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Molecular advances in medullary thyroid cancer diagnostics
Clinica Chimica Acta 370 (2006) 2 – 8 www.elsevier.com/locate/clinchim
Invited critical review
Molecular advances in medullary thyroid cancer diagnostics Richard A. Hubner ⁎, Richard S. Houlston Institute of Cancer Research, Cancer Genetics, 15 Cotswold Road, Sutton, SM2 5NG, United Kingdom Received 5 January 2006; received in revised form 24 January 2006; accepted 25 January 2006 Available online 7 March 2006
Abstract Germline activating mutations in the RET proto-oncogene cause inherited medullary thyroid cancer (MTC) and the multiple endocrine neoplasia type 2 (MEN2) syndrome. Identification of a RET mutation in an individual with MEN2 allows pre-symptomatic genetic testing of other at-risk family members, and guides early intervention to prevent death and serious morbidity from MTC. Developments in the understanding of downstream RET receptor signalling pathways and how activating mutations disturb receptor function has led to insights into the possible molecular mechanisms underlying the different MEN2 phenotypes. Mutation analysis of RET in individuals with MEN2 has identified a number of different mutations, and correlation with cancer biology and clinical outcome has led to tailoring of management according to the mutation detected. © 2006 Elsevier B.V. All rights reserved. Keywords: Medullary thyroid cancer; MEN2; RET proto-oncogene; Tyrosine kinase
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . . . . Clinical syndromes . . . . . . . . . . . . . . . Natural history of medullary thyroid carcinoma . The RET proto-oncogene . . . . . . . . . . . . 4.1. Normal structure and function . . . . . . 4.2. RET proto-oncogene mutations . . . . . 5. Genotype–phenotype correlations in MEN2. . . 6. Molecular basis for disease phenotype in MEN2 7. Genetic testing for inherited MTC. . . . . . . . 8. Clinical management based on mutation testing. 9. Conclusion. . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Medullary thyroid cancer (MTC) arises from the parafollicular or C-cells of the thyroid gland, and was first recognised as a distinct clinicopathologic entity in 1959 [1]. MTC is rare and accounts for only 3% to 10% of thyroid cancer, equating to
⁎ Corresponding author. Tel.: +44 020 87224385. E-mail address: [email protected] (R.A. Hubner). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.01.029
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approximately 1000 new diagnoses in the US each year [2,3]. In 60% to 75% of cases MTC occurs sporadically, whilst in the remainder it is inherited in an autosomal dominant fashion and may be associated with other endocrine tumours as part of the multiple endocrine neoplasia type 2 (MEN2) syndrome. MEN2 affects 1 in 30,000 individuals, and to date around 500 families with members affected by MTC as part of MEN2 have been identified worldwide [4]. Germline activating mutations of the RET proto-oncogene were first recognised to cause inherited MTC in 1993 [5,6], since
R.A. Hubner, R.S. Houlston / Clinica Chimica Acta 370 (2006) 2–8
then mutation analysis in MEN2 families with MTC has identified over 30 different missense mutations segregating with the disease [7]. In MEN2 families with an identified germline RET mutation pre-symptomatic genetic testing of unaffected at-risk family members can be performed, enabling prophylactic surgical treatment to be directed to those carrying a mutation [8]. It has been suggested that there are distinct genotype-phenotype correlations, and that the biological aggressiveness of MTC can be predicted according to the underlying germline RET mutation. International consortia have studied the outcomes of individuals within families carrying different mutations and have concluded that mutation carriers may be placed into three risk levels according to the RET mutation carried, and that treatment should be tailored accordingly [4,9]. This review will focus on recent advances in the understanding of how RET mutations in inherited MTC affect RET proto-oncogene functioning, current information concerning genotype–phenotype correlations, and how both of these are refining the use of genotype in the diagnosis and management of MTC. 2. Clinical syndromes Inherited MTC can occur either in isolation, in which case it is termed Familial MTC (FMTC), or in combination with other endocrine neoplasms as part of the MEN2 syndrome. The most common form of inherited MTC, accounting for up to 80% of cases, is multiple endocrine neoplasia type 2A (MEN2A), or Sipple Syndrome [10]. Individuals with MEN2A develop MTC (100% of cases), phaeochromocytoma (50% of cases), and hyperparathyroidism (10–20% of cases). MTC is by far the commonest clinical presentation of MEN2A [11]. Two rare variants of MEN2A have also been documented, MEN2A with Hirschsprung's disease, and MEN2A with cutaneous lichen amyloidosis [11]. Hirschprung's disease is characterised by the absence of autonomic ganglia in the terminal hindgut, resulting in colonic dilatation, constipation, and obstruction in neonates. Cutaneous lichen amyloidosis presents as a pruritic rash between the shoulder blades in the second or third decade [12]. Multiple endocrine neoplasia type 2B (MEN2B) is less common than MEN2A, accounting for 5% of MEN2 cases, but is clinically more distinctive. It is characterised by aggressive MTC (100% of cases), phaeochromocytoma (50% of cases), a Marfanoid body habitus (without the vascular and ophthalmologic abnormalities of Marfan's syndrome), the presence of distinctive mucosal neuromas on the distal tongue, lips, and subconjunctival areas, and diffuse ganglioneuromas of the gastrointestinal tract. Parathyroid disease does not occur in MEN2B. Children with MEN2B may be identified by their characteristic facial and oral features, but gastrointestinal symptoms with intermittent diarrhoea, constipation, and obstruction are the most common initial presentation [11]. The majority of MEN2B cases are the result of spontaneous new RET mutations, thus most patients lack a family history and do not undergo screening or prophylactic thyroidectomy,
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and therefore may present late with mucosal neuromas or a palpable thyroid mass [13]. Familial MTC accounts for 5% to 10% of inherited MTC, and is defined as the presence of MTC in families with four or more affected members and with objective evidence of the absence of adrenal medullary or parathyroid involvement [11]. However the 50% or lower penetrance of adrenal medullary and parathyroid manifestations in MEN2A can make distinguishing FMTC from MEN2A difficult, particularly in families with few affected individuals, or mainly young affected individuals. 3. Natural history of medullary thyroid carcinoma The normal thyroid is composed of follicular cells involved in the production of thyroid hormone, and parafollicular C-cells which account for 0.1% of thyroid cells, produce the hormone calcitonin, and are the cell of origin for MTC [3]. Calcitonin is secreted by MTCs and promotes the absorption of calcium by the skeletal system and inhibits the resorption of bone by osteoclasts [14]. C-cell hyperplasia is believed to be a precursor of MTC, particularly in inherited MTC where it is virtually always present [15]. Inherited MTC, as with other tumours in inherited syndromes, is more often multicentric and bilateral than sporadic MTC. Metastatic spread is initially to the cervical lymph nodes, and subsequently outside the neck to liver, lung, and bone [3]. The aggressiveness of the disease varies with the clinical setting. In inherited MTC, where individuals have not undergone screening, the average age of presentation is 20 to 40years, whilst for sporadic MTC it is 50 to 60years [11]. Patients with MEN2A may develop MTC as early as age 5years and C cell hyperplasia at an even earlier age [13]. Early metastasis is particularly a feature of MEN2B where cervical lymph node metastases have been observed in patients as young as three years [16]. Familial medullary thyroid cancer tends to have the least aggressive course. Metastatic MTC does not generally respond well to conventional radiotherapy or chemotherapy. Thus the optimum strategy for effective management is screening of at-risk individuals and early surgical intervention prior to the development of the primary tumour [3,17]. 4. The RET proto-oncogene 4.1. Normal structure and function The RET (REarranged during Transfection) gene was first identified in 1985 as a proto-oncogene that can undergo activation by DNA rearrangement [18]. The RET gene is located near the centromere on the long arm of chromosome 10, has 21 exons, and encodes a receptor tyrosine kinase expressed mainly in neural crest and urogential precursor cells, including thyroid parafollicular C-cells, adrenal medullary cells, parasympathetic, sympathetic, and colonic ganglia, and parathyroid cells [18–20]. Gene knockout studies in mice have shown that the RET gene is essential for the development of the enteric nervous system and kidney [21–23].
