RET proto-oncogene mutations in multiple endocrine neoplasia type 2 and medullary thyroid carcinoma

8 RET proto-oncogene mutations in multiple endocrine neoplasia type 2 and medullary thyroid carcinoma GILBERT J. COTE NELSON W O H L L K D O U G L A S EVANS H E L M U T H GOEPFERT ROBERT F. G A G E L

Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant genetic syndrome characterized by the presence of medullary thyroid carcinoma (MTC), phaeoehromocytoma and hyperparathyroidism. Several variants of this syndrome exist. The most common is MEN type 2A (MEN2A), the association of MTC, which develops in over 90% of gene carriers, phaeochromocytoma, either unilateral or bilateral, which occurs in 50% of gene carriers, and hyperparathyroidism, which occurs in approximately 10-20% of gene carriers (Cance and Wells, 1985; Gagel, 1992, 1994). A second and less common variant of this syndrome is MEN type 2B (MEN2B), in which there is the association of MTC in nearly 100% of gene carriers and phaeochromocytoma in 50% and the absence of hyperparathyroidism. Individuals with MEN2B also have a Marfanoid habitus and prominent mucosal neuromas, which affect the conjunctival surfaces of the eyes and the tongue and are distributed throughout the gastrointestinal tract (Williams and Pollock, 1966; Carney et al, 1978). The third variant of MEN2 is the familial MTC syndrome (FMTC), in which MTC is the only manifestation found (Farndon et al, 1986). In small families with MTC only, it is frequently difficult to differentiate between FMTC and MEN2A, because the other manifestations of MEN2A may not be completely penetrant. An uncommon variant is the association of MEN2A with cutaneous lichen amyloidosis, which has been identified in fewer than 15 families world wide (Gagel et al, 1989; Nunziata et al, 1989). Individuals with this variant have pruritus and characteristic skin lesions located over the upper back. The clinical management of MEN2 has been the subject of many previous reviews (Cance and Wells, 1985; Gagel, 1992, 1994a,b). What has changed in the past 2 years is the identification of molecular defects of the RET proto-oncogene, which are believed to be causative for each of these Bailli~re's Clinical Endocrinology and Metabolism-609 Vol. 9, No. 3, July 1995 ISBN 0-7020-1945-3

Copyright © 1995, by BailliSre Tindall All rights of reproduction in any form reserved

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clinical variants. Somatic mutations of the RET proto-oncogene (those mutations that occur only in the tumour and are not germ line) have also been found in sporadic MTC. The importance of these mutations in the causation of sporadic MTC is unclear, but enough information can be gleaned from available studies to indicate that abnormalities of RET are found for 15-25% of sporadic MTCs. This review will provide an update on the current understanding of the molecular causes for MEN2 and FMTC and begin to consider the options for use of this information in the clinical management of families with this disease. It will also focus on the potential role for analysis of the RET proto-oncogene in sporadic MTC. MAPPING THE MEN2 GENE Efforts aimed at linking MEN2 to a particular chromosomal location began as early as 1970. These efforts were focused primarily on the HLA locus and were unsuccessful. It was not until the refinement of linkage techniques through the use of DNA potymorphisms in the human genome that MEN2 was targeted for focused investigation. MEN2 was considered an excellent candidate for study because of the many well-characterized families with this disorder. Progress was initially slow. Studies presented at the First International Workshop on Multiple Endocrine Neoplasia Type 2 in 1984 provided the first reports on efforts to map the causative gene (Kidd et al, 1984). By the time the Second International Workshop was held in 1986, the genetic locus had not been identified, but significant portions of the human genome had been excluded from linkage (Simpson and Kidd, 1987). Simultaneous reports from two groups, one led by Bruce Ponder and the second a collaborative effort between Kenneth Kidd and Nancy Simpson, first mapped the causative gene to a centromeric chromosome 10 locus in 1987 (Mathew et al, 1987; Simpson et al, 1987). Progress slowed thereafter, for two reasons. Firstly, there was a scarcity of polymorphic DNA sequences within the centromeric chromosome 10 region and, secondly, the rate of recombination around the centromere was very low, making it difficult to use recombinant events in the families studied to further localize the gene. Six years were to pass before two recombinant events in a single extended family mapped the causative gene to a 0.5 cM (0.5 Mb) region (Gardner et al, 1993), with the RET proto-oncogene centrally located in this region (Lairmore et al, 1993; Mole et al, 1993). Very quickly thereafter, reports from Mulligan et al (1993) and, within several months, DonisKeller et al (1993) identified mutations within two exons of the RET protooncogene. THE ROLE OF THE RET PROTO-ONCOGENE IN NEOPLASIA The RET proto-oncogene was a logical candidate for involvement in this genetic syndrome. It had been considered to be a candidate gene but was

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excluded erroneously because of unpublished reports indicating a recombination between MEN2 and a polymorphic sequence within this gene. In fact, the recombination had been observed with the papillary thyroid carcinoma (PTC) oncogene, a rerranged form of RET that brought sequences from elsewhere on chromosome 10 into fusion with coding CADHERIN

