**Abstract**

Variants of MTC result from different mutations in exons of the RET gene. RET proto-oncogene is activated by a DNA rearrangement and it is one of the first tyrosine kinase receptor (RTK) proteins found to play a role in neoplasia. Early detection using genetic screening has become the gold standard of therapy, followed by prophylactic thyroidectomy. RET-kinase inhibitors have been developed recently for the treatment of MTC and are currently at various phases of pre- and clinical trials. Numerous autosomal dominantly inherited mutations have been demonstrated to activate RET constitutively. These mutations in separate populations are believed to be correlated with a rather heterogeneous prototype across countries. As such, one objective of this study was to demonstrate a geographical pattern of RET mutations in various populations. Advances in RET genetic screening have facilitated for the rapid recognition of hereditary MTCs and prophylactic thyroidectomy for relatives who may not show signs of the disease. In this chapter, we will discuss oncogenic RET signaling, RET inhibitors and the major RET mutations found in MTC and the necessity of RET genetic screening for the early diagnosis of MTC patients, using American Thyroid Association guidelines and genotype-phenotype correlation.

**Keywords:** medullary thyroid cancer, RET proto-oncogene, RET mutation, RET signaling, RET inhibitors

#### **1. Introduction**

The medullary thyroid carcinoma (MTC) is one of the most aggressive kinds of thyroid cancer. It is a neuroendocrine tumor and is notably distinct from differentiated thyroid carcinoma. MTC accounts for 5–10% of all thyroid malignancies and occurs in both sporadic (75%) and inherited (25% of cases) forms [1, 2]. The latter exhibits an autosomal dominant inheritance pattern with varying expressivity and age-dependent penetrance [3, 4]. RET (REarranged during Transfection) protooncogene is essential for the molecular pathogenesis of hereditary MTCs [5].

The human RET proto-oncogene encodes a transmembrane receptor tyrosine kinase that transmits growth and differentiation signals. Extracellular binding of ligands and coreceptors, receptor dimerization via the cysteine-rich domain, and intracellular autophosphorylation of the tyrosine kinase catalytic domain are required for RET function. RET can be activated oncogenically *in vivo* and *in vitro* by cytogenetic rearrangement. RET gene mutations have been identified in a variety of human disorders, including PTC (Papillary thyroid cancer), MTC, MEN2A, and MEN2B. Consequently, RET is referred to as the one implicated in numerous disorders [6, 7]. RET proto-oncogene is known as a molecular therapeutic target in thyroid cancer. Numerous factors, including as sex, age, tumor stage, and tumor grade can influence the prognosis. Therefore, the average survival rate for patients with thyroid gland cancers (95 percent) is approximately 10 years. Patients with regional and metastatic illness stages are estimated to have an overall survival rate of 75%, according to estimates. When a patient is diagnosed with distant metastasis, the prognosis is poor, with a 10-year survival rate of only 40% [8, 9]. Extra thyroidal metastasis and stage upon diagnosis are the only independent predictors of MTC patients' life expectancy. In other words, those detected at an early stage and patients without detectable recurrence had a life expectancy [10]. Postoperative calcitonin level and tumor extension have also been identified as significant prognostic variables for identifying MTC patients at high risk for disease recurrence [11]. We proposed that RET genetic mutations may be different in distinct populations. Therefore, in our recent study we found a geographical pattern of RET mutations in different populations [12].

This chapter is a summary of the current understanding of RET mutations and the most advanced therapeutic methods for RET-dependent thyroid tumors. We will discuss oncogenic RET signaling, RET inhibitors, and the major RET mutations found in MTC, as well as the necessity of RET genetic screening for the early diagnosis of MTC patients, in accordance with American Thyroid Association guidelines and genotype-phenotype correlation.