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R.A. Hubner, R.S. Houlston / Clinica Chimica Acta 370 (2006) 2–8 RET receptor Cadherin-like domain
Cysteine-rich domain
Tyrosine kinase domain
Tyrosine kinase domain
RET gene
Codon mutation
Clinical syndrome
532 533
FMTC FMTC
609 611 618 620
FMTC/MEN2A FMTC/MEN2A FMTC/MEN2A MEN2A/FMTC
630 634 635 637
FMTC MEN2A/FMTC MEN2A MEN2A
768 790 791
FMTC FMTC/MEN2A FMTC/MEN2A
Exon 14
804 V804M+Y806C V804M+S904C 844
FMTC/MEN2A MEN2B MEN2B FMTC
Exon 15
883 891
MEN2B FMTC/MEN2A
912 918
FMTC MEN2B
Exon 8 Exon 10 Exon 11
Exon 13
Exon 16
Fig. 1. Schematic diagram of the RET receptor, RET gene, and location of reported codon mutations and their associated clinical syndromes.
The RET protein has four cadherin-like repeats and a cysteine-rich region in the extracellular domain, a transmembrane domain, and two intracellular tyrosine kinase domains (Fig. 1). The extracellular cysteine-rich region is important for receptor dimerization, whilst the intracellular tyrosine kinase domains are involved in signal transduction. Alternative splicing of the 3′ region of RET generates three protein isoforms, short (RET9), middle (RET43), and long (RET51), which differ in the amino acid sequence of the C-terminal tail [24,25]. RET9 and RET51 are the main products in vivo, and animal studies have shown different roles for these two isoforms in kidney and enteric nervous system development with RET9 being essential for kidney morphogenesis and enteric nervous development, while RET51 is essential for later kidney differentiation [26,27]. The activation of different signalling pathways by the different isoforms has also been demonstrated [28]. Four members of the glial cell line-derived neurotrophic factor (GDNF) family have been shown to be RET receptor ligands (GDNF, neurturin, artemin, and persephin) [29–32]. RET activation by each ligand is mediated through specific glycosyl-phosphatidylinositol-anchored co-receptors (GFRα1– 4) [33]. Interaction of one of the RET receptor ligands with its specific GFRα co-receptor induces RET dimerization which results in tyrosine autophosphorylation of the intracellular domain at specific tyrosine residues. These phosphorylated tyrosines serve as binding sites for a number of transduction molecules to activate downstream signalling pathways. The RET9 and RET43 contain 16 tyrosine residues, whilst RET51 has two additional tyrosines in the C-terminal end. Among these tyrosine residues, Y1062 has been shown to be the binding site for at least five different proteins, including SHC, FRS2, DOK4/5, IRS1/2, and Enigma, and the activation of the rat sarcoma oncogene/extracellular signal-regulated kinase (RAS/ ERK), phosphatidylinositol-3-kinase (PI3K)/AKT, p38 mito-
gen-activated protein kinase (p38MAPK), and c-Jun N-terminal kinase (JNK) pathways occurs mainly through Y1062 [34]. 4.2. RET proto-oncogene mutations Somatic and germline RET mutations are responsible for several human diseases. Germline inactivating mutations result in Hirschprung's disease, and approximately 23% of dominantly inherited Hirschprung's patients carry germline inactivating mutations of one allele of the RET proto-oncogene [11]. Germline activating point mutations are responsible for the development of MEN2A, MEN2B, and FMTC, and since RET is a proto-oncogene a single activating mutation of one allele is sufficient for neoplastic transformation. The mutations in MEN2 are located mainly in the extracellular cysteine-rich domain corresponding to exons 10 and 11, or the intracellular tyrosine kinase domains corresponding to exons 13 to 16 [35– 37]. Cysteine mutations identified in MEN2A and FMTC families activate RET by inducing ligand-independent, disulphide-linked homodimerization of two RET mutants resulting in constitutive activation [38,39]. It is thought that replacement of a cysteine with another amino acid due to a FMTC or MEN2A mutation allows an adjacent cysteine, that would normally be involved in forming an intramolecular disulphide bond, to become free and to induce an aberrant intermolecular disulphide bond between two adjacent mutant RET receptors [40]. Mutations in the tyrosine kinase domain induce a conformational change of the catalytic core which alters substrate specificity and allows RET activation without dimerization [39,41]. Somatic activating RET mutations in similar regions to germline activating mutations have been identified in a quarter of patients with sporadic MTC, and somatic RET mutations may also occur in patients with sporadic phaeochromocytoma [13]. Sporadic and radiation-associated papillary thyroid
R.A. Hubner, R.S. Houlston / Clinica Chimica Acta 370 (2006) 2–8 Table 1 Risk groups in hereditary medullary thyroid carcinoma
Codon mutation
Clinical syndrome (% of risk level) a
a
Level 1 (intermediate risk)
Level 2 (high risk)
Level 3 (highest risk)
609 768 790 791 804 891 MEN2A (11%)
611 618 620 634
883 918
MEN2A (68%)
MEN2B (100%)
FMTC (33%) Unclassified (56%)
FMTC (14%) Unclassified (18%)
Data from Brandi et al. [4] and Yip et al. [9].
