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The genomic revolution of the past 20 years has led to the identification of the genetic mechanisms that are responsible for a wide variety of human cancers, and has made a huge impact on our ability to recognize, diagnose and, more recently, treat patients with these diseases. Genetic diagnosis of heritable forms of cancer holds out the potential for presymptomatic detection in familial cases and raises the possibility of prophylactic treatments that would decrease morbidity and mortality and improve the quality of life for those affected. While the potential impact of these advances is huge, to date, our understanding of the underlying genetic events that contribute to the familial cancers has only allowed us to significantly modify our management of the disease in a few instances. The paradigm for such genetically based molecular management is the multiple endocrine neoplasia type 2 (MEN 2) syndromes.

Multiple endocrine neoplasia type 2 (MEN 2)

As described in previous chapters (1), MEN 2 is an inherited cancer syndrome characterized by medullary thyroid carcinoma (MTC) and its precursor lesion, C-cel! hyperplasia. These phenotypes are clinically recognizable in >90% of all cases (2). Traditionally, MEN 2 has been divided into three disease subtypes, based on the presence of other associated phenotypes. The most common subtype, MEN 2A accounts for about 85% of MEN 2 cases. In addition to MTC, MEN 2A is characterized by pheochromocytoma (PC), tumours of the adrenal chromaffin cells, in about 50% of cases, and hyperparathyroidism (HPT) in 15-30% of individuals. The most aggressive of the MEN 2 subtypes, MEN 2B, occurs in about 5% of cases and has a median age of tumour onset that is 10 years earlier than other forms of MEN 2 (<10 years) (3, 4). Approximately 50% of MEN 2B are de novo cases with no previous family history (5). As in MEN 2A, PC occurs in about 50% of individuals with MEN 2B but HPT is rare and patients also have a variety of other developmental anomalies such as buccal neuromas, marfanoid habitus, ganglioneuromas of the gut, and thickened corneal nerves (3, 6). The final disease subtype, familial MTC (FMTC) is characterized only by thyroid tumors and has no other associated anomalies. This disease form is the least aggressive MEN 2 subtype and may have a lower penetrance and later disease onset (7). As a result, FMTC families are frequently small and may be phenotypically quite difficult to distinguish from MEN 2A families in which cases of PC or HPT have not yet manifested. Because of this, stringent definitions have been suggested in which the diagnosis of FMTC requires a minimum of 4 (8, 9) or even 10 (10) family members with MTC in the absence of other phenotypes.

RET and the genetics of MEN 2

MEN 2 is inherited as an autosomal dominant disease and, as a result, all first-degree relatives of an affected individual are at 50% risk of inheriting the disease causing mutation. Although they differ in phenotype and aggressiveness, all three MEN 2 subtypes are caused by mutations of the RET (Rearranged in Transfection) oncogene (8, 9). RET encodes a cell surface receptor tyrosine kinase normally required for development of neuroendocrine cell types, the peripheral nervous system, and kidney (1, 11). RET mutations are identified in more than 95% of all MEN 2 families and there is no evidence of families in which the MEN 2 phenotype is not linked to RET. In each case, RET mutations are single amino acid substitutions that result in inappropriate activation of the RET receptor (12, 13). Mutations are clustered in "hot spots" in the extracellular domain (exons 10 and 11) or in the tyrosine kinase domain (exons 13-16) of the receptor (8, 9) (Figure 1).

Because >99% of mutations occur in only 10 codons of RET, direct DNA testing in MEN 2 is simple, widely available, and very efficient and is recommended for all at-risk individuals (Discussed below).

