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© 1999 American Society for Clinical Oncology
RET Proto-Oncogene in the Development of Human CancerFrom the Translational Research Laboratory, Department of Adult Oncology, Charles A. Dana Human Cancer Genetics Unit, Dana-Farber Cancer Institute; Department of Medicine, Harvard Medical School, Boston, MA; Cancer Research Campaign Human Cancer Genetics Research Group, University of Cambridge, Cambridge, United Kingdom; and Human Cancer Genetics Program, Ohio State University, Columbus, OH. ABSTRACT The RET proto-oncogene, located on chromosome subband 10q11.2, encodes a receptor tyrosine kinase expressed in tissues and tumors derived from neural crest. Germline (present in every cell of the body) mutations in RET cause multiple endocrine neoplasia type 2 (MEN 2), an inherited cancer syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytoma (PC), and hyperparathyroidism (HPT). This knowledge has allowed molecular diagnosis and presymptomatic DNA-based testing to become possible. RET testing is considered the standard of care in MEN 2 families because clinical decisions are made based on the results of such gene testing. There appears to be a correlation between specific RET mutation type and organ-specific tumor development. Such knowledge might be useful in tailoring targeted surveillance in the near future. Somatic (in the tumor only) RET mutations have been found in a proportion of sporadic MTCs and PCs. Whether the presence of somatic RET mutation is associated with a poor prognosis is currently being investigated as another tool for molecular medicine. THE RET PROTO-ONCOGENE is the susceptibility gene for the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2), as well as a major susceptibility gene for the seemingly unrelated syndrome of congenital absence of enteric ganglia, or Hirschsprung disease (HSCR). Somatic (tumor-specific) mutations in RET have also been found in a variety of sporadic tumors. RET PROTO-ONCOGENE The RET proto-oncogene, localized to chromosome subband 10q11.2, comprises 21 exons, which encodes the protein RET (REarranged during Transfection), a receptor tyrosine kinase (RTK) expressed in derivatives and tumors of neural crest origin.1-7 Receptor tyrosine kinases transduce the extracellular signals for processes as diverse as cell growth, differentiation, survival, and programmed cell death. In response to binding of extracellular ligands, RTKs generally form homodimers or heterodimers. On dimerization, autophosphorylation occurs, followed by intracellular signal transduction through effectors that recognize and interact with the phosphorylated form of the RTK. Although the downstream signaling pathways activated by these steps may be shared by different receptors, the ligand-receptor interaction itself is very specific. In some cases, however, high-affinity ligand-RTK interactions can be modulated by the presence of other, low-affinity, nonsignaling accessory molecules at the cell surface.
To date, two related ligands for RET have been identified: glial cell line–derived neurotrophic factor (GDNF) and neurturin (NTN).8-14 The receptor for GDNF or NTN comprises one of at least two membrane-bound adaptor molecules and RET.15 Glial cell line–derived neurotrophic factor preferentially binds GFR
Like most other RTKs, RET is a relatively large molecule and comprises an extracellular ligand-binding domain, a cysteine-rich domain, a dynamic transmembrane domain, and intracellular tyrosine kinase domains. It is believed that the cysteines form intramolecular covalent bonds with one another to help engender the tertiary structure of RET as it folds around the ligand-binding pocket. Several alternative 5' and 3' splicing variants that could lead to various forms of the RET protein have been described.7,20 SOMATIC RET/PTC TRANSLOCATION IN SPORADIC PAPILLARY THYROID CARCINOMA The RET proto-oncogene is involved in the genesis of both medullary thyroid tumors and nonmedullary thyroid tumors in different ways. Shortly after RET was discovered, a variable proportion of sporadic and radiation-associated papillary thyroid carcinomas (PTCs) were found to have somatic translocations involving the 3' half of RET, containing the tyrosine kinase (TK) or business end, and the 5' end of several genes.21-26 Instead of being restricted to cells of neural crest origin, the fused RET/PTC products were uniformly expressed in PTCs, which are not derived from neural crest. Interestingly, the 5' end of the novel fusion gene always derives from genes that are normally expressed in the thyroid follicular cells, hence, "allowing" RET to be expressed in a "foreign" environment. These genes usually encode motifs that allow the new fusion proteins to consitutively dimerise, and hence, cause constitutive activation of the TK of RET, leading to PTC formation. CLASSIFICATION OF MEN 2 The C cells of the thyroid are derived from neural crest and are believed to be the precursor cell from which medullary thyroid carcinoma (MTC) can arise. As many as 75% of all MTCs are sporadic; the remainder are hereditary. The hereditary form of MTC, MEN 2, is divided into three subtypes depending on the organs involved (Table 1). The sine qua non of all three subtypes is MTC. Multiple endocrine neoplasia type 2A comprises the classic triad of MTC, pheochromoctyoma (PC) in approximately 50% of cases, and hyperparathyroidism (HPT) in 15% to 30% of cases.27 Multiple endocrine neoplasia type 2A is the most common, likely accounting for more than 90% of all MEN 2 cases. Figure 2 shows the age and incidence curves for clinical presentation and screening evidence of MEN 2A.28,29 Approximately 30% of individuals will not have symptoms referable to MTC, PC, or HPT by the time they reach 70 years of age. This observation of incomplete penetrance of the gene has implications for detection and follow-up of families at risk (see Predictive Testing and the Practice of Molecular Medicine).
