01.031

[37] Dionigi G, Bacuzzi A, Barczynski M, Biondi A, Boni L, Chiang FY, et al. Implementation of systematic neuromonitoring training for thyroid surgery. Updates in Surgery. 2011;**63**:201-207. DOI: 10.1007/

[38] Calò PG, Medas F, Conzo G, Podda F, Canu GL, Gambardella C, et al. Intraoperative neuromonitoring in thyroid surgery: Is the two-staged thyroidectomy justified? International Journal of Surgery. 2017;**41**(Suppl 1): S13-S20. DOI: 10.1016/j.ijsu.2017.02.001

CT.2014.1744

10.1002/bjs.6417

s13304-011-0098-z

Oncology. 2014;**12**:152. DOI: 10.1186/1477-7819-12-152

[27] Lamadé W, Ulmer C, Friedrich C, Rieber F, Schymik K, Gemkow HM, et al. Signal stability as key requirement

[28] Schneider R, Randolph GW, Sekulla C, Phelan E, Thanh PN, Bucher M, et al. Continuous intraoperative vagus nerve stimulation for identification of imminent recurrent laryngeal nerve injury. Head & Neck. 2013;**35**: 1591-1598. DOI: 10.1002/hed.23187

[29] Dionigi G, Wu CW, Kim HY, Rausei S, Boni L, Chiang FY. Severity of recurrent laryngeal nerve injuries in thyroid surgery. World Journal of Surgery. 2016;**40**:1373-1381. DOI: 10.1007/s00268-016-3415-3

[30] Randolph GW, Kobler JB, Wilkins J. Recurrent laryngeal nerve identification and assessment during thyroid surgery: Laryngeal palpation. World Journal of Surgery. 2004;**28**:755-760. DOI: 10.1007/

[31] Tomoda C, Hirokawa Y, Uruno T, Takamura Y, Ito Y, Miya A, et al. Sensitivity and specificity of intraoperative recurrent laryngeal nerve stimulation test for predicting vocal cord palsy after thyroid surgery. World Journal of Surgery. 2006;**30**:1230-1233. DOI: 10.1007/s00268-005-0351-z

[32] Calò PG, Pisano G, Medas F, Marcialis J, Gordini L, Erdas E, et al. Total thyroidectomy without prophylactic central neck dissection in clinically node-negative papillary

for continuous intraoperative neuromonitoring. Der Chirurg. 2011;**82**:913-920. DOI: 10.1007/

s00104-011-2080-1

s00268-004-7348-x

**82**

## Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid Carcinogenesis

Syed Mudassar, Mosin S. Khan, Shariq R. Masoodi, Mahboob Ul Hussain and Khurshid I. Andrabi

#### Abstract

Thyroid cancers are malignant tumors in the thyroid gland. DNA polymorphisms are playing a decisive role in unscrambling the genomic basis of tumor formation and development in cancer. Thyroid cancer is influenced in a polygenic and low-penetrance manner by RET gene polymorphisms and this part of the world (North India) has not recorded any study regarding RET alterations in this very cancer. We assessed RET G691S (rs1799939), L769L (rs1800861) and S904S (rs1800863) polymorphisms by restriction fragment length polymorphism (RFLP) in order to explain their potential role in the diagnosis and prognosis of Papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). In RET G691S polymorphism, the total dissemination of variant alleles (GA + AA) was 62.9% in cases as related to 44.5% in controls (P < 0.05). RET L769L variant alleles (TG + GG) was 70% in cases versus 88% in controls (P < 0.05). In RET S904S, occurrence of variant alleles (CG + GG) was 56% in cases versus 44% in controls (P < 0.05). G691S and L769L polymorphism advocate a "Dominant mode of inheritance". The S904S polymorphism approves an "Additive mode of inheritance". In conclusion, there was an over-representation of RET G691S/S904S polymorphisms and underrepresentation of L769L polymorphism in PTC and FTC patients. Additionally, our data suggest that some haplotypes (A T G, G T G and A T C) of RET may act as low penetrance alleles for predisposition of thyroid cancer.

Keywords: thyroid cancer, rearranged during transfection, RET, polymorphism, papillary thyroid cancer, follicular thyroid cancer

#### 1. Introduction

Cancer is a large group of diseases that vary in their age of onset, rate of growth, state of cellular differentiation, diagnostic detectability, invasiveness, metastatic potential, response to treatment, and prognosis. However, cancer may be a relatively small number of diseases caused by similar molecular defects in cell function

resulting from common types of alterations to a cell's genes as per molecular and cell biological point of view. Ultimately, abnormal gene expression causes cancer which may occur due to mutation, translocation, amplification, deletion, loss of heterozygosity, etc. Cell replication and cell death in a tumor cell population gets imbalanced leading to an expansion of tumor tissue [1, 2].

of association of various SNPs G691S (G2071A), L769L (T2307G) and S904S

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid…

importance through visualizing thousands of DNA polymorphisms.

The ability to visualize sequence differences directly in DNA is one of the most important tools underlying the revolution in medical genetics. Polymorphisms are these differences in DNA sequences when studied in the context of a population which may be present in exons (coding regions) or introns (noncoding) regions of genes. Family studies have been undergone by studying the genes of medical

Characteristically, polymorphisms denote sequence variations which confer no deleterious effects and are present in the general population. However, as molecular epidemiological studies were performed and the human genome project was deciphered it became vibrant that some "polymorphisms" were not entirely harmless. Genes for many disorders with a clear pattern of Mendelian inheritance were located and identified by this technique, such as, muscular dystrophies, cystic fibrosis and neurodegenerative disorders and This technique also assists in finding genes that predispose people to diseases in which inheritance patterns are complex, such as diabetes, atherosclerosis, and hypertension. These polymorphisms are crucial in the identification of genes important for susceptibility to common cancers, such as colon cancer, as well as susceptibility to less common childhood tumors, such as retinoblastoma and Wilms' tumor [23]. Over 3.1 million sequence variations have been mapped in the human genome in which, 25–35% of the total estimated SNPs are present [15, 24]. Individual susceptibility is likely due to genetic factors modulating the environmental risk otherwise differentiated thyroid cancer is described by a strong hereditability, hence, the identification of genetic variations is important for understanding the possible mechanisms tangled in thyroid carcinogenesis. In thyroid cancer many single nucleotide polymorphisms have been reported in different genes and functional analysis of many single nucleotide polymorphisms have been carried out. It has been reported that these DNA polymorphisms in various genes predispose a person to higher risk of thyroid cancer and also has a marked effect on various clinicopathological characteristics of thyroid

Located on chromosome 10q11.2 near the centromere, the RET gene includes 21 exons. RET (rearranged during transfection) was first identified by Takahashi et al. in 1985 as a proto-oncogene that can undergo activation by cytogenic rearrangement [25]. RET gene was cloned by the same investigators 3 years later [26]. This gene encodes the RET receptor, a plasma membrane-bound tyrosine kinase enzyme. RET gene is expressed by neuroendocrine and neural cells, including parasympathetic, sympathetic and colonic ganglia, cells of the urogenital tract, thyroid C cells, adrenal medullary cells and parathyroid cells derived from branchial arches [27, 28]. The RET protein contains two intracellular tyrosine kinase domains, a transmembrane domain, an extracellular region (four cadherin-like repeats, a calcium binding site, and a cysteine-rich domain) and N terminal signal peptide. The cysteine-rich extracellular domain is central for receptor dimerization,

(C2712G) of RET gene in thyroid cancer.

DOI: http://dx.doi.org/10.5772/intechopen.86902

cancer patients [23].

85

3. Structure and biology of RET receptor

2. DNA polymorphisms in thyroid cancer

The most common malignancy of the endocrine system is Thyroid cancer. 2% of all diagnosed cancer cases and majority of endocrine cancer related deaths each year are due to this cancer type [3–5].

The rearranged during transfection (RET) proto-oncogene expressed in cells of neural crest origin and encodes a membrane tyrosine-kinase receptor [6]. However, thyroid follicular cells may also express the RET tyrosine kinase domain [7]. Somatic RET translocations were found in some sporadic and radiation-induced PTCs. Papillary thyroid cancer contains RET/PTC chromosomal rearrangement [8]. In this gene rearrangement, a portion of the RET gene is fused to one of several genes. RET/PTC1 and RET/PTC3 are the most common rearrangement types in which RET is fused to CCDC6 (also known as H4) or NCOA4 (also known as ELE1 or RFG) respectively [9, 10]. RET tyrosine kinase domain gets constitutively activated due to RET rearrangement that can lead to PTC [11, 12]. 10–20% of sporadic PTC's contain RET/PTC rearrangements. Patients with the history of radiation exposure (50–80%), PTC's from children and young adults (40–70%) have higher frequency of RET/PTC rearrangements [13, 14].

Genetic basis of tumor formation and cancer progression is being unraveled by DNA polymorphisms. The human genome as a whole (in which over 3.1 million sequence variations have been mapped), represent 25–35% of the total estimated SNPs [15]. Predisposition to several human cancers is due to polymorphisms. Apart from RET rearrangements, the coding sequence of RET display polymorphisms in exon 2 (G135A, A45A), in exon 7 (G1296A, A432A), in exon 11 (G2071A, G691S), in exon 13 (T2307G, L769L), in exon 14 (C2508T, S836S), and in exon 15 (C2712G, S904S) [16, 17]. Etiology of sporadic Hirschsprung disease (HSCR) and MTC has been associated with RET polymorphisms [18–20]. Silent RET polymorphisms; A45A in exon 2 and L769L in exon 13 may represent low-penetrance risk in PTC [20]. Risk of differentiated thyroid cancer with reference to RET Polymorphic Haplotypes was also reported by some studies [21]. The mechanisms by which the silent polymorphisms may act in the development of cancer include transcript stability, RNA splicing, and DNA protein binding and protein folding.

The valley of Kashmir is one of the divisions of Jammu and Kashmir State, situated in the Himalayas. Kashmir, regarded worldwide as paradise on earth, with over 07 million populations is heavily burdened with different organ cancers. In Kashmir valley where incidences of almost all types of organ cancers have shown a drastic increase in last couple of decades particularly GIT and lung cancers, the thyroid cancer figures no less in this deadly race. Thyroid cancer is the 8th most common cancer and 7th most common cancer among women in Kashmir valley. The frequency of thyroid cancer has increased from 2.3% in 1995 to 5.4% in 2010 in Kashmir valley [22]. Also, owing to the fact that there is no data on genetic alterations in thyroid cancer available in our population and given the backdrop of a significant presence of thyroid cancer patients, it is the first initiative to study the gene alterations in Thyroid cancer patients of Kashmir Valley. In view of these observations this study was designed to address the disease pathology associated with the thyroid cancer through the Polymorphic analysis of RET gene SNPs— G691S (G2071A), L769L (T2307G) and S904S (C2712G) in order to observe pattern Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid… DOI: http://dx.doi.org/10.5772/intechopen.86902

of association of various SNPs G691S (G2071A), L769L (T2307G) and S904S (C2712G) of RET gene in thyroid cancer.

