RET Gene Mutations (Genotype and Phenotype) of Multiple Endocrine Neoplasia Type 2 and Familial Medullary Thyroid Carcinoma

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1 Review Article RET Gene Mutations (Genotype and Phenotype) of Multiple Endocrine Neoplasia Type 2 and Familial Medullary Thyroid Carcinoma Geoffrey W. Krampitz, MD; and Jeffrey A. Norton, MD The rapid technical advances in molecular biology and accelerating improvements in genomic and proteomic diagnostics have led to increasingly personalized strategies for cancer therapy. Such an approach integrates the genomic, proteomic, and molecular information unique to the individual to provide an accurate genetic diagnosis, molecular risk assessment, informed family counseling, therapeutic profiling, and early preventative management that best fits the particular needs of each patient. The discovery of mutations in the RET proto-oncogene resulting in variable onset and severity of multiple endocrine neoplasia type 2 (MEN2) was the first step in developing direct genetic testing for at-risk individuals. Patients with germline RET mutations may undergo risk assessment and appropriate intervention based on specific mutations. Moreover, family members of affected individuals receive counseling based on understanding of the genetic transmission of the disease. Increasingly, clinicians are able to make therapeutic choices guided by an informative biomarker code. Improvements in detection and management of patients with MEN2 resulting from understanding of the RET proto-oncogene are evidence of the benefits of personalized cancer medicine. This review describes the discovery of the RET proto-oncogene, the association between genotype and phenotype, and the role of mutation analysis on diagnosis and treatment of MEN2. Cancer 2014;120: VC 2014 American Cancer Society. KEYWORDS: RET, calcitonin, medullary thyroid cancer, pheochromocytoma. INTRODUCTION Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant genetic syndrome caused by missense mutations in the RET proto-oncogene with different penetrance producing 3 variants, MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC). MEN2 is a rare syndrome with an incidence of 1 in 200,000 live births. The principal feature of all MEN2 subtypes is medullary thyroid carcinoma (MTC), a cancer of the parafollicular (C, or calcitonin-secreting) cells. Patients with MEN2 have a 70% to 100% risk of developing MTC by age 70 years, and MTC is the most important determinant of mortality in these patients. MTC has a poor prognosis once it has spread beyond the thyroid. However, patient survival is greatly improved when MTC is treated early in the course of the disease. Therefore, much of the emphasis of treating patients with MEN2 has focused on identifying and treating MTC. 1 As such, recognition of at-risk patients, early diagnosis, genetic risk stratification, timely surveillance, and appropriate treatment are crucial. Identifying mutations in the RET proto-oncogene as the causative factor in MEN2 has led to the development of direct genetic testing for at-risk individuals. Patients with germline RET mutations may undergo risk assessment based on particular mutations and may be offered thyroidectomy prior to developing clinically evident MTC or early in the course of the disease when cancer is limited to the thyroid gland. Family members may also receive counseling based on understanding of the genetic transmission of the disease. The initial discovery of the RET proto-oncogene as the causative factor in MEN2 was the first step in establishing the highly personalized approach to the diagnosis and treatment of MEN2. MEN2 John Sipple recognized the hereditary nature of the syndrome in However, the first description of pheochromocytoma by Felix Frankle in 1886 may be the initial account of a patient with MEN2. The descendants of the patient, who had a bilateral pheochromocytoma, were later genotyped and found to have the C634R (Cys634Arg) RET mutation. 2,3 The first description of MEN2A was by Alton Steiner and his colleagues in In 1980, Glen W. Sizemore recognized Corresponding author: Jeffrey A. Norton, MD, The Robert L. and Mary Ellenburg Professor in Surgery, Chief of Surgical Oncology and General Surgery, Stanford University School of Medicine, 300 Pasteur Drive, H3591, Stanford, CA ; Fax: (650) ; janorton@stanford.edu Stanford University School of Medicine, Stanford, California DOI: /cncr.28661, Received: December 31, 2013; Revised: February 7, 2014; Accepted: February 12, 2014, Published online April 3, 2014 in Wiley Online Library (wileyonlinelibrary.com) 1920 Cancer July 1, 2014

2 Personalized Cancer Medicine/Krampitz and Norton TABLE 1. Characteristics of MEN2 Subtypes Syndrome MEN2A MEN2B FMTC Characteristic Features Medullary thyroid cancer Pheochromocytoma Primary hyperparathyroidism (May include Hirshsprung s disease and cutaneous lichen amyloidosis) Medullary thyroid cancer Pheochromocytoma Marfanoid habitus Intestinal neuromas Mucosal ganlioneuromas Medullary thyroid cancer Abbreviations: FMTC, familial medullary thyroid cancer; MEN, multiple endocrine neoplasia. that MEN2 was composed of 2 variants that he called MEN2A and MEN2B. 5 Common features of the 2 groups are multicentric MTC, occurring in > 90% of patients, and bilateral pheochromocytomas, occurring in 50% of patients. 6 In 1986, Farndon working with Samuel Wells described FMTC, a third variant containing only MTC. The characteristics of the MEN2 subtypes are summarized in Table 1. MEN2A is the most common manifestation of MEN2, accounting for the majority of cases. MTC is often the first manifestation of MEN2A, usually occurring between the ages of 20 and 30 years. MEN2A is characterized by MTC and pheochromocytoma plus primary hyperparathyroidism, manifesting in 20% to 30% of patients. 1 In contrast to MEN2B, patients with MEN2A have a normal physical appearance. MEN2B is the most rare of the variants, accounting for approximately 5% to 10% of all cases. MEN2B is marked by earlier onset of disease compared to other forms of MEN2. Usually MTC develops within the first year of life, and the clinical course is usually more aggressive due to more advanced disease at presentation. 7 Approximately 40% to 50% of patients with MEN2B have pheochromocytoma. MEN2B lacks parathyroid pathology, but instead features Marfanoid habitus, mesodermal abnormalities, labial and mucosal neuromas, and intestinal ganglioneuromas, often leading to constipation and megacolon. 1,5,6,8 It was soon recognized that the MTC in MEN2B was the most aggressive from of hereditary MTC, characterized by rapid spread to regional and distant sites and early mortality. 