Multiple endocrine neoplasia type 2B accounts for approximately 5% of all MEN 2 cases. The features of MEN 2B are similar to those of MEN 2A, except that the average age of tumor onset is 10 years earlier, clinically evident parathyroid disease is absent, and developmental abnormalities such as ganglioneuromatosis, medullated corneal nerves, and marfanoid habitus are present (Table 2).30
It has been suggested that MEN 2B has a poorer prognosis from MTC. However, this is controversial. In addition, low reproductive rate and/or potential is observed possibly due to increased mortality from the disease, impotence secondary to neurologic problems, infertility, and low marriage rates. This leads to a high proportion of apparently new mutation cases of MEN 2B. Familial MTC comprises families with MTC as their only disease phenotype.31 It is believed that the course of the MTC in these families is more benign and prognosis is good.31 CLINICAL DIAGNOSIS Medullary thyroid carcinoma is, by far, the most common clinical presentation of MEN 2A. In contrast to the average age of presentation of a patient with sporadic MTC (6th decade), that of a patient with familial MTC is 20 to 40 years. Medullary thyroid carcinoma can present locally as a mass in the neck or as metastatic disease. Because of the possible coexistence of PC (see below), it is essential to make the histologic diagnosis of MTC before thyroidectomy so that a screen for PC may be performed before surgery. A fine-needle biopsy confirms the diagnosis. A serum calcitonin greater than 1,000 pg/mL associated with an elevated serum carcinoembryonic antigen is also diagnostic of MTC. In addition, MTC can have associated paraneoplastic syndromes, characterized by the production of adrenocorticotrophin (most common), beta-endorphin, somatostatin, vasoactive intestinal polypeptide, dopamine, dopadecarboxylase, histaminase, serotonin, and prostaglandins (reviewed by Thakker and Ponder32 and Eng and Ponder33), and by an unusual associated feature called cutaneous lichen amyloidosis.34 It is not known what cutaneous lichen amyloidosis really is, but it manifests as mild brawny induration and pruritis, most commonly in the intrascapular region. A few cases of MTC are incidentally noted at thyroidectomy for other diagnoses. Less commonly, patients initially present with PC with its attendant signs, including tachycardia, hypertension, and flushing, or HPT with hypercalcemia or renal stones. It would be unusual for patients with MEN 2A to present initially with unilateral PC and/or parathyroid adenoma. This combination is probably not sufficient to justify biochemical screening without other evidence suggestive of the syndrome, such as a family history, multifocal disease, or the presence of a mutation in the RET proto-oncogene characterizing MEN 2A (see RET Proto-Oncogene Mutations in MEN 2). Multiple endocrine neoplasia type 2B should be recognizable early in life by the characteristic facial appearance, mucosal neuromas, and constipation or diarrhea, with failure to thrive secondary to ganglioneuromatosis of the gut. Other nonspecific findings include failure to feed and hypotonia in the neonatal period. Confirmation of these diagnoses can be made by biopsy of the mucosal neuromas, rectal biopsy, or slit-lamp examination for medullated corneal nerves. Medullated corneal nerve fibers are graded on a scale of 1+ to 4+. This feature is often overcalled, and hence, the presence of medullated corneal nerve fibers, especially graded 1+ and 2+, cannot be considered key features when making the diagnosis of MEN 2B. If there is a suspicion that MEN 2B is the diagnosis, biochemical testing for MTC by measurement of pentagastrin-stimulated calcitonin levels and direct mutation testing should be performed (see Screening of Individuals-at-Risk in the Pre-DNA Era). If there are many members of a family (eg, four or more) affected with MTC in the absence of PC and parathyroid involvement after meticulous screening, then that family has FMTC (familial MTC). However, when there are three or fewer cases of MTC in a family, it is difficult to determine whether this is classic FMTC, MEN 2A without PC development yet, or coincidental sporadic cases. Unfortunately, direct mutation analysis of the RET proto-oncogene is not always helpful in these smaller FMTC families (see Genotype-Phenotype Correlations in MEN 2 and Targeted Surveillance). Nonetheless, the clinician must always keep in mind the rarity of MTC, and so the coincidental coexistence of two truly sporadic MTCs, without an heritable component, is rare. PATHOLOGY Like other inherited cancer syndromes, MEN 2 neoplasms are characterized by multifocality and bilaterality. Thus, multifocal MTC, PC, and/or