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Figure 1. RET proto-oncogene rearrangements in papillary thyroid carcinoma. Three different rearrangements of the RET proto-oncogene have been identified in papillary thyroid carcinoma, which have been designated PTC 1, PTC 2 and PTC 3. In each of these examples, there is an intrachromosomal rearrangement that brings the coding sequence of the RET proto-oncogene under the control of another constitutively expressed gene.

sequences for RET (Figure 1). The recombination was actually observed with the translocated DNA rather than with RET sequences. The RET proto-oncogene was first identified as an oncogene in 1985 by an American group (Takahashi et al, 1985) and subsequently identified as the PTC oncogene by Italian (Bongarzone et al, 1989; Donghi et al, 1989; Ishizaka et al, 1990; Lanzi et al, 1992; Sozzi et al, 1992) and Japanese (Ishizaka et al, 1990) investigators. The PTC oncogene was identified by transforming activity of DNA derived from a PTC, a tumour type derived from follicular epithelium, where the RET gene is not normally expressed. The PTC oncogene is a rearranged form of RET (Figure 1). These rearrangements cause promoter sequences from one of three different genes to cause expression of the coding sequence for the PET proto-oncogene tyrosine kinase. The breakpoint for the RET translocation occurs, in each case, within intron 11 of the RET proto-oncogene (Bongarzone et al, 1994) and results in continuous activation of the tyrosine kinase in cell types in which the promoter sequences are expressed. Approximately 15-30% of PTCs in the USA and Europe have one of these three translocations, whereas this rearrangement is far less common in Japan. Further evidence that RET is oncogenic is provided by the development of melanomas in transgenic mice that overexpress the gene (Iwamoto et al, 1990). Although the exact role that the PTC oncogene plays in the development of PTC remains unclear, the frequency of this mutation suggests a central role.

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RET P R O T O - O N C O G E N E MUTATIONS IDENTIFIED IN MEN2 AND MTC The RET proto-oncogene encodes a tyrosine kinase (TK) receptor. This receptor has several unique features, including an extracellular cadherin region, a cysteine-rich region adjacent to the membrane and two intracellular TK regions (Kwok et al, 1993) (Figure 1). The RET receptor is expressed constitutively in thyroid C cells and adrenal medulla (Pachnis et al, 1993); its ligand is unknown. Studies in transgenic mice in whom both copies of the RET gene have been deleted indicate a role in neuronal and renal development (Schuchardt et al, 1994). Indeed, inactivating mutations of RET are causative for familial Hirschsprung's disease (Edery et al, 1994; Romeo et al, 1994). Mutations of the RET proto-oncogene have been discovered in two regions of the gene. The first type of mutation changes highly conserved cysteines in the extracellular region of the receptor to another amino acid. Five specific codons (609, 611, 618, 620 and 634) are affected (DonisKeller et al, 1993; Mulligan et al, 1993, 1994a) (Figure 2, Table 1). The second class of mutations affects the intracellular TK regions. The first is a mutation at codon 768, which changes a glutamine to an aspartate in the first TK region (Eng et al, in press) (Figure 2, Tables 1 and 2). The second is a mutation at codon 918, which changes a methionine to a threonine in the second TK region (Carlson et al, 1994; Hofstra et al, 1994) (Figure 2, Tables 1 and 2).

Table 1. Specific mutations of the RET proto-oncogene associated with hereditary MTC. Codon

Normal amino acid

Mutant amino acid

Clinical syndrome

609 611 618 620 634 768 918

Cysteine Cysteine Cysteine Cysteine Cysteine Glutamine Methionine

* * * * * Aspartate Threonine

MEN2A/FMTC MEN2A/FMTC MEN2A/FMTC MEN2A/FMTC MEN2A FMTC MEN2B

Approximate percentage of all MEN2 mutations 0-1 2-3 3-5 6-8 80-90 0-1 10-20

* Specific mutations are shown in Table 3.

Table 2. Specific mutations of the RET proto-oncogene associated with sporadic MTC. Codon

Normal amino acid

Mutant amino acid

768

Glutamine

Aspartate

918

Methionine

Threonine

Clinical syndrome Somatic mutation in sporadic MTC Somatic mutation in sporadic MTC

Approximate percentage of all MEN2 mutations Unknown 10-25

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CLINICAL S Y N D R O M E S ASSOCIATED WITH SPECIFIC MUTATIONS MEN2A Mutations of five codons have been associated with MEN2A. These are codons 609, 611,618, 620 and 634 (Figure 2, Table 3). These mutations change a highly conserved cysteine to another amino acid. The most common mutation occurs at codon 634, and the most frequent mutation at this site is a cysteine to arginine mutation. The mutations at codon 634 are most commonly associated with classical MEN2A, although a number of cases of FMTC have been described (Donis-KeUer et al, 1993; Mulligan et al, in press). There are reports that a codon 634 mutation, converting a cysteine to an arginine, is most likely to be associated with parathyroid disease (Mulligan et al, 1994a), but there is abundant evidence that para-

Table 3. Exon 10 and 11 mutations in MEN2A and familial MTC. Codon 611 AAC

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The symbol ---) indicates a mutation that has been reported in the context of MEN2A or FMTC. These mutations were collected from abstracts from several meetings, mutational analysis derived from patients at the MD Anderson Cancer Center or published reports. Other mutations shown are possible but have not been reported.