7 FMTC is the mildest subtype and features MTC without any of the extrathyroid characteristics of the other MEN 2 subtypes. 9 FMTC, as it was originally described, is uncommon. Although some studies attribute up to 35% of MEN2 cases to FMTC, most families thought to have FMTC become MEN2A families when an isolated member develops either a pheochromocytoma or hyperparathyroidism. Nevertheless, the term FMTC is now commonly used to describe families who have hereditary MTC with a rare patient having one of the other endocrinopathies. Discovery of RET In 1985, Masahide Takahashi transfected NIH 3T3 fibroblast cells with sonicated human lymphoma DNA segments, which resulted in transformation of these cells. Subsequent cloning and homology analysis suggested that the new transforming gene was unlike previously identified transforming genes. Using blot hybridization analysis, Takahashi determined that the transforming sequence encompassed 34 kilobases and represented a rearrangement of 2 unlinked human DNA segments. Molecular cloning indicated that the biologically active gene was generated by recombination of 2 separate normal DNA segments. Northern blot analysis indicated that the new gene produced a new transcriptional unit. Because the new transforming gene was the product of gene rearrangement during transfection of 3T3 fibroblasts, the gene was named REarranged during Transfection, or RET. 10 RET Proto-Oncogene The proto-oncogene RET is composed of 21 exons located on chromosome 10 (10q11.2) and encodes for a transmembrane receptor tyrosine kinase for members of the glial cell line derived neurotrophic factor family (GDNF) and associated ligands (artemin, neuturin, persephin) (Fig. 1A) The RET protein is composed of 3 functional domains, including an extracellular ligandbinding domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain (Fig. 1B). The extracellular domain contains 4 cadherin-like repeats and a cysteine-rich region. The highly conserved cysteine-rich region is important for disulfide bond formation needed for maintaining the native tertiary structure allowing for receptor dimerization. The intracellular domain contains 2 tyrosine kinase subdomains that are involved in several intracellular signal transduction pathways. 15 Coupling RET, its coreceptors, GDNF-family receptor alpha (GFRa1 togfra4), 16 and GDNF-family ligands leads to RET dimerization to form a heterohexamer complex that results in transcellular kinase activation and signaling. RET is involved in a number of cellular signaling pathways during development regulating the survival, proliferation, differentiation, and migration of the enteric nervous system progenitor cells, as well as the survival and Cancer July 1,

3 Review Article Figure 1. RET proto-oncogene is the cause of MEN2. (A) The proto-oncogene RET is composed of 21 exons located on chromosome 10 (10q11.2) and encodes for a transmembrane receptor tyrosine kinase. (B) The RET protein is composed of 3 functional domains,including an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. The extracellular domain contains a signal peptide that is cleaved, 4 cadherin-like repeats, and a cysteine-rich region critical for disulfide bond formation needed in dimerization. (C) The locations of known RET mutations are highlighted according to American Thyroid Association risk classification (blue 5 rating A; green 5 rating B; orange 5 rating C; red 5 rating D). (D) Mutations are grouped by the MEN (multiple endocrine neoplasia) subtype they cause. regeneration of neural and kidney cells Mice homozygous for a targeted mutation in RET demonstrate renal agenesis or severe dysgenesis and lack enteric neurons throughout the digestive tract, suggesting that RET is a component of a signaling pathway required for renal organogenesis and enteric neurogenesis. 25,26 Subsequently, inactivating mutations in RET associated with Hirschprung s disease and renal agenesis were identified. 25,26,33-37 Mutations in RET Cause MEN2 In 1987, genetic linkage analysis mapped the MEN2A locus to a region on chromosome ,39 In subsequent years, the location of the MEN2A locus was further refined. In 1990, Massimo Santoro found increased expression of the RET gene in both familial and sporadic human pheochromocytomas and MTC. Because the RET gene was mapped to a location on chromosome 10 in close proximity to the gene that predisposes patients to 1922 Cancer July 1, 2014

4 Personalized Cancer Medicine/Krampitz and Norton the MEN2A syndrome, he suggested that this region of chromosome 10 might be involved in the proliferation and differentiation of neuroectodermal tissues. 40 Wells knew that even pentagastrin- and calcium-stimulated serum levels of calcitonin failed to reliably diagnose patients with curable familial forms of MTC, so he decided to try to identify the exact gene defect responsible for these syndromes. Furthermore, he realized that family members without the inherited mutation could be told that they did not have the syndrome and that they would not need serial screening as had been done with the pentagastrinstimulated calcitonin levels. In 1993, Helen Donis-Keller, working with Wells, identified 7 RET mutations in exons 7 and 8 associated with MEN2A and FMTC. They screened a panel of genomic DNA from 7 MEN2A, MEN2B, and FMTC families and normal controls. Using polymerase chain reaction (PCR) and sequence analysis approaches, a total of 7 single-strand conformational variants (SSCVs) were observed in MEN2 families and not in DNA from normal individuals. Six of these were identified in exon 7 and 1 in exon 8, and all mutations affected codons specifying cysteine residues. SSCVs present in germline DNA were also found in MTC and pheochromocytoma from familial cases. However, these mutations were not found in 3 families with MEN2A, suggesting that these were not the only mutations responsible for MEN2. 41 Almost simultaneously, Lois Mulligan working with Bruce Ponder generated complementary DNA from 4 MTCs and 3 pheochromocytomas from patients with MEN2A and sporadic tumors. Using chemical cleavage mismatch procedures and sequencing, they identified specific missense mutations in the RET gene at codons encoding for cysteine residues within the transition point between the extracellular and transmembrane domains. These mutations were found in 20 of 23 apparently distinct MEN2A families, but not in 23 normal controls. Furthermore, 19 of these 20 mutations affected the same conserved cysteine residue at the boundary of the RET extracellular and transmembrane domains. 42 Subsequently, Robert Hofstra showed that MEN2B is associated with a T644M (Thr644Met) mutation affecting the intracellular tyrosine kinase domain of the RET protein. Using single-strand conformational polymorphism analysis, he found a variant pattern in all 9 unrelated MEN2B patients and 6 of 18 sporadic MTC patients in his cohort. 