parathyroid hyperplasia/adenoma within their respective glands and/or bilaterality are hallmarks of MEN 2. However, without viewing the C cells on the microscopic level, little difference can be seen between a sporadic tumor and that of an MEN 2 case. C-cell hyperplasia (CCH) is believed to be the precursor of MTC. Hence, it was believed that CCH is pathognomonic for hereditary forms of MTC, ie, MEN 2.35 Although it is an important indicator that heredity may be playing a role, false-positives have been amply documented.36,37 In addition, the recognition of CCH is not always straightforward, because there seems to be some overlap between the upper limits of normal physiological findings and the earliest stages of CCH. CLASSIC GENETICS Multiple endocrine neoplasia type 2 is characterized by an autosomal dominant mode of inheritance. So, any child of an affected individual has a one in two (or 50%) chance of inheriting the susceptibility gene. This mode of transmission seems to be equal through males and females and severity is not correlated with sex. There does seem to be considerable variation among and even within families in the age of tumor onset and the incidence of PC and HPT. SCREENING OF INDIVIDUALS-AT-RISK IN THE PRE-DNA ERA First-degree relatives (children, parents, siblings) of affected individuals are at 50% risk of inheriting the mutated gene. Before DNA-based predictive testing, all unaffected individuals at 50% risk were subjected to annual screening for MTC, PC, and HPT from the age of 6 years to the age of 35 years. This involves pentagastrin-stimulated calcitonin levels, 24-hour urinary levels for catecholamines, and serum calcium and parathyroid levels. Many centers advocated prophylactic thyroidectomy before the age of 6 years in individuals who are first-degree relatives of affected individuals, for two reasons: first, the youngest age at diagnosis reported for MTC in MEN 2A is approximately 6 years,38,39 and second, MTC can be lethal. RET PROTO-ONCOGENE MUTATIONS IN MEN 2 Analysis of RET in MEN 2A and FMTC families revealed germline missense mutations in affected individuals but not in unaffected individuals or normal controls.40-44 (Reviewed by Eng45 and Eng and Mulligan.46) In each case, one of five particular cysteine codons in exon 10 (C609, C611, C618, and C620) and exon 11 (C634) was found to be mutated (Fig 3).42-44,47-52 These residues are located in the extracellular, juxtamembrane cysteine-rich domain. Mutations were detected in 98% of unrelated classic MEN 2A families and were also found in 85% of FMTC families.44 Mutations in the gene encoding one of the RET ligands, GDNF, did not seem to account for RET mutation–negative MEN 2 families.53
Unlike the susceptibility genes of most inherited cancer syndromes thus far characterized, RET is a proto-oncogene (ie, a single, activating mutation on one allele should be necessary and sufficient to cause transformation). At least three studies have shown that codon 634 mutations render the RTK constitutively active by the tendency of mutant RET monomers to undergo dimerization,54-56 a situation that mimics ligand binding. It is believed that cysteine residues normally involved in intramolecular disulphide bonds in wild-type RET are mutated to leave unpaired residues. An unpaired cysteine from each mutant RET monomer forms a disulfide bond with its unpaired counterpart from another mutant RET. Simplistically, the same general mechanism may be proposed to explain the effects of mutations in the other cysteine codons. However, this generalized constitutive activation does not fully explain the mechanism underlying the correlation between some mutations and the range of tissue involvement (see MEN 2/HSCR Dilemma Resolved: A Unifying Hypothesis). In MEN 2B, however, a single-point mutation at codon 918 (exon 16) has been identified in 95% of cases (Fig 3).43,44,57-60 The codon 918 mutation results in a methionine to threonine change (M918T). Until recently, a few rare families showing classic clinical manifestations of MEN 2B but having no codon 918 mutation were subjected to exhaustive examination of RET, and no mutations were detected.44,58,60,61 In 1997, two groups found a total of four distinct MEN 2B families, originating from the United Kingdom, Australia, and Germany, who had a novel germline RET mutation at codon 883 (exon 15) that altered the normal alanine to the mutant phenylalanine (A883F).62,63 Why more than 95% of MEN 2B cases are accounted for by germline M918T,44 but fewer than 4% are accounted for by A883F,62 is unknown. Codon 918 is located in the region thought to encode the substrate recognition pocket of the TK catalytic core.64 Constitutive activation and an alteration in the range of