carcinomas may be caused by somatic rearrangements involving RET. Ten such rearranged forms of RET have been identified; in each case, the intracellular domain of RET is fused to the amino-terminal sequence of activating genes resulting in ligand-independent dimerization and constitutive activation of the chimeric protein, and aberrant expression in thyroid follicular cells [42,43]. 5. Genotype–phenotype correlations in MEN2 Germline mutations have been identified in more than 95% of families with MEN2, and specific RET codon mutations are associated with the different clinical syndromes (Fig. 1). MEN2A mutations are found more commonly in the extracellular cysteine-rich domain and infrequently in the intracellular tyrosine kinase domains [44]. Mutations of codon 634 (exon 11, cysteine-rich domain) occur in 85% of cases of MEN2A, and all cases of MEN2A with cutaneous lichen amyloidosis. Among codon 634 mutations the most frequent alteration is TGC (cysteine) to CGC (arginine) (C634R) accounting for more than 50% of cases [44, 45]. Mutations of codons 609, 611, 618, and 620 (exon 10, cysteine-rich domain) account for a further 10% to 15% of MEN2A and all cases of MEN2A with Hirschprung's disease [44,46]. Hyperparathyroidism in MEN2A is most commonly associated with codon 634 mutations, and in particular with the C634R mutation [46]. Rare mutations associated with MEN2A include codons 630, 635 and 637 (exon 11, cysteine-rich domain), 790 and 791 (exon 13, tyrosine kinase domain), and V804L and 891 (exons 14 and 15, tyrosine kinase domain) [13]. In FMTC germline mutations are distributed with a more even frequency throughout the RET gene and include mutations in codons 532, 533 (exon 8), 609, 611, 618, 620 (exon 10), 630, 634 (exon 11), 768, 790, 791 (exon 13), V804M, 844 (exon 14), 891 (exon 15), and 912 (exon 16) [44,47]. Of these, mutations at codons 532, 533, 768, 844, and 912 have been identified only in families with FMTC, whilst the remainder have also been identified in families with MEN2A [47,48]. 30% of FMTC mutations occur in codon 634 compared to 85% in MEN2A [44]. More than 95% of MEN2B patients have a mutation in codon 918 (exon 16, tyrosine kinase domain) resulting in
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substitution of methionine by threonine [11]. Most of the remainder carry a mutation in codon 883 (exon 15, tyrosine kinase domain), and MEN2B patients with compound heterozygous mutations of V804M with Y806C, and V804M with S904C have also been reported [49]. Phaeochromocytomas in MEN2 are most frequently associated with mutations in codons 634 and 918; however, associations between phaeochromocytoma and all other MEN2 mutations apart from codon 791 have been reported [13]. Follow-up of MEN2 patients with known RET mutations has allowed correlation of specific mutations with aggressiveness of hereditary MTC, and stratification of RET mutations into three risk levels (levels 1 to 3) based on earliest age of development of MTC (Table 1) [4,9]. Patients with level 1, or intermediate risk mutations, are those with mutations in codons 609, 768, 790, 791, 804, 891. Level 2 (high risk) mutations are those in codons 611, 618, 620, and 634, and level 3 (highest risk) mutations occur in codons 883 and 918. The level 3 mutations are therefore confined to MEN2B, and the commonest MEN2A mutation (codon 634) falls into level 2. A recent singleinstitution study based on 86 patients from 47 families with MEN2 or FMTC undergoing mutation analysis and thyroidectomy found that the risk of having advanced (stage III or IV) MTC at diagnosis increased by 12% per year of age at thyroidectomy, and increased 14-fold for each step in MTC risk level (from level 1 to level 3), confirming the predictive power of stratification by mutation in determining MTC biology [9]. 6. Molecular basis for disease phenotype in MEN2 Although it is established that RET mutations in MEN2 cause constitutive activation by either ligand-independent dimerization (cysteine-rich domain mutations) or conformational changes to the catalytic core of the kinase domain (tyrosine kinase domain mutations), the exact mechanism operating within the downstream signalling cascade resulting in the different phenotypes in FMTC, MEN2A, and MEN2B remains undefined. Studies have indicated possible changes in RET substrate specificity due to MEN2B mutations, for example, increases in phosphorylation of paxillin and other proteins associated with the signalling proteins Crk and Nck has been demonstrated by MEN2B mutated RET (RET–MEN2B) compared to wild-type RET [50]. Increased activation of the PI3/AKT and JNK pathways by RET–MEN2B compared to RET–MEN2A has also been reported [51]. Adaptor proteins that preferentially bind to RET–MEN2B compared to RET– MEN2A include Dok1, which is highly phosphorylated in cells expressing RET–MEN2B, and JNK is strongly activated through its association with Dok1 [52]. Animal studies have also demonstrated that the JNK pathway is involved in the ability of RET–MEN2B cells to metastasise [53]. Together these findings suggest that activation of the PI3/AKT and JNK pathways might be responsible for the aggressive MTC phenotype in MEN2B. Differences in transforming activity of RET receptors carrying different mutations within the cysteine-rich domain
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may be due to differences in the ability of the particular cysteine mutation to induce intermolecular disulphide bridge-mediated homodimerization, or to differences in cell surface expression of the mutated RET receptors. Enhanced constitutive homodimerization and consequent activation due to codon 634 mutation compared to codon 620 mutation has been reported, and cell surface expression of RET receptors with codon 609, 611, 618, or 620 mutations has been shown to be very low compared with that of RET receptors with codon 634 mutation, indicating that the former four mutations might impair transport of mutant RET receptors to the plasma membrane [54,55]. On this basis, mutations in codon 634 would be expected to be more frequently associated with the more aggressive MEN2A phenotype rather than FMTC, and vice versa for mutations in codons 609, 618, and 620. This pattern is observed in clinical practice. 7. Genetic testing for inherited MTC First-degree relatives of individuals with inherited MTC are at 50% risk of inheriting the mutated gene and developing MTC. Prior to the identification of RET proto-oncogene mutations in inherited MTC the only available screening tool to direct prophylactic thyroid surgery for at risk individuals in MEN2 families was measurement of basal and pentagastrin stimulated plasma calcitonin levels. Differentiating results of calcitonin level testing in normal children and those with early C-cell abnormalities could however be very difficult, resulting in significant false-positive rates potentially leading to unnecessary operations, and also false-negatives [12]. DNA-based predictive testing for RET mutations in MEN2 families is now the standard of care worldwide [11]. Direct sequencing following amplification of selected exons (usually exons 10, 11, 13, 14, 15, and 16) known to be mutated in MEN2 families, or sequencing of all exons, is considered the most accurate technique, and has the advantage of possible identification of new unreported mutations. In a MEN2 family with a known RET mutation, identification of the same mutation in an at-risk individual indicates that they have MEN2, whilst failure to find the mutation indicates that they do not have MEN2 and prophylactic thyroidectomy and biochemical screening can be avoided. At least 6% of patients with apparently sporadic MTC are also found to have germline RET mutations, and this is particularly likely when MTC is multifocal or diagnosed at a young age [56,57]. This has led to the recommendation that all individuals with sporadic MTC undergo RET mutation testing as identification of mutations will allow testing of other at-risk family members [4]. Genetic counselling is essential for patients and their families prior to embarking on genetic testing. The potential implications of both positive and negative test results, for both the individual and other at-risk family members, need to be fully explained. 8. Clinical management based on mutation testing In individuals where pre-symptomatic RET mutation testing is positive, the optimum strategy for the prevention of MTC is