#### 2. DNA polymorphisms in thyroid cancer

resulting from common types of alterations to a cell's genes as per molecular and cell biological point of view. Ultimately, abnormal gene expression causes cancer which may occur due to mutation, translocation, amplification, deletion, loss of heterozygosity, etc. Cell replication and cell death in a tumor cell population gets imbalanced

The most common malignancy of the endocrine system is Thyroid cancer. 2% of all diagnosed cancer cases and majority of endocrine cancer related deaths each year

The rearranged during transfection (RET) proto-oncogene expressed in cells of neural crest origin and encodes a membrane tyrosine-kinase receptor [6]. However, thyroid follicular cells may also express the RET tyrosine kinase domain [7]. Somatic RET translocations were found in some sporadic and radiation-induced PTCs. Papillary thyroid cancer contains RET/PTC chromosomal rearrangement [8]. In this gene rearrangement, a portion of the RET gene is fused to one of several genes. RET/PTC1 and RET/PTC3 are the most common rearrangement types in which RET is fused to CCDC6 (also known as H4) or NCOA4 (also known as ELE1 or RFG) respectively [9, 10]. RET tyrosine kinase domain gets constitutively activated due to RET rearrangement that can lead to PTC [11, 12]. 10–20% of sporadic PTC's contain RET/PTC rearrangements. Patients with the history of radiation exposure (50–80%), PTC's from children and young adults (40–70%) have higher

Genetic basis of tumor formation and cancer progression is being unraveled by DNA polymorphisms. The human genome as a whole (in which over 3.1 million sequence variations have been mapped), represent 25–35% of the total estimated SNPs [15]. Predisposition to several human cancers is due to polymorphisms. Apart from RET rearrangements, the coding sequence of RET display polymorphisms in exon 2 (G135A, A45A), in exon 7 (G1296A, A432A), in exon 11

(G2071A, G691S), in exon 13 (T2307G, L769L), in exon 14 (C2508T, S836S), and in exon 15 (C2712G, S904S) [16, 17]. Etiology of sporadic Hirschsprung disease (HSCR) and MTC has been associated with RET polymorphisms [18–20]. Silent RET polymorphisms; A45A in exon 2 and L769L in exon 13 may represent low-penetrance risk in PTC [20]. Risk of differentiated thyroid cancer with reference to RET Polymorphic Haplotypes was also reported by some studies [21]. The mechanisms by which the silent polymorphisms may act in the development of cancer include transcript stability, RNA splicing, and DNA protein binding

The valley of Kashmir is one of the divisions of Jammu and Kashmir State, situated in the Himalayas. Kashmir, regarded worldwide as paradise on earth, with over 07 million populations is heavily burdened with different organ cancers. In Kashmir valley where incidences of almost all types of organ cancers have shown a drastic increase in last couple of decades particularly GIT and lung cancers, the thyroid cancer figures no less in this deadly race. Thyroid cancer is the 8th most common cancer and 7th most common cancer among women in Kashmir valley. The frequency of thyroid cancer has increased from 2.3% in 1995 to 5.4% in 2010 in Kashmir valley [22]. Also, owing to the fact that there is no data on genetic alterations in thyroid cancer available in our population and given the backdrop of a significant presence of thyroid cancer patients, it is the first initiative to study the gene alterations in Thyroid cancer patients of Kashmir Valley. In view of these observations this study was designed to address the disease pathology associated with the thyroid cancer through the Polymorphic analysis of RET gene SNPs— G691S (G2071A), L769L (T2307G) and S904S (C2712G) in order to observe pattern

leading to an expansion of tumor tissue [1, 2].

frequency of RET/PTC rearrangements [13, 14].

are due to this cancer type [3–5].

Knowledges on Thyroid Cancer

and protein folding.

84

The ability to visualize sequence differences directly in DNA is one of the most important tools underlying the revolution in medical genetics. Polymorphisms are these differences in DNA sequences when studied in the context of a population which may be present in exons (coding regions) or introns (noncoding) regions of genes. Family studies have been undergone by studying the genes of medical importance through visualizing thousands of DNA polymorphisms.

Characteristically, polymorphisms denote sequence variations which confer no deleterious effects and are present in the general population. However, as molecular epidemiological studies were performed and the human genome project was deciphered it became vibrant that some "polymorphisms" were not entirely harmless. Genes for many disorders with a clear pattern of Mendelian inheritance were located and identified by this technique, such as, muscular dystrophies, cystic fibrosis and neurodegenerative disorders and This technique also assists in finding genes that predispose people to diseases in which inheritance patterns are complex, such as diabetes, atherosclerosis, and hypertension. These polymorphisms are crucial in the identification of genes important for susceptibility to common cancers, such as colon cancer, as well as susceptibility to less common childhood tumors, such as retinoblastoma and Wilms' tumor [23]. Over 3.1 million sequence variations have been mapped in the human genome in which, 25–35% of the total estimated SNPs are present [15, 24]. Individual susceptibility is likely due to genetic factors modulating the environmental risk otherwise differentiated thyroid cancer is described by a strong hereditability, hence, the identification of genetic variations is important for understanding the possible mechanisms tangled in thyroid carcinogenesis. In thyroid cancer many single nucleotide polymorphisms have been reported in different genes and functional analysis of many single nucleotide polymorphisms have been carried out. It has been reported that these DNA polymorphisms in various genes predispose a person to higher risk of thyroid cancer and also has a marked effect on various clinicopathological characteristics of thyroid cancer patients [23].

#### 3. Structure and biology of RET receptor

Located on chromosome 10q11.2 near the centromere, the RET gene includes 21 exons. RET (rearranged during transfection) was first identified by Takahashi et al. in 1985 as a proto-oncogene that can undergo activation by cytogenic rearrangement [25]. RET gene was cloned by the same investigators 3 years later [26]. This gene encodes the RET receptor, a plasma membrane-bound tyrosine kinase enzyme. RET gene is expressed by neuroendocrine and neural cells, including parasympathetic, sympathetic and colonic ganglia, cells of the urogenital tract, thyroid C cells, adrenal medullary cells and parathyroid cells derived from branchial arches [27, 28]. The RET protein contains two intracellular tyrosine kinase domains, a transmembrane domain, an extracellular region (four cadherin-like repeats, a calcium binding site, and a cysteine-rich domain) and N terminal signal peptide. The cysteine-rich extracellular domain is central for receptor dimerization,

whereas, the extracellular cadherin-like domains are key for cell-cell signaling. The RET C-terminal tail shows three splicing variants producing three protein isoforms with 9 (RET9; short isoform), 43 (RET43; middle isoform), or 51 (RET51; long

isoform) distinct amino acids at their C termini [29, 30] (Figure 1). Four ligands for the RET receptor have been recognized so far [31, 32]. These ligands are persephin, artemin, neurturin and the glial cell line-derived neurotrophic factor (GDNF) [33, 34].

RET polymorphisms are might be associated with an increased relative risk for the development of disorders derived from neural crest cells and are believed to be genetic modifiers. High penetrant germline RET mutations have a key role in disease development. Various disease phenotypes have relatively strong association

nonsynonymous variant in exon 11 (G691S; G2071A) and the synonymous variants in exon 13 (L767L; T2307G), in exon 14 (S836S; C2508T) and in exon 15 (S904S; C2712G), are being denoted as disease modifiers due to their presence in patients with sporadic MTC and DTC [35–37]. Disease-associated germline mutations might interact with these polymorphisms or other genetic variants, tempering the age of onset or disease phenotype. Polymorphisms could bestow a much higher attributable risk on the general population as compared with rare mutations in highpenetrance RET gene. G691S and S904S polymorphisms of RET have a transformer effect on the age of onset of MEN2A [38]. Sporadic MTC has been associated with several RET polymorphisms [39]. A low-penetrance RET haplotype comprising the wild-type allele at IVS1-126 and IVS1-1463 and a 16-basepair intron-1 deletion of these SNPs is strongly associated with and over represented in sporadic pheochromocytoma [40]. Hirschsprung disease has disease associated polymorphisms linked to it [41, 42]. Two closely located SNPs, rs2435357 and rs2506004, in intron 1 has been observed by two groups as disease-causing candidates on the basis of comparative genomics, functional assays and association studies [43, 44]. G691S and S904S linkage have been proposed previously [45]. G691S/S904S cosegregated together as haplotype (P < 0.001) in one of the studies, proposing that these polymorphisms

5. RET polymorphisms and haplotypes in differentiated thyroid cancers

Presence of RET ligands in this microenvironment is highly reasonable because C cells express the RET receptor. Follicular cell derived thyroid cancers contain RET mRNA which may be activated in the presence of specific ligands [7]. So the role of RET gene polymorphism in differentiated thyroid cancers came into existence. Some variants within RET could represent low penetrant alleles for the PTC phenotype. A study revealed the toughest association of A45A (G135A) and L769L (T2307G) with PTC [20]. Borrego et al. described the seven most frequent haplotypes in cases and controls and some of which differed in their distribution [19]. The G G C C haplotype is over-represented in both populations of sporadic PTC.

Follicular and parafollicular-type C cells were shown to have some interconnections. The MTC cells provide the microenvironment that has the capacity to stimulate the proliferation of follicular cells, resulting in hyperplastic and adenomatous follicles which could ultimately acquire a fully developed neoplastic phenotype. Some patients with Hashimoto thyroiditis had C-cell hyperplasia [47, 48]. RET receptor has been shown to express in thyroid follicular component,

which may be activated in the existence of precise ligands in the thyroid

Figure 2 summarizes various RET mutations along with disease phenotype.

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid…

with RET polymorphisms. The most common RET polymorphisms; the

4. Polymorphisms and haplotypes in RET

DOI: http://dx.doi.org/10.5772/intechopen.86902

are in linkage disequilibrium with each other [46].

microenvironment.

87

#### Figure 1.

The RET protein, its functional domains, ligands and co-receptors. Left, functional domains of the three RET isoforms. Right, canonical (unbroken lines) and noncanonical (broken lines) interactions of the RET ligands GDNF, neurturin (NRTN), persephin (PSPN) and artemin (ARTN) with their GFRa co-receptors. Lipid rafts are depicted as a purple box in the plasma membrane.

#### Figure 2.

Germline missense mutations in RET associated with MEN2 and Hirschsprung disease (HSCR). Shown are the structure of the RET mRNA and protein. The codons mutated, the associated clinical entities, and the location of these mutations in relation to the exons and structural domains are indicated.

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid… DOI: http://dx.doi.org/10.5772/intechopen.86902

isoform) distinct amino acids at their C termini [29, 30] (Figure 1). Four ligands for the RET receptor have been recognized so far [31, 32]. These ligands are persephin, artemin, neurturin and the glial cell line-derived neurotrophic factor (GDNF) [33, 34]. Figure 2 summarizes various RET mutations along with disease phenotype.

#### 4. Polymorphisms and haplotypes in RET

whereas, the extracellular cadherin-like domains are key for cell-cell signaling. The RET C-terminal tail shows three splicing variants producing three protein isoforms with 9 (RET9; short isoform), 43 (RET43; middle isoform), or 51 (RET51; long

The RET protein, its functional domains, ligands and co-receptors. Left, functional domains of the three RET isoforms. Right, canonical (unbroken lines) and noncanonical (broken lines) interactions of the RET ligands GDNF, neurturin (NRTN), persephin (PSPN) and artemin (ARTN) with their GFRa co-receptors. Lipid

Germline missense mutations in RET associated with MEN2 and Hirschsprung disease (HSCR). Shown are the structure of the RET mRNA and protein. The codons mutated, the associated clinical entities, and the location of

these mutations in relation to the exons and structural domains are indicated.

rafts are depicted as a purple box in the plasma membrane.

Figure 1.

Knowledges on Thyroid Cancer

Figure 2.

86

RET polymorphisms are might be associated with an increased relative risk for the development of disorders derived from neural crest cells and are believed to be genetic modifiers. High penetrant germline RET mutations have a key role in disease development. Various disease phenotypes have relatively strong association with RET polymorphisms. The most common RET polymorphisms; the nonsynonymous variant in exon 11 (G691S; G2071A) and the synonymous variants in exon 13 (L767L; T2307G), in exon 14 (S836S; C2508T) and in exon 15 (S904S; C2712G), are being denoted as disease modifiers due to their presence in patients with sporadic MTC and DTC [35–37]. Disease-associated germline mutations might interact with these polymorphisms or other genetic variants, tempering the age of onset or disease phenotype. Polymorphisms could bestow a much higher attributable risk on the general population as compared with rare mutations in highpenetrance RET gene. G691S and S904S polymorphisms of RET have a transformer effect on the age of onset of MEN2A [38]. Sporadic MTC has been associated with several RET polymorphisms [39]. A low-penetrance RET haplotype comprising the wild-type allele at IVS1-126 and IVS1-1463 and a 16-basepair intron-1 deletion of these SNPs is strongly associated with and over represented in sporadic pheochromocytoma [40]. Hirschsprung disease has disease associated polymorphisms linked to it [41, 42]. Two closely located SNPs, rs2435357 and rs2506004, in intron 1 has been observed by two groups as disease-causing candidates on the basis of comparative genomics, functional assays and association studies [43, 44]. G691S and S904S linkage have been proposed previously [45]. G691S/S904S cosegregated together as haplotype (P < 0.001) in one of the studies, proposing that these polymorphisms are in linkage disequilibrium with each other [46].