intracellular phosphorylated substrates were detected over the wild-type.54,56,65 In support of this, in vitro studies have demonstrated that the M918T mutation results in altered substrate specificity, such that it recognizes and phosphorylates substrates preferred by nonreceptor tyrosine kinases such as c-src and c-abl.66 It is not known how A883F affects function. However, residue 883 is located in a subdomain of RET that defines substrate preference.62 It is possible that alteration of substrate preference is the common etiologic thread that underlies the pathogenesis of MEN 2B. Two novel mutations associated with FMTC have been found in the intracellular tyrosine kinase domain of RET (Fig 3). A missense mutation in codon 768 (exon 13), altering a glutamate to an aspartate (E768D), was initially identified in four FMTC families.67-69 Also, a mutation at codon 804, predicted to alter a valine to a leucine (V804L), was found in exon 14. Subsequently, another mutation at codon 804 that alters the valine to methionine (V804M) has been found in FMTC families as well70 (Gimm O and Eng C, unpublished data). Although numbers are small, it seems that the codon 804 mutations occur in FMTC families in which fewer than four members are affected, and where the ages of diagnosis are more advanced (Eng C, unpublished observations). The effects of the codon 768 and 804 mutations are, as yet, unclear. In the case of E768D, computer modeling has suggested that the kinase activity is modified by altering the substrate specificity or the ATP-binding capacity.68 Given its location within the tyrosine kinase domain, the substitution of either a leucine or a methionine at position 804 may exert an activating effect by altering the kinetics of interactions with normal cellular substrates or by modifying the range of substrates that are phosphorylated. Apart from the common hotspot mutations that define MEN 2, a few rare mutations have been identified as well. Point mutations at codons 630, a cysteine codon, 790, 791, and 891, and a 12 bp duplication that results in an insertion of four amino acids between cysteine 634 and residue 635, have been described in FMTC and MEN 2A families.71-74 The codon 630 and 891 germline mutations have been described only once each, occurring in FMTC families.71,73 Codons 790 and 791 have been reported as "novel hotspots" for mutation in MEN 2.72 Interestingly, however, the codon 790 and 791 mutations have never been reported outside Germany, despite many academic centers and commercial laboratories performing RET mutation analysis around the world. Because codons 790 and 791 are located in the same exon as codon 768, a mutation that is commonly tested for since 1995,68 it is difficult to imagine that the lack of reports of codon 790 and 791 mutations outside Germany is due entirely to lack of examination of those codons. This is especially underscored by no families with these two novel mutations in the Netherlands (Hofstra RMW, personal communication, January 1998) and by the lack of reports emanating from neighboring Austria and France. Even more tantalizing is the lack of these mutations from at least one center in Germany as well (Gimm O, Neumann HPH, and Eng C, unpublished observations). Perhaps, the codon 790 and 791 mutations result from a founder effect peculiar only to particular regions of Germany. GENOTYPE-PHENOTYPE CORRELATIONS IN MEN 2 AND TARGETED SURVEILLANCE When a small series of MEN 2 patients were analyzed for genotype-phenotype correlations in order to determine if specific mutations predict for development of specific component tumors in a given family, it revealed that mutations at codon 634, the most commonly affected codon in MEN 2A, correlated with the presence of PC and HPT in a family.42 In addition, a specific mutation at codon 634, altering a TGC (cysteine) to a CGC (arginine) (C634R), was associated with the development of HPT in particular.42 This latter correlation was controversial, however.49,75
Although these preliminary genotype-phenotype correlations showed promise in terms of the applicability to clinical practice in this era of data-based, cost-effective medicine, it was deemed prudent by the international MEN 2 community to pool the worldwide RET mutation data for joint genotype-phenotype analysis while using uniform operational definitions for phenotypic features (Table 3).43,44 To that end, the International RET Mutation Consortium was first convened at the Fifth International MEN Workshop held in Stockholm, Sweden, in June 1994.43 To date, 18 centers from across North America, Europe, Australia, and Asia submitted data from 477 MEN 2 families.44 Of these 477 MEN 2 families, 42.6% were considered classic MEN 2A, 16.6% were considered MEN 2B, and 7.1% were considered FMTC. The remaining were operationally classified into an "other" category comprising "small" FMTC (