prophylactic thyroidectomy and removal of the central neck lymph nodes prior to the development of the cancer [8]. The stratification of RET mutations into three risk levels for MTC development has allowed the timing and extent of prophylactic surgery to be tailored to the specific mutation carried by individual patients [4]. Such ‘codon-directed’ prophylactic surgery is recommended based on the youngest age at first diagnosis of MTC according to the identified mutated codon [58]. Prophylactic thyroidectomy in children is not without risk. Surgical risks include unilateral or bilateral recurrent laryngeal nerve injury, permanent hypoparathyroidism, and postoperative cervical haematoma. Reported complication rates following thyroid surgery in adults are lower than those in children, and it is possible that there is a relationship between complication rate and age of patient and size of their cervical structures [58]. Thus delaying prophylactic surgery is desirable if the risk of MTC at young age is low. The decision to also remove the central neck lymph glands at initial thyroidectomy is also important since it may increase morbidity associated with the procedure; however, sequential thyroidectomy followed by lymph node dissection should metastatic disease develop is also undesirable since the scar tissue from the original operation will obscure delicate structures such as the recurrent laryngeal nerves making damage more likely [58]. In patients with known or suspected MEN2 preoperative screening for phaeochromocytoma should be performed to prevent the risk of serious haemodynamic disturbances during the operation. Current guidelines suggest patients with level 3 mutations (codons 918 and 883, MEN2B) who have the highest risk of aggressive MTC should undergo prophylactic thyroidectomy before the age of six months and preferably in the first month [4]. Patients with level 2 mutations, including the most frequent MEN2A codon 634 mutation, should have prophylactic thyroid surgery by the age of 5 years. Prophylactic central neck node dissection may also be performed if ultrasound findings or basal calcitonin levels raise suspicion of occult lymph node metastases [13]. There is currently no consensus on the management of patients with level 1 mutations who generally develop MTC later in life and with low metastatic potential, with opinions on optimum timing of prophylactic thyroidectomy ranging from 5 to 10 years of age [4,8]. However, there is heterogeneity in the biological behaviour of MTCs due to level 1 mutations and if thyroidectomy is delayed patients are at risk of developing lymph node and distant metastases [9]. It is possible for a level 1 mutation to be associated with no MTC-related deaths when present in one FMTC family, but when present in a different family to correlate with more aggressive behaviour and MTCrelated deaths [10]. It should also be noted that there is considerably less clinical experience with level 1 mutations compared to the other two groups [12]. Follow-up for patients carrying germline RET mutations who have undergone prophylactic thyroidectomy should include yearly screening for phaeochromocytoma (MEN2A/FMTC and MEN 2B) and hyperparathyroidism (MEN2A/FMTC only). Although the likelihood of development of phaeochromocytoma and hyperparathyroidism may be indicated by the underlying
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RET mutation, it would seem prudent to continue such screening even in individuals carrying mutations that have not so far been associated with the conditions. 9. Conclusion The biological behaviour of MTC can now be usefully stratified by molecular diagnostics to guide clinical management. RET mutation testing should allow both reduced mortality from MTC, and reduced morbidity from unnecessarily early or aggressive prophylactic surgery. Advances in our understanding of the molecular mechanisms underlying the different clinical phenotypes within MEN2 may enable further refining of genotype–phenotype correlations and hence further tailoring of treatment. References [1] Hazard JB, Hawk WA, Crile Jr G. Medullary (solid) carcinoma of the thyroid; a clinicopathologic entity. J Clin Endocrinol Metab 1959; 19:152–61. [2] Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10–30. [3] DeVita V, Hellman S, Rosenberg S. Principles and practice of oncology. seventh ed. Philadelphia: Lippincott Williams and Wilkinson; 2005. [4] 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; 86:5658–71. [5] Mulligan LM, Kwok JB, Healey CS, et al. Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458–60. [6] Donis-Keller H, Dou S, Chi D, et al. Mutations in the RET protooncogene are associated with MEN 2A and FMTC. Hum Mol Genet 1993;2:851–6. [7] Eng C, Mulligan LM. Mutations of the RET proto-oncogene in the multiple endocrine neoplasia type 2 syndromes, related sporadic tumours, and hirschsprung disease. Hum Mutat 1997;9:97–109. [8] Machens A, Niccoli-Sire P, Hoegel J, et al. Early malignant progression of hereditary medullary thyroid cancer. N Engl J Med 2003; 349:1517–25. [9] Yip L, Cote GJ, Shapiro SE, et al. Multiple endocrine neoplasia type 2: evaluation of the genotype–phenotype relationship. Arch Surg 2003; 138:409–16. [10] Larsen P, Kronenberg H, Melmed S, Polonsky K. Multiple endocrine neoplasia. tenth ed. Philadelphia: Saunders; 2003. [11] Eng C. RET proto-oncogene in the development of human cancer. J Clin Oncol 1999;17:380–93. [12] Jimenez C, Gagel RF. Genetic testing in endocrinology: lessons learned from experience with multiple endocrine neoplasia type 2 (MEN2). Growth Horm IGF Res 2004;14(Suppl A):S150–7. [13] Kouvaraki MA, Shapiro SE, Perrier ND, et al. RET proto-oncogene: a review and update of genotype–phenotype correlations in hereditary medullary thyroid cancer and associated endocrine tumors. Thyroid 2005;15:531–44. [14] Cotran RS, Kumar V, Collins T. Robbins pathologic basis of disease. sixth ed. Philadelphia: Saunders; 1999. [15] Leboulleux S, Baudin E, Travagli JP, Schlumberger M. Medullary thyroid carcinoma. Clin Endocrinol (Oxf) 2004;61:299–310. [16] Kaufman FR, Roe TF, Isaacs Jr H, Weitzman JJ. Metastatic medullary thyroid carcinoma in young children with mucosal neuroma syndrome. Pediatrics 1982;70:263–7. [17] Carlomagno F, Vitagliano D, Guida T, et al. ZD6474, an orally available inhibitor of KDR tyrosine kinase activity, efficiently blocks oncogenic RET kinases. Cancer Res 2002;62:7284–90.