Although all MEN 2 subtypes are associated with RET mutations, specific mutations confer much higher risks for some phenotypes. For example, mutations of RET codon 634 are strongly correlated with HPT and PC and thus, not surprisingly, represent 85% of MEN 2A mutations (14, 15). In MEN 2A, mutations generally alter specific cysteine residues in the extracellular domain of RET (residues 609, 611, 618, 620, 634), resulting in a ligand-independent constitutively activated molecule (Figure 1). The mutations found in patients with FMTC have a broader range of functional effects, and may include both the same type of mutations as those seen in MEN 2A as well as mutations in the tyrosine kinase domain (residues 768, 804, 891) that appear to alter ATP binding (16). More than 95% of MEN 2B patients share the same amino acid substitution (Met918Thr) in the binding pocket of the RET kinase

Figure 1. Schematic diagram of the RET receptor showing the relative positions of the more common mutations found in MEN 2.

domain (8, 9), although rare mutations of codon 883 are also detected (17, 18). Both mutations appear to alter the substrates of RET, thereby changing the downstream signals it sends (16) (Figure 1). The strong associations of specific mutations with each of the disease phenotypes can provide us with an additional tool for predicting patient prognosis and guiding management strategies (Discussed below).

In contrast to the activating RETmutations found in MEN 2, inactivating mutations of RET are identified in patients with Hirschsprung disease (HSCR), a congenital abnormality of gut innervation*19'. HSCR mutations are found throughout the RET gene and result in reduced levels of functional RET protein (20, 21). In rare cases, both the MEN 2 and HSCR phenotypes are associated with a single RET mutation (22, 23). These are generally single amino acid substitutions found in cysteine residues in the extracellular domain of RET (exon 10) (22). These oncogenic mutations may be as frequent as 1% in the HSCR population (24).

Diagnosis and prediction of MEN 2

Diagnosis in the "pre-genotyping" era

Before the identification of disease causing mutations in RET, MEN 2 disease status in at-risk individuals was established by biochemical screening. Generally, this involved measurement of calcitonin peptide release by C-cells in response to a provocative agent, such as pentegastrin or calcium (25). Elevated levels of calcitonin indicated the presence of an increased number of C-cells (C-cell hyperplasia) or of MTC and these individuals would be offered prophylactic thyroidectomy. This strategy, while clinically important, had several drawbacks, not the least of which was that diagnosis was dependent on identification of early hyperplastic changes and frequently was not made until MTC or even metastatic disease was already present (26, 27). Further, a negative screen result did not indicate that a patient did not carry the MEN 2 disease mutation, only that they had no detectable disease at that time. Thus, repeated screening of all at-risk individuals was required annually, or at regular intervals, in order to detect all MEN 2 cases. As a result, 50% of at-risk individuals who had no MEN 2 mutation would be repeatedly and unnecessarily screened. Because every at-risk individual needed to be repeatedly tested, biochemical screening for MEN 2 was a relatively costly strategy. Further, after several negative tests compliance could be a significant issue. For those undergoing regular testing, borderline or difficult to interpret biochemical screening results occasionally resulted in unnecessary thyroidectomy in individuals who did not carry the MEN 2 mutation (28). The incidence of false positives may have been as high as 5-10% (10).

In individuals with a confirmed diagnosis of MEN 2 additional screening for other associated phenotypes such as PC and HPT was required. Patients would be screened for PC on a regular basis by measurement of plasma metanephrines or levels of catecholamines or metanephrines in 24 h urine collection (29). A positive screen would lead to unilateral or, if necessary bilateral adrenalectomy. In general, prophylactic adrenalec-tomy would not be recommended due to risks from adrenal insufficiency (30). HPT is rarely symptomatic in MEN 2 but can be detected by measurement of calcium or parathyroid hormone levels. Biochemical screening for PC and HPT is recommended annually for individuals diagnosed with MEN 2.

MEN 2 and RET mutation testing

The identification of RET mutations as the underlying cause of MEN 2 has changed the management of the individual and family with MEN 2 significantly. Genetic testing, scanning the RET codons known to be frequently mutated, is now the preferred method for confirming the clinical diagnosis of MEN 2 and is considered the standard of good practice (9, 10, 31). The rate of false negative (2-5%) (10) and false positive (<0.1%) results in DNA testing is a dramatic improvement over biochemical screening methods. Individuals at-risk for MEN 2 should now be screened at birth or at the earliest possible time for RET mutations and the results should provide the basis for recommending thyroidectomy. Prophylactic thyroidectomy for individuals carrying a germline RET mutation has dramatically reduced morbidity and mortality due to MTC, and perceived quality of life is much better in individuals at risk for MTC than for those at risk of other cancers where diagnosis and management are less clear cut (32).