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thyroid disease occurs with other amino acid substitutions at this codon (Donis-Keller et al, 1993; Gagel et al, in press; Mulligan et al, in press). It is difficult to know the relevance of the reported relationship between the codon 634 arginine mutation and hyperparathyroidism, because this particular mutation accounts for approximately 45% of all mutations in MEN2A, raising the possibility that a sampling bias could lead to this reported association. Further data are being collected by a constortium established to examine the clinical features of a larger number of patients.* Phaeochromocytoma is also most likely to be associated with a codon 634 mutation (Mulligan et al, in press). The MEN2A cutaneous lichen amyloidosis syndrome is an uncommon variant of MEN2A, in which there is a characteristic form of cutaneous lichen amyloidosis located over the upper back. A total of 15 families have been identified with this particular variant of MEN2A. The clinical features in this variant include MTC and phaeochromocytoma; parathyroid disease has been an uncommon manifestation. All families reported to date with this variant have had a codon 634 mutation (Ceccherini et al, 1994; Robinson et al, 1994), although it seems unlikely that this mutation can be the sole explanation for this clinical syndrome, because no more than 15 out of 145 families with a codon 634 mutations have cutaneous lichen amyloidosis. A codon 634 mutation seems to be necessary but not sufficient for the development of the cutaneous lesion. A more detailed examination of the RET proto-oncogene has not been reported. Mutations of the four codons (609, 611,618 and 620) in exon 10 are most likely to be associated with FMTC (Donis-Keller et al, 1993; Mulligan et al, in press), although considerable overlap exists between FMTC and MEN2A. Collectively, the exon 10 mutations account for no more than 20-30% of all mutations associated with hereditary MTC. Approximately 3% of families with MEN2A do not have an identifiable mutation. One of these families is an unusual variant, in which classic MEN2A has been associated with corneal nerve thickening; no mutation of the RET proto-oncogene has been identified it~ this family, although only codons 609, 611,618, 620, 634 and 918 have been analysed (Kane et al, in press). Another uncommon variant of MTC is the association of MEN2A with Hirsehsprung's disease, several families having been identified with this variant. Preliminary results have identified codon 618 and 620 mutations (Borst et al, 1994; Lacroix et al, 1994). Although these mutations may explain the development of hereditary MTC, it is intellectually dissatisfying to consider these point mutations as the sole explanation for the Hirschsprung's disease-like manifestations.

* The consortium is being organized by Lois Mulligan, PhD, Department of Paediatrics, 20 Barrie St, Queen's University, Kingston, Ontario K7L 3N6, Canada (FAX: +1 613 548 1348; E-mail: [email protected]) and Charis Eng, MD, PhD, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom (FAX: +44 1223 333 875; Email: ceel001 @cus.eam.ac.uk). They can provide details for submission of information to the consortium.

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MEN2B Germline mutations of codon 918 of the RET proto-oncogene are found in more than 94% of patients with MEN2B (Carlson et al, 1994; Hofstra et al, 1994). We have examined nine patients with MEN2B and have found codon 918 mutations in all. The point mutations in MEN2B are frequently examples of new mutations, neither parent exhibiting evidence of disease or peripheral blood mutation. Recent studies indicate that the mutated allele is derived from the father in all examples in which detailed information is available, even though the father does not carry a germline mutant allele (Carlson et al, 1994). One supposition in these cases is that the mutation occurred during spermatogenesis in the father. The nature of the mutation in the 5-6% of individuals with MEN2B who do not have codon 918 mutations is unclear. It is postulated that a mutation elsewhere in the RET gene, the ligand for the RET receptor, a downstream component of the tyrosine kinase signalling system or another regulatory factor important in this pathway may be involved in this handful of patients. FMTC FMTC is most commonly associated with an exon 10 (codon 609, 611,618 or 620) mutation, although some patients with FMTC have codon 634 mutations (Donis-Keller et al, 1993; Mulligan et al, 1994a, in press). It is possible that the distinction between FMTC and MEN2A will be clearer once more detailed information is available on the family size of kindreds studied. For example, since phaeochromocytoma is found in only 50% of MEN2A patients, it is possible that MTC could be the only manifestation noted in a small family, leading to incorrect assignment of clinical status. The identification of the exon 10 mutations may also have some prognostic significance, since individuals with FMTC generally have a better prognosis with respect to their thyroid carcinoma than do patients with MEN2A or 2B. Somewhat surprisingly, approximately 13% of families with FMTC do not have identifiable mutations (Mulligan et al, in press). One family with FMTC has been found to have a codon 768 glutamine to aspartate mutation in the first TK domain of the RET proto-oncogene (Eng et al, in press). The evidence that this mutation is causative for MTC in this family is limited to co-segregation of the molecular defect and MTC. However, the location of the mutation within the ATP binding region, and the observation of the same mutation in 4 out of 10 sporadic MTCs, provides support for its importance (Eng et al, in press).