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[18] Takahashi M, Ritz J, Cooper GM. Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell 1985;42:581–8. [19] Takahashi M, Buma Y, Iwamoto T, Inaguma Y, Ikeda H, Hiai H. Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene 1988; 3:571–8. [20] Pachnis V, Mankoo B, Costantini F. Expression of the c-ret proto-oncogene during mouse embryogenesis. Development 1993;119:1005–17. [21] 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;367:380–3. [22] Taraviras S, Marcos-Gutierrez CV, Durbec P, et al. Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system. Development 1999;126:2785–97. [23] Enomoto H, Crawford PA, Gorodinsky A, Heuckeroth RO, Johnson Jr EM, Milbrandt J. RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. Development 2001;128:3963–74. [24] Tahira T, Ishizaka Y, Itoh F, Sugimura T, Nagao M. Characterization of ret proto-oncogene mRNAs encoding two isoforms of the protein product in a human neuroblastoma cell line. Oncogene 1990;5:97–102. [25] Myers SM, Eng C, Ponder BA, Mulligan LM. Characterization of RET proto-oncogene 3′ splicing variants and polyadenylation sites: a novel Cterminus for RET. Oncogene 1995;11:2039–45. [26] de Graaff E, Srinivas S, Kilkenny C, et al. Differential activities of the RET tyrosine kinase receptor isoforms during mammalian embryogenesis. Genes Dev 2001;15:2433–44. [27] Lee DC, Chan KW, Chan SY. RET receptor tyrosine kinase isoforms in kidney function and disease. Oncogene 2002;21:5582–92. [28] Tsui-Pierchala BA, Ahrens RC, Crowder RJ, Milbrandt J, Johnson Jr EM. The long and short isoforms of Ret function as independent signaling complexes. J Biol Chem 2002;277:34618–25. [29] Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F. GDNF: a glial cell linederived neurotrophic factor for midbrain dopaminergic neurons. Science 1993;260:1130–2. [30] Kotzbauer PT, Lampe PA, Heuckeroth RO, et al. Neurturin, a relative of glial-cell-line-derived neurotrophic factor. Nature 1996;384:467–70. [31] Milbrandt J, de Sauvage FJ, Fahrner TJ, et al. Persephin, a novel neurotrophic factor related to GDNF and neurturin. Neuron 1998; 20:245–53. [32] Baloh RH, Tansey MG, Lampe PA, et al. Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFRalpha3–RET receptor complex. Neuron 1998; 21:1291–302. [33] Airaksinen MS, Saarma M. The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci 2002;3:383–94. [34] Hayashi H, Ichihara M, Iwashita T, et al. Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor. Oncogene 2000;19:4469–75. [35] Akhand AA, Ikeyama T, Akazawa S, et al. Evidence of both extra- and intracellular cysteine targets of protein modification for activation of RET kinase. Biochem Biophys Res Commun 2002;292:826–31. [36] 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;367:375–6. [37] Eng C, Smith DP, Mulligan LM, et al. A novel point mutation in the tyrosine kinase domain of the RET proto-oncogene in sporadic medullary thyroid carcinoma and in a family with FMTC. Oncogene 1995;10:509–13. [38] Asai N, Iwashita T, Matsuyama M, Takahashi M. Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations. Mol Cell Biol 1995;15:1613–9. [39] 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;267:381–3. [40] Kodama Y, Asai N, Kawai K, et al. The RET proto-oncogene: a molecular therapeutic target in thyroid cancer. Cancer Sci 2005;96:143–8. [41] Iwashita T, Asai N, Murakami H, Matsuyama M, Takahashi M. Identification of tyrosine residues that are essential for transforming
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Invited critical review
Molecular advances in medullary thyroid cancer diagnostics Richard A. Hubner ⁎, Richard S. Houlston Institute of Cancer Research, Cancer Genetics, 15 Cotswold Road, Sutton, SM2 5NG, United Kingdom Received 5 January 2006; received in revised form 24 January 2006; accepted 25 January 2006 Available online 7 March 2006
Abstract Germline activating mutations in the RET proto-oncogene cause inherited medullary thyroid cancer (MTC) and the multiple endocrine neoplasia type 2 (MEN2) syndrome. Identification of a RET mutation in an individual with MEN2 allows pre-symptomatic genetic testing of other at-risk family members, and guides early intervention to prevent death and serious morbidity from MTC. Developments in the understanding of downstream RET receptor signalling pathways and how activating mutations disturb receptor function has led to insights into the possible molecular mechanisms underlying the different MEN2 phenotypes. Mutation analysis of RET in individuals with MEN2 has identified a number of different mutations, and correlation with cancer biology and clinical outcome has led to tailoring of management according to the mutation detected. © 2006 Elsevier B.V. All rights reserved. Keywords: Medullary thyroid cancer; MEN2; RET proto-oncogene; Tyrosine kinase
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . . . . Clinical syndromes . . . . . . . . . . . . . . . Natural history of medullary thyroid carcinoma . The RET proto-oncogene . . . . . . . . . . . . 4.1. Normal structure and function . . . . . . 4.2. RET proto-oncogene mutations . . . . . 5. Genotype–phenotype correlations in MEN2. . . 6. Molecular basis for disease phenotype in MEN2 7. Genetic testing for inherited MTC. . . . . . . . 8. Clinical management based on mutation testing. 9. Conclusion. . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Medullary thyroid cancer (MTC) arises from the parafollicular or C-cells of the thyroid gland, and was first recognised as a distinct clinicopathologic entity in 1959 [1]. MTC is rare and accounts for only 3% to 10% of thyroid cancer, equating to
⁎ Corresponding author. Tel.: +44 020 87224385. E-mail address: [email protected] (R.A. Hubner). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.01.029
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approximately 1000 new diagnoses in the US each year [2,3]. In 60% to 75% of cases MTC occurs sporadically, whilst in the remainder it is inherited in an autosomal dominant fashion and may be associated with other endocrine tumours as part of the multiple endocrine neoplasia type 2 (MEN2) syndrome. MEN2 affects 1 in 30,000 individuals, and to date around 500 families with members affected by MTC as part of MEN2 have been identified worldwide [4]. Germline activating mutations of the RET proto-oncogene were first recognised to cause inherited MTC in 1993 [5,6], since
R.A. Hubner, R.S. Houlston / Clinica Chimica Acta 370 (2006) 2–8
then mutation analysis in MEN2 families with MTC has identified over 30 different missense mutations segregating with the disease [7]. In MEN2 families with an identified germline RET mutation pre-symptomatic genetic testing of unaffected at-risk family members can be performed, enabling prophylactic surgical treatment to be directed to those carrying a mutation [8]. It has been suggested that there are distinct genotype-phenotype correlations, and that the biological aggressiveness of MTC can be predicted according to the underlying germline RET mutation. International consortia have studied the outcomes of individuals within families carrying different mutations and have concluded that mutation carriers may be placed into three risk levels according to the RET mutation carried, and that treatment should be tailored accordingly [4,9]. This review will focus on recent advances in the understanding of how RET mutations in inherited MTC affect RET proto-oncogene functioning, current information concerning genotype–phenotype correlations, and how both of these are refining the use of genotype in the diagnosis and management of MTC. 2. Clinical syndromes Inherited MTC can occur either in isolation, in which case it is termed Familial MTC (FMTC), or in combination with other endocrine neoplasms as part of the MEN2 syndrome. The most common form of inherited MTC, accounting for up to 80% of cases, is multiple endocrine neoplasia type 2A (MEN2A), or Sipple Syndrome [10]. Individuals with MEN2A develop MTC (100% of cases), phaeochromocytoma (50% of cases), and hyperparathyroidism (10–20% of cases). MTC is by far the commonest clinical presentation of MEN2A [11]. Two rare variants of MEN2A have also been documented, MEN2A with Hirschsprung's disease, and MEN2A with cutaneous lichen amyloidosis [11]. Hirschprung's disease is characterised by the absence of autonomic ganglia in the terminal hindgut, resulting in colonic dilatation, constipation, and obstruction in neonates. Cutaneous lichen amyloidosis presents as a pruritic rash between the shoulder blades in the second or third decade [12]. Multiple endocrine neoplasia type 2B (MEN2B) is less common than MEN2A, accounting for 5% of MEN2 cases, but is clinically more distinctive. It is characterised by aggressive MTC (100% of cases), phaeochromocytoma (50% of cases), a Marfanoid body habitus (without the vascular and ophthalmologic abnormalities of Marfan's syndrome), the presence of distinctive mucosal neuromas on the distal tongue, lips, and subconjunctival areas, and diffuse ganglioneuromas of the gastrointestinal tract. Parathyroid disease does not occur in MEN2B. Children with MEN2B may be identified by their characteristic facial and oral features, but gastrointestinal symptoms with intermittent diarrhoea, constipation, and obstruction are the most common initial presentation [11]. The majority of MEN2B cases are the result of spontaneous new RET mutations, thus most patients lack a family history and do not undergo screening or prophylactic thyroidectomy,