43 At the same time, Kerri Carlson working with Helen Donis-Keller found a single missense T918M (Thr918Met) mutation in the tyrosine kinase catalytic domain of the RET proto-oncogene associated with MEN2B. They performed sequence analysis of germline DNA from 34 unrelated MEN2B patients that revealed the mutation not found in 93 unaffected individuals, including the normal parents of 14 de novo MEN2B patients. 44 In 1995, Santoro demonstrated the RET mutations responsible for MEN2A and MEN2B acted by constitutively activating the kinase, rather than through a loss of tumor suppression. By transfecting fibroblast cells with engineered vectors containing different RET mutations in the extracellular domain responsible for MEN2A and FMTC (C634Y, C634A, C634W) and MEN2B (M918T) he was able to demonstrate the increased RET kinase activity and transforming efficiency. MEN2A mutations altered the disulfide bond between receptor monomers, creating an activating homodimer. On the other hand, the MEN2B mutation altered the substrate specificity for the receptor. 45 Of the cases with MTC, 75% are sporadic and 25% are familial. Virtually all patients with hereditary MTC have germline RET mutations, whereas up to 75% of patients with sporadic MTC have somatic RET mutations, the presence of which appears to be associated with a more aggressive clinical phenotype compared to RETnegative sporadic patients. 46 However, approximately 2% to 5% of MEN2 families do not have RET mutations, and many cases of sporadic MTC do not carry RET mutations. 47,48 The absence of RET mutations suggests the existence of additional loci predisposing to MEN2 and sporadic MTC. Other genes, including those encoding components of the retinoblastoma (RB) and P53 tumorsuppressor pathways and RAS proto-oncogene may be involved in MTC formation Moreover, work done in transgenic mouse models of MTC suggest that even in the setting of RET mutations, secondary genetic alterations are required for development of MTC. 55,56 Furthermore, C-cell hyperplasia is associated with MTC in inherited MEN2 syndromes. Germline mutations in succinate dehydrogenase subunit D have been found in families with C-cell hyperplasia without MTC. 57 These patients with familial C-cell hyperplasia may conceivably be misdiagnosed as having FMTC and may account for cases attributed to FMTC in absence of RET mutations. 58 Biochemical Diagnosis The hormone calcitonin was discovered by Douglas Harold Copp in 1962, 59 and it was subsequently shown by Hirsch and Munson that the hormone was secreted by the thyroid gland. 60 The group of Armen Tashjian Jr studied a large kindred of MEN2A patients and discovered that Cancer July 1,

5 Review Article TABLE 2. American Thyroid Association Risk Assessment 69 ATA Risk Level Genetic Testing Neck Ultrasound Serum Calcitonin Thyroidectomy A <3-5 y >3-5 y >3-5 y May delay beyond age 5 if normal annual calcitonin and neck ultrasound, indolent MTC history, family preference B <3-5 y >3-5 y >3-5 y Consider before age 5 C <3-5 y >3-5 y >3-5 y Before age 5 D Immediately Immediately Immediately Immediately the thyroid C-cells secreted calcitonin and that a calcium infusion stimulated release of calcitonin. This was an excellent provocative test for the early diagnosis of MTC. Tashjian s group developed the first radioimmunoassay for calcitonin and performed early thyroidectomy based on detection of elevated serum calcitonin levels in family members at risk for MEN2A. 61 Wells studied several kindreds with MEN2A and realized that most of the elders in the families died of metastatic MTC. In 1975, his group worked on a method for the early diagnosis of MTC so that family members who had inherited a mutated RET allele could be identified and possibly cured by early thyroidectomy. Studies in pigs showed that a combined infusion of calcium and pentagastrin was the most potent provocative test for calcitonin release. 62 Wells and colleagues applied this method clinically and measured serum calcitonin levels after provocative testing. In some patients thyroidectomy and central lymph node dissection was curative, however, in others stimulated serum calcitonin levels were elevated postoperatively, indicating residual disease. Because provocative testing could not identify patients prior to onset of disease, methods to prospectively select at-risk patients and subsequently tailor appropriate treatment strategies were needed. Genetic Testing and Risk Stratification Since the identification of mutations in the RET gene as the causative agents in MEN2, genetic tests have been developed and refined to clinically detect these defects Chi and Wells were the first to develop predictive testing for MEN2 based on genetic mutations in the RET proto-oncogene. They extracted genomic DNA from 96 members of a MEN2A kindred and PCR-amplified known RET mutations resulting in new restriction endonuclease sites. These new sites could then be detected by digestion with the appropriate enzyme. They then verified the inheritance of the mutation with a previously established genetic linkage test. They found that mutations vary among kindreds but are consistently inherited within kindreds. In addition, they determined that their new direct genetic analysis supplanted an established linkagebased test, because the latter was precluded by recombination events and required the selection of informative genetic markers. The group was able to detect 43 mutations in the 43 affected individuals, thus establishing an invariable correlation between mutation and disease. Importantly, their test identified 2 genetically affected individuals who were presymptomatic. Thus, the new direct genetic analysis offered a predictive diagnostic test for the disorder. Today, genetic testing detects nearly 100% of mutation carriers and is considered the standard of care for all first-degree relatives of patients with newly diagnosed hereditary MTC. However, due to the varying clinical effects of RET mutations, strategies based on clinical phenotype, age of onset, and aggressiveness of MTC were needed to guide therapy. The first classification system was put forth after the Seventh International Workshop on MEN in 2001, which provided guidelines for the age of genetic testing and prophylactic thyroidectomy. 67 This stratification was revised when the American Thyroid Association (ATA) convened a panel of experts in 2009 to create evidence-based guidelines to assist in the clinical care of patients with MTC. Recommendations on the diagnostic workup and timing of prophylactic thyroidectomy and extent of surgery are based on a classification into 4 risk levels utilizing the genotype-phenotype correlation. 68 The ATA classified mutations according to the risk of developing early, aggressive MTC from A to D in increasing levels, and made diagnostic and treatment recommendations based on patient risk stratification (Table 2). 69 In 2010, the North American Neuroendocrine Tumor Society published consensus guidelines for the diagnosis and management of MTC. 70 These guidelines were developed by classifying RET mutations into 3 groups based on aggressiveness of MTC or level of risk (Table 3). Level 1 RET mutations (codons 609, 768s, 1924 Cancer July 1, 2014