#### **1.1** *RET* **protein kinase structure and activation mechanism**

RET is predominantly expressed in peripheral enteric, sympathetic, and sensory neurons, in addition to central motor, dopamine, and noradrenaline neurons. It is also expressed in branching ureteric buds and differentiating spermatogenia during embryogenesis [13]. RET contains three distinct transcripts, each of which encodes RET isoforms. RET exon 19 is present in all transcripts; however, the 3' end of exon 19 undergoes variable splicing, resulting in transcripts in which exon 19 is unspliced, spliced to exon 20, or spliced to exon 21. These transcripts encode RET isoforms with 9 (RET9), 51 (RET51), or 43 (RET43) amino acid c-terminal ends. RET9 and RET51, composed of 1072 and 1114 amino acids, are the predominant isoforms *in vivo*. These two isoforms are co-expressed in the majority of tissues but have differential developmental roles and gene expression profiles, suggesting possible discrepancies in cellcell contact pathway regulation [14].

Tyrosine (Y1062), the last amino acid shared by all three isoforms, is phosphorylated during RET activation. Thus, alternate splicing inserts Y1062 in distinct contexts of amino acids in the three RET isoforms, imparting distinct binding potentials. An Nterminal extracellular portion of RET contains a ligand-binding domain, a cadherin (Ca2+-dependent cell adhesion)-like domain, and a cysteine-rich domain (near the cell membrane). This domain is a ligand for glial cell-derived neurotropic factor (GDNF), an activator protein [15]. A hydrophobic transmembrane domain and an intracellular TK domain are the other two domains. The TK domain contains several

#### *RET Proto-Oncogene Mutations: Impact on Diagnosis,Treatment and Prognosis of MTC DOI: http://dx.doi.org/10.5772/intechopen.108941*

tyrosine residues (16 in RET9 and 18 in RET51), two of which are unique to RET51 at locations 1019 and 1051. The transmembrane domain ensures the close proximity of RET monomers via noncovalent interactions between receptors. Two TK subdomains, which are phosphorylated upon receptor activation and are important in the activation of intracellular signaling pathways, are present in the intracellular region [16, 17].

GDNF, NRTN, ARTN, and PSPN are ligands of the RET receptor TK that belong to GFLs. RET is unphosphorylated and inactive in the absence of these ligands. Multiple signaling pathways are activated as a result of the activation of receptor dimerization and autophosphorylation caused by the binding of ligand to the extracellular domain of the RET receptor by GFR co-receptors [18]. In other words, after GFL binds to the RET receptor, an intracytoplasmic domain within the upstream portion of RET is autophosphorylated, stabilizing the protein and necessitating subsequent downstream activity of the RET autophosphorylation cascade. In fact, phosphorylation of Tyr981, in addition to Tyr1015, Tyr1062, and Tyr1096, is crucial for beginning intracellular signal transduction cascades [19]. It is believed that RET signaling provides growth and survival signals through the RAF-MEK-ERK and PI3K-AKT-mTOR pathways [20, 21].

#### **1.2 Intracellular signaling pathway of RET mutations**

The RET gene is located on chromosome 10q11.2, is approximately 55,000 base pairs in length, includes 21 exons, and encodes a single-pass transmembrane receptor tyrosine kinase (RTK) that is mostly expressed in neural crest and urogenital tract precursor cells [10, 22]. The RET proto-oncogene encodes a receptor tyrosine kinase with four cadherin-related motifs and a cysteine-rich region in the extracellular domain, and its four ligands mentioned above. When these neurotrophic factors are administered, they activate a unique receptor system that consists of the GFR1–4 coreceptor, which is the receptor for the ligand-binding component, and the GFR2–4 coreceptor, which is responsible for the signaling component [19, 23].

Alternate 3'-spicing generates three splicing variants of RET, including RET9, RET43, and RET51. Of these, RET9 and RET51 have the most significant isoforms, each with 1072 amino acids. Through the GFR1–4 (GDNF family receptors 1–4), GFL activation of RET can be induced. These ligands activate intrinsic tyrosine kinase activity when they interact with GFR1–4 [20, 23]. In order to activate RET, the ligand must first form a complex with the necessary co-receptor. This co-receptor then interacts on the cell membrane with the RET protein, which leads to the dimerization of the receptor and the beginning of intracellular signaling via the tyrosine kinase domains [24].