Germline mutations at codons 609, 611, 618, 620, and 634 were associated with MEN 2A.44 Eighty-five percent of MEN2A mutations occurred at codon 634. Among codon 634 mutations, the most frequent alteration was a TGC (cysteine) to CGC (arginine) (C634R). The presence of any germline mutation at codon 634 was found to be highly associated with the development of PC and HPT in a given MEN 2A family.44 In the consortium data-set as a whole, C634R was associated with specific development of HPT. However, if the original Cambridge data-set42 were removed, the association was no longer significant.44 This was initially explained by geographic differences or differences in criteria for the diagnosis of HPT between the United Kingdom families and those of continental Europe,44 although another alternative, but not mutually exclusive, explanation is possible. A recent French population-based study examining the risk and penetrance of HPT in MEN 2A families again demonstrated a lack of association between C634R and the development of HPT on a family-as-unit basis but revealed a highly significant association between C634R and the development of HPT on an individual basis.76 Additionally, the risk for HPT development increased sharply after the age of 30 years. Hence, it is possible that the members of MEN 2 families ascertained in the United Kingdom were, on average, older than were those from continental Europe. From a clinical point of view, the C634R-HPT correlation is somewhat moot; the presence of any codon 634 mutation should alert the clinician to the risk of both PC and HPT. Germline RET mutations in FMTC were distributed over the codons in a more uniform relative frequency.44 For example, 30% of all mutations found in FMTC families occurred at codon 634 in contrast with 85% of mutations for MEN 2A. Interestingly, among this modest group of FMTC families, no C634R mutations were noted. Clinicians should, therefore, be alerted if they find an apparent FMTC family with a C634R mutation; they should rigorously pursue PC and HPT screening in all affected or mutation-positive members. A novel correlation was found: E768D and V804L seemed to be associated with FMTC and small FMTC only. Whether such families can forego PC and HPT surveillance in the future, as the consortium accrues larger numbers of families with codon 768 and 804 mutations and has longer follow-up of existing families, is as yet unknown. PREDICTIVE TESTING AND THE PRACTICE OF MOLECULAR MEDICINE IN MEN 2 Before DNA-based genetic testing, MEN 2 was either diagnosed when an individual presented with the signs and symptoms of the syndrome or when biochemical screening with pentagastrin-stimulated calcitonin levels were measured in clinically at-risk individuals.33 Because the sensitivity of pentragastrin screening is age-dependent and the youngest age of diagnosis of MEN 2 MTC is before the age of 6 years, all clinically at-risk individuals were either subjected to prophylactic thyroidectomy and/or annual biochemical screening before the age of 6 years.77,78 Because MEN 2 is inherited as an autosomal dominant trait, any offspring of an affected person has a 50% chance of inheriting the trait. Thus, the strategy of early prophylactic thyroidectomy is lifesaving in one half of these clinically at-risk individuals, but would represent a senseless and not trivial procedure, condemning a child to lifelong thyroid replacement, in the other one half. The strategy of pentagastrin screening, on the other hand, seems to be less invasive, but the trade-off is lack of sensitivity when it is needed most—before the adolescent years. False-positives and false-negatives are well documented with pentagastrin screening.36,37,79,80 Because mutations of the RET proto-oncogene have been identified in the majority of MEN 2 families, DNA-based testing is possible. This has distinct advantages, such as not having age-dependent sensitivity, being useful as a molecular diagnostic test to confirm a clinical diagnosis of MEN 2, and most importantly, being used as a predictive test for asymptomatic clinically at-risk individuals. In a known MEN 2 family, a clinically at-risk individual should undergo DNA testing before the age of 6 years38 or certainly before surgery. In an MEN 2 family with a known family-specific RET mutation, no prophylactic surgery should be performed in an as yet unaffected individual without proof of mutation-positive status. Predictive testing for RET mutations in MEN 2 kindreds is performed using polymerase chain reaction–based protocols; target exonic sequences are amplified either for direct