Sporadic M T C The term 'sporadic MTC' implies that the tumour has arisen as a result of a new mutation in an individual C cell. In fact, a preliminary analysis of patients (Wohllk et al, unpublished data) with sporadic medullary thyroid carcinoma evaluated at the MD Anderson Cancer Center indicates that at least five out of 88 patients with sporadic MTC had germline mutations

RET MUTATIONS IN MEN2 AND MTC

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more commonly associated with MEN2A. Two of these patients were examples of new (codon 634) mutations, neither parent having a mutation, despite demonstrated paternity. The other three were codon 609 and 611 mutations; one was the first identified member of a new kindred, the other family has not been studied. There are, however, at least two additional examples of patients with apparent sporadic MTC presenting with new germline mutations of the RET proto-oncogene normally associated with MEN2A that have been described in the literature (Mulligan et al, 1994a). These results suggest that new mutation MEN2A may be more common than previously thought. This conclusion is supported by an independent analysis of a large number of kindreds with MEN2A, demonstrating lack of a common haplotype for many families with the same mutation, indicating that these mutations arose independently. The second mutation identified in sporadic MTC is a codon 918 mutation (Figure 2 and Table 2). This somatic mutation occurs in up to 25% of all sporadic MTCs (Hofstra et al, 1994). Of particular interest is the suggestion that tumours with codon 918 mutations pursue a more aggressive course, with earlier metastasis and death. A preliminary evaluation of patients with this mutation followed at the MD Anderson Cancer Center supports this suggestion (Wohllk et al, unpublished data), although the total number of reported patients is too small to draw meaningful conclusions. The generally aggressive nature of the MTC in MEN2B provides further support for the aggressive nature of these tumours. More recently, somatic mutations of codon 768 have been identified in 4 out of 10 sporadic MTCs (Figure 2 and Table 2) (Eng et al, in press). There is currently no information regarding the clinical course of MTC in these patients and no estimate of the frequency of these mutations. E V I D E N C E THAT RET P R O T O - O N C O G E N E MUTATIONS ARE C A U S A T I V E F O R MTC AND MEN2A There are several lines of evidence that support a role for RET proto-oncogene mutations in the causation of MTC and MEN2. The first type of evidence is genotype to phenotype correlation. In over 400 families with MEN2 where a RET mutation has been identified, there is perfect correlation between the presence of a mutation (genotype) and FMTC or MEN2 (phenotype). To define this more specifically, in adults who have a RET mutation, either MTC or an abnormal pentagastrin-stimulated calcitonin response has been found in all cases studied. Additionally, all adults with proven MTC in genotyped kindreds have a RET mutation. We have examined over 35 families with FMTC or MEN2A at the MD Anderson Cancer Center and have identified mutations in all (Gagel et al, in press). There are, of course, children who have RET proto-oncogene mutations, indicative of gene cartier status, but no evidence of MTC. These children will be discussed more fully in the section on the clinical use of mutational information. A second type of evidence supporting an oncogenic role for the RET gene is the identification of new mutation forms of MTC. The most

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common example is MEN2B, in which the majority of patients diagnosed with this disorder represent new mutation cases. There are two published cases of new mutation MTC associated with codon 634 mutations (Mulligan et al, 1994b; Zedenius et al, 1994) and, at the MD Anderson Cancer Center, we have identified four individuals with sporadic MTC who have germline mutations of codon 634 of the RET proto-oncogene (unpublished observations), two of whom have proven to be examples of new mutations. The finding of MTC and germline mutations in these individuals with no evidence of prior familial MTC or RET mutation is another piece of evidence supporting a causative role. Thirdly, there is ample evidence for the oncogenic potential of the RET proto-oncogene in other tumours. For example, the PTC oncogene has proven oncogenic potential in PTC and was identified by its ability to transform NIH 3T3 fibroblasts. Overexpression of a RET proto-oncogene driven by the mouse metalothionein promoter in transgenic mice results in the development of melanoma (Takahashi et al, 1992). It seems likely that experimental paradigms demonstrating the oncogenic potential of MEN2 mutations associated with MEN2 will follow in the next year or two. These experiments can be envisaged to be of several types: experiments in which it is demonstrated that overexpression of RET in a tissue-specific manner (in the C cell for example) in transgenic animals or in primary culture of C cells results in transformation; and a second approach in which reversal of transformation is effected by neutralization of the RET mutation in a human cell line that carries a mutant allele. The latter approach also suggests a strategy for several of the oncogenic effects of the mutation. IDENTIFICATION OF RET PROTO-ONCOGENE POINT MUTATIONS