#### 5. RET polymorphisms and haplotypes in differentiated thyroid cancers

Follicular and parafollicular-type C cells were shown to have some interconnections. The MTC cells provide the microenvironment that has the capacity to stimulate the proliferation of follicular cells, resulting in hyperplastic and adenomatous follicles which could ultimately acquire a fully developed neoplastic phenotype. Some patients with Hashimoto thyroiditis had C-cell hyperplasia [47, 48]. RET receptor has been shown to express in thyroid follicular component, which may be activated in the existence of precise ligands in the thyroid microenvironment.

Presence of RET ligands in this microenvironment is highly reasonable because C cells express the RET receptor. Follicular cell derived thyroid cancers contain RET mRNA which may be activated in the presence of specific ligands [7]. So the role of RET gene polymorphism in differentiated thyroid cancers came into existence. Some variants within RET could represent low penetrant alleles for the PTC phenotype. A study revealed the toughest association of A45A (G135A) and L769L (T2307G) with PTC [20]. Borrego et al. described the seven most frequent haplotypes in cases and controls and some of which differed in their distribution [19]. The G G C C haplotype is over-represented in both populations of sporadic PTC.

G allele of exon 2 and the G allele of exon 13 are included in the G G C C haplotype. A432A and S836S polymorphisms had strongest association with DTC as per the study conducted by Lesueur et al. Yet, the scale of the detected effect between DTC and RET SNPs was modest [20].

polymorphisms that deregulates the MAP kinase or Akt pathway, hence

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid…

Blood samples of 140 cases were collected from thyroid cancer patients attending Department of Nuclear Medicine, at Sher-I-Kashmir Institute of Medical Sciences (SKIMS), besides blood samples were obtained from 180 healthy controls from the Out Patients Departments of SKIMS. The cases and controls gave written pre-informed consent. Questionnaire was used to record demographic and clinicopathological characteristics of each patient. This study was approved by the Ethical

0.5 ml of peripheral blood was obtained from each subject in EDTA containing

Salting out method was used for the extraction of DNA from blood samples. Automated DNA sequencing of the RAS genes revealed one frequent SNP at codon 27 (T81C SNP) of HRAS. This SNP along with three other SNPs mentioned above were conducted in our study by PCR-RFLP which is the simple, cheap and common

Restriction enzymes (REs) are called molecular scissors. They recognize and cut specific sequences. The restriction endonuclease type II for SNP detection is selected, such that it recognizes and cleaves one of the polymorphic bases. Upon incubation at optimum temperature and for optimum time with a buffer, the enzyme restricts the DNA, at a specific site. Electrophoresis of the digested products yields fragments of sizes based on the cleavage pattern. If both the alleles harbor the base recognized by the enzyme, fragments of sizes accounting cumulative to the undigested product are obtained (homozygous for that base). If one of the allele harbors a different base, then the single allele is cleaved resulting usually in 3 fragments—the original

undigested product, and two digested fragments of sizes cumulatively accounting to the original PCR product (heterozygous). If more than one restriction site is present within the allele, more than two fragments are made from the same allele and the number of fragments depends on number of restriction sites present within the allele. Absence of the base recognized by the enzyme does not result in digestion thereby

Oligonucleotide Primers and the corresponding Restriction enzymes for codon 691, codon 769, codon 904 of RET SNPs along with Annealing temperatures are elucidated Table 1. The PCR products were then checked on 2% agarose gel as

10 μl of the PCR products were subjected to restriction digestion by BanI,TaqI,

and RsaI for codons 691,769 and 904 of RET SNPs respectively. The reaction conditions were set up according to the supplier of restriction enzymes (Table 2). For RET codon 691 the homozygous wild type (GG) has one BanI site and is branded by 267 and 187 bp fragments while the (AA) homozygote (variant) offered a single fragment of 454 bp and heterozygous form (G/A) showed 454, 267 and

retaining the original PCR product (Homozygous).

7.1 Restriction digestion procedure

vials (200 μl of 0.5 M, pH = 8.0) and stored at 20°C till use.

7. Genotyping by restriction fragment length polymorphism

predisposing a person to thyroid cancer [18, 45].

DOI: http://dx.doi.org/10.5772/intechopen.86902

6. Patients and controls

committee of the SKIMS.

(PCR-RFLP)

genotyping method.

described earlier.

89

The mechanism by which these silent polymorphisms act and develop RET related diseases is still a question of debate. First, stability of protein synthesis could be disturbed by such genetic sequence variations through influence on RNA splicing, hence, a new cryptic splice acceptor, donor, or enhancer site could be formed which could result in a receptor that did not bind ligand well or in a truncated protein [49]. RET A45A polymorphism may result in alternative splicing and produce a mRNA isoforms as per Borrego et al. [49]. They further hypothesized that decreased protein expression on the cell surface could be due to these RNA isoforms, erroneous ligand binding, microRNA binding, change of structure/copies and mRNA stability synthesis of incomplete proteins and also the change in the structure of proteins caused by slowing down of translation [50, 51]. However, homozygosity confers the phenotypically evident changes [49]. Second, other nearby mutations could be influenced by these silent polymorphisms. Third, the polymorphism may incline to decreased expression of the variant allele, thus leading to low level functional haplo insufficiency. Fourth, these polymorphisms may lie in linkage disequilibrium with other sequences that may directly confer low level predisposition to or protection against disease. Fifth, slightly decreased efficiency of RET translation may be due to preferential usage of tRNA molecules. The variant(s) is less favored and the wild type would be the favored sequence. Sixth, RET gene will be susceptible to damage by environmental insults such as radiation by these silent polymorphisms [21]. Finally, mRNA conservation in the case of presence of various polymorphic variants in the RET gene based on bioinformatics methods is the answer. Codon usage bias refers to differences among organisms in the frequency of occurrence of synonymous codons in mRNA. Faster translation rates and higher accuracy is achieved by optimal codons. If the translation rate changes before the process of beta sheet formation is finished, newly synthesized sequence influences the structure earlier (or later) than usual and may have an effect on the folding of the protein as the folding of the beta sheet occurs slower than the alpha helix formation [52]. Translation or protein folding disorders because of ribosome stalling (pause) may occur if the mutation is a change from optimal to less frequent codon. S904S SNP gives rise to less frequent codons, so ribosome stalling can happen. In the case of SNP L679L where the codon with higher codon usage appears, the sheet may not finish creating the structure when the helix appears [53]. As a consequence, kinase activity and/or specificity get changed. These postulated mechanisms are not mutually exclusive [54]. The mutant Y791F reduces the energy of the wild type by 7% and L769L (T > G) variant the by 17%, concluding that the L769L polymorphism reduces the MFE of small RET mRNA [45, 55]. Because of its cosegregation of S904S with G691S, the results obtained could be interpreted as a founder effect without influence as genetic modifier [38].

The G691S SNP occurs close to the residue Y687 in the cytoplasmic tail of the RET amidst transmembrane region. Two scenarios are possible to explain the G691S polymorphism exerting an effect in PTC without activation by RET rearrangement. Firstly, although RET expressed in the parafollicular C-cells and hence might influence the microenvironment of the follicular cells [56]. Alternatively, the two amino acids, glycine in the wild-type RET protein and serine in the polymorphic RET variant, confer different electrochemical and conformational structures to the RET protein, and accordingly effect the subcellular localization, folding, processing or function of the protein [7, 57, 58]. So lot of changes in RET proto-oncogene at mRNA level or at protein level can be conferred by single nucleotide

polymorphisms that deregulates the MAP kinase or Akt pathway, hence predisposing a person to thyroid cancer [18, 45].

#### 6. Patients and controls

G allele of exon 2 and the G allele of exon 13 are included in the G G C C haplotype. A432A and S836S polymorphisms had strongest association with DTC as per the study conducted by Lesueur et al. Yet, the scale of the detected effect between DTC

The mechanism by which these silent polymorphisms act and develop RET related diseases is still a question of debate. First, stability of protein synthesis could be disturbed by such genetic sequence variations through influence on RNA splicing, hence, a new cryptic splice acceptor, donor, or enhancer site could be formed which could result in a receptor that did not bind ligand well or in a truncated protein [49]. RET A45A polymorphism may result in alternative splicing and produce a mRNA isoforms as per Borrego et al. [49]. They further hypothesized that decreased protein expression on the cell surface could be due to these RNA

isoforms, erroneous ligand binding, microRNA binding, change of structure/copies and mRNA stability synthesis of incomplete proteins and also the change in the structure of proteins caused by slowing down of translation [50, 51]. However, homozygosity confers the phenotypically evident changes [49]. Second, other nearby mutations could be influenced by these silent polymorphisms. Third, the polymorphism may incline to decreased expression of the variant allele, thus leading to low level functional haplo insufficiency. Fourth, these polymorphisms may lie in linkage disequilibrium with other sequences that may directly confer low level predisposition to or protection against disease. Fifth, slightly decreased efficiency of RET translation may be due to preferential usage of tRNA molecules. The variant(s) is less favored and the wild type would be the favored sequence. Sixth, RET gene will be susceptible to damage by environmental insults such as radiation by these silent polymorphisms [21]. Finally, mRNA conservation in the case of presence of various polymorphic variants in the RET gene based on bioinformatics methods is the answer. Codon usage bias refers to differences among organisms in the frequency of occurrence of synonymous codons in mRNA. Faster translation rates and higher accuracy is achieved by optimal codons. If the translation rate changes before the process of beta sheet formation is finished, newly synthesized sequence influences the structure earlier (or later) than usual and may have an effect on the folding of the protein as the folding of the beta sheet occurs slower than the alpha helix formation [52]. Translation or protein folding disorders because of ribosome stalling (pause) may occur if the mutation is a change from optimal to less frequent codon. S904S SNP gives rise to less frequent codons, so ribosome stalling can happen. In the case of SNP L679L where the codon with higher codon usage appears, the sheet may not finish creating the structure when the helix appears [53]. As a consequence, kinase activity and/or specificity get changed. These postulated mechanisms are not mutually exclusive [54]. The mutant Y791F reduces the energy of the wild type by 7% and L769L (T > G) variant the by 17%, concluding that the L769L polymorphism reduces the MFE of small RET mRNA [45, 55]. Because of its cosegregation of S904S with G691S, the results obtained could be interpreted as a

founder effect without influence as genetic modifier [38].

mRNA level or at protein level can be conferred by single nucleotide

88

The G691S SNP occurs close to the residue Y687 in the cytoplasmic tail of the RET amidst transmembrane region. Two scenarios are possible to explain the G691S polymorphism exerting an effect in PTC without activation by RET rearrangement. Firstly, although RET expressed in the parafollicular C-cells and hence might influence the microenvironment of the follicular cells [56]. Alternatively, the two amino acids, glycine in the wild-type RET protein and serine in the polymorphic RET variant, confer different electrochemical and conformational structures to the RET protein, and accordingly effect the subcellular localization, folding, processing or function of the protein [7, 57, 58]. So lot of changes in RET proto-oncogene at

and RET SNPs was modest [20].

Knowledges on Thyroid Cancer

Blood samples of 140 cases were collected from thyroid cancer patients attending Department of Nuclear Medicine, at Sher-I-Kashmir Institute of Medical Sciences (SKIMS), besides blood samples were obtained from 180 healthy controls from the Out Patients Departments of SKIMS. The cases and controls gave written pre-informed consent. Questionnaire was used to record demographic and clinicopathological characteristics of each patient. This study was approved by the Ethical committee of the SKIMS.