3
and therefore may present late with mucosal neuromas or a palpable thyroid mass [13]. Familial MTC accounts for 5% to 10% of inherited MTC, and is defined as the presence of MTC in families with four or more affected members and with objective evidence of the absence of adrenal medullary or parathyroid involvement [11]. However the 50% or lower penetrance of adrenal medullary and parathyroid manifestations in MEN2A can make distinguishing FMTC from MEN2A difficult, particularly in families with few affected individuals, or mainly young affected individuals. 3. Natural history of medullary thyroid carcinoma The normal thyroid is composed of follicular cells involved in the production of thyroid hormone, and parafollicular C-cells which account for 0.1% of thyroid cells, produce the hormone calcitonin, and are the cell of origin for MTC [3]. Calcitonin is secreted by MTCs and promotes the absorption of calcium by the skeletal system and inhibits the resorption of bone by osteoclasts [14]. C-cell hyperplasia is believed to be a precursor of MTC, particularly in inherited MTC where it is virtually always present [15]. Inherited MTC, as with other tumours in inherited syndromes, is more often multicentric and bilateral than sporadic MTC. Metastatic spread is initially to the cervical lymph nodes, and subsequently outside the neck to liver, lung, and bone [3]. The aggressiveness of the disease varies with the clinical setting. In inherited MTC, where individuals have not undergone screening, the average age of presentation is 20 to 40years, whilst for sporadic MTC it is 50 to 60years [11]. Patients with MEN2A may develop MTC as early as age 5years and C cell hyperplasia at an even earlier age [13]. Early metastasis is particularly a feature of MEN2B where cervical lymph node metastases have been observed in patients as young as three years [16]. Familial medullary thyroid cancer tends to have the least aggressive course. Metastatic MTC does not generally respond well to conventional radiotherapy or chemotherapy. Thus the optimum strategy for effective management is screening of at-risk individuals and early surgical intervention prior to the development of the primary tumour [3,17]. 4. The RET proto-oncogene 4.1. Normal structure and function The RET (REarranged during Transfection) gene was first identified in 1985 as a proto-oncogene that can undergo activation by DNA rearrangement [18]. The RET gene is located near the centromere on the long arm of chromosome 10, has 21 exons, and encodes a receptor tyrosine kinase expressed mainly in neural crest and urogential precursor cells, including thyroid parafollicular C-cells, adrenal medullary cells, parasympathetic, sympathetic, and colonic ganglia, and parathyroid cells [18–20]. Gene knockout studies in mice have shown that the RET gene is essential for the development of the enteric nervous system and kidney [21–23].
4
R.A. Hubner, R.S. Houlston / Clinica Chimica Acta 370 (2006) 2–8 RET receptor Cadherin-like domain
Cysteine-rich domain
Tyrosine kinase domain
Tyrosine kinase domain
RET gene
Codon mutation
Clinical syndrome
532 533
FMTC FMTC
609 611 618 620
FMTC/MEN2A FMTC/MEN2A FMTC/MEN2A MEN2A/FMTC
630 634 635 637
FMTC MEN2A/FMTC MEN2A MEN2A
768 790 791
FMTC FMTC/MEN2A FMTC/MEN2A
Exon 14
804 V804M+Y806C V804M+S904C 844
FMTC/MEN2A MEN2B MEN2B FMTC
Exon 15
883 891
MEN2B FMTC/MEN2A
912 918
FMTC MEN2B
Exon 8 Exon 10 Exon 11
Exon 13
Exon 16
Fig. 1. Schematic diagram of the RET receptor, RET gene, and location of reported codon mutations and their associated clinical syndromes.
The RET protein has four cadherin-like repeats and a cysteine-rich region in the extracellular domain, a transmembrane domain, and two intracellular tyrosine kinase domains (Fig. 1). The extracellular cysteine-rich region is important for receptor dimerization, whilst the intracellular tyrosine kinase domains are involved in signal transduction. Alternative splicing of the 3′ region of RET generates three protein isoforms, short (RET9), middle (RET43), and long (RET51), which differ in the amino acid sequence of the C-terminal tail [24,25]. RET9 and RET51 are the main products in vivo, and animal studies have shown different roles for these two isoforms in kidney and enteric nervous system development with RET9 being essential for kidney morphogenesis and enteric nervous development, while RET51 is essential for later kidney differentiation [26,27]. The activation of different signalling pathways by the different isoforms has also been demonstrated [28]. Four members of the glial cell line-derived neurotrophic factor (GDNF) family have been shown to be RET receptor ligands (GDNF, neurturin, artemin, and persephin) [29–32]. RET activation by each ligand is mediated through specific glycosyl-phosphatidylinositol-anchored co-receptors (GFRα1– 4) [33]. Interaction of one of the RET receptor ligands with its specific GFRα co-receptor induces RET dimerization which results in tyrosine autophosphorylation of the intracellular domain at specific tyrosine residues. These phosphorylated tyrosines serve as binding sites for a number of transduction molecules to activate downstream signalling pathways. The RET9 and RET43 contain 16 tyrosine residues, whilst RET51 has two additional tyrosines in the C-terminal end. Among these tyrosine residues, Y1062 has been shown to be the binding site for at least five different proteins, including SHC, FRS2, DOK4/5, IRS1/2, and Enigma, and the activation of the rat sarcoma oncogene/extracellular signal-regulated kinase (RAS/ ERK), phosphatidylinositol-3-kinase (PI3K)/AKT, p38 mito-
gen-activated protein kinase (p38MAPK), and c-Jun N-terminal kinase (JNK) pathways occurs mainly through Y1062 [34]. 4.2. RET proto-oncogene mutations Somatic and germline RET mutations are responsible for several human diseases. Germline inactivating mutations result in Hirschprung's disease, and approximately 23% of dominantly inherited Hirschprung's patients carry germline inactivating mutations of one allele of the RET proto-oncogene [11]. Germline activating point mutations are responsible for the development of MEN2A, MEN2B, and FMTC, and since RET is a proto-oncogene a single activating mutation of one allele is sufficient for neoplastic transformation. The mutations in MEN2 are located mainly in the extracellular cysteine-rich domain corresponding to exons 10 and 11, or the intracellular tyrosine kinase domains corresponding to exons 13 to 16 [35– 37]. Cysteine mutations identified in MEN2A and FMTC families activate RET by inducing ligand-independent, disulphide-linked homodimerization of two RET mutants resulting in constitutive activation [38,39]. It is thought that replacement of a cysteine with another amino acid due to a FMTC or MEN2A mutation allows an adjacent cysteine, that would normally be involved in forming an intramolecular disulphide bond, to become free and to induce an aberrant intermolecular disulphide bond between two adjacent mutant RET receptors [40]. Mutations in the tyrosine kinase domain induce a conformational change of the catalytic core which alters substrate specificity and allows RET activation without dimerization [39,41]. Somatic activating RET mutations in similar regions to germline activating mutations have been identified in a quarter of patients with sporadic MTC, and somatic RET mutations may also occur in patients with sporadic phaeochromocytoma [13]. Sporadic and radiation-associated papillary thyroid
R.A. Hubner, R.S. Houlston / Clinica Chimica Acta 370 (2006) 2–8 Table 1 Risk groups in hereditary medullary thyroid carcinoma
Codon mutation
Clinical syndrome (% of risk level) a
a
Level 1 (intermediate risk)
Level 2 (high risk)
Level 3 (highest risk)
609 768 790 791 804 891 MEN2A (11%)
611 618 620 634
883 918
MEN2A (68%)
MEN2B (100%)
FMTC (33%) Unclassified (56%)
FMTC (14%) Unclassified (18%)
Data from Brandi et al. [4] and Yip et al. [9].