6 Personalized Cancer Medicine/Krampitz and Norton TABLE 3. North American Neuroendocrine Tumor Society Consensus Guideline for the Diagnosis and Management of Neuroendocrine Tumors: RET Mutations Associated With Hereditary Medullary Thyroid Carcinoma (MTC) 70 NANETS Risk Level for MTC Most Common Codon Mutations Age at Prophylactic Thyroidectomy Level 1 (High) 609 By 5-10 y of age Level 2 (Higher) 611 By 5 y of age Level 3 (Highest) 883 Within the first 6 mo of life (preferably in the first month of life) 790, 791, 804, and 891) are the lowest risk for aggressive MTC marked by later onset of tumor development and a more indolent biological course. Patients with Level 1 mutations rarely develop tumors before the age of 10 years of age. However, because of variability in tumor onset in some families with these mutations and because thyroidectomy can be curative if performed early, many experts advocate performing a prophylactic operation by age 5 years whenever possible. Level 2 RET mutations (codons 611, 618, 620, and 634 mutations) are considered high risk for aggressive MTC. Patients with level 2 RET mutations should undergo thyroidectomy before age 5 years. Level 3 RET mutations (codons 883, 918, and 922) are the most aggressive of all the RET mutations. Patients with Level 3 mutations can have metastasis in the first years of life. 71 Consequently, thyroidectomy is recommended within the first 6 months and preferably within the first month of life. Genotype and Phenotype Since the initial discovery of RET mutations responsible for MEN2, as many as 50 different point mutations across 7 exons (exons 8, 10, 11, 13-16) have been identified (Fig. 1C and Table 4). 72 Different mutations in the RET gene produce varying phenotypes for the disease, including age of onset and aggressiveness of MTC, and the presence or absence of other endocrine neoplasms, such as pheochromocytoma or hyperparathyroidism (Fig. 1D and Table 4). Approximately 85% of patients with MEN2 have a mutation of exon 11 codon 634, whereas mutations in codons 609, 611, 618, and 620 account for 10% to 15% of cases. 68 Particularly early aggressive behavior and metastasis in MEN2A and MEN2B are associated with C634 and M918T mutations, respectively, requiring early intervention. 22 On the other hand, A883F mutation displays a more indolent form of MTC compared with a M918T mutation for MEN2B. 73 In addition, polymorphism at codon 836 is associated with early metastases in patients with hereditary or sporadic MTC. 74 Prophylactic Thyroidectomy Untreated, patients with MEN2 ultimately develop MTC, which is the most common cause of death in the MEN2 and related syndromes. Although the calcium pentagastrin test is the most sensitive test for the diagnosis of MTC, it is associated with significant side effects and requires annual testing of each family member. Because early detection and intervention are paramount to patient survival, genetic tests were needed to prospectively identify MEN2 patients at-risk for accelerated MTC and requiring immediate treatment. In 1995, shortly after the discovery that mutations in RET caused all forms of hereditary MTC, Wells group began performing thyroidectomies based on identification of mutated RET alleles in kindred members at direct risk for inheriting hereditary MTC. Using PCR-based testing (confirmed by haplotype analysis) for 19 known RET mutations, Wells studied 132 members of 7 kindreds with MEN2A. Twenty-one of the 58 subjects at risk for the disease had germline mutations in the RET oncogene associated with MEN2A. Plasma calcitonin levels were elevated in 9 of the 21 subjects. However, 12 subjects had normal levels of calcitonin. After undergoing genetic counseling, 13 of the 21 subjects with detected germline mutations in RET, including 6 with normal serum calcitonin levels, underwent total thyroidectomies with central lymph node dissections. All of the surgically resected thyroid glands demonstrated C-cell hyperplasia, many with evidence of MTC. However, there was no evidence of lymph node metastasis in any patient at the time of surgery. In addition, postoperative stimulated serum calcitonin levels were undetectable. Wells concluded that for family members who have germline mutations in the RET proto-oncogene, total thyroidectomy is indicated irrespective of plasma calcitonin levels. 75 Genetic screening and timely thyroidectomy in kindred members who have germline mutated RET alleles characteristic of MEN2 can prevent MTC, the most common cause of death in these syndromes. 15 More recently, a number of studies have confirmed the significance of age-appropriate thyroidectomy on Cancer July 1,

7 Review Article TABLE 4. Genotype to Phenotype Correlation 69 ATA risk Mutation MEN subtype Phenotype A G321R 106, 107 MEN2A, FMTC MTC 531/9 base pair duplication 108 MEN2A, FMTC MTC 532 duplication 109 MEN2A, FMTC MTC C515S 110 MEN2A, FMTC MTC G533C 111, 112 MEN2A, FMTC MTC, PHE R600Q 113, 114 MEN2A, FMTC MTC K603E 115 MEN2A, FMTC MTC Y606C 107, 116 MEN2A, FMTC MTC 635/insertion ELCR;T636P 107 MEN2A, FMTC MTC, PHE S649L 117, 118 MEN2A, FMTC MTC, HPT K666E 107 MEN2A, FMTC MTC, PHE E768D 119 MEN2A, FMTC MTC, PHE, HPT N777S 120 MEN2A, FMTC MTC L790F 121 MEN2A, FMTC MTC, PHE, HPT Y791F 121 MEN2A, FMTC MTC, PHE, HPT V804L 119 MEN2A, FMTC MTC, PHE, HPT V804M 119 MEN2A, FMTC MTC, PHE, HPT G819K 117 MEN2A, FMTC MTC R833C 122 MEN2A, FMTC MTC R844Q 123 MEN2A, FMTC MTC R866W 124 MEN2A, FMTC MTC S891A 117, 125, 126 MEN2A, FMTC MTC, PHE, HPT R912P 117 MEN2A, FMTC MTC B C609F/R/G/S/Y 119 MEN2A, FMTC MTC, PHE, HPT, HSC C611R/G/F/S/W/Y 119 MEN2A, FMTC MTC, PHE, HPT, HSC C618R/G/F/S/Y 119 MEN2A, FMTC MTC, PHE, HPT, HSC C620R/G/F/S/W/Y 119 MEN2A, FMTC MTC, PHE, HPT, HSC C630R/F/S/Y 127 MEN2A, FMTC MTC, PHE, HPT D631Y 123 MEN2A, FMTC MTC 633/9 base pair duplication 128 MEN2A, FMTC MTC, HPT 634/12 base pair duplication 129 MEN2A, FMTC MTC, HPT V804M1V778I 130 MEN2A, FMTC MTC C C634R 131 MEN2A MTC, PHE, HPT, CLA C634G/F/S/W/Y 131 MEN2A, FMTC MTC, PHE, HPT, CLA D V804M1E805K 132 MEN2B MTC, PHE V804M1Y806C 133 MEN2B MTC, PHE V804M1S904C 134 MEN2B MTC, HPT A883F 135, 136 MEN2B MTC, PHE M918T 119 MEN2B MTC, PHE Abbreviations: CLA, cutaneous lichen amyloidosis; FMTC, familial medullary thyroid carcinoma; HPT, hyperparathyroidism; MEN2, multiple endocrine neoplasia type 2; PHE, pheochromocytoma. disease-free survival. Machens et al conducted a European multicenter study in which 207 patients from 145 families were enrolled. 