Oncogenic RET proteins activate a complex network of signal transduction pathways that contributes to cellular transformation. Binding of the ligand GFR complex to RET triggers its homo dimerization, phosphorylation of tyrosine residues and subsequent intracellular signaling; subsequently, RET activation leads to increased proliferation through a complex network of second messengers, and the molecular partners and/or targets include Jun N-terminal kinase (JNK); mammalian target of rapamycin (m-TOR); phosphatidyl- inositol 3 kinase (PI3K), son of seven less (SOS); vascular endothelial growth factor (VEGF); growth actor receptor bound protein 2 (GRB2), hypoxia inducible factor 1a (HIF1a), extracellular signal-regulated kinase

(ERK), protein kinase C (PKC), pyruvate dehydrogenase kinase (PDK), phospholipase Cγ (PLCγ) [24].

The intracellular domain of RET contains autophosphorylation sites, and phosphorylated tyrosine serve as docking sites for signaling molecules [25, 26]. Phosphorylated tyrosine 1062, also known as Y1062, is one of these residues. It serves as a binding site for several different adaptor proteins, including Shc, FRS2, Dok1/4/5, IRS1/2, and Enigma, and it is critical to the capacity of mutant RET to transform cells. In addition, it was discovered that tyrosine 905 binds to Grb7/10, tyrosine 981 binds to Src, tyrosine 1015 binds to phospholipase Cγ (PLCγ), and tyrosine 1096 binds to Grb2; all of these findings were independently confirmed by other researchers [27]. Interestingly, RAS/ERK, (PI3K)/AKT, p38MAPK, and JNK pathways are activated mainly through tyrosine 1062. When the adaptor protein Shc binds to phosphorylated tyrosine 1062, it recruits the Grb2-Gab1 and Grb2-Sos complexes that then activate the PI3K/AKT and RASERK pathways, respectively (**Figure 1**) [23, 28].

#### **Figure 1.**

*RET Intracellular signaling pathway. homodimeric GFLs activate the transmembrane RET tyrosine kinase by binding to different GFR receptors; binding of the ligand GFRα complex to RET initiate the homodimerization, phosphorylation of tyrosine residues and subsequent intracellular signaling. RET activation leads to high proliferation through a complex-network of second and third messengers that is developmentally-dependent and tissue-specific.*

#### **1.3 Oncogenic RET inhibitors**

Preclinical models and early phase clinical trials have explored targeted therapy through inhibition of RET and downstream signaling pathways. Phase III trials of the multi kinase inhibitors Vandetanib and Cabozantinib showed improvement in PFS (progression-free survival) but with many adverse events, which led to a trial of lower- dose Vandetanib [29].

#### *RET Proto-Oncogene Mutations: Impact on Diagnosis,Treatment and Prognosis of MTC DOI: http://dx.doi.org/10.5772/intechopen.108941*

In recent years, selective RET inhibitors have been created in an effort to obtain increased potency while also achieving lower toxicity (**Figure 2)**. Pralsetinib (BLU-667) and Selpercatinib (LOXO-292) are examples of such next-generation small molecule inhibitors that have been rapidly developed and introduced into clinical testing. Both inhibitors are capable of blocking a wide spectrum of RET changes, including M918T, C634W, gatekeeper mutations V804L and V804M, KIF5B-RET, and CCDC6- RET, according to the functional tests that were conducted utilising a variety of in vitro and in vivo models. It is important to note that LOXO-292 and BLU-667 have substantially less activity against VEGFR2 in comparison to modifications in RET, which could potentially reduce their toxicity [30].