0.5 ml of peripheral blood was obtained from each subject in EDTA containing vials (200 μl of 0.5 M, pH = 8.0) and stored at 20°C till use.

#### 7. Genotyping by restriction fragment length polymorphism (PCR-RFLP)

Salting out method was used for the extraction of DNA from blood samples. Automated DNA sequencing of the RAS genes revealed one frequent SNP at codon 27 (T81C SNP) of HRAS. This SNP along with three other SNPs mentioned above were conducted in our study by PCR-RFLP which is the simple, cheap and common genotyping method.

Restriction enzymes (REs) are called molecular scissors. They recognize and cut specific sequences. The restriction endonuclease type II for SNP detection is selected, such that it recognizes and cleaves one of the polymorphic bases. Upon incubation at optimum temperature and for optimum time with a buffer, the enzyme restricts the DNA, at a specific site. Electrophoresis of the digested products yields fragments of sizes based on the cleavage pattern. If both the alleles harbor the base recognized by the enzyme, fragments of sizes accounting cumulative to the undigested product are obtained (homozygous for that base). If one of the allele harbors a different base, then the single allele is cleaved resulting usually in 3 fragments—the original undigested product, and two digested fragments of sizes cumulatively accounting to the original PCR product (heterozygous). If more than one restriction site is present within the allele, more than two fragments are made from the same allele and the number of fragments depends on number of restriction sites present within the allele. Absence of the base recognized by the enzyme does not result in digestion thereby retaining the original PCR product (Homozygous).

Oligonucleotide Primers and the corresponding Restriction enzymes for codon 691, codon 769, codon 904 of RET SNPs along with Annealing temperatures are elucidated Table 1. The PCR products were then checked on 2% agarose gel as described earlier.

#### 7.1 Restriction digestion procedure

10 μl of the PCR products were subjected to restriction digestion by BanI,TaqI, and RsaI for codons 691,769 and 904 of RET SNPs respectively. The reaction conditions were set up according to the supplier of restriction enzymes (Table 2). For RET codon 691 the homozygous wild type (GG) has one BanI site and is branded by 267 and 187 bp fragments while the (AA) homozygote (variant) offered a single fragment of 454 bp and heterozygous form (G/A) showed 454, 267 and


gene. Table 3 shows the risk and demographic factors of study group. Mean age and smoking was not having significant differences among cases and controls. The mean age of the controls and patients were 38 14.6 years and 35 13.4 years respectively. Almost 71% (100 of 140) were <45 years of age and 29% (40 of 140) were ≥45 years of age. Only 19% (26 of 140) of the cases were females and 81% (114 of 140) were males. There was a difference between cases and controls with respect to their gender. Only 11% (16 of 140) of thyroid cancer patients were smokers and 89% (124 of 140) were non-smokers with no significant difference between the groups (P > 0.05). Based on the histology 84% (118 of 140) of the included cases

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid…

were papillary thyroid cancers and 16% (22 of 140) were follicular type.

9.1 Analysis of RET codon G691S, L769L and S904S polymorphism

In RET G691S (rs1799939), the overall dissemination of variant alleles (GA + AA) in cases was 62.9% as compared to 44.5% in controls (P < 0.05; OR = 2.1). Table 5 shows the Link between RET G691S phenotypes and clinicopathological characteristics. For further classification, our study found higher distribution of variant alleles (GA + AA) in female cases as equated to healthy

In RET L769L (rs1800861), the overall dissemination of variant alleles (TG + GG) in controls 88% as against 70% in cases (P < 0.05; OR = 0.3). Table 6 represents the connection between RET L769L phenotypes and clinicopathological characteristics of patients. For further organization, our study found lower distribution of variant alleles (TG + GG) in <45 years old patients as compared to healthy controls (68 vs. 88%; P < 0.05). A significant difference was found between variant alleles (TG + GG) of cases (males—61.5%, females—71.9%) and controls (males— 89.5%, females—87%) (P < 0.05). Non-smoker controls had higher frequency of variant allele as compared to cases with no smoking status (87 vs. 67.7%; P < 0.05). A higher frequency of variant alleles (82.1%) (62%) was found in thyroid cancer patients having no history of benign thyroid disease as compared to patients with history of benign thyroid disease (62%; P < 0.05). RET gene L769L polymorphism was not found to be associated with any other clinicopathological characteristics

In RET S904S (rs1800863), the overall dissemination of variant alleles (CG + GG) in controls was 44% as against 56% in cases (P < 0.05; OR = 1.6). Table 7 lists the connection between RET S904S phenotypes and clinicopathological characteristics of study cases and controls. For further arrangement, our study found lower distribution of variant alleles (CG + GG) in controls of ≥45 years of age as compared to matched thyroid cancer cases (28 vs. 45%; P < 0.05). We found a higher distribution of variant alleles (CG + GG) in male thyroid cancer patients as compared to healthy controls (69 vs. 57%; P < 0.05). A lower frequency of variant alleles was found in thyroid cancer patients having no history of benign thyroid disease when compared to patients having benign thyroid disease (43 vs. 63%; P < 0.05). No association was found between any other clinicopathological

characteristic and RET gene L769L polymorphism (Table 7).

phisms (P < 0.05) (Table 4).

DOI: http://dx.doi.org/10.5772/intechopen.86902

(Table 6).

91

clinicopathological characteristics (Table 5).

Table 4 represents the genotype distributions of RET codon L691S, L769L and S904S polymorphisms in the cases and controls. There was a significant difference in the genotype distributions between cases and controls in all the three polymor-

controls (63.2 vs. 47%; P < 0.05). Higher frequency of variant genotype was found in cases with no smoking status as compare to non-smoker controls (71.4 vs. 42.9%; P < 0.05). No significance was found between G691S polymorphism and any other

Table 1. Conditions and consumables used for screening RET SNPs.


#### Table 2.

Composition of RD mixture for codons 691, 769 and 904 of RET.

187 bp. For RET codon 769 the homozygous wild type (TT) has one TaqI site and is branded by 270 and 190 bp fragments while the (GG) homozygote (variant) offered a single fragment of 460 bp and heterozygous form (T/G) showed 460, 270 and 190 bp. In case of RET codon 904 the homozygous wild type (CC) offered a single fragment of 332 bp and the (GG) homozygote (variant) has one RsaI site and is branded by 224 and 108 bp fragments while as heterozygous form (C/G) showed 332, 224 and 108 bp fragments.

The PCR products were visualized on a 3% agarose gel containing 0.5 μg/ml ethidium bromide and photographed.

#### 8. Statistical analysis

Statistical analysis was performed by using SPSS software (V.16.0). Statistical significance was considered when P ≤ 0.05 [59].

#### 9. Polymorphic analysis of codon G691S, L769L and S904S of RET gene

A total of 140 cases (thyroid cancer patients) and 180 normal healthy controls were studied for polymorphic analysis of codon G691S, L769L and S904S of RET

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid… DOI: http://dx.doi.org/10.5772/intechopen.86902

gene. Table 3 shows the risk and demographic factors of study group. Mean age and smoking was not having significant differences among cases and controls. The mean age of the controls and patients were 38 14.6 years and 35 13.4 years respectively. Almost 71% (100 of 140) were <45 years of age and 29% (40 of 140) were ≥45 years of age. Only 19% (26 of 140) of the cases were females and 81% (114 of 140) were males. There was a difference between cases and controls with respect to their gender. Only 11% (16 of 140) of thyroid cancer patients were smokers and 89% (124 of 140) were non-smokers with no significant difference between the groups (P > 0.05). Based on the histology 84% (118 of 140) of the included cases were papillary thyroid cancers and 16% (22 of 140) were follicular type.

#### 9.1 Analysis of RET codon G691S, L769L and S904S polymorphism

Table 4 represents the genotype distributions of RET codon L691S, L769L and S904S polymorphisms in the cases and controls. There was a significant difference in the genotype distributions between cases and controls in all the three polymorphisms (P < 0.05) (Table 4).

In RET G691S (rs1799939), the overall dissemination of variant alleles (GA + AA) in cases was 62.9% as compared to 44.5% in controls (P < 0.05; OR = 2.1). Table 5 shows the Link between RET G691S phenotypes and clinicopathological characteristics. For further classification, our study found higher distribution of variant alleles (GA + AA) in female cases as equated to healthy controls (63.2 vs. 47%; P < 0.05). Higher frequency of variant genotype was found in cases with no smoking status as compare to non-smoker controls (71.4 vs. 42.9%; P < 0.05). No significance was found between G691S polymorphism and any other clinicopathological characteristics (Table 5).

In RET L769L (rs1800861), the overall dissemination of variant alleles (TG + GG) in controls 88% as against 70% in cases (P < 0.05; OR = 0.3). Table 6 represents the connection between RET L769L phenotypes and clinicopathological characteristics of patients. For further organization, our study found lower distribution of variant alleles (TG + GG) in <45 years old patients as compared to healthy controls (68 vs. 88%; P < 0.05). A significant difference was found between variant alleles (TG + GG) of cases (males—61.5%, females—71.9%) and controls (males— 89.5%, females—87%) (P < 0.05). Non-smoker controls had higher frequency of variant allele as compared to cases with no smoking status (87 vs. 67.7%; P < 0.05). A higher frequency of variant alleles (82.1%) (62%) was found in thyroid cancer patients having no history of benign thyroid disease as compared to patients with history of benign thyroid disease (62%; P < 0.05). RET gene L769L polymorphism was not found to be associated with any other clinicopathological characteristics (Table 6).

In RET S904S (rs1800863), the overall dissemination of variant alleles (CG + GG) in controls was 44% as against 56% in cases (P < 0.05; OR = 1.6). Table 7 lists the connection between RET S904S phenotypes and clinicopathological characteristics of study cases and controls. For further arrangement, our study found lower distribution of variant alleles (CG + GG) in controls of ≥45 years of age as compared to matched thyroid cancer cases (28 vs. 45%; P < 0.05). We found a higher distribution of variant alleles (CG + GG) in male thyroid cancer patients as compared to healthy controls (69 vs. 57%; P < 0.05). A lower frequency of variant alleles was found in thyroid cancer patients having no history of benign thyroid disease when compared to patients having benign thyroid disease (43 vs. 63%; P < 0.05). No association was found between any other clinicopathological characteristic and RET gene L769L polymorphism (Table 7).

187 bp. For RET codon 769 the homozygous wild type (TT) has one TaqI site and is branded by 270 and 190 bp fragments while the (GG) homozygote (variant) offered a single fragment of 460 bp and heterozygous form (T/G) showed 460, 270 and 190 bp. In case of RET codon 904 the homozygous wild type (CC) offered a single fragment of 332 bp and the (GG) homozygote (variant) has one RsaI site and is branded by 224 and 108 bp fragments while as heterozygous form (C/G) showed

The PCR products were visualized on a 3% agarose gel containing 0.5 μg/ml

Statistical analysis was performed by using SPSS software (V.16.0). Statistical

9. Polymorphic analysis of codon G691S, L769L and S904S of RET gene

A total of 140 cases (thyroid cancer patients) and 180 normal healthy controls were studied for polymorphic analysis of codon G691S, L769L and S904S of RET

332, 224 and 108 bp fragments.

8. Statistical analysis

ethidium bromide and photographed.

significance was considered when P ≤ 0.05 [59].

Amplicon Primer sequence AT



F-5CAGAGCATACGCAGCCTGTAC-3 R-5-GCCTCGTCTGCCCAGCGTTG-3

F-5-CCTGTCCACTGATCCCAAAG-3 R-5-CACTCAGCCCGTGGACTC-3

F-5-GGTCTCACCAGGCCGCTAC-3 R-5-TCGGTATCTTTCCTAGGCTTC-3

F 5<sup>0</sup>

Conditions and consumables used for screening RET SNPs.