76 Patients were included in the study if they were 20 years or younger, asymptomatic, and had undergone total thyroidectomy after confirmation of a germline RET point mutation. There was a significant age-related progression from C-cell hyperplasia to MTC and, ultimately, nodal metastasis in patients whose RET mutations were grouped according to the extracellularand intracellular-domain codons affected and in those with the codon 634 genotype. Skinner and associates evaluated 50 consecutive asymptomatic children having prophylactic thyroidectomy after genetic screening had identified that they had inherited MEN2A. Physical examination and a calcium/pentagastrin stimulation test were performed at least 5 years following thyroidectomy. There was no physical evidence of MTC in any child and stimulated serum calcitonin did not increase over undetectable baseline levels in 44 (88%) of the them indicating that C-cells were absent. All 22 children having thyroidectomy prior to 8 years of age were cured, however, 6 of 28 children older than 8 years of age, had elevated stimulated plasma calcitonin levels within 5 years of thyroidectomy documenting persistent or recurrent MTC. 77 Frank-Raue et al analyzed 46 patients that were RET mutation carriers and underwent totally thyroidectomy. 78 The asymptomatic patients were stratified into 2 groups (levels 1 and 2) based on the biological aggressiveness of MTC. All patients with level 1 mutations were cured, whereas 14% of patients with level 2 mutations were not cured. The results support the relevance of treatment according to the guidelines stratifying management of MTC according to 1926 Cancer July 1, 2014

8 Personalized Cancer Medicine/Krampitz and Norton genetic information. In 2010, Schreinemakers et al conducted a case-control study to identify prognostic factors associated with recurrent MTC. 79 A total of 93 MEN2 patients were identified as RET-mutation carriers who underwent total thyroidectomy before 20 years of age. Disease-free patients were compared to those with recurrent disease. On multivariate analysis, only age of surgery was associated with persistent disease. In 2013, Shepet et al analyzed 28 patients who underwent age-appropriate surgery for hereditary MTC compared to those who had thyroidectomy past the recommended age according to the NANETS guidelines. 80 They found that patients who underwent surgery according to the NANETS guidelines were cured with no disease recurrence, whereas patients who underwent surgery past the recommended age had a 42% recurrence rate. The excellent prognosis associated with identification of MTC at its earliest stage underscores the importance of early diagnosis by RET-mutation analysis for patients at risk for familial MTC. Advanced Disease Unfortunately, the majority of patients with hereditary MTC and all patients with sporadic MTC present with a thyroid nodule and most of them already have metastases to regional lymph nodes or distant sites including brain, bone, lung, and liver. In these patients, thyroidectomy with nodal clearance is rarely curative, even though some patients have repeat operations to remove residual tumor. Distant metastases limited to a single organ may be considered for curative surgical resection or another treatment modality, such as radiofrequency ablation or external beam radiation therapy. Chemotherapy is relatively ineffective in patients with MTC, and the infrequent responses that occur are short lived. External beam radiotherapy may improve locoregional disease control, although an improvement in overall survival has not been established. 81 Consequently, effective therapy for patients with systemic disease is sorely needed and recently several molecular targeted therapeutics (MTTs) have been used in clinical trials of patients with locally advanced or metastatic MTC. There have been several clinical trials of MTTs in various formats of patients with MTC The most effective agents are the multi-tyrosine kinase inhibitors vandetanib and carbozantinib, approved by the FDA for patients with advanced MTC in 2011 and 2012, respectively. 88 Vandetanib selectively targets RET, vascular endothelial growth factor receptor-2 and -3 (VEGFR2/3), and epidermal growth factor receptor (EGFR) signaling that contribute to the growth and invasiveness of MTC. 89 Wells et al. conducted a phase 3 trial comparing vandetanib and placebo in 331 patients with advanced MTC. 90 Patients receiving vandetanib had significant improvement in median progression-free survival (estimated 30 versus 19.3 months; hazard ratio, 0.46; 95% confidence interval [CI], ; P <.001; median follow-up 24 months), objective response rate (45% versus 13%; P <.001), disease control rate (87% versus 71%; P 5.001), and biochemical response (69% versus 3%; P <.001) compared to placebo. However, nearly all patients experienced at least one adverse event and 55% experienced a grade 3 or higher adverse event. Cabozantinib selectively targets hepatocyte growth factor receptor (HGFR/MET), VEGFR2, and RET that promote invasive growth and angiogenesis in MTC Elisei et al conducted a phase 3 trial comparing cabozantinib with placebo in 330 patients with progressive metastatic MTC. 94 Patients receiving cabozantinib had improved median progression-free survival (11.2 versus 4.0 months; hazard ratio, 0.28; 95% CI, ; P <.001; median follow-up 13.9 months) and objective response rate (28% versus 0%; P <.001) compared to placebo. However, adverse events resulted in dose reductions in 79%, holds in 65%, and treatment discontinuation in 16% of cabozantinib-treated patients. Both cabozantinib and vandetanib are approved as first-line therapies for advanced MTC independent of RET-mutational status. Other multikinase inhibitors sorafenib, 84,95 axitinib, 82 and motesanib 83 have been investigated in phase 2 trials with moderate response rates. Preliminary data from a phase 2 trial evaluating the efficacy of lenvatinib in 59 patients with unresectable MTC and disease progression was presented at the 2012 ASCO Annual Meeting. 96 Lenvatinib is an oral multi-tyrosine kinase inhibitor targeting RET, VEGFR1 through VEGFR3, fibroblast growth factor receptors 1 through 4, (FGFR1 through FGFR4), stem cell factor receptor (CD117/SCFR/KIT), and platelet-derived growth factor receptor beta (PDGFRb). There was a median progression-free survival of 9.0 months (46% events observed), an objective response rate of 35% in patients receiving prior anti-vegf therapy and response rate of 36% in patients that did not receive prior anti-vegf therapy. Importantly, there were no clear differences in treatment responses based on RET-mutational status. However, 54% of patients required dose reduction and 22% required treatment discontinuation due to drug toxicity. Other agents are currently under study and various investigators are initiating trials of combinatorial therapy for patients with advanced MTC as well as other thyroid Cancer July 1,