The RET receptor can be inhibited effectively and selectively by LOXO-292. Both RET mutations, as observed in MTC, and RET fusions are the targets of this medication (seen in PTC, PDTC, and ATC). The RET V804 gatekeeper mutation, which is linked to resistance to RET-targeted kinase inhibitors, was the primary focus of the research that went into the development of this medication. Because LOXO-292 is able to pass the blood-brain barrier and achieve therapeutic concentrations in the central nervous system, it has the potential to be used as a therapy for brain metastases caused by RET mutation or fusion. At the annual meeting of the American Society of Clinical Oncology in 2018, preliminary findings from the LIBRETTO-001 phase 1 dose escalation and expansion trial were presented. Fatigue ranging from grade 1 to 2, diarrhea, constipation, dry mouth, nausea, and dyspnea were the side effects that occurred the most frequently (10 percent to 20 percent). Asymptomatic increase of the alanine aminotransferase level and a case of tumor lysis syndrome were the two conditions in question here. The maximum dose that the patient could tolerate was not achieved. The LIBRETTO-001 clinical trial is currently in the expansion phase, during which additional patients with RET-mutated MTC and RET-fusion malignancies, such as PTC (papillary thyroid cancer), PDTC (poorly differentiated thyroid cancer), and ATC (anaplastic thyroid cancer), are being enrolled.

**Figure 2.** *Oncogenic RET signaling and selective RET inhibitors*

Additionally, BLU-292 is an extremely selective and highly effective RET inhibitor. It works in a manner very similar to that of LOXO-292, in that it targets RET fusions as well as RET mutations, such as the RET V804 mutation. A phase 1 escalation/ expansion clinical trial of BLU-667 is currently being conducted, and the results of the trial were reported at the annual meeting of the American Association for Cancer Research in 2018. This study's objectives include determining the maximum tolerated dose, assessing safety, analyzing pharmacokinetics, and evaluating preliminary anticancer activity. Constipation, elevated alanine aminotransferase and aspartate aminotransferase, hypertension, leukopenia, headache, sleeplessness, and exhaustion were the only side effects of the low toxicity that was seen [31].

BOS172738, TPX- 0046, and TAS0953/HM06 are likewise in the early phases of development as selective RET inhibitors. In addition to the RET V804M gatekeeper mutation, multiple alternative pathways of acquired resistance to MKIs have been described. Mechanisms of resistance to selective RET remain a major field of study. According to a preclinical investigation, the unique solvent front mutation KIF5B-RET G810R may develop on-target resistance to Selpercatinib and Pralsetinib, however it remains vulnerable to TPX-0046, a selective RET inhibitor built with a macrocyclic structure to target active RET confirmation [30].

In conclusion, over the past three decades, the involvement of RET activating mutations and rearrangements in carcinogenesis has been proven. With the emergence of extremely selective RET inhibitors, there is significant enthusiasm in the RET sector. In preliminary phase I/II trials, the next-generation selective RET inhibitors Selpercatinib and Pralsetib displayed excellent clinical efficacy and safety. Both agents have obtained breakthrough designations from the FDA. Unanswered questions include the PFS, DOR (duration of response), and OS (overall survival) with these drugs; if all RET aberrant tumors respond similarly for a tissue-agnostic indication; and the mechanisms of acquired resistance to the potent RET inhibitors. In addition, combination therapies that investigate the simultaneous inhibition of RET and associated pathways will shed light on the clinical efficacy of such techniques [30].

According to clinical and preclinical studies, initiation of RET kinase activity has been characterized as a target for a number of tyrosine kinase inhibitors [32]. The finding of molecular targets in thyroid cancer has led to the development of treatments for patients who have advanced forms of the disease. These treatments include FDA-approved drugs such as Cabozantinib and Vandetanib for MTC and Sorafenib and Lenvatinib for differentiated thyroid cancer [33, 34].

Strong inhibition of the target proteins VEGFR-2, MET, RET, KIT, AXL, and TIE2 is provided by Cabozantinib. Because of its potent ability to inhibit RET; Cabozantinib was identified as a particularly promising candidate for treatment in MTC patients. Cabozantinib, in contrast to Vandetanib, does not reduce EGFR activity to a significant degree. Other tyrosine kinase receptor inhibitors, such as ZD6474, which are medicines that are active when taken orally, have an effect on VEGFR-2 and limit the actions of RET tyrosine kinase. In patients with metastatic familial MTC who were participating in a clinical research, it was revealed that ZD6474 therapy triggered some degree of cure [12].