(μl)

1

Composition of RD mixture for codons 691, 769 and 904 of RET.

R 5<sup>0</sup>

Knowledges on Thyroid Cancer

HRAS Codon 27 (T81C)

RET Codon 691 (G2071A)

Codon 769 (T2307G)

Codon 904 (C2712G)

RE, restriction enzyme. \*\*DP, digestion product.

Reagents Volume

Water 18

10� buffer R 2 PCR product 10

Restriction enzyme

Table 2.

90

\*

Table 1.

(°C)

Incubation

Incubation temperature and time for BanI,TaqI and RsaI was 37°C for 1–16 h

Product (bp)

60 186 DraIII 128 and 58

60 454 BanI 267 and 187

64 460 TaqI 190 and 270

62 332 RsaI 224 and 108

RE\* DP\*\* (bp)


9.2 Mode of inheritance in genetic association studies of RET polymorphisms

S904S polymorphism. The results are presented in Tables 8–10.

SNP Cases

DOI: http://dx.doi.org/10.5772/intechopen.86902

G691S (G2071A)

Genotype

Allele type

Genotype

Allele type

Genotype

Allele type

Table 4.

93

BTD, benign thyroid disease.

S904S (C2712G)

L769L (T2307G)

n = 140 (%)

GG 52 (37.1) 100 (55.5) 1.0 (ref.)

AA 24 (17.2) 16 (09) 2.9 (1.2–6.6)

G 168(60) 264 (73.3) 1.0 (ref.)

TT 42 (30) 22 (12) 1.0 (ref.)

GG 28 (20) 48 (27) 0.30 (0.15–0.6)

T 154(55) 154 (42) 1.0 (ref.)

CC 62 (44) 102 (56) 1.0 (ref.)

GG 14 (10) 08 (04) 2.8 (1.1–7.0)

C 188(67) 274 (76) 1.0 (ref.)

Genotype frequencies of cases and controls in RET polymorphisms.

Controls n =180 (%)

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid…

GA 64 (45.7) 64 (35.5) 1.9 (1.1–3.2) <0.05

A 112(40) 96 (26.7) 1.8 (1.2–2.5) <0.05

TG 70 (50) 110 (61) 0.32 (0.17–0.57) <0.05

G 126(45) 206 (58) 0.61 (0.44–0.82) <0.05

CG 64 (46) 70(40) 1.5 (0.93–2.4) <0.05

G 92(33) 86 (24) 1.5 (1.0–2.1) <0.05 TSH, thyroid stimulating hormone; WD, well differentiated thyroid cancer; PD, poorly differentiated thyroid cancer;

OR (95% CI) P-value

9.3 Association between RET haplotypes and thyroid cancer risk

The risk of thyroid cancer with respect to gender and smoking status with RET G691S (G2071A), L769L (T2307G) and S904S (C2712G) polymorphisms. Recessive, dominant, co-dominant and additive inheritance models were used to assess Adjusted ORs. The inheritance model matching individual SNP data shall have lowest P-value. For RET G691S and L769L polymorphism dominant inheritance model is appropriate while as additive inheritance model is appropriate for RET

In order to evaluate the combined effect of the three polymorphisms on thyroid cancer risk Haplotype analyses were conducted. Among both cases and controls all haplotypes have frequencies >5%. G2071/2307G/ C2712 (GGC) was the most

### Table 3.

Details of thyroid cancer cases and controls for the study.


Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid… DOI: http://dx.doi.org/10.5772/intechopen.86902

TSH, thyroid stimulating hormone; WD, well differentiated thyroid cancer; PD, poorly differentiated thyroid cancer; BTD, benign thyroid disease.

#### Table 4.

Characteristics Cases

Knowledges on Thyroid Cancer

Age group

Sex

Dwelling

Smoking

TSH levels

Histological types

Tumor grade

Stage, <45 years

Stage, 45 years

Vascular/capsular invasion

Lymph node metastasis

Table 3.

92

Benign thyroid disease

n = 140 (%)

≥45 40 (29) 50 (28)

Male 26 (19) 76 (42)

Urban 28 (20) 82 (45.6)

Ever 16 (11) 40 (22.2)

Yes 84 (60) No 56 (40)

Elevated 100 (71) Normal 40 (29)

Papillary 118 (84) Follicular 22 (16)

WD 134 (96) PD 06 (04)

Stage I 94(67) Stage II 06 (4.3)

Stage I and II 36 (25.7) Stage III and above 04 (03)

Yes 68 (48.5) No 72 (51.5)

Yes 52 (37) No 88 (63)

Details of thyroid cancer cases and controls for the study.

<45 100 (71) 130 (72) 0.025 >0.05

Female 114 (81) 104 (58) 20.2 <0.05

Rural 112 (80) 98 (54.4) 22.8 <0.05

Never 124 (89) 140 (77.8) 6.3 <0.05

TSH, thyroid stimulating hormone; WD, well differentiated thyroid cancer; PD, poorly differentiated thyroid cancer.

Controls n = 180 (%) χ2


Genotype frequencies of cases and controls in RET polymorphisms.

#### 9.2 Mode of inheritance in genetic association studies of RET polymorphisms

The risk of thyroid cancer with respect to gender and smoking status with RET G691S (G2071A), L769L (T2307G) and S904S (C2712G) polymorphisms. Recessive, dominant, co-dominant and additive inheritance models were used to assess Adjusted ORs. The inheritance model matching individual SNP data shall have lowest P-value. For RET G691S and L769L polymorphism dominant inheritance model is appropriate while as additive inheritance model is appropriate for RET S904S polymorphism. The results are presented in Tables 8–10.

#### 9.3 Association between RET haplotypes and thyroid cancer risk

In order to evaluate the combined effect of the three polymorphisms on thyroid cancer risk Haplotype analyses were conducted. Among both cases and controls all haplotypes have frequencies >5%. G2071/2307G/ C2712 (GGC) was the most


Cases n (%)

thyroid disease.

Overall genotype

Age group

Sex

Dwelling

Smoking

BTD

TSH levels

Histological types

Tumor grade

Stage, <45 years

95

Table 5.

Yes 52 (37) 18 (34.6) 34 (65.4)

DOI: http://dx.doi.org/10.5772/intechopen.86902

Cases n (%)

GG n (%) GA + AA n (%)

Clinicopathological characteristics vs. RET G691S (G2071A) genotypes.

TT n (%)

<45 100 (71) 32 (32) 68 (68) 130 (72) 16

≥45 40 (29) 10 (25) 30 (75) 50 (28) 06

Female 114 (81) 32 (28.0) 82 (71.9) 104 (58) 14

Male 26 (19) 10 (38.4) 16 (61.5) 76 (42) 08

Rural 112 (80) 36 (32.1) 76 (67.8) 98 (54) 10

Urban 28 (20) 06 (21.4) 22 (78.5) 82 (46) 12

Never 124 (89) 40 (32.2) 84 (67.7) 140 (78) 18

Ever 16 (11) 02 (12.5) 14 (87.5) 40 (22) 04

No 56 (40) 10 (17.8) 46 (82.1)

Normal 40 (29) 12 (30) 28 (70)

Follicular 22 (16) 06 (27.2) 16 (72.7)

PD 06 (04) 02 (33.3) 04 (66.6)

TG + GG n (%)

n = 140 42 (30) 98 (70) n = 180 22

Controls n (%)

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid…

No 88 (63) 34 (38.6) 54 (61.4) 1.2 (0.5–2.7) >0.05 TSH, thyroid stimulating hormone; WD, well differentiated thyroid cancer; PD, poorly differentiated thyroid cancer; BTD, benign

> Controls n (%)

TT n (%)

(12)

(12)

(12)

(13)

(10.5)

(10)

(15)

(13)

(10)

Yes 84 (60) 32 (38) 52 (62) 0.35(0.15–0.78) <0.05

Elevated 100 (71) 30 (30) 70 (70) 0.25(0.1–0.62) >0.05

Papillary 118 (84) 36 (30.5) 82 (69.4) 0.85 (0.3–2.2) >0.05

WD 134 (96) 40 (29.8) 94 (70.14) 1.17 (0.19–6.5) >0.05

Stage I 94 (67) 28 (29.8) 66 (70.2) 4.7 (0.8–26.8) >0.05

TG + GG n (%)

GG n (%) GA + AA n (%)

OR (95% CI) P value

OR (95% CI) P value

158 (88) 0.3 (0.17–0.6) <0.05

114 (88) 0.3 (0.14–0.5) <0.05

44 (88) 0.4 (0.13–1.2) >0.05

90 (87) 0.4 (0.18–0.85) <0.05

68 (89.5) 0.2 (0.07–0.6) <0.05

88 (90) 0.23 (0.1–0.5) <0.05

70(85) 0.6 (0.2–1.8) >0.05

122 (87) 0.3 (0.16–0.55) <0.05

36 (90) 1.1 (0.16–6.2) >0.05

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid… DOI: http://dx.doi.org/10.5772/intechopen.86902


#### Table 5.

Cases n (%)

Knowledges on Thyroid Cancer

Overall genotype

Age group

Sex

Dwelling

Smoking

BTD

TSH levels

Histological types

Tumor grade

Stage, <45 years

Stage, 45 years Stage I and II

Stage III and above

Vascular/ capsular invasion

Lymph node metastasis

94

GG n (%)

Never 124 (89) 42 (28.6) 82 (71.4) 140

Yes 84 (60) 30 (35.7) 54 (64.2)

Elevated 100 (71) 40 (40) 60 (60)

Papillary 118(84) 44(37.2) 74(62.8)

WD 134 (96) 50 (37.3) 84 (62.7)

Stage I 94 (67) 28 (29.7) 66 (70.3)

Yes 68 (48.5) 22 (32.3) 46 (67.7)

36 (25.7) 20 (55.5) 16 (44.5)

GA + AA n (%)

Controls n (%)

<45 100 (71) 30 (30) 70 (70) 130 (72) 62 (48) 68 (52) 2.1 (1.2–3.6) <0.05 ≥45 40 (29) 22 (55) 18 (45) 50 (28) 38 (76) 12 (24) 2.6 (3.6–6.2) <0.05

Female 114 (81) 42 (36.8) 72 (63.2) 104 (58) 60 (58) 44 (42) 2.3 (1.3–3.9) <0.05 Male 26 (19) 10 (38.5) 16 (61.5) 76 (42) 40 (53) 36 (47) 1.8 (0.72–4.4) >0.05

Rural 112 (80) 44 (39.3) 68 (60.7) 98 (54.4) 56 (57.1) 42 (42.9) 2.0 (1.2–3.4) <0.05 Urban 28 (20) 08 (28.5) 20 (71.5) 82 (45.6) 44 (53.6) 38 (46.4) 2.9 (1.1–7.2) <0.05

(77.8)

Ever 16 (11) 10 (62.5) 06 (37.5) 40 (22.2) 20 (50) 20 (50) 0.6 (0.1–2.8) >0.05

No 56 (40) 22 (39.2) 34 (60.8) 1.2 (0.8–1.9) >0.05

Normal 40 (29) 12 (30) 28 (70) 0.6 (0.2–1.5) >0.05

Follicular 22(16) 08(36.3) 14(63.7) 1.0 (0.3–3.2) >0.05

PD 06 (04) 02 (33.3) 04 (66.7) 0.8 (0.06–9.7) >0.05

Stage II 06 (4.3) 02 (33.3) 04 (66.7) 1.2 (0.1–15.3) >0.05

No 72 (51.5) 30 (41.6) 42 (58.4) 1.5 (0.75–3) >0.05

04 (03) 02 (50) 02 (50) 0.8 (0.09–6.3) >0.05

GG n (%)

n = 140 52 (37.1) 88 (62.9) n = 180 100 (55.5) 80 (44.5) 2.1 (1.3–3.2) <0.05

GA + AA n (%)

80 (57.1) 60 (42.9) 2.6 (1.5–4.5) <0.05

OR (95% CI) P value

Clinicopathological characteristics vs. RET G691S (G2071A) genotypes.