9 Review Article cancers. Recent evidence suggests that aberrations in the PI3K/AKT, MAPK, Notch, and the glycogen synthase kinase-3 pathways may contribute to tumor growth in MTC Small molecule inhibitors of these pathways are also currently being investigated as potential therapies for MTC The use of MTTs in patients with the various types of thyroid cancer offers a new therapeutic modality for patients with advanced disease, where formerly there had been nothing. Summary MEN2 is a genetic syndrome caused by mutations in the RET proto-oncogene with different penetrance producing 3 variants, MEN2A, MEN2B, and FMTC, each of which is characterized by MTC. The discovery of RET mutations that cause MEN2 lead to the development of genetic testing that enabled personalized approaches to diagnosis, risk stratification, and appropriate treatment. FUNDING SUPPORT No specific funding was disclosed. CONFLICT OF INTEREST DISCLOSURES The authors made no disclosures. REFERENCES 1. Moline J, Eng C. Multiple endocrine neoplasia type 2: An overview. Genet Med Frankel F. Ein Fall von doppelseitigem, v ollig latent verlaufenen Nebennierentumor und gleichzeitiger Nephritis mit Ver anderungen am Circulationsapparat und Retinitis. Arch Pathol Anat Physiol Klin Med. 1886;103: Neumann HP, Vortmeyer A, Schmidt D, et al. Evidence of MEN- 2 in the original description of classic pheochromocytoma. N Engl J Med. 2007;357: Steiner AL, Goodman AD, Powers SR. Study of a kindred with pheochromocytoma, medullary thyroid carcinoma, hyperparathyroidism and Cushing s disease: multiple endocrine neoplasia, type 2. Medicine (Baltimore). 1968;47: Sizemore GW, Health H, 3rd, Carney JA. Multiple endocrine neoplasia type 2. Clin Endocrinol Metab. 1980;9: Howe JR, Norton JA, Wells SA, Jr. Prevalence of pheochromocytoma and hyperparathyroidism in multiple endocrine neoplasia type 2A: results of long-term follow-up. Surgery. 1993;114: Norton JA, Froome LC, Farrell RE, Wells SA, Jr. Multiple endocrine neoplasia type IIb: the most aggressive form of medullary thyroid carcinoma. Surg Clin North Am. 1979;59: Carney JA, Go VL, Sizemore GW, Hayles AB. Alimentary-tract ganglioneuromatosis. A major component of the syndrome of multiple endocrine neoplasia, type 2b. N Engl J Med. 1976;295: Farndon JR, Leight GS, Dilley WG, et al. Familial medullary thyroid carcinoma without associated endocrinopathies: a distinct clinical entity. Br J Surg. 1986;73: Takahashi M, Ritz J, Cooper GM. Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell. 1985;42: Durbec P, Marcos-Gutierrez CV, Kilkenny C, et al. GDNF signalling through the Ret receptor tyrosine kinase. Nature. 1996;381: Robertson K, Mason I. The GDNF-RET signalling partnership. Trends Genet. 1997;13: Nosrat CA, Tomac A, Hoffer BJ, Olson L. Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mrnas. Exp Brain Res. 1997;115: Trupp M, Arenas E, Fainzilber M, et al. Functional receptor for GDNF encoded by the c-ret proto-oncogene. Nature. 1996;381: Wells SA, Jr., Santoro M. Targeting the RET pathway in thyroid cancer. Clin Cancer Res. 2009;15: Jing S, Wen D, Yu Y, et al. GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF. Cell. 1996;85: Golden JP, Hoshi M, Nassar MA, et al. RET signaling is required for survival and normal function of nonpeptidergic nociceptors. J Neurosci. 2010;30: Tee JB, Choi Y, Shah MM, et al. Protein kinase A regulates GDNF/RET-dependent but not GDNF/Ret-independent ureteric bud outgrowth from the Wolffian duct. Dev Biol. 2010;347: Ryu H, Jeon GS, Cashman NR, Kowall NW, Lee J. Differential expression of c-ret in motor neurons versus non-neuronal cells is linked to the pathogenesis of ALS. Lab Invest. 2011;91: Ohgami N, Ida-Eto M, Sakashita N, et al. Partial impairment of c- Ret at tyrosine 1062 accelerates age-related hearing loss in mice. Neurobiol Aging Pachnis V, Mankoo B, Costantini F. Expression of the c-ret protooncogene during mouse embryogenesis. Development. 1993;119: Moore SW, Zaahl MG. Multiple endocrine neoplasia syndromes, children, Hirschsprung s disease and RET. Pediatr Surg Int. 2008; 24: Reginensi A, Clarkson M, Neirijnck Y, et al. SOX9 controls epithelial branching by activating RET effector genes during kidney development. Hum Mol Genet. 2011;20: Brantley MA, Jr., Jain S, Barr EE, Johnson EM, Jr., Milbrandt J. Neurturin-mediated ret activation is required for retinal function. J Neurosci. 2008;28: Schuchardt A, D Agati V, Larsson-Blomberg L, Costantini F, Pachnis V. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature. 1994;367: Schuchardt A, D Agati V, Larsson-Blomberg L, Costantini F, Pachnis V. RET-deficient mice: an animal model for Hirschsprung s disease and renal agenesis. J Intern Med. 1995;238: Rungby J. [RET, a gene responsible for familial endocrine neoplasias and Hirschsprung disease]. Ugeskr Laeger. 1994;156: Lyonnet S, Edery P, Mulligan LM, et al. [Mutations of RET proto-oncogene in Hirschsprung disease]. C R Acad Sci III. 1994; 317: Edery P, Lyonnet S, Mulligan LM, et al. Mutations of the RET proto-oncogene in Hirschsprung s disease. Nature. 1994;367: Romeo G, Ronchetto P, Luo Y, et al. Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung s disease. Nature. 1994;367: Smith DP, Eng C, Ponder BA. Mutations of the RET proto-oncogene in the multiple endocrine neoplasia type 2 syndromes and Hirschsprung disease. J Cell Sci Suppl. 1994;18: Attie T, Edery P, Lyonnet S, Nihoul-Fekete C, Munnich A. [Identification of mutation of RET proto-oncogene in Hirschsprung disease]. C R Seances Soc Biol Fil. 1994;188: Jeanpierre C, Mace G, Parisot M, et al. RET and GDNF mutations are rare in fetuses with renal agenesis or other severe kidney development defects. J Med Genet. 2011;48: Jain S. The many faces of RET dysfunction in kidney. Organogenesis. 2009;5: Cancer July 1, 2014