The identification of 7 mutated codons associated with hereditary MTC, and 2 of these 7 associated with sporadic MTC, makes it feasible to perform straightforward molecular analyses. Several different techniques have been used for the detection of these mutations; all are based on polymerase chain reaction (PCR) methodology (Gagel and Cote 1994; Khorana et al, 1994). The authors routinely perform direct DNA sequencing of PCR products of exons 10, 11, 13 and 16 (Khorana et al, 1994; Gagel et al, in press) (Figure 3). Specific mutations are then confirmed by restriction enzyme analysis. In an unknown family, our strategy has been to focus initially on the exon 11 codon 634 mutation, because 80% of all mutations in MEN2A affect this codon. DNA sequence analysis makes it feasible to determine the specific mutation and to determine the presence of both a normal and mutant allele (Figure 3). It is possible to confirm the presence of all mutations at codon 634 by restriction enzyme analysis (Gagel et al, in press). We find a combination of direct DNA sequence analysis and restriction enzyme analysis to be a highly reproducible combination for determining the presence of a particular mutation. Direct DNA sequencing from PCR products is sometimes associated with variable quality of the

RET M U T A T I O N S IN M E N 2 A N D MTC

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sequencing ladder. A simplified method, using an agarose gel purification of amplified DNA, has improved the quality of our sequencing results (Khorana et al, 1994), but nonetheless, we routinely confirm a specific mutation by restriction analysis. Other techniques, including denaturing gradient gel electrophoresis (Decker et al, 1994), allele-specific hybridization and single-stranded conformational polymorphism (Gagel et al, in press) have been applied to the detection of mutations. Each of these techniques has its proponents, and there are advantages and disadvantages of each. The authors prefer the approach outlined above because of the ability to quickly identify the specific mutation and confirm it by a second independent technique. Other approaches encompassing these same qualities could be envisioned, and current techniques are likely to be replaced by even simpler and more specific techniques over the next several years. We have been concerned about the possibility of false positive or false negative test results because of their implications for decision-making in the management of this disorder. Retrospective analysis of kindreds previously screened by pentagastrin testing has identified multiple examples of false positive pentagastrin test results (Lichter et al, 1992; Gagel et al, in press), resulting in unnecessary thyroidectomy. The reasons for false positive pentagastrin test results remain unclear but provide an example we would like to avoid in the application of genetic screening. In our experience, over the past one and one-half years, we have identified two examples of false negative tests based on a codon 691 polymorphism of the RET proto-oncogene (Gagel et al, in press). In these particular examples, one of the primers used for the amplification of genomic DNA annealed to the site of the polymorphism, resulting in an inefficient or absent amplification of the allele containing the polymorphism. Since this particular polymorphism is relatively common in the population, we have observed examples of failure to amplify both normal (over 15 examples) or mutant (2 examples) alleles (unpublished observation). The identification of this particular polymorphism makes it unlikely that this specific error will be repeated in the future. Although it is possible that other rarer polymorphisms exist that could affect amplification, this is unlikely to be a major problem. There is also concern about the possibility of false positive results. PCR is very sensitive, and a few copies of a mutant gene in a laboratory that routinely performs these analyses could result in contamination of a normal sample, a problem that has plagued the use of DNA analysis in forensic analysis (Lewontin, 1994). We have approached this problem by observing strict protocol with appropriate controls in each reaction to prevent or detect contamination. We also insist that a second analysis on a separate blood sample be performed on all positive or negative test results and suggest it may be appropriate for this analysis to be performed in a separate laboratory. Results of genetic tests should be viewed no differently from there of any other laboratory test. False positive or negative test results are likely to occur as a result of technical difficulties, sample mix-up or incorrect tabulation or reporting of results. The clinical implications of

RET MUTATIONS IN MEN2 AND MTC

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either a false positive or negative test result may be enormous and will be discussed below. The codon 768 (exon 13) glutamine to aspartate mutation is detected by amplification of exon 13 and the demonstration that an Alu I restriction site is destroyed (Eng et al, in press). Detection of a codon 918 (exon 16) mutation is best accomplished by amplification of exon 16, followed by restriction analysis with Fok I (Hofstra et al, 1994). The methionine to threonine mutation at codon 918 results in destruction of this restriction site. It is important to include, in each assay, a negative and positive control to confirm that conditions for enzymatic digestion are appropriate. Since only one mutation has been identified for both codons 768 and 918, sequence analysis is probably not necessary.