Cases n (%)

DOI: http://dx.doi.org/10.5772/intechopen.86902

WD 134 (96) 60 (45) 74 (55)

Stage I 94 (67) 40 (42.5) 54 (57.5)

Stage I and II 36 (25.7) 18 (50) 18 (50)

Yes 68 (48.5) 32 (47) 36 (53)

Yes 52 (37) 28 (54) 24 (46)

Genotypes and alleles (patients vs.

Recessive model (AA vs. GA + GG)

Dominant model (AA+ GA vs. GG)

Co-dominant model (GA vs. AA + GG)

G691S (G2071A) polymorphism association with thyroid cancer.

Additive model (AA vs. GG)

Clinicopathological characteristics vs. RET S904S (C2712G) genotypes.

Tumor grade

Stage, < 45 years

Stage, 45 years

Stage III and above

Vascular/ capsular invasion

Lymph node metastasis

thyroid disease.

controls)

Table 7.

Table 8.

97

CC n (%) CG + GG n (%)

Follicular 22 (16) 06 (27) 16 (73) 0.4 (0.15–

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid…

Controls n (%)

PD 06 (04) 02 (33) 04 (67) 0.6 (0.10–3.4) >0.05

Stage II 06 (4.3) 02 (33) 04 (67) 0.7 (0.12–4.0) >0.05

No 72 (51.5) 30 (41.7) 42 (58.3) 0.8 (0.4–1.56) >0.05

No 88 (63) 34 (39) 54 (61) 0.5 (0.25–1.0) >0.05 TSH, thyroid stimulating hormone; WD, well differentiated thyroid cancer; PD, poorly differentiated thyroid cancer; BTD, benign

> Cases (n = 140)

AA 24 16 2.12 (1.0–4.7) 0.027

AA + GA 88 80 2.11 (1.3–3.3) 0.001

GA 64 64 1.5 (1.0–2.3) 0.066

AA 24 16 2.8 (1.4–5.7) 0.003

GA + GG 116 164 1.0 (ref.)

GG 52 100 1.0 (ref.)

AA + GG 76 116 1.0 (ref.)

GG 52 100 1.0 (ref.)

Controls (n = 180) OR (95% CI) P value

04 (03) 02 (50) 02 (50) 1.0 (0.12–7.9) >0.05

CC n (%) CG + GG n (%)

OR (95% CI) P-value

>0.05

1.08)

TSH, thyroid stimulating hormone; WD, well differentiated thyroid cancer; PD, poorly differentiated thyroid cancer; BTD, benign thyroid disease.

#### Table 6.

Clinicopathological characteristics vs. RET L769L (T2307G) genotypes.



Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid… DOI: http://dx.doi.org/10.5772/intechopen.86902

TSH, thyroid stimulating hormone; WD, well differentiated thyroid cancer; PD, poorly differentiated thyroid cancer; BTD, benign thyroid disease.

#### Table 7.

Cases n (%)

Knowledges on Thyroid Cancer

Stage, 45 years

Stage III and above

Vascular/ capsular invasion

Lymph node metastasis

thyroid disease.

Overall genotype

Age group

Sex

Dwelling

Smoking

BTD

TSH levels

Histological types

96

Table 6.

Stage II 06 (4.3) 04 (66.6) 02 (33.3)

No 72 (51.5) 20 (27.8) 52 (72.2)

No 88 (63) 24 (27.2) 64 (72.8)

Cases n (%)

Yes 84 (60) 30 (36) 54 (64)

Elevated 100 (71) 40 (40) 60 (60)

Papillary 118 (84) 56 (47) 62 (53)

TT n (%)

04 (03) 02 (50) 02 (50)

Clinicopathological characteristics vs. RET L769L (T2307G) genotypes.

CG + GG n (%)

CC n (%) TG + GG n (%)

Controls n (%)

Stage I and II 36 (25.7) 08 (22.2) 28 (77.8) 3.5 (0.42–28.7) >0.05

Yes 68 (48.5) 22 (32.3) 46 (67.7) 0.8 (0.4–1.6) >0.05

Yes 52 (37) 18 (34.6) 34 (65.4) 0.7 (0.32–1.4) >0.05

TSH, thyroid stimulating hormone; WD, well differentiated thyroid cancer; PD, poorly differentiated thyroid cancer; BTD, benign

Controls n (%)

<45 100 (71) 40 (64.5) 60 (35.5) 130 (72) 66 (51) 64 (49) 1.5 (0.9–2.9) >0.05 ≥45 40 (29) 22 (55) 18 (45) 50 (28) 36 (72) 14 (28) 2.1 (0.84–5.0) <0.05

Female 114 (81) 54 (47) 60 (53) 104 (58) 58 (56) 46 (44) 1.4 (0.8–2.4) >0.05 Male 26 (19) 08 (31) 18 (69) 76 (42) 44 (43) 32 (57) 3.1 (1.2–8.0) <0.05

Rural 112 (80) 46 (41) 66(59) 98 (54) 54 (55) 44 (45) 1.7 (0.96–2.9) >0.05 Urban 28 (20) 16 (57) 12(43) 82 (46) 48 (58.5) 34 (41.5) 1.05 (0.4–2.4) >0.05

Never 124 (89) 56 (45) 68 (55) 140 (78) 78 (56) 62 (44) 1.5 (0.9–2.4) >0.05 Ever 16 (11) 06 (37.5) 10 (62.5) 40 (22) 24 (60) 16 (40) 2.5 (0.75–8.2) >0.05

No 56 (40) 32 (57) 24 (43) 2.4 (1.2–4.8) <0.05

Normal 40 (29) 22 (55) 18 (45) 1.8 (0.7–3.7) >0.05

CC n (%)

n = 140 62 (44) 78 (56) n = 180 102 (56) 78 (44) 1.6 (1.0–2.4) <0.05

CG + GG n (%)

TT n (%) TG + GG n (%)

OR (95% CI) P value

OR (95% CI) P-value

Clinicopathological characteristics vs. RET S904S (C2712G) genotypes.


#### Table 8.

G691S (G2071A) polymorphism association with thyroid cancer.


as the reference group and haplotype-specific ORs are estimated by the haplotypebased logistic regression method [60]. The frequencies for the estimated 3-marker

Cases Controls Cumulative

G G C 0.3061 0.2498 0.3337 0.3061 1.00 (ref) – G T C 0.2122 0.2004 0.2321 0.5183 0.94 (0.48–1.84) 0.86 A G C 0.1224 0.1084 0.1503 0.6407 1.26 (0.55–2.87) 0.59 A T G 0.0999 0.1442 0.0657 0.7406 0.24 (0.10–0.57) 0.0012 A T C 0.0843 0.1129 0.0506 0.8249 0.38 (0.17–0.83) 0.016 G T G 0.0817 0.0925 0.0738 0.9066 0.23 (0.08–0.64) 0.0051 G G G 0.075 0.0573 0.0938 0.9816 1.59 (0.59–4.31) 0.36 A G G 0.0184 0.0345 0 1 0.05 (0.00–1.37) 0.076 The OR (95% CI) of thyroid cancer associated with each haplotype was estimated by comparison with the common reference haplotype.

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid…

frequency

OR (95% CI) P value

Thyroid tumors signify a proper model for the study of epithelial neoplastic transformation. At the genomic level, thyroid cancers gather a number of changes and it has been projected that genomic instability has a vital role in the development

With erudite genetic tools producing a treasure of information, we have added improved vision into the mechanisms driving thyroid tumor development. Recognition of these features is crucial to the management of patients with TC. New therapeutics is being designed based on our improved understanding of this disease

In Kashmiri population we studied genetic alterations of RET genes in thyroid

• In RET G691S (rs1799939), the total distribution of variant alleles (CA + AA) in controls was 44.5% as against 62.9% in controls which revealed a 2-fold higher risk of variant allele (TC + CC) in cases against the controls (P < 0.05).

• In RET L769L (rs1800861), the total distribution of variant alleles (TG + GG)

• In RET S904S (rs1800863), the total distribution of variant alleles (CG + GG)

in controls was 88% as against 70% in cases (P < 0.05; OR = 0.3).

in controls was 44% as against 56% in cases (P < 0.05; OR = 1.6).

• We found higher distribution of variant alleles (CG + GG) in TC cases of ≥45 years of age and male gender (45 and 69%) as compared to matched healthy controls (28 and 57%) (P < 0.05). Association was also observed with

• RET G691S and L769L polymorphisms follow "Dominant inheritance model" while as "Additive inheritance model" is appropriate for analysis of RET S904S

haplotypes among patients and controls are shown in Table 11.

Haplotype frequencies and its association with thyroid cancer.

cancer patients. Following are the major findings of our study;

10. Conclusion

ref: reference.

Table 11.

course.

of thyroid neoplasms [61].

G2071A T2307G C2712G Total

DOI: http://dx.doi.org/10.5772/intechopen.86902

frequency

BTD (P < 0.05).

polymorphism.

99

#### Table 9.

Genetic model for L769L (T2307G) polymorphism.

common haplotype, with frequencies of 24% in cases and 33% in controls. The complete dissemination of various haplotypes between cases and controls presented a clear difference (P < 0.0001). Haplotype pattern for the three SNPs is shown in Table 11. Haplotypes with frequency <1% was omitted from the study. After stratified by gender, age and smoking status the haplotype frequencies were estimated from the genotyping data. In our study, the most common haplotype is taken


#### Table 10.

Genetic model for S904S (C2712G) polymorphism.

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid… DOI: http://dx.doi.org/10.5772/intechopen.86902


The OR (95% CI) of thyroid cancer associated with each haplotype was estimated by comparison with the common reference haplotype. ref: reference.

#### Table 11.

common haplotype, with frequencies of 24% in cases and 33% in controls. The complete dissemination of various haplotypes between cases and controls presented a clear difference (P < 0.0001). Haplotype pattern for the three SNPs is shown in Table 11. Haplotypes with frequency <1% was omitted from the study. After stratified by gender, age and smoking status the haplotype frequencies were estimated from the genotyping data. In our study, the most common haplotype is taken

> Cases (n = 140)

GG 14 08 2.4 (0.97–5.8) 0.051

GG + CG 78 78 1.64 (1.0–2.6) 0.028

CG 64 70 1.3 (0.8–2.0) 0.22

GG 14 08 2.8 (1.1–7.2) 0.021

CG + CC 126 172 1.0 (ref.)

CC 62 102 1.0 (ref.)

GG + CC 76 110 1.0 (ref.)

CC 62 102 1.0 (ref.)

Controls (n = 180) OR (95% CI) P-value

Cases (n = 140)

GG 28 48 0.7 (0.4–1.2) 0.17

GG + TG 98 158 0.32 (0.2–0.6) 0.000

TG 70 110 0.63 (0.4–1.0) 0.047

GG 28 48 0.3 (0.15–0.6) 0.001

TG + TT 112 132 1.0 (ref.)

TT 42 22 1.0 (ref.)

GG + TT 70 70 1.0 (ref.)

TT 42 22 1.0 (ref.)

Controls (n = 180) OR (95% CI) P value

Genotypes and alleles (patients vs.

Recessive model (GG vs. TG + TT)

Knowledges on Thyroid Cancer

Dominant model (GG + TG vs. TT)

Co-dominant model (TG vs. GG + TT)

Genetic model for L769L (T2307G) polymorphism.

Genotypes and alleles (patients vs.

Recessive model (GG vs. CG + CC)

Dominant model (GG + CG vs. CC)

Co-dominant model (CG vs. GG + CC)

Genetic model for S904S (C2712G) polymorphism.

Additive model (GG vs. CC)

Table 10.

98

Additive model (GG vs. TT)

Table 9.

controls)

controls)

Haplotype frequencies and its association with thyroid cancer.

as the reference group and haplotype-specific ORs are estimated by the haplotypebased logistic regression method [60]. The frequencies for the estimated 3-marker haplotypes among patients and controls are shown in Table 11.