10 Personalized Cancer Medicine/Krampitz and Norton 35. Skinner MA, Safford SD, Reeves JG, Jackson ME, Freemerman AJ. Renal aplasia in humans is associated with RET mutations. Am J Hum Genet. 2008;82: Gestblom C, Sweetser DA, Doggett B, Kapur RP. Sympathoadrenal hyperplasia causes renal malformations in Ret(MEN2B)-transgenic mice. Am J Pathol. 1999;155: Schuchardt A, D Agati V, Pachnis V, Costantini F. Renal agenesis and hypodysplasia in ret-k- mutant mice result from defects in ureteric bud development. Development. 1996;122: Mathew CG, Chin KS, Easton DF, et al. A linked genetic marker for multiple endocrine neoplasia type 2A on chromosome 10. Nature. 1987;328: Simpson NE, Kidd KK, Goodfellow PJ, et al. Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage. Nature. 1987;328: Santoro M, Rosati R, Grieco M, et al. The ret proto-oncogene is consistently expressed in human pheochromocytomas and thyroid medullary carcinomas. Oncogene. 1990;5: Donis-Keller H, Dou S, Chi D, et al. Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet. 1993;2: Mulligan LM, Kwok JB, Healey CS, et al. Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature. 1993;363: Hofstra RM, Landsvater RM, Ceccherini I, et al. A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature. 1994;367: Carlson KM, Dou S, Chi D, et al. Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B. Proc Natl Acad Sci U S A. 1994;91: Santoro M, Carlomagno F, Romano A, et al. Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science. 1995;267: Elisei R, Cosci B, Romei C, et al. Prognostic significance of somatic RET oncogene mutations in sporadic medullary thyroid cancer: a 10-year follow-up study. J Clin Endocrinol Metab. 2008;93: Lodish MB, Stratakis CA. RET oncogene in MEN2, MEN2B, MTC and other forms of thyroid cancer. Expert Rev Anticancer Ther. 2008;8: Montero-Conde C, Martin-Campos JM, Lerma E, et al. Molecular profiling related to poor prognosis in thyroid carcinoma. Combining gene expression data and biological information. Oncogene. 2008;27: Harvey M, Vogel H, Lee EY, Bradley A, Donehower LA. Mice deficient in both p53 and Rb develop tumors primarily of endocrine origin. Cancer Res. 1995;55: Coxon AB, Ward JM, Geradts J, Otterson GA, Zajac-Kaye M, Kaye FJ. RET cooperates with RB/p53 inactivation in a somatic multi-step model for murine thyroid cancer. Oncogene. 1998;17: Ziebold U, Lee EY, Bronson RT, Lees JA. E2F3 loss has opposing effects on different prb-deficient tumors, resulting in suppression of pituitary tumors but metastasis of medullary thyroid carcinomas. Mol Cell Biol. 2003;23: Cerrato A, De Falco V, Santoro M. Molecular genetics of medullary thyroid carcinoma: the quest for novel therapeutic targets. J Mol Endocrinol. 2009;43: Nakagawa T, Mabry M, de Bustros A, Ihle JN, Nelkin BD, Baylin SB. Introduction of v-ha-ras oncogene induces differentiation of cultured human medullary thyroid carcinoma cells. Proc Natl Acad Sci U S A. 1987;84: Johnston D, Hatzis D, Sunday ME. Expression of v-ha-ras driven by the calcitonin/calcitonin gene-related peptide promoter: a novel transgenic murine model for medullary thyroid carcinoma. Oncogene. 1998;16: Smith-Hicks CL, Sizer KC, Powers JF, Tischler AS, Costantini F. C-cell hyperplasia, pheochromocytoma and sympathoadrenal malformation in a mouse model of multiple endocrine neoplasia type 2B. EMBO J. 2000;19: Cranston AN, Ponder BA. Modulation of medullary thyroid carcinoma penetrance suggests the presence of modifier genes in a RET transgenic mouse model. Cancer Res. 2003;63: Lima J, Teixeira-Gomes J, Soares P, et al. Germline succinate dehydrogenase subunit D mutation segregating with familial non-ret C cell hyperplasia. J Clin Endocrinol Metab. 2003;88: Mulligan LM. From genes to decisions. In: Farid NR, editor. Molecular Basis of Thyroid Cancer. New York: Kluwer Academic Publishers, Copp DH, Cheney B. Calcitonin-a hormone from the parathyroid which lowers the calcium-level of the blood. Nature. 1962;193: Hirsch PF, Voelkel EF, Munson PL. Thyrocalcitonin: Hypocalcemic Hypophosphatemic Principle of the Thyroid Gland. Science. 1964;146: Melvin KE, Miller HH, Tashjian AH, Jr. Early diagnosis of medullary carcinoma of the thyroid gland by means of calcitonin assay. N Engl J Med. 1971;285: Wells SA, Jr., Baylin SB, Linehan WM, Farrell RE, Cox EB, Cooper CW. Provocative agents and the diagnosis of medullary carcinoma of the thyroid gland. Ann Surg. 1978;188: Unger K, Malisch E, Thomas G, et al. Array CGH demonstrates characteristic aberration signatures in human papillary thyroid carcinomas governed by RET/PTC. Oncogene. 2008;27: Xue F, Yu H, Maurer LH, et al. Germline RET mutations in MEN 2A and FMTC and their detection by simple DNA diagnostic tests. Hum Mol Genet. 1994;3: Chi DD, Toshima K, Donis-Keller H, Wells SA, Jr. Predictive testing for multiple endocrine neoplasia type 2A (MEN 2A) based on the detection of mutations in the RET protooncogene. Surgery. 1994;116: ; discussion Pazaitou-Panayiotou K, Kaprara A, Sarika L, et al. Efficient testing of the RET gene by DHPLC analysis for MEN 2 syndrome in a cohort of patients. Anticancer Res. 2005;25: Brandi ML, Gagel RF, Angeli A, et al. Guidelines for diagnosis and therapy of MEN type 1 and type 2. J Clin Endocrinol Metab. 2001;86: Raue F, Frank-Raue K. Update multiple endocrine neoplasia type 2. Fam Cancer. 2010;9: Kloos RT, Eng C, Evans DB, et al. Medullary thyroid cancer: management guidelines of the American Thyroid Association. Thyroid. 2009;19: Chen H, Sippel RS, O Dorisio MS, et al. The North American Neuroendocrine Tumor Society consensus guideline for the diagnosis and management of neuroendocrine tumors: pheochromocytoma, paraganglioma, and medullary thyroid cancer. Pancreas. 2010;39: Roman S, Lin R, Sosa JA. Prognosis of medullary thyroid carcinoma: demographic, clinical, and pathologic predictors of survival in 1252 cases. Cancer. 2006;107: Frank-Raue K, Rondot S, Schulze E, Raue F. Change in the spectrum of RET mutations diagnosed between 1994 and Clin Lab. 2007;53: Jasim S, Ying AK, Waguespack SG, et al. Multiple endocrine neoplasia type 2B with a RET proto-oncogene A883F mutation displays a more indolent form of medullary thyroid carcinoma compared with a RET M918T mutation. Thyroid. 2011;21: Siqueira DR, Romitti M, da Rocha AP, et al. The RET polymorphic allele S836S is associated with early metastatic disease in patients with hereditary or sporadic medullary thyroid carcinoma. Endocr Relat Cancer. 2010;17: Wells SA, Jr., Chi DD, Toshima K, et al. Predictive DNA testing and prophylactic thyroidectomy in patients at risk for multiple endocrine neoplasia type 2A. Ann Surg. 1994;220: ; discussion Machens A, Niccoli-Sire P, Hoegel J, et al. Early malignant progression of hereditary medullary thyroid cancer. N Engl J Med. 2003;349: Cancer July 1,