sequencing and/or restriction endonuclease digestion, where mutations would create or cause loss of specific sites.36,50,81 Such tests are reproducible, accurate, and allow presymptomatic identification of at-risk individuals,36,80 permitting more effective and timely surgical intervention or initiation of screening, although the course advocated by many is prophylactic thyroidectomy before the age of 6 years.38 In a known MEN 2 family with an identified family-specific mutation, therefore, the detection of the same mutation in a clinically at-risk individual indicates that that person has MEN 2. Conversely, if a mutation is not detected, that individual will not have MEN 2. Barring administrative errors, DNA-based predictive testing is 100% accurate. Thus, individuals found to carry a germline RET mutation can be subjected to targeted screening for the presence of PC and HPT annually from the age of 6 years and offered prophylactic thyroidectomy (reviewed in Learoyd et al82 and Eng and Ponder33). The only exception is the discovery of the MEN 2B-specific M918T or A883F mutations; such patents should be offered prophylactic thyroidectomy at a younger age, ie, at infancy. Those clinically at-risk individuals who test mutation-negative can be reassured, do not have to be subjected to annual biochemical screening, and can be spared prophylactic thyroidectomy. RET testing has been recommended by the American Society of Clinical Oncology83 and is the clinical standard of care for MEN 2 in this and other countries worldwide. This is because the great majority (> 95%) of MEN 2 cases have germline RET mutations and because the determination of mutation status does influence medical management.84 In general, in the United States, the standard practice is to perform a prophylactic thyroidectomy between the ages of 5 and 10 years, preferably before 6 years, in mutation-positive asymptomatic individuals, except in individuals with the MEN 2B-specific mutations, M918T or A883F; in such cases, prophylactic thyroidectomy should occur well before the age of 3 years. This strategy is advocated, and seems obvious, for the highly penetrant mutations at codons 918, 883, and 634, or in families in which the diagnosis of MTC has occurred at a young age. However, recent clinical genetic and preliminary functional data have suggested that mutations such as the exon 10 mutations, codon 768 mutations, and codon 804 mutations may have low penetrance.85-88 These types of mutations tend to occur only in FMTC and small FMTC families, which is already an indication that the full-blown syndrome with PC and HPT is not manifested; second, the codon 804 mutations seem to confer a later age of onset (Gimm O and Eng C, unpublished observations). If these anecdotes and in vitro studies can be proved "in the field," then clinical management might actually be tailored, such as targeted biochemical surveillance or later prophylactic surgery. In a syndrome where more than 95% of cases have identified germline mutations, a clinician may be uncomfortable when faced with a RET mutation–negative MEN 2 family. The mutation-negative family will most likely tend to be a small FMTC family. Because MTC is rare, the occurrence of even two MTCs in a single family in unusual. So, the chances that this has occurred entirely by coincidence are low. It would, therefore, be most conservative to treat these sorts of families as we would any MEN 2 family before the era of DNA-based diagnosis. RET PROTO-ONCOGENE TESTING IN APPARENTLY SPORADIC MTC Medullary thyroid carcinoma accounts for approximately 10% of all thyroid malignancies.33 Hence, there are approximately 1,000 new cases of MTC per year in the United States alone. Of these, 25% or 250 cases will be hereditary (ie, MEN 2), and the rest sporadic.33 Without an obvious family history or stigmata of MEN 2, hereditary cases may not be able to be distinguished from truly sporadic ones without RET testing. The majority of MTCs will be truly sporadic. DNA testing can be helpful in determining if an apparently sporadic MTC case is hereditary or nonhereditary. Before the discovery of the susceptibility gene for MEN 2, pentagastrin screening of first-degree relatives of patients with apparently sporadic MTC revealed that 10% of such cases were hereditary.89 With the discovery that RET is the susceptibility gene for MEN 2, four series examined the frequency of occult or de novo germline RET mutations in MTC cases. One series examined consecutive cases of MTC without regard to family history or the presence of C-cell hyperplasia and found a 25% occult RET mutation frequency.90 The other three series examined cases of apparently sporadic MTC without known family history and without features