#### 10. Conclusion

Thyroid tumors signify a proper model for the study of epithelial neoplastic transformation. At the genomic level, thyroid cancers gather a number of changes and it has been projected that genomic instability has a vital role in the development of thyroid neoplasms [61].

With erudite genetic tools producing a treasure of information, we have added improved vision into the mechanisms driving thyroid tumor development. Recognition of these features is crucial to the management of patients with TC. New therapeutics is being designed based on our improved understanding of this disease course.

In Kashmiri population we studied genetic alterations of RET genes in thyroid cancer patients. Following are the major findings of our study;


• In RET codon G691S, L769L and S904S polymorphism the most overrepresented haplotype is A T G followed by G T G and A T C depending upon their Akaike information criteria (P-value).

References

Publishing; 2001

Publishers; 2005

2010;2:885-912

237-264

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DOI: http://dx.doi.org/10.5772/intechopen.86902

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid…

rearranged version of the RET protooncogene in a human thyroid papillary carcinoma. Oncogene. 1994;9:509-516

[11] Eng C. RET proto-oncogene in the development of human cancer. Journal of Clinical Oncology. 1999;17:380-393

[12] Tallini G, Asa SL. RET oncogene activation in papillary thyroid carcinoma. Advances in Anatomic

[13] Fenton CL, Lukes Y, Nicholson D, et al. The RET/PTC mutations are common in sporadic papillary thyroid carcinoma of children and young adults. The Journal of Clinical Endocrinology and Metabolism. 2000;85:1170-1175

[14] Nikiforov YE. Molecular analysis of thyroid tumors. Modern Pathology.

[15] Frazer KA et al. A second generation human haplotype map of over 3.1 million SNPs. Nature. 2007;449:851-861

[16] Stephens LA, Powell NG, Grubb SJ, Jeremiah JA, et al. Investigation of loss of heterozygosity and SNP frequencies in the RET gene in papillary thyroid carcinoma. Thyroid. 2005;15:100-105

[17] Adjadj E, Schlumberger M, de Vathaire F. Germ-line DNA

polymorphisms and susceptibility to differentiated thyroid cancer. The Lancet Oncology. 2009;10:181-190

[18] Fitze G, Schreiber M, Kuhlisch E, Schackert HK, et al. Association of RET protooncogene codon 45 polymorphism with Hirschsprung disease. American Journal of Human Genetics. 1999;65:

[19] Borrego S, Ruiz A, Saez ME, Gimm O, Gao X, Lopez-Alonso M, et al. RET

genotypes comprising specific

Pathology. 2001;8:345-354

2011;24:S34-S43

1469-1473

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[3] Sarlis NJ, Benvenga S. Molecular signaling in thyroid cancer. Cancer Treatment and Research. 2004;122:

[4] Sarlis NJ. Expression patterns of cellular growth-controlling genes in non-medullary thyroid cancer: Basic aspects. Reviews in Endocrine and Metabolic Disorders. 2000;1:183-196

[5] Hundahl SA, Fleming ID, Fremgen AM, Menck HR. A National Cancer Data Base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985– 1995. Cancer. 1998;83:2638-2648

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[7] Bunone G, Uggeri M, Mondellini P, et al. RET receptor expression in thyroid follicular epithelial cell-derived tumors. Cancer Research. 2000;60:2845-2849

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[9] Grieco M, Santoro M, et al. PTC is a novel rearranged form of the ret

protooncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell. 1990;60:557-563

characterization of RET/PTC3: A novel

[10] Santoro M et al. Molecular

101

In conclusion, our study shows that among different polymorphisms predisposing to thyroid tumors, RET gene polymorphism is among the prominent ones. Our results support the earlier reports of the G691S/S904S polymorphism in RET gene as a marked predisposing factor for the risk of developing thyroid tumors in our population with G691S variant showing an increased risk for the nonsmokers but this needs to be authenticated in a large sample study in the future to determine the course of thyroid cancer. Further we conclude L769L polymorphism to be protective in our series of thyroid cancer patients. Apart from it our data suggest that some specific haplotypes (A T G, G T G, and A T C) of RET are overrepresented and may act as low penetrance alleles in the predisposition to thyroid cancer.

### Author details

Syed Mudassar<sup>1</sup> \*†, Mosin S. Khan1†, Shariq R. Masoodi<sup>2</sup> , Mahboob Ul Hussain<sup>3</sup> and Khurshid I. Andrabi<sup>3</sup>

1 Department of Clinical Biochemistry, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, Kashmir, India

2 Department of Endocrinology, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, Kashmir, India

3 Department of Biotechnology, University of Kashmir, Srinagar, Kashmir, India

\*Address all correspondence to: syed.mudassar@skims.ac.in

† Both authors (first two) have contributed equally.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid… DOI: http://dx.doi.org/10.5772/intechopen.86902

#### References

• In RET codon G691S, L769L and S904S polymorphism the most over-

In conclusion, our study shows that among different polymorphisms predisposing to thyroid tumors, RET gene polymorphism is among the prominent ones. Our results support the earlier reports of the G691S/S904S polymorphism in RET gene as a marked predisposing factor for the risk of developing thyroid tumors in our population with G691S variant showing an increased risk for the nonsmokers but this needs to be authenticated in a large sample study in the future to determine the course of thyroid cancer. Further we conclude L769L polymorphism to be protective in our series of thyroid cancer patients. Apart from it our data suggest that some specific haplotypes (A T G, G T G, and A T C) of RET are over-

represented and may act as low penetrance alleles in the predisposition to

\*†, Mosin S. Khan1†, Shariq R. Masoodi<sup>2</sup>

1 Department of Clinical Biochemistry, Sher-I-Kashmir Institute of Medical

2 Department of Endocrinology, Sher-I-Kashmir Institute of Medical Sciences,

3 Department of Biotechnology, University of Kashmir, Srinagar, Kashmir, India

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: syed.mudassar@skims.ac.in

Both authors (first two) have contributed equally.

provided the original work is properly cited.

, Mahboob Ul Hussain<sup>3</sup>

their Akaike information criteria (P-value).

thyroid cancer.

Knowledges on Thyroid Cancer

Author details

Syed Mudassar<sup>1</sup>

†

100

and Khurshid I. Andrabi<sup>3</sup>

Srinagar, Kashmir, India

Sciences, Srinagar, Kashmir, India

represented haplotype is A T G followed by G T G and A T C depending upon

[1] Corner J, Bailey C. Cancer Nursing Care in Context. Oxford: Blackwell Publishing; 2001

[2] Yarbro C, Frogge M, Goodman M. Cancer Nursing: Principles and Practice. 6th ed. Boston, MA: Jones and Bartlett Publishers; 2005

[3] Sarlis NJ, Benvenga S. Molecular signaling in thyroid cancer. Cancer Treatment and Research. 2004;122: 237-264

[4] Sarlis NJ. Expression patterns of cellular growth-controlling genes in non-medullary thyroid cancer: Basic aspects. Reviews in Endocrine and Metabolic Disorders. 2000;1:183-196

[5] Hundahl SA, Fleming ID, Fremgen AM, Menck HR. A National Cancer Data Base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985– 1995. Cancer. 1998;83:2638-2648

[6] Grogan RH, Mitmaker EJ, Clark OH. The evolution of biomarkers in thyroid cancer-from mass screening to a personalized biosignature. Cancers. 2010;2:885-912

[7] Bunone G, Uggeri M, Mondellini P, et al. RET receptor expression in thyroid follicular epithelial cell-derived tumors. Cancer Research. 2000;60:2845-2849

[8] Santoro M et al. Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype. The Journal of Clinical Investigation. 1992;89:1517-1522

[9] Grieco M, Santoro M, et al. PTC is a novel rearranged form of the ret protooncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell. 1990;60:557-563

[10] Santoro M et al. Molecular characterization of RET/PTC3: A novel rearranged version of the RET protooncogene in a human thyroid papillary carcinoma. Oncogene. 1994;9:509-516

[11] Eng C. RET proto-oncogene in the development of human cancer. Journal of Clinical Oncology. 1999;17:380-393

[12] Tallini G, Asa SL. RET oncogene activation in papillary thyroid carcinoma. Advances in Anatomic Pathology. 2001;8:345-354

[13] Fenton CL, Lukes Y, Nicholson D, et al. The RET/PTC mutations are common in sporadic papillary thyroid carcinoma of children and young adults. The Journal of Clinical Endocrinology and Metabolism. 2000;85:1170-1175

[14] Nikiforov YE. Molecular analysis of thyroid tumors. Modern Pathology. 2011;24:S34-S43

[15] Frazer KA et al. A second generation human haplotype map of over 3.1 million SNPs. Nature. 2007;449:851-861

[16] Stephens LA, Powell NG, Grubb SJ, Jeremiah JA, et al. Investigation of loss of heterozygosity and SNP frequencies in the RET gene in papillary thyroid carcinoma. Thyroid. 2005;15:100-105

[17] Adjadj E, Schlumberger M, de Vathaire F. Germ-line DNA polymorphisms and susceptibility to differentiated thyroid cancer. The Lancet Oncology. 2009;10:181-190

[18] Fitze G, Schreiber M, Kuhlisch E, Schackert HK, et al. Association of RET protooncogene codon 45 polymorphism with Hirschsprung disease. American Journal of Human Genetics. 1999;65: 1469-1473

[19] Borrego S, Ruiz A, Saez ME, Gimm O, Gao X, Lopez-Alonso M, et al. RET genotypes comprising specific

haplotypes of polymorphic variants predispose to isolated Hirschsprung disease. Journal of Medical Genetics. 2000;37:572-578

[20] Lesueur F, Corbex M, McKay JD, et al. Specific haplotypes of the RET proto-oncogene are over-represented in patients with sporadic papillary thyroid carcinoma. Journal of Medical Genetics. 2002;39:260-265

[21] Ho T, Li G, Zhao C, Wei Q, Sturgis EM. RET polymorphisms and haplotypes and risk of differentiated thyroid cancer. Laryngoscope. 2005;115: 1035-1041

[22] Pandith AA, Siddiqi MA. Burden of cancers in the valley of Kashmir: 5 year epidemiological study reveals a different scenario. Tumor Biology. 2012;33: 1629-1637

[23] Kleiman DA, Buitrago D, Crowley MJ, Housman D. Human DNA polymorphism. The New England Journal of Medicine. 1995;332:318-320

[24] The International Hap Map Consortium. The International Hap Map project. Nature. 2003;426:789-796

[25] Takahashi M, Ritz J, Cooper GM. Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell. 1985;42:581-588

[26] Takahashi M, Buma Y, et al. Cloning and expression of the ret protooncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene. 1988;3:571-578

[27] Pachnis V, Mankoo B, Costantini F. Expression of the c-ret proto-oncogene during mouse embryogenesis. Development. 1993;119:1005-1017

[28] Tsuzuki T, Takahashi M, Asai N, Iwashita T, Matsuyama M, Asai J. Spatial and temporal expression of the retproto-oncogene product in

embryonic, infant and adult rat tissues. Oncogene. 1995;10:191-198

signaling pathway and susceptibility to sporadic medullary thyroid carcinoma. Journal of Clinical

DOI: http://dx.doi.org/10.5772/intechopen.86902

[44] Plaza-Menacho I, Burzynski GM, de Groot JW, et al. Activated ras and ret oncogenes induce over-expression of cmet (hepatocyte growth factor receptor) in human thyroid epithelialcells. Oncogene. 1997;14:

[45] Borrego S, Saez ME, Ruiz A, Gimm O, Lopez-Alonso M, Antiñolo G, et al. Specific polymorphisms in the RET proto-oncogene are over-represented in patients with Hirschsprung disease and

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modifiers of MEN 2A. Cancer Research.