C L I N I C A L USE OF G E N E T I C I N F O R M A T I O N Although the identification of RET proto-oncogene mutations occurred less than two years ago, a clear picture is beginning to emerge about the usefulness of this information. Three potential applications can be envisioned. The first is the use of this information to determine which children of parents with hereditary MTC are at risk for the development of MTC or MEN2. The second is to identify cases of hereditary MTC who present as cases of apparent sporadic MTC. Thirdly, there are preliminary data that suggest that particular mutations may be of use in assessing prognosis. Each of these examples will be discussed. Decision-making for M E N 2 A or FMTC

There are several reasons to perform genetic analysis in families with known hereditary MTC and a detectable mutation. Firstly, it is possible to exclude 50% of family members with no mutation from further screening. In such individuals, the RET mutational analysis should be performed on more than one blood sample, preferably in different laboratories, for the reasons mentioned in the preceding section. If a mutation has been readily detected in the parent and is not found in the child, it does not seem reasonable to continue extensive and unpleasant pentagastrin testing in children, although unfamiliarity and unease with this transition to DNAbased testing will undoubtedly lead to some continuation of testing. The identification of two false negative test results by our laboratory has led us to recommend repeat genetic testing at some future date with (hopefully) better technology. This recommendation is based on the possibility that there are technical issues creating false negative results that we cannot currently understand. It seems likely that our understanding of these mutations and the technology for detecting them will evolve over a period of several years to a decade. It would be prudent to consider repeat testing at a future date. A second reason for obtaining mutational analysis on children at risk is

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to identify gene carriers. Two types of action could be envisaged in children who inherit a mutated allele. The first is to focus pentagastrin, adrenal and parathyroid testing on children with mutations, using preexisting criteria for decision-making regarding surgical intervention. The proponents of this approach argue that annual screening by pentagastrin testing has been successfully used to identify MTC in children at risk (Lips et al, 1994); follow-up studies demonstrate that more than 90% of these children have been cured by thyroidectomy. The disadvantages of continued pentagastrin testing are obvious. The test is unpleasant, expensive (two separate genetic analyses cost less than a single pentagastrin test) and there is the possibility that a child may miss an annual test for a variety of reasons, leading to potential delay in removal of the thyroid gland. An alternative approach advocated by others is to use genetic testing and information about the clinical course of the disease to make decisions regarding surgical removal of the thyroid gland (Chi et al, 1994; Wells et al, 1994; Gagel et al, in press). It is known that more than 90% of those who inherit the causative gene for hereditary MTC will develop MTC at some point during their lives. The average age for surgical removal in several prospective series has ranged from 10 to 13 years of age, and improvements in assay sensitivity have resulted in the gradual reduction of the age of detection to about 10 years. It is also known that the youngest child to present with metastatic MTC was 6 years old. Using this type of information, some clinicians have argued that it makes sense to remove the thyroid gland by the age of 6 years in children with RET mutations. There are other considerations in this decision. It is necessary for children to take thyroid hormone replacement, although this has been accomplished with relative ease in most young children. A potential problem of greater import is the development of hypoparathyroidism or recurrent laryngeal nerve damage in a young child. Concern has been expressed that the incidence of these complications may be greater in younger children, in whom the size of the thyroid gland and surrounding structures is smaller. In fact, there is no available evidence that suggests this view to be true, although, when queried, most endocrine surgeons would prefer to postpone the surgical procedure until the age of 5 years, a time when the surgical anatomy is clearly defined. An important consideration in the decision to use pentagastrin testing to decide when to operate is the belief that pentagastrin testing will detect MTC before metastasis has occurred. The available long-term follow-up data provide convincing evidence that 85-90% of children who received prospective thyroidectomy based on pentagastrin testing have normal or non-detectable plasma calcitonin (CT) values 15-20 years after surgery (Cance and Wells, 1985; Telander et al, 1986; Gagel et al, 1988). There are, however, a few examples of abnormal follow-up pentagastrin test results in children who have received thyroidectomy based on annual pentagastrin screening (Cance and Wells, 1985; Telander et al, 1986; Gagel et al, 1988), and the authors are aware of other unreported examples. It seems appropriate to take these cases into account in the

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623

decision-making process and to acknowledge the possibility that 5-10% of children are not cured by thyroidectomy at the time the pentagastrin test first becomes positive. In the interpretation of these follow-up data, it is important to recognize that abnormal CT values that develop after surgery could be the result of either metastasis prior to surgery or inadequate primary surgery, leaving a remnant of thyroid tissue behind along with small numbers of C cells, some of which later become hyperplastic and develop into MTC. If one believes that metastasis can occur early, surgery at the age of 5-6 years would be beneficial. On the other hand, if the problem is inadequate surgical removal of the thyroid gland, earlier surgery may not alter the course of the disease unless the surgery is more extensive than routinely performed. The major concern related to an adequate total thyroidectomy (one that removes the posterior capsule to ensure complete removal of C cells) is the likelihood that an unacceptable percentage of children will develop hypoparathyroidism. Wells et al (1994) have addressed this issue by performing a total thyroidectomy, central node dissection and transplantation of parathyroid tissue to the non-dominant arm. In their initial series, they reported no cases of hypoparathroidism in 13 children. Although this type of approach will undoubtedly afford the greatest chance of eliminating all C cells from the neck, not all practicioners agree that this aggressive approach is required. At present, the only clinical indication of recurrent disease in children screened and thyroidectomized using more standard approaches over the past two decades is the presence of elevated CT values in 5-10% of patients despite follow-up periods of as long as 15-20 years (Gagel et al, 1988; Lips et al, 1994). None of the children the authors have followed has any other evidence of recurrent disease. It is possible that these elevated calcitonin values (in most cases less than 100pg/ml after pentagastrin) will not evolve into clinically evident disease that shortens the life span. Since the longest follow-up is 20 years, additional time will be required before definitive conclusions can be drawn. The lack of available data and the low number of such patients provide areas in which collaborative approaches involving several centres might yield greater insight. We have considered all of the aforementioned issues and have decided to offer surgical removal of the thyroid gland to children older than 5 years (Gagel et al, in press). We believe that the potential for development of metastatic disease outweighs the small risk of hypoparathyroidism or recurrent laryngeal nerve damage. In each of the cases, we have also offered the parents the possibility of continued pentagastrin testing. The youngest child to undergo thyroidectomy was 3~ years, a decision based on both positive genetic and abnormal pentagastrin tests (Gagel et al, 1993). Studies from Wells et al (1994) describe a similar experience in 21 family members with positive genetic tests, 13 of whom decided to have surgical removal of the thyroid gland. Six of their children had a normal pentagastrin test at the time of surgery, with a mean age of 12 years and a range of 6-21 years. Lips et al (1994) have reported thyroidectomies for five children with positive genetic and normal pentagastrin tests but continue to advocate penta-