suggestive of MEN 2.91-93 The occult or de novo mutation frequency among these apparently sporadic cases ranged from 2.5% to 7%. Before 1997, the more conservative practitioners among the MEN 2 community and those who wished to practice evidence-based medicine recommended germline RET testing in apparently isolated MTC cases when the age of diagnosis was particularly young, when C-cell hyperplasia or multifocality was noted on the resection specimen, and/or when a suggestive family history was uncovered.33,91,94 However, given the recent realization that low penetrance RET mutations, particularly codon 804 mutations, might exist in MEN 2, it might be prudent for all individuals who present with MTC to undergo RET testing. The discovery of an occult germline RET mutation in an apparently sporadic MTC case means that the individual has MEN 2 and should be managed accordingly. However, the interpretation and genetic counselling involved in the setting of an individual with apparently isolated MTC and no germline RET mutation is not straightforward. Therefore, such testing should be performed in centers familiar with RET testing and MEN 2 in all its nuances. SOMATIC RET MUTATIONS IN SPORADIC MEN 2 COMPONENT TUMORS Somatic mutations have been detected in a proportion of sporadic MTCs and PCs. An MEN 2B-like M918T mutation accounts for the largest proportion of RET mutations detected in MTCs, with estimates ranging from 23% to 70%48,57,58,71,79,95-98 (reviewed by Eng and Mulligan46 and Marsh et al99). Interestingly, when carefully microdissected subpopulations of MTC were examined for the presence of somatic RET mutation using a highly sensitive detection technique,100 somatic mutation status was found to be heterogeneous even within a single MTC and among metastases.101 At least 80% of all sporadic MTCs studied in this manner had at least one subpopulation within the tumor that harbored a somatic RET mutation, specificially M918T.101 One tumor had metastases with both M918T and A883F. These data might reflect clonal evolution within MTC, which is a relatively slow-growing tumor, and/or a polyclonal origin.101 The latter hypothesis has been corroborated by an independent study using X-chromosome inactivation.102 Studies from two centers, each comprising small numbers of patients, suggest that the presence of somatic codon 918 mutation in sporadic MTC is an indicator of poor prognosis with respect to metastases and recurrence,79,98 but this correlation could not be confirmed in another series.97 However, ascertainment, numbers, and end points differ among the three studies. Studies using neuroblastoma cell lines might lend credence to the claim that the presence of somatic M918T portends a poor prognosis.103 Expression of MEN2B-M918T RET in the cell lines led to altered cell adhesion in vitro and increased metastatic behavior in vivo.103 Despite competent surgery, approximately one half of all localized sporadic MTC cases will recur. Currently, there is no reliable way to predict which cases will relapse. Thus, it would be desirable to perform a definitive analysis based on large numbers to determine whether the presence of somatic M918T in sporadic MTC predicts for a poorer prognosis. Mutations of other RET codons are occasionally found (C611Y, C634R, C634W, deletion-insertion 634, E768D, and A883F).58,95,97,98,101,104,105 A study originating from Uppsala found a complex 9-bp deletion which encompasses codon 634 in 14 of 15 sporadic MTCs.106 This type of somatic mutation has yet to be reported by other centers. Interestingly, sporadic MTCs presumably originating from the catchment area in and around Stockholm have never been shown to have this type of mutation (Zedenius J, personal communication, January 1998). Perhaps, the complex 9-bp deletion is associated with a particular environmental carcinogen specific to the Uppsala region. Mutation analysis of sporadic PCs has revealed a smaller percentage with RET mutations. It appears that PCs have a wider spectrum of RET mutation, including infrequent mutations at M918T (10% to 20%), C620, C630, and C634, and an exon 9 splice donor site mutation58,107-109 compared with sporadic MTCs where M918T mutations predominate. Somatic RET mutations have yet to be identified in PCs from MEN 2 patients.58 Recently, Marsh and colleagues100 have detected somatic M918T mutations in three of 15 MTCs and one sample with C-cell hyperplasia, all from MEN 2 patients with well-defined germline RET mutations. Since the samples were derived from archival sources, it was not possible to determine if the germline and somatic mutations were syntenic. Because a single RET mutation has been shown to be sufficient to cause transformation in vitro,54-56 the significance