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[40] McWhinney SR et al. Intronic single nucleotide polymorphisms in the RET protoonco gene are associated with a subset of apparently sporadic

pheochromocytoma and may modulate age of onset. The Journal of Clinical Endocrinology and Metabolism. 2003;

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[42] Garcia-Barcelo M et al. TTF-1 and RET promoter SNPs: Regulation of RET transcription in Hirschsprung's disease. Human Molecular Genetics. 2005;14:

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90:6268-6274

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88(10):4911-4916

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850-858

103

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[30] Myers SM, Eng C, Ponder BA, Mulligan LM. Characterization of RET proto-oncogene splicing variants and polyadenylation sites: A novel Cterminus for RET. Oncogene. 1995;11: 2039-2045

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[34] Milbrandt J, de Sauvage FJ, Fahrner TJ, et al. Persephin, a novel neurotrophic factor related to GDNF and neurturin. Neuron. 1998;20:245-253

[35] Baumgartner-Parzer SM, Lang R, Wagner L, et al. Polymorphisms in exon 13 and intron 14 of the RET protooncogene: Genetic modifiers of medullary thyroid carcinoma? Journal of Clinical Endocrinology and Metabolism. 2005;90:6232-6236

[36] Cebrian A, Lesueur F, Martin S, et al. Polymorphisms in the initiators of RET (rearranged during transfection)

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid… DOI: http://dx.doi.org/10.5772/intechopen.86902

signaling pathway and susceptibility to sporadic medullary thyroid carcinoma. Journal of Clinical Endocrinology and Metabolism. 2005; 90:6268-6274

haplotypes of polymorphic variants predispose to isolated Hirschsprung disease. Journal of Medical Genetics.

Knowledges on Thyroid Cancer

embryonic, infant and adult rat tissues.

Sugimura T, Nagao M. Characterization of ret proto-oncogene mRNAs encoding two isoforms of the protein product in a

Oncogene. 1995;10:191-198

[29] Tahira T, Ishizaka Y, Itoh F,

human neuroblastoma cell line. Oncogene. 1990;5:97-102

[30] Myers SM, Eng C, Ponder BA, Mulligan LM. Characterization of RET proto-oncogene splicing variants and polyadenylation sites: A novel Cterminus for RET. Oncogene. 1995;11:

[31] Jing S, Wen D, Yu Y, Holst PL, Luo Y, Fang M, et al. GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF. Cell. 1996;85:

[32] Treanor JJ, Goodman L, et al. Characterization of a multicomponent receptor for GDNF. Nature. 1996;382:

[33] Baloh RH, Tansey MG, Lampe PA, Fahrner TJ, et al. Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFR alpha3– RET receptor complex. Neuron. 1998;21:

[34] Milbrandt J, de Sauvage FJ, Fahrner

neurotrophic factor related to GDNF and neurturin. Neuron. 1998;20:245-253

[35] Baumgartner-Parzer SM, Lang R, Wagner L, et al. Polymorphisms in exon

protooncogene: Genetic modifiers of medullary thyroid carcinoma? Journal of Clinical Endocrinology and Metabolism.

[36] Cebrian A, Lesueur F, Martin S, et al. Polymorphisms in the initiators of RET (rearranged during transfection)

TJ, et al. Persephin, a novel

13 and intron 14 of the RET

2005;90:6232-6236

2039-2045

1113-1124

80-83

1291-1302

[20] Lesueur F, Corbex M, McKay JD, et al. Specific haplotypes of the RET proto-oncogene are over-represented in patients with sporadic papillary thyroid carcinoma. Journal of Medical Genetics.

[21] Ho T, Li G, Zhao C, Wei Q, Sturgis

[22] Pandith AA, Siddiqi MA. Burden of cancers in the valley of Kashmir: 5 year epidemiological study reveals a different scenario. Tumor Biology. 2012;33:

[23] Kleiman DA, Buitrago D, Crowley

Consortium. The International Hap Map project. Nature. 2003;426:789-796

[25] Takahashi M, Ritz J, Cooper GM.

[26] Takahashi M, Buma Y, et al. Cloning

[27] Pachnis V, Mankoo B, Costantini F. Expression of the c-ret proto-oncogene

[28] Tsuzuki T, Takahashi M, Asai N, Iwashita T, Matsuyama M, Asai J. Spatial and temporal expression of the

MJ, Housman D. Human DNA polymorphism. The New England Journal of Medicine. 1995;332:318-320

[24] The International Hap Map

Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell. 1985;42:581-588

and expression of the ret protooncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene. 1988;3:571-578

during mouse embryogenesis. Development. 1993;119:1005-1017

retproto-oncogene product in

102

EM. RET polymorphisms and haplotypes and risk of differentiated thyroid cancer. Laryngoscope. 2005;115:

2000;37:572-578

2002;39:260-265

1035-1041

1629-1637

[37] Machens A, Frank-Raue K, Lorenz K, Rondot S, Raue F, Dralle H. Clinical relevance of RET variants G691S, L769L, S836S and S904S to sporadic medullary thyroid cancer. Clinical Endocrinology. 2012;76:691-697

[38] Robledo M et al. Polymorphisms G691S/S904S of RET as genetic modifiers of MEN 2A. Cancer Research. 2003;63:1814-1817

[39] Elisei R, Cosci B, Romei C, Bottici V, Sculli M, Lari R, et al. RET exon 11 (G691S) polymorphism is significantly more frequent in sporadic medullary thyroid carcinoma than in the general population. The Journal of Clinical Endocrinology and Metabolism. 2004; 89:3579-3584

[40] McWhinney SR et al. Intronic single nucleotide polymorphisms in the RET protoonco gene are associated with a subset of apparently sporadic pheochromocytoma and may modulate age of onset. The Journal of Clinical Endocrinology and Metabolism. 2003; 88(10):4911-4916

[41] Fitze G et al. Functional haplotypes of the RET proto-oncogene promoter are associated with Hirschsprung disease (HSCR). Human Molecular Genetics. 2003;12:3207-3214

[42] Garcia-Barcelo M et al. TTF-1 and RET promoter SNPs: Regulation of RET transcription in Hirschsprung's disease. Human Molecular Genetics. 2005;14: 191-204

[43] Burzynski GM et al. Identifying candidate Hirschsprung disease associated RET variants. American Journal of Human Genetics. 2005;76: 850-858

[44] Plaza-Menacho I, Burzynski GM, de Groot JW, et al. Activated ras and ret oncogenes induce over-expression of cmet (hepatocyte growth factor receptor) in human thyroid epithelialcells. Oncogene. 1997;14: 2417-2423

[45] Borrego S, Saez ME, Ruiz A, Gimm O, Lopez-Alonso M, Antiñolo G, et al. Specific polymorphisms in the RET proto-oncogene are over-represented in patients with Hirschsprung disease and may represent loci modifying phenotypic expression. Journal of Medical Genetics. 1999;36:771-774

[46] Gil L, Azanedo M, et al. Genetic analysis of RET, GFR alpha 1 and GDNF genes in Spanish families with multiple endocrine neoplasia type 2A. International Journal of Cancer. 2002; 99:299-304

[47] Tsui-Pierchala BA, Milbrandt J, Johnson EM. NGF utilizes c-Ret via a novel GFL-independent, inter-RTK signaling mechanism to maintain the trophic status of mature sympathetic neurons. Neuron. 2002;33:261-273

[48] Sipple JH. The association of pheochromocytoma with carcinoma of the thyroid gland. The American Journal of Medicine. 1961;31:163-166

[49] Borrego S, Eng C, Sánchez B, Sáez ME, Navarro E, Antiñolo G. Molecular analysis of RET and GDNF genes in a family with multiple endocrine neoplasia type 2A and Hirschsprung disease. The Journal of Clinical Endocrinology and Metabolism. 1998; 83:3361-3364

[50] Parmley JL, Hurt LD. How do synonymous mutations affect fitness? Bioassay. 2007;29:515-519

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MDR1 gene changes substrate specificity. Science. 2007;26:525-528

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[54] Sromek M, Czetwertyńska M, et al. The frequency of selected polymorphic variants of the RET gene in patients with medullary thyroid carcinoma and in the general population of Central Poland. Endocrine Pathology. 2010;21: 178-185

[55] Ceolin L, Siqueira DR, et al. Additive effect of RET polymorphisms on sporadic medullary thyroid carcinoma susceptibility and tumor aggressiveness. European Journal of Endocrinology. 2012;11:1-29

[56] Fabien N, Paulin C, Santoro M, Berger N, et al. Detection of RET oncogene activation in human papillary thyroid carcinomas by in situ hybridisation. British Journal of Cancer. 1992;66:1094-1098

[57] Fluge O, Haugen DR, Akslen LA, Marstad A, et al. Expression and alternative splicing of c-ret RNA in papillary thyroid carcinomas. Oncogene. 2001;20:885-892

[58] Leviev I, Negro F, James RW. Two alleles of the human paraoxonase gene produce different amounts of mRNA. An explanation for differences in serum concentrations of paraoxonase associated with the (Leu-Met54) polymorphism. Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2935-2939

[59] Rao J, Scott A. On chi-squared tests for multiway contingency tables with cell proportions estimated from survey data. The Annals of Statistics. 1984;12: 46-60

[60] Akaike H. Information measures and model selection. Bulletin of the International Statistical Institute. 1983; 50:277-290

[61] Sobrinho-Simoes M et al. Molecular pathology of well-differentiated thyroid carcinomas. Virchows Archiv. 2005; 447:787-793

MDR1 gene changes substrate specificity. Science. 2007;26:525-528

Knowledges on Thyroid Cancer

Nature. 1997;390:196-199

334-340

178-185

2012;11:1-29

[52] Muñoz V, Thompson PA, Hofrichter J, Eaton WA. Folding dynamics and mechanism of β-hairpin formation.

[59] Rao J, Scott A. On chi-squared tests for multiway contingency tables with cell proportions estimated from survey data. The Annals of Statistics. 1984;12:

[60] Akaike H. Information measures and model selection. Bulletin of the International Statistical Institute. 1983;

[61] Sobrinho-Simoes M et al. Molecular pathology of well-differentiated thyroid carcinomas. Virchows Archiv. 2005;

46-60

50:277-290

447:787-793

[53] Nakamura Y, Gojobori T, Ikemura T. Codon usage tabulated fromthe international DNA sequence databases. Nucleic Acids Research. 2007;26:

[54] Sromek M, Czetwertyńska M, et al. The frequency of selected polymorphic variants of the RET gene in patients with medullary thyroid carcinoma and in the general population of Central Poland. Endocrine Pathology. 2010;21:

[55] Ceolin L, Siqueira DR, et al. Additive

effect of RET polymorphisms on sporadic medullary thyroid carcinoma susceptibility and tumor aggressiveness. European Journal of Endocrinology.

[56] Fabien N, Paulin C, Santoro M, Berger N, et al. Detection of RET oncogene activation in human papillary

hybridisation. British Journal of Cancer.

[57] Fluge O, Haugen DR, Akslen LA, Marstad A, et al. Expression and alternative splicing of c-ret RNA in papillary thyroid carcinomas. Oncogene.

[58] Leviev I, Negro F, James RW. Two alleles of the human paraoxonase gene produce different amounts of mRNA. An explanation for differences in serum

concentrations of paraoxonase associated with the (Leu-Met54) polymorphism. Arteriosclerosis, Thrombosis, and Vascular Biology.

thyroid carcinomas by in situ

1992;66:1094-1098

2001;20:885-892

1997;17:2935-2939

104

## *Edited by Omer Engin*

Thyroid cancer is being increasingly diagnosed nowadays. This situation has attracted the attention of scientists and physicians alike and new applications in diagnosis and treatment are being developed and used. There are many cases associated with thyroid cancer and in this book, thyroid cancer is examined in various aspects.

Published in London, UK © 2019 IntechOpen © Dr\_Microbe / iStock

Knowledges on Thyroid Cancer

Knowledges on

Thyroid Cancer

*Edited by Omer Engin*