Because the basis and expectations associated with genetic testing need to be clearly understood in order for at-risk individuals to understand the implications of a RET mutation test and for them to use that information to make informed decisions about disease management, it is essential that all DNA testing be accompanied by appropriate genetic counseling. This would generally take the form of pretest counseling to explain the implications and risks of the test and one or more post-test counseling sessions involving delivery of results and discussion of their implications as necessary.

INDICATIONS FOR RET MUTATION TESTING. Screening for RET mutations is now the basis of all management strategies for MEN 2, replacing reliance on biochemical screening. In families where the disease causing mutation has already been established by previous screening of affected individuals, all at-risk individuals should be genetically screened for the familial mutation. Individuals who do not carry this mutation are not at risk of MEN 2 and can be excluded from further testing. Individuals with the mutation are at high risk for MEN 2 phenotypes and should be managed accordingly. If diagnosis is made in a child, thyroidectomy should be performed before age 5 for MEN 2A and FMTC families and before age 6 months in individuals with MEN 2B mutations which are associated with earlier tumour development (10).

Early, genetically based identification of mutation carriers permits surgery before the usual onset of malignant disease and carries the optimal possibility of preventing metastatic disease. Older patients diagnosed with RET mutations will be offered thyroidectomy as soon as possible accompanied by biochemical monitoring for the presence of metastatic disease. Mutation positive individuals will be monitored throughout life for other tumour types or anomalies associated with their specific MEN 2 subtype, as described above. The regime for these screening protocols may, in theory, be modified based on the occurrence and age of onset of these phe-notypes in other family members. However, variability in these, even within a single family, suggests that caution should be used when relaxing screening protocols (33, 34).

If an individual represents a new case/family diagnosed for MEN 2, RET mutation screening should be performed in a known affected family member. This also holds true for all individuals diagnosed with apparently sporadic MTC, as 1-7% of these have germline RET mutations and represent new MEN 2 families (35). Interestingly, early studies had suggested that germline RET mutations were very rare in sporadic PC but recent studies showing they may occur in up to 5% of cases (36) have suggested that all patients with these tumours should also be screened for RET mutations (10). Initial screening must include RET exons in which mutations are most frequently found, (exons 10, 11, 13-16) however these may be prioritized based on the patient phenotype. For example, 85% of MEN 2A families have mutations in exon 11 and almost 95% of MEN 2B cases have mutations of codon 918 in exon 16. If mutations of these exons are not identified a broader screen, including all RET exons may be necessary. Once a RETmutation is identified, all other at-risk family members may be screened specifically for that change and individuals carrying the mutation are managed as described above.

In a few instances, a RET mutation may not be identified in a putative MEN 2 individual. These cases are rare and frequently, although not always, involve families that are quite small with few or a single affected individual. In the past, some of these families may have resulted from conservative diagnosis of FMTC in families that did not have clear features of MEN 2. For example, studies have shown that up to 5% of the population may have C-cell hyperplasia but do not have RET mutations nor a true MEN 2 phenotype (28). Recent studies suggest that at least some of these cases may be related to mutations of the succinate dehydrogenase subunit D gene (chromosome 11q23) and not to RET (37).

This highlights the necessity of accurate and unambiguous clinical diagnosis in cases where RET mutations have not been identified. If families with clearly defined MEN 2 but no RET mutation have sufficient confirmed affected family members available, linkage analysis using polymorphic sites in or near the RET gene (chromosome 10q11.2) may provide an alternative to direct mutation detection. In this method, a haplotype for a series of polymorphisms over the region of RET which presumably includes a disease mutation may be constructed based on the genotype of multiple affected family members (38-41). Inheritance of this "disease haplo-type" can be used to identify individuals who have also inherited the predicted RET mutation and are therefore assumed to be MEN 2 carriers. This method is less amenable to diagnosis than is direct mutation testing since it is dependent on the availability of a suitable family structure, the participation of multiple family members and relies on the assumption that the clinical diagnosis is correct in suggesting MEN 2.