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gastrin testing after genetic testing to determine the need for thyroid removal. Table 4 details the published information in nine children with RET mutations who were found to have normal stimulated CT concentrations from the MD Anderson Cancer Center, Washington University and a combined Dutch experience. Of particular importance is the finding of microscopic MTC in 1 out of 2 patients operated on at the MD Anderson Cancer Center 3 out of 6 operated on at Washington University and 5 out of 8 patients in the Dutch experience with normal pentagastrin test results. These results clearly demonstrate that microscopic or macroscopic MTC may develop in the presence of a normal pentagastrin test response. These results are buttressed by the finding of microscopic MTC in more than 50% of patients who received thyroidectomy at the time of pentagastrin test conversion from normal to abnormal in an earlier series (Gagel et al, 1988), suggesting that pentagastrin testing is a relatively insensitive method for the detection of early disease. There exists the possibility that metastasis occurred prior to thyroidectomy in the 5-10% of these children with postoperative elevations of calcitonin. We believe that these findings provide additional support for a decision to intervene earlier

Table 4. Identification of gross or microscopic MTC in thyroid glands removed from 16 children with normal pentagastrin tests who had RET proto-oncogene mutations: combined MD Anderson Cancer

Center (MDACC), Washington University and Netherlands experience. Peak

Histology

Institution

Age/sex

Calcitonin (pg/ml)

Mutation

right/left lobes

MDACC

23/M

105"

CCH/GMTC

6/M

223]

Wash. Univ.

12/M

192~

Wash. Univ.

14/M

57t"

Netherlands Total 5/8 Netherlands

12/M

150:~

10/M

1305

Netherlands

9/F

190~:

Netherlands

7/M

220~

Netherlands

4/F

1405

Codon 634 CYS---->ARG Codon 634 CYS---~TYR Codon 618 CYS---)PHE Codon 620 CYS---)TYR Codon 634 CYS---~ARG Codon 634 CYS--+ARG Codon 634 CYS---)ARG Codon 634 CYS---~ARG Codon 634 CYS---)ARG

Total 1/2 Wash. Univ.

Total 3/6

MMTC/CCH GMTC/CCH MMTC/CCH MMTC MMTC MMTC MMTC MMTC

* Calcitonin assay performed using radioimmunoassay kit from Nichols Institute, San Juan Capistrano, CA; upper limit of normal for pentagastrin injection 0.5 gg/kg body weight in males is 105 pg/ml. Data from Gagel et al (1988, New England Journal of Medicine 318: 478-484). t Calcitonin assay performed using radioimmunoassay kit from Nichols Institute, San Juan Capistrano, CA; upper limit of normal for males after combined calcium/pentagastrin is 350pg/ml. Data from Wells et al (1994, Annals of Surgery 220: 237-250). Mutational information for the individuals from Washington University were provided by Samuel Wells Jr. :~ The upper limit of normal after pentagastrin 0.5 gg/kg body weight is 300 pg/ml. CCH = C cell hyperplasia; MMTC = microscopic MTC; GMTC = gross or macroscopic MTC.

RET MUTATIONS IN MEN2 AND MTC

625

in the course of hereditary MTC. Although a case could be made for performance of thyroidectomy even before the age of 5 years, the available facts do not support such action at present.

Thyroidectomy should be performed early in MEN2B The diagnosis of MEN2B is most commonly based on the clinical identification of the mucosal neuroma syndrome or gastrointestinal problems caused by the presence of neuromas and abnormal gastrointestinal innervation. MTC in MEN2B has been found at birth (Stjernholm et al, 1980; Samaan et al, 1991), and there is general agreement that thyroidectomy should be performed as early as possible after birth in children with this disorder. In cases in which clinical features are ambiguous, it may be appropriate to obtain a RET codon 918 analysis before committing to surgical removal of the thyroid, although surgery should not be delayed for more than a month or two to obtain this information. The finding of an occasional patient with a very mild neuronal phenotype (Sciubba et al, 1987) suggests that parents and siblings should be studied for the codon 918 mutation.