carcinomas may be caused by somatic rearrangements involving RET. Ten such rearranged forms of RET have been identified; in each case, the intracellular domain of RET is fused to the amino-terminal sequence of activating genes resulting in ligand-independent dimerization and constitutive activation of the chimeric protein, and aberrant expression in thyroid follicular cells [42,43]. 5. Genotype–phenotype correlations in MEN2 Germline mutations have been identified in more than 95% of families with MEN2, and specific RET codon mutations are associated with the different clinical syndromes (Fig. 1). MEN2A mutations are found more commonly in the extracellular cysteine-rich domain and infrequently in the intracellular tyrosine kinase domains [44]. Mutations of codon 634 (exon 11, cysteine-rich domain) occur in 85% of cases of MEN2A, and all cases of MEN2A with cutaneous lichen amyloidosis. Among codon 634 mutations the most frequent alteration is TGC (cysteine) to CGC (arginine) (C634R) accounting for more than 50% of cases [44, 45]. Mutations of codons 609, 611, 618, and 620 (exon 10, cysteine-rich domain) account for a further 10% to 15% of MEN2A and all cases of MEN2A with Hirschprung's disease [44,46]. Hyperparathyroidism in MEN2A is most commonly associated with codon 634 mutations, and in particular with the C634R mutation [46]. Rare mutations associated with MEN2A include codons 630, 635 and 637 (exon 11, cysteine-rich domain), 790 and 791 (exon 13, tyrosine kinase domain), and V804L and 891 (exons 14 and 15, tyrosine kinase domain) [13]. In FMTC germline mutations are distributed with a more even frequency throughout the RET gene and include mutations in codons 532, 533 (exon 8), 609, 611, 618, 620 (exon 10), 630, 634 (exon 11), 768, 790, 791 (exon 13), V804M, 844 (exon 14), 891 (exon 15), and 912 (exon 16) [44,47]. Of these, mutations at codons 532, 533, 768, 844, and 912 have been identified only in families with FMTC, whilst the remainder have also been identified in families with MEN2A [47,48]. 30% of FMTC mutations occur in codon 634 compared to 85% in MEN2A [44]. More than 95% of MEN2B patients have a mutation in codon 918 (exon 16, tyrosine kinase domain) resulting in
5
substitution of methionine by threonine [11]. Most of the remainder carry a mutation in codon 883 (exon 15, tyrosine kinase domain), and MEN2B patients with compound heterozygous mutations of V804M with Y806C, and V804M with S904C have also been reported [49]. Phaeochromocytomas in MEN2 are most frequently associated with mutations in codons 634 and 918; however, associations between phaeochromocytoma and all other MEN2 mutations apart from codon 791 have been reported [13]. Follow-up of MEN2 patients with known RET mutations has allowed correlation of specific mutations with aggressiveness of hereditary MTC, and stratification of RET mutations into three risk levels (levels 1 to 3) based on earliest age of development of MTC (Table 1) [4,9]. Patients with level 1, or intermediate risk mutations, are those with mutations in codons 609, 768, 790, 791, 804, 891. Level 2 (high risk) mutations are those in codons 611, 618, 620, and 634, and level 3 (highest risk) mutations occur in codons 883 and 918. The level 3 mutations are therefore confined to MEN2B, and the commonest MEN2A mutation (codon 634) falls into level 2. A recent singleinstitution study based on 86 patients from 47 families with MEN2 or FMTC undergoing mutation analysis and thyroidectomy found that the risk of having advanced (stage III or IV) MTC at diagnosis increased by 12% per year of age at thyroidectomy, and increased 14-fold for each step in MTC risk level (from level 1 to level 3), confirming the predictive power of stratification by mutation in determining MTC biology [9]. 6. Molecular basis for disease phenotype in MEN2 Although it is established that RET mutations in MEN2 cause constitutive activation by either ligand-independent dimerization (cysteine-rich domain mutations) or conformational changes to the catalytic core of the kinase domain (tyrosine kinase domain mutations), the exact mechanism operating within the downstream signalling cascade resulting in the different phenotypes in FMTC, MEN2A, and MEN2B remains undefined. Studies have indicated possible changes in RET substrate specificity due to MEN2B mutations, for example, increases in phosphorylation of paxillin and other proteins associated with the signalling proteins Crk and Nck has been demonstrated by MEN2B mutated RET (RET–MEN2B) compared to wild-type RET [50]. Increased activation of the PI3/AKT and JNK pathways by RET–MEN2B compared to RET–MEN2A has also been reported [51]. Adaptor proteins that preferentially bind to RET–MEN2B compared to RET– MEN2A include Dok1, which is highly phosphorylated in cells expressing RET–MEN2B, and JNK is strongly activated through its association with Dok1 [52]. Animal studies have also demonstrated that the JNK pathway is involved in the ability of RET–MEN2B cells to metastasise [53]. Together these findings suggest that activation of the PI3/AKT and JNK pathways might be responsible for the aggressive MTC phenotype in MEN2B. Differences in transforming activity of RET receptors carrying different mutations within the cysteine-rich domain
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may be due to differences in the ability of the particular cysteine mutation to induce intermolecular disulphide bridge-mediated homodimerization, or to differences in cell surface expression of the mutated RET receptors. Enhanced constitutive homodimerization and consequent activation due to codon 634 mutation compared to codon 620 mutation has been reported, and cell surface expression of RET receptors with codon 609, 611, 618, or 620 mutations has been shown to be very low compared with that of RET receptors with codon 634 mutation, indicating that the former four mutations might impair transport of mutant RET receptors to the plasma membrane [54,55]. On this basis, mutations in codon 634 would be expected to be more frequently associated with the more aggressive MEN2A phenotype rather than FMTC, and vice versa for mutations in codons 609, 618, and 620. This pattern is observed in clinical practice. 7. Genetic testing for inherited MTC First-degree relatives of individuals with inherited MTC are at 50% risk of inheriting the mutated gene and developing MTC. Prior to the identification of RET proto-oncogene mutations in inherited MTC the only available screening tool to direct prophylactic thyroid surgery for at risk individuals in MEN2 families was measurement of basal and pentagastrin stimulated plasma calcitonin levels. Differentiating results of calcitonin level testing in normal children and those with early C-cell abnormalities could however be very difficult, resulting in significant false-positive rates potentially leading to unnecessary operations, and also false-negatives [12]. DNA-based predictive testing for RET mutations in MEN2 families is now the standard of care worldwide [11]. Direct sequencing following amplification of selected exons (usually exons 10, 11, 13, 14, 15, and 16) known to be mutated in MEN2 families, or sequencing of all exons, is considered the most accurate technique, and has the advantage of possible identification of new unreported mutations. In a MEN2 family with a known RET mutation, identification of the same mutation in an at-risk individual indicates that they have MEN2, whilst failure to find the mutation indicates that they do not have MEN2 and prophylactic thyroidectomy and biochemical screening can be avoided. At least 6% of patients with apparently sporadic MTC are also found to have germline RET mutations, and this is particularly likely when MTC is multifocal or diagnosed at a young age [56,57]. This has led to the recommendation that all individuals with sporadic MTC undergo RET mutation testing as identification of mutations will allow testing of other at-risk family members [4]. Genetic counselling is essential for patients and their families prior to embarking on genetic testing. The potential implications of both positive and negative test results, for both the individual and other at-risk family members, need to be fully explained. 8. Clinical management based on mutation testing In individuals where pre-symptomatic RET mutation testing is positive, the optimum strategy for the prevention of MTC is