#### **1.4** *RET* **proto-oncogene mutations**

There have been a total of 100 different mutations found in the RET gene so far, and with the exception of a few that cause dual phenotypes, the majority of them can be classified as either having a loss of function or a gain of function. Gain-of-function mutations in RET are primarily what cause RET-related malignancies, and these

#### *RET Proto-Oncogene Mutations: Impact on Diagnosis,Treatment and Prognosis of MTC DOI: http://dx.doi.org/10.5772/intechopen.108941*

mutations may be broken down into two categories: those that modify cysteine residues in the cysteine-rich domain, and those that alter residues in the RET-KD. Within the first group, the mutated residue that occurs most frequently in MEN2A patients is Cys634. This occurs because the removal of one-half of an intra-molecular disulfide bond makes it possible to form an intermolecular disulfide bond with a second mutant molecule. This results in constitutive receptor dimerization and aberrant signaling. It is not known if activating mutations within RET-KD directly lead to constitutive dimer formation or whether the mechanism for activating mutations is more diverse. RET transformation can be produced by a wide variety of mutations, including L790F, Y791F, S891A, and R844L, but the resulting symptoms are only moderately severe MTC and MEN2A. In contrast, the M918T mutation has a very high capacity for transformation and is present in 95% of MEN2B patients. This mutation is responsible for the disease. A number of mucosal, ophthalmic, and skeletal disorders are included in the MEN2B phenotype. In addition to the thyroid and adrenal glands, this phenotype affects the skeleton. In stark contrast to the MEN2A dimerizing mutations, in which Tyr905 is necessary for oncogenesis, the M918T RET mutation does not require this residue in order to become activated [35]. This implies that various underlying mechanisms disrupt RET activation's normal control in MEN2A and MEN2B. Furthermore, M918T RET specifically targets novel substrates like STAT3 that may aid in cell transformation [36]. In addition to transforming mutations that occur inside intact RET, chromosome translocations have the potential to produce oncogenic fusions that include the RET kinase domain (RET/PTC oncogenes). These oncogenes are responsible for the development of PTC. RET/PTC fusion proteins are found in the cytoplasm and contain RET-KD from the beginning of exon 12 (which begins at Glu713) all the way through the C terminus. The N-terminal domain of RET/PTC is often a dimerization domain derived from the fusion partner in many instances. Notably, reducing the converting potential of RET/PTC by mutating the residue that corresponds to Tyr905 in wild-type RET results in less transformation [37, 38].

As previously mentioned, RET missense mutations in the germline are linked to MEN2A, MEN2B, and FMTC, whereas sporadic MTC is thought to result from a somatic mutation in the tumor cells, RET mutations are primarily missense and located in exons 10, 11, 13, 14, 15, and 16 (RET's extracellular domain) (in the TK domain) [5, 39, 40]. A ligand-independent dimerization of receptor molecules, increased phosphorylation of intracellular substrates, and cell transformation can be caused by a mutation of the extracellular cysteine in codon 634 of exon 11 of RET. A mutation in the intracellular TK (for example, codon 918) has no effect on receptor dimerization, but it does promote constitutive activation of intracellular signaling pathways, which in turn culminates in cellular transformation [21, 41].

Exons 10 and 11 have the FMTC-specific mutations as well. Exon 8 (codons 532 and 533), exon 13, (codons 768, 790, and 791), (codons 804 and 844), (codon 891), and exon 16 have also been shown to have non-cysteine point mutations (codon 912) [42]. According to a recent meta-analysis, 39 distinct RET germline mutations have been discovered in FMTC patients from various families since 1993. All mutations were missense type and dispersed among exons 5, 8, 10, 11, 13, 14, 15, and 16 with the exception of a 9-bp duplication (after codon 531, exon 8). In FMTC, age-specific penetrance of cancer growth and nodal metastasis were strongly linked with particular germline RET mutations [43]. Overall, mutations in codons 609, 611, 618, and 620 of exon 10, codon 768 of exon 13, and codon 804 of exon 14 are most frequently related with FMTC. When FMTC is related with mutations in codon 634 of exon 11, C634R is nearly never observed, while C634Y is the most prevalent variant [21].