Where suitable family members are not available, or a haplotype cannot be constructed, MEN 2 families without RET mutations will be treated as they were before the advent ofDNA mutation testing, using repeated biochemical screening of all at-risk individuals and offering surgery at the first sign of a positive test result.

who should be offered screening?RET mutation screening is considered the standard of care for all individuals at-risk for MEN 2, irrespective of their age. Ideally, at-risk individuals in known MEN 2 families would be screened for RET mutations at birth or shortly thereafter. This is somewhat different from mutation testing in many other cancer syndromes where minors are not automatically screened. In the case of MEN 2, the penetrance of the disease is very high (>90% have clinically detectable disease) and onset is very young, the earliest recognition of MEN 2B being reported in children under age 5 (24, 42, 43) necessitating very early detection to allow presymptomatic intervention. Further, unlike many other cancer syndromes of childhood, there are clear, well tolerated, prophylactic options available which are demonstrably valuable in decreasing the burden of the disease in affected individuals. The advent of early childhood mutation detection has greatly reduced the incidence of MTC in families with known MEN 2 and will, with time, virtually eliminate the morbidity and mortality associated with metastatic disease in known MEN 2 families.

RET mutation screening is also recommended for individuals with sporadic MTC and also PC, although there has been no indications that RET mutations contribute to sporadic HPT (44). In each case, it is important to remember that RET mutations can occur somatically in these tumours (45-49) and that these should not be confused with germline MEN 2-RETmutations.

Approximately 1% of individuals with HSCR have a RET mutation in exon 10 which may also confer risk for MEN 2 phenotypes (24). While this may seem rare, RET mutations in HSCR are widely distributed throughout the gene and exon 10 mutations, primarily in codons 609, 618 and 620, represent one of the largest clusterings of mutations. Because of the oncogenic risk associated with these mutations, RET exon 10 mutation screening is recommended for all children diagnosed with HSCR. Individuals identified with any of these mutations should be treated as a potential MEN 2 case and screening of at-risk family members and surgical intervention should be offered.

Diagnosis and management: The evolving genetic contribution

While the advent of genetic testing for RET mutations has significantly improved our ability to manage patients and families with MEN 2, the potential exists for additional refinement that may improve the prospects even further. Several exciting options for this refinement are currently being evaluated and the accumulating body of experience with RET genotype and phenotype are allowing us to improve the accuracy of disease prediction.

Genotype-phenotype associations in genetically based management

Our 10 year experience of RET mutation and MEN 2 disease phenotype correlation has shown us that not all RET mutations carry equal associated risk and that the traditional definitions of disease phenotype may in future not be as useful to us as genetically or mutation based disease risk estimates.

We have long known that some RET mutations conferred higher risk of PC (e.g. Cys634Arg or Met918Thr) (8, 9) while others are associated with a less aggressive disease phenotype (e.g. Glu768Arg or Val804Met). Recent studies have begun to investigate the potential of using specific RET mutation data to guide management strategies. Three general categories of high, medium, and low risk RET mutations may be defined based on their relative penetrance, the specific disease phenotypes associated with them, and the aggressiveness of these phenotypes (Table 1) (10, 31). Mutations in the highest disease risk group include those found in MEN 2B (Met 918Thr, Ala883Phe). The lowest risk mutations are those found most frequently in FMTC and those associated with later disease onset (Table 1) (50).

These risk groupings are an attractive tool to supplement clinical diagnosis for defining individuals with high risk who need early thyroidectomy and/or stringent biochemical screening regimes. By defining a subgroup of higher risk patients genetically predisposed to more, or more severe, disease phenotypes we may be able to focus health care resources more effectively and reduce patient stress associated with disease management. In future, the specific RET mutation found in a patient is likely, to act

Table 1. Relative risk groups in MEN 2 associated with specific RET mutations

Re la rive risk

Mutant RET codons

Disease phenotype

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