prophylactic thyroidectomy and removal of the central neck lymph nodes prior to the development of the cancer [8]. The stratification of RET mutations into three risk levels for MTC development has allowed the timing and extent of prophylactic surgery to be tailored to the specific mutation carried by individual patients [4]. Such ‘codon-directed’ prophylactic surgery is recommended based on the youngest age at first diagnosis of MTC according to the identified mutated codon [58]. Prophylactic thyroidectomy in children is not without risk. Surgical risks include unilateral or bilateral recurrent laryngeal nerve injury, permanent hypoparathyroidism, and postoperative cervical haematoma. Reported complication rates following thyroid surgery in adults are lower than those in children, and it is possible that there is a relationship between complication rate and age of patient and size of their cervical structures [58]. Thus delaying prophylactic surgery is desirable if the risk of MTC at young age is low. The decision to also remove the central neck lymph glands at initial thyroidectomy is also important since it may increase morbidity associated with the procedure; however, sequential thyroidectomy followed by lymph node dissection should metastatic disease develop is also undesirable since the scar tissue from the original operation will obscure delicate structures such as the recurrent laryngeal nerves making damage more likely [58]. In patients with known or suspected MEN2 preoperative screening for phaeochromocytoma should be performed to prevent the risk of serious haemodynamic disturbances during the operation. Current guidelines suggest patients with level 3 mutations (codons 918 and 883, MEN2B) who have the highest risk of aggressive MTC should undergo prophylactic thyroidectomy before the age of six months and preferably in the first month [4]. Patients with level 2 mutations, including the most frequent MEN2A codon 634 mutation, should have prophylactic thyroid surgery by the age of 5 years. Prophylactic central neck node dissection may also be performed if ultrasound findings or basal calcitonin levels raise suspicion of occult lymph node metastases [13]. There is currently no consensus on the management of patients with level 1 mutations who generally develop MTC later in life and with low metastatic potential, with opinions on optimum timing of prophylactic thyroidectomy ranging from 5 to 10 years of age [4,8]. However, there is heterogeneity in the biological behaviour of MTCs due to level 1 mutations and if thyroidectomy is delayed patients are at risk of developing lymph node and distant metastases [9]. It is possible for a level 1 mutation to be associated with no MTC-related deaths when present in one FMTC family, but when present in a different family to correlate with more aggressive behaviour and MTCrelated deaths [10]. It should also be noted that there is considerably less clinical experience with level 1 mutations compared to the other two groups [12]. Follow-up for patients carrying germline RET mutations who have undergone prophylactic thyroidectomy should include yearly screening for phaeochromocytoma (MEN2A/FMTC and MEN 2B) and hyperparathyroidism (MEN2A/FMTC only). Although the likelihood of development of phaeochromocytoma and hyperparathyroidism may be indicated by the underlying
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RET mutation, it would seem prudent to continue such screening even in individuals carrying mutations that have not so far been associated with the conditions. 9. Conclusion The biological behaviour of MTC can now be usefully stratified by molecular diagnostics to guide clinical management. RET mutation testing should allow both reduced mortality from MTC, and reduced morbidity from unnecessarily early or aggressive prophylactic surgery. Advances in our understanding of the molecular mechanisms underlying the different clinical phenotypes within MEN2 may enable further refining of genotype–phenotype correlations and hence further tailoring of treatment. References [1] Hazard JB, Hawk WA, Crile Jr G. Medullary (solid) carcinoma of the thyroid; a clinicopathologic entity. J Clin Endocrinol Metab 1959; 19:152–61. [2] Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10–30. [3] DeVita V, Hellman S, Rosenberg S. Principles and practice of oncology. seventh ed. Philadelphia: Lippincott Williams and Wilkinson; 2005. [4] 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; 86:5658–71. [5] Mulligan LM, Kwok JB, Healey CS, et al. Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458–60. [6] Donis-Keller H, Dou S, Chi D, et al. Mutations in the RET protooncogene are associated with MEN 2A and FMTC. Hum Mol Genet 1993;2:851–6. [7] Eng C, Mulligan LM. Mutations of the RET proto-oncogene in the multiple endocrine neoplasia type 2 syndromes, related sporadic tumours, and hirschsprung disease. Hum Mutat 1997;9:97–109. [8] Machens A, Niccoli-Sire P, Hoegel J, et al. Early malignant progression of hereditary medullary thyroid cancer. N Engl J Med 2003; 349:1517–25. [9] Yip L, Cote GJ, Shapiro SE, et al. Multiple endocrine neoplasia type 2: evaluation of the genotype–phenotype relationship. Arch Surg 2003; 138:409–16. [10] Larsen P, Kronenberg H, Melmed S, Polonsky K. Multiple endocrine neoplasia. tenth ed. Philadelphia: Saunders; 2003. [11] Eng C. RET proto-oncogene in the development of human cancer. J Clin Oncol 1999;17:380–93. [12] Jimenez C, Gagel RF. Genetic testing in endocrinology: lessons learned from experience with multiple endocrine neoplasia type 2 (MEN2). Growth Horm IGF Res 2004;14(Suppl A):S150–7. [13] Kouvaraki MA, Shapiro SE, Perrier ND, et al. RET proto-oncogene: a review and update of genotype–phenotype correlations in hereditary medullary thyroid cancer and associated endocrine tumors. Thyroid 2005;15:531–44. [14] Cotran RS, Kumar V, Collins T. Robbins pathologic basis of disease. sixth ed. Philadelphia: Saunders; 1999. [15] Leboulleux S, Baudin E, Travagli JP, Schlumberger M. Medullary thyroid carcinoma. Clin Endocrinol (Oxf) 2004;61:299–310. [16] Kaufman FR, Roe TF, Isaacs Jr H, Weitzman JJ. Metastatic medullary thyroid carcinoma in young children with mucosal neuroma syndrome. Pediatrics 1982;70:263–7. [17] Carlomagno F, Vitagliano D, Guida T, et al. ZD6474, an orally available inhibitor of KDR tyrosine kinase activity, efficiently blocks oncogenic RET kinases. Cancer Res 2002;62:7284–90.
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