#### **1.5 Germline screening of RET mutations**

The genetic testing for RET germline mutation has demonstrated 100 percent sensitivity and specificity in identifying persons at risk for developing MTC. In comparison to the current standard of annual biochemical monitoring, such as blood calcitonin, this genetic assay allows for earlier and more conclusive diagnosis and clinical management of people who have a familial risk for MTC. Once a person is identified as having a RET mutation, they must receive thorough counseling. In order to give a preventive thyroidectomy to asymptomatic individuals who are diagnosed as RET mutation carriers, it is necessary to identify and test at-risk family members [4].

Since prophylactic thyroidectomy can prevent hMTC, the American Thyroid Association suggests that all patients with MTC be offered germline RET testing [44]. Based on a model that categorizes mutations into risk levels using genotypephenotype correlations, recommendations for the scheduling of prophylactic thyroidectomy and the extent of surgical resection are made (A-D). The highest risk of MTC is associated with ATA level D (ATA-D) mutations. Codons 883 (exon 15) and 918 (exon 16) are two of these mutations that are linked to the lowest age of onset, the highest risk of metastasis, and the highest fatality rate. A lower but still significant prevalence of aggressive MTC is linked to ATA level C (ATA-C) mutations, which include codon 634 changes (exon 11). ATA-B mutations, which include mutations at codons 609, 611, 618, 620 (exon 10) and 630, are associated with a decreased risk for severe MTC mutations (exon 11). ATA-A mutations are associated with the "least severe" risk. When they have preventative thyroidectomy at age 4 years, these patients have lower serum calcitonin levels, a lower tumor stage, and a better rate of biochemical cure compared to ATA-B mutation carriers of the same age [45]. RET mutations can be found at codons 768, 790, 791 (exon 13), 804 (exon 14), and 891 in ATA-A mutations (exon 15). ATA made the decision to develop specialized MTC Clinical Guidelines in order to compile and update the vast amount of MTC-related literature, as well as to integrate this information with evidence-based medicine and the feedback of a panel of experienced physicians [21, 46].

There are limited reports of these mutations in Iranian families with MTC in the literature [47, 48]. In our recent study, we tested individuals with MTC and their MTC-affected first-degree relatives for RET exon10 mutations. In our latest investigation, 14 individuals with sMTC and FMTC were found to have six distinct mutations in exon10 of RET that were confined to codons 611, 618, and 620, but not codon 609. This data revealed an atypical distribution of RET exon10 mutations in comparison to other groups. In our study population, exon10 of the RET proto-oncogene was mutation-free in MEN2A, MEN2B, and pheochromocytoma. However, exon10 mutations in MEN2A have been found in numerous populations. C611Y and C620R were the most prevalent mutations in exon10 among patients with FMTC and sMTC, respectively [49, 50].

Codon 620 of exon 10 has generally been shown to include eight different variants, including seven missense mutations and one synonymous mutation. The codon in exon 10 with the greatest frequency of mutations during our analysis was codon 620. In other words, more over 50% of the mutations in our investigation were caused by codon 620. Additionally, neither synonymous nor nonsense mutations in exon 10 of the RET proto-oncogene were found in our study population. None of the cysteine codons in exon 10 had any mutations. The findings of this study suggest that mutations in exon 10 of the RET proto-oncogene are limited to three critical cysteine codons (611, 618, and 620), which were only identified in Iranian patients with FMTC

#### *RET Proto-Oncogene Mutations: Impact on Diagnosis,Treatment and Prognosis of MTC DOI: http://dx.doi.org/10.5772/intechopen.108941*

and likely sMTC. All patients with exon 10 mutations, with the exception of one, had the haplotype G691S/S904S. In the current analysis, no mutation in the RET protoexon oncogene's 10 in the syndromic type of MTC was found [50].