The impact of genetic screening on the management of other manifestations of MEN2 Genetic testing will have the important effect of excluding 50% of family members from the screening process, whereas it will have little impact on the management of adrenal medullary or parathyroid neoplasia in gene carriers, except in those instances in which there is a documented history of adrenal medullary malignancy. The current approach to the detection of adrenal medullary abnormalities is to obtain an annual urine or serum catecholamine determination and to investigate more intensively those individuals who have abnormalities or clinical features of phaeochromocytoma, such as palpitations, headaches or jitteriness (Gagel et al, 1988). It does not seem likely that genetic testing will change these recommendations. In families with a proven history of adrenal medullary malignancy, it may be prudent to consider early bilateral adrenalectomy (van Heerden et al, 1984), although in other patients with no family history of malignant phaeochromocytoma, there is controversy over appropriate surgical management (Gagel, 1994b).

Screening apparent cases of sporadic MTC to detect hereditary disease The experience at the MD Anderson Cancer Center indicates that approximately 5% of patients with apparent sporadic MTC carry a peripheral blood mutation consistent with hereditary MTC. In these individuals, the possibility of transmission of the mutant allele to a child must be considered. It is important to offer genetic screening to such individuals to determine whether an affected individual is an example of a new mutation

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COTE ET AL

or a member of a larger kindred. Testing for hereditary disease is important because hereditary MTC may pursue a variable course, with little or no morbidity in one generation followed by a more aggressive pattem in a subsequent generation. Although current practice guidelines do not make it mandatory to obtain this type of analysis in every case of sporadic MTC, it seems likely that it will become a routine part of an evaluation for MTC in the future. For the purposes of excluding hereditary transmission, it is probably adequate to perform a peripheral blood DNA RET analysis focused on exons 10, 11 and 13. In individuals with clinical features of MEN2B, an exon 16 analysis on peripheral blood DNA should also be performed. Although there is the hypothetical possibility that an individual with MEN2A or 2B could be mosaic, i.e. have the mutation in the thyroid and germ cells but not in peripheral blood, the probability is so low that routinely analysing all children is probably unnecessary. Correlation of particular RET proto-oncogene mutations with the clinical course of MTC There is currently insufficient information to indicate that tissue mutational analysis can be correlated with a particular clinical course or tumour virulence. The suggestion has been made, however, that MTC with a codon 918 mutation, associated with either MEN2B or sporadic MTC, has a more virulent clinical course (Zedenius et al, 1994). There is currently no information on tumour virulence available with respect to codon 768 mutation associated with both hereditary and sporadic MTC (Eng et al, in press). Finally, there is the general belief that FMTC, generally associated with exon 10 mutations, has a more benign clinical course than that of MTC associated with codon 634 mutations. The data regarding such correlations are of insufficient strength at the present time to make any firm recommendations regarding examination of tumour tissue for specific mutations.

R E T P R O T O - O N C O G E N E M U T A T I O N S ASSOCIATED W I T H SPORADIC PHAEOCHROMOCYTOMA Reports from several investigative groups make it clear that 5-15% of sporadic phaeochromocytomas have RET proto-oncogene mutations (Beldjord et al, in press; Lindor et al, in press). Within this group of sporadic phaeochromocytomas, some individuals were found to be examples of MEN2, although the majority had somatic mutations. When this information is combined with the recent identification of specific mutations of the Von Hippel-Lindau gene associated with phaeochromocytoma (Chen et al, in press) and mutations of the NF gene in phaeochromoctyomas in neurofibromatosis, it becomes reasonable to suggest that mutational analyses of these three genes should be performed in patients with apparent sporadic phaeochromocytoma.

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627

CONCLUSION The identification of RET proto-oncogene mutations in hereditary and sporadic MTC and phaeochromocytoma is likely to have a great impact on the clinical management of these two disorders. As more information becomes available on the mechanisms by which these mutations cause endocrine tumour formation, it seems likely that clinical management will be improved and strategies will be developed to reverse or prevent these tumour types.

SUMMARY The identification of RET proto-oncogene mutations in patients with MEN2 2 years ago was a watershed event in the management of this genetic cancer syndrome. The identification of a finite number of mutations that together cause more than 95% of hereditary and 15-25% of sporadic MTC has made it possible to develop simple and definitive tests to screen individuals at risk for this tumour syndrome. The impact of this technology is enormous. It is now possible to reassure 50% of family members at risk that they, and their children, do not have to worry about developing MTC. In the other 50% who are gene carriers, it is now possible to approach clinical management with greater certainty and plot strategies that are likely to result in a greater percentage of curative therapy. It seems likely that this technology will also have an impact on the management of sporadic MTC, although it is still too early to define a specific role for mutational analysis in these patients, except to exclude hereditary disease. The identification of specific mutations causative for MTC makes it possible to conceive future strategies for the treatment or prevention of MTC and to further extend the impact of these exciting findings.

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