11 Review Article 77. Skinner MA, Moley JA, Dilley WG, Owzar K, Debenedetti MK, Wells SA, Jr. Prophylactic thyroidectomy in multiple endocrine neoplasia type 2A. N Engl J Med. 2005;353: Frank-Raue K, Buhr H, Dralle H, et al. Long-term outcome in 46 gene carriers of hereditary medullary thyroid carcinoma after prophylactic thyroidectomy: impact of individual RET genotype. Eur J Endocrinol. 2006;155: Schreinemakers JM, Vriens MR, Valk GD, et al. Factors predicting outcome of total thyroidectomy in young patients with multiple endocrine neoplasia type 2: a nationwide long-term follow-up study. World J Surg. 2010;34: Shepet K, Alhefdhi A, Lai N, Mazeh H, Sippel R, Chen H. Hereditary medullary thyroid cancer: age-appropriate thyroidectomy improves disease-free survival. Ann Surg Oncol. 2013;20: Brierley J, Tsang R, Simpson WJ, Gospodarowicz M, Sutcliffe S, Panzarella T. Medullary thyroid cancer: analyses of survival and prognostic factors and the role of radiation therapy in local control. Thyroid. 1996;6: Cohen EE, Rosen LS, Vokes EE, et al. Axitinib is an active treatment for all histologic subtypes of advanced thyroid cancer: results from a phase II study. J Clin Oncol. 2008;26: Schlumberger MJ, Elisei R, Bastholt L, et al. Phase II study of safety and efficacy of motesanib in patients with progressive or symptomatic, advanced or metastatic medullary thyroid cancer. J Clin Oncol. 2009;27: Lam ET, Ringel MD, Kloos RT, et al. Phase II clinical trial of sorafenib in metastatic medullary thyroid cancer. J Clin Oncol. 2010; 28: Ravaud A, de la Fouchardiere C, Asselineau J, et al. Efficacy of sunitinib in advanced medullary thyroid carcinoma: intermediate results of phase II THYSU. Oncologist. 2010;15: ; author reply Kurzrock R, Patnaik A, Aisner J, et al. A phase I study of weekly R1507, a human monoclonal antibody insulin-like growth factor-i receptor antagonist, in patients with advanced solid tumors. Clin Cancer Res. 2010;16: Wells SA, Jr., Robinson BG, Gagel RF, et al. Vandetanib in Patients With Locally Advanced or Metastatic Medullary Thyroid Cancer: A Randomized, Double-Blind Phase III Trial. J Clin Oncol Haddad RI. New developments in thyroid cancer. J Natl Compr Canc Netw. 2013;11: Rodriguez-Antona C, Pallares J, Montero-Conde C, et al. Overexpression and activation of EGFR and VEGFR2 in medullary thyroid carcinomas is related to metastasis. Endocr Relat Cancer. 2010; 17: Wells SA, Jr., Robinson BG, Gagel RF, et al. Vandetanib in patients with locally advanced or metastatic medullary thyroid cancer: a randomized, double-blind phase III trial. J Clin Oncol. 2012; 30: Papotti M, Olivero M, Volante M, et al. Expression of Hepatocyte Growth Factor (HGF) and its Receptor (MET) in Medullary Carcinoma of the Thyroid. Endocr Pathol. 2000;11: Soh EY, Duh QY, Sobhi SA, et al. Vascular endothelial growth factor expression is higher in differentiated thyroid cancer than in normal or benign thyroid. J Clin Endocrinol Metab. 1997;82: Cassinelli G, Favini E, Degl Innocenti D, et al. RET/PTC1-driven neoplastic transformation and proinvasive phenotype of human thyrocytes involve Met induction and beta-catenin nuclear translocation. Neoplasia. 2009;11: Elisei R, Schlumberger MJ, Muller SP, et al. Cabozantinib in progressive medullary thyroid cancer. J Clin Oncol. 2013;31: Ahmed M, Barbachano Y, Riddell A, et al. Analysis of the efficacy and toxicity of sorafenib in thyroid cancer: a phase II study in a UK based population. Eur J Endocrinol. 2011;165: Schlumberger M JB, Cabanillas M, et al. A phase II trial of the multitargeted kinase inhibitor lenvatinib (E7080) in advanced medullary thyroid cancer (MTC). Annals of Surgical Oncology Annual Meeting, Sippel RS, Kunnimalaiyaan M, Chen H. Current management of medullary thyroid cancer. Oncologist. 2008;13: Kunnimalaiyaan M, Vaccaro AM, Ndiaye MA, Chen H. Inactivation of glycogen synthase kinase-3beta, a downstream target of the raf-1 pathway, is associated with growth suppression in medullary thyroid cancer cells. Mol Cancer Ther. 2007;6: Kunnimalaiyaan M, Chen H. Tumor suppressor role of Notch-1 signaling in neuroendocrine tumors. Oncologist. 2007;12: Kunnimalaiyaan M, Ndiaye M, Chen H. Apoptosis-mediated medullary thyroid cancer growth suppression by the PI3K inhibitor LY Surgery. 2006;140: ; discussion Vaccaro A, Chen H, Kunnimalaiyaan M. In-vivo activation of Raf- 1 inhibits tumor growth and development in a xenograft model of human medullary thyroid cancer. Anticancer Drugs. 2006;17: Adler JT, Cook M, Luo Y, et al. Tautomycetin and tautomycin suppress the growth of medullary thyroid cancer cells via inhibition of glycogen synthase kinase-3beta. Mol Cancer Ther. 2009;8: Adler JT, Hottinger DG, Kunnimalaiyaan M, Chen H. Inhibition of growth in medullary thyroid cancer cells with histone deacetylase inhibitors and lithium chloride. J Surg Res. 2010;159: Cook MR, Luo J, Ndiaye M, Chen H, Kunnimalaiyaan M. Xanthohumol inhibits the neuroendocrine transcription factor achaetescute complex-like 1, suppresses proliferation, and induces phosphorylated ERK1/2 in medullary thyroid cancer. Am J Surg. 2010; 199: ; discussion Truong M, Cook MR, Pinchot SN, Kunnimalaiyaan M, Chen H. Resveratrol induces Notch2-mediated apoptosis and suppression of neuroendocrine markers in medullary thyroid cancer. Ann Surg Oncol. 2011;18: Dvorakova S, Vaclavikova E, Duskova J, Vlcek P, Ryska A, Bendlova B. Exon 5 of the RET proto-oncogene: a newly detected risk exon for familial medullary thyroid carcinoma, a novel germline mutation Gly321Arg. J Endocrinol Invest. 2005;28: Ahmed SA, Snow-Bailey K, Highsmith WE, Sun W, Fenwick RG, Mao R. Nine novel germline gene variants in the RET proto-oncogene identified in twelve unrelated cases. J Mol Diagn. 2005;7: Pigny P, Bauters C, Wemeau JL, et al. A novel 9-base pair duplication in RET exon 8 in familial medullary thyroid carcinoma. J Clin Endocrinol Metab. 1999;84: Niccoli-Sire P, Murat A, Rohmer V, et al. When should thyroidectomy be performed in familial medullary thyroid carcinoma gene carriers with non-cysteine RET mutations? Surgery. 2003;134: ; discussion Fazioli F, Piccinini G, Appolloni G, et al. A new germline point mutation in Ret exon 8 (cys515ser) in a family with medullary thyroid carcinoma. Thyroid. 2008;18: Da Silva AM, Maciel RM, Da Silva MR, Toledo SR, De Carvalho MB, Cerutti JM. A novel germ-line point mutation in RET exon 8 (Gly(533)Cys) in a large kindred with familial medullary thyroid carcinoma. J Clin Endocrinol Metab. 2003;88: Bethanis S, Koutsodontis G, Palouka T, et al. A newly detected mutation of the RET protooncogene in exon 8 as a cause of multiple endocrine neoplasia type 2A. Hormones (Athens). 2007;6: Saez ME, Ruiz A, Cebrian A, et al. A new germline mutation, R600Q, within the coding region of RET proto-oncogene: a rare polymorphism or a MEN 2 causing mutation? Hum Mutat. 2000; 15: Raue F, Kraimps JL, Dralle H, et al. Primary hyperparathyroidism in multiple endocrine neoplasia type 2A. J Intern Med. 1995;238: Rey JM, Brouillet JP, Fonteneau-Allaire J, et al. Novel germline RET mutation segregating with papillary thyroid carcinomas. Genes Chromosomes Cancer. 2001;32: Cancer July 1, 2014