Since the research of other exons within the same gene has received less attention, we investigated the incidence of germ line mutations in exon 2 of the RET protooncogene in Iranian patients with MTC. The RET gene has the nucleotide substitutions c135G>A/A45A (rs1800858) in exon 2 and c.337+9G>A (rs2435351) and c.337 +137G>T (rs2505530) in the intronic region. Among patients and relatives, the genotype and allele frequencies with the highest and lowest frequencies, respectively, were c.337+137G>T (rs2505530) and c135G>A/A45A (rs1800858). Also, no link was found between identified nucleotide alterations and disease phenotype, gender, or race. No mutations resulting in altered amino acid sequences in exon 2 or exon-intron splice sites were identified. However, additional research is advised to determine the likely correlation between discovered variants and the presence or absences of other mutations in other RET major exons, as well as to determine the haplotype association with the disease [51].

217 people were included in order to study the spectrum of prominent RET germline mutations in exons 10, 11, and 16 in hereditary MTC in the Iranian population. Leukocytes' genomic DNAs were isolated utilizing the Salting Out/Proteinase K technique. The mutations were detected using PCR-RFLP and DNA sequencing. In 217 subjects, 43 missense mutations were found in exons 10, 11, and 16 (6 percent, 13 percent, and 16 percent, respectively) (0.9 percent). In addition, a new germline mutation was found in exon 11 (S686N). In addition, eight individuals had four distinct SNPs in intron 16. The data revealed the frequency profile of RET mutations in Iranian patients with MTC (19.8 percent). In our population, C634G was the most prevalent mutation, but in most populations it was C634R. Collectively, these data highlight the significance of the genetic background of family members of any MTC patient [5].

Finally, it is advised that other RET exons, particularly those with a high frequency of mutations, such as exons 13, 14, and 15, be studied. Additionally, direct sequencing analysis is a reliable tool for detecting unknown RETS mutations. In addition, the transformative activity and functional effect(s) of novel RET mutations such as S686N and intronic polymorphisms have yet to be determined (**Figure 3**) [5].

#### **1.6 The relationship between RET tyrosine kinase inhibition and MTC treatment**

Recent RET-kinase inhibitors for the treatment of MTC are through various levels of preclinical and clinical testing [52]. A group has launched a phase II clinical research assessing the efficacy of oral ZD6474 (Zactima®) in patients with locally advanced or metastatic MTC: of the 20 patients accrued to date, around 30% have seen objective remissions. Other inhibitors of RET activity targeting various areas of its molecular biology and signaling pathway are in development [24, 53, 54]. The most important drugs for MTC treatment is listed in **Figure 4**.

Patients with MTC are evaluated using tumor markers (calcitonin and carcinoembryonic antigen; CEA), a complete and precise ultrasonography of the neck, and genetic testing. Cross-sectional imaging may be obtained for surgical planning or when suspected distant metastases are present. Biochemical testing is required to exclude primary hyperparathyroidism and PHEO (pheochromocytoma). After this, a total thyroidectomy with central neck dissection is often advised, and in rare instances, more extensive surgery may be required (if indicated by the preoperative

#### **Figure 4.**

*The most important FDA-approved drugs for MTC according to their approved years or current phases of clinical trials.*

assessment). External beam radiation therapy may improve loco regional control if the patient has a high risk of recurrence [34]. Since the response to first therapy influences survival and recurrence risk, an experienced multidisciplinary team should be included from the start [55]. Many of these concerns are addressed in the new American Thyroid Association (ATA) guidelines for MTC management [56].

Watchful waiting, surgery, radiation, cryo ablation, and chemoembolization are treatment options for asymptomatic residual, recurrent, and distant metastatic disease in MTC and differentiated thyroid carcinoma (DTC). In order to minimize skeletal-related occurrences in MTC patients, receptor activator of nuclear factor kappa B ligand (RANKL) inhibitors or intravenous bisphosphonate are often administered [12, 57].