of this observation remains to be determined. In the last of the triad of MEN 2 component tumors, parathyroid hyperplasia and adenoma, several studies have not detected somatic mutations in exons 10, 11, or 16 in a panel comprising primary hyperplasia, adenoma, carcinoma, and parathyromatosis.98,110,111 Further, no somatic GDNF mutation has been detected in parathyroid adenomas either.53 SOMATIC RET MUTATIONS IN OTHER SPORADIC NEUROENDOCRINE TUMORS Because RET is believed to play a role in neural crest development, it is plausable that somatic mutations in the gene might contribute to the pathogenesis of other noncomponent neuroendocrine tumors, which are known or believed to derive from the neural crest. In a tentalizing preliminary report, Futami et al reported a mutation in codon 664 (alanine to aspartic acid) (exon 11) in two of six small-cell lung carcinoma cell lines.112 However, in subsequent studies of a large number of primary small-cell lung carcinomas, no mutations in exons 10, 11, 13, 15, or 16 have been found.113,114 Further, when one of the ligands of RET, GDNF, was examined, no alterations could be found either.53,114 Somatic RET mutations have not been found in pituitary adenomas (n = 8), pancreatic neuroendocrine tumors (n = 17), pulmonary and gastrointestinal carcinoids (n = 21), neuroblastomas (n = 5), malignant melanomas (n = 10), and schwannomas (n = 4).113 However, the expression of RET in these tumors is not well established. In neuroblastomas, which express RET at high levels,115 mutations have not been found in the MEN 2-specific exons of RET. It is possible that mutations elsewhere in this gene or its promoter cause overexpression or stabilization of the RET message. Alternatively, neuroblastoma may represent a clonal outgrowth of a precurser cell with normally high RET expression. RET MUTATIONS IN HIRSCHSPRUNG DISEASE Hirschsprung disease (HSCR) is a congenital absence of enteric innervation which results in intestinal obstruction.116 Inactivating mutations of one allele of the RET proto-oncogene have been detected in approximately 23% of dominantly inherited cases of HSCR (estimates range from 10% to 40% in three different series) and in one third of sporadic HSCR cases (reviewed by Eng45 and Eng and Mulligan46). The mutations are varied and scattered throughout the RET coding sequence, and include gross and microdeletions and a variety of point mutations within the RET gene itself (reviewed by Eng and Mulligan46). In contrast to the gain of function MEN 2 RET mutations, RET mutations associated with HSCR are truncating mutations, those that cause haploinsufficiency or those that cause a loss of function.117,118 GAIN OF FUNCTION MUTATIONS IN FAMILIES WITH MEN 2 AND HSCR Although germline mutations in RET have been found in MEN 2 and in HSCR, the two syndromes usually occur separately, and the types of RET mutations are different between them. Whereas gain of function mutations are associated with MEN 2, loss of function mutations are associated with HSCR (reviewed by Eng and Mulligan46). However, in rare families, FMTC or MEN 2A cosegregates with HSCR. In five such kindreds where phenotypic cosegregation of MEN 2 and HSCR was demonstrated, germline RET mutations in codons 618 and 620 have been detected.119,120 Further, several families with HSCR as the only disease phenotype that bear RET mutations characteristic of the MEN 2 syndromes have been reported.119,121,122 MEN 2/HSCR DILEMMA RESOLVED: A UNIFYING HYPOTHESIS While the genetic study of RET in MEN 2 and HSCR has progressed at a rapid pace, biochemical and functional clues to how RET works have been accumulating in recent years. Both transfection experiments and biochemical analysis have shown that the cysteine codon mutations characteristic of MEN 2A cause constitutive dimerisation and activation, and hence, transformation.54-56,123 Are all cysteine codon mutations created equally? The genotype-phenotype association data (see Genotype-Phenotype Correlations in MEN 2 and Targeted Surveillance) would suggest otherwise. Because codon 634 mutations are overwhelmingly associated with MEN 2A, and the more 5' cysteine codon mutations (eg, codons 609 and 611) are associated with FMTC, it might be predicted that the 5' cysteine codon mutations would cause a weaker activation than the ones nearer the transmembrane domain. This prediction has been borne out by functional analyses. Non–634 cysteine codon mutations appear to be more weakly transforming in transfection studies as well as in biochemical assays of kinase activity.85,87 Additionally, mutations of the more 5' cysteine codons seem to result in a decreased proportion of receptor molecules on the cell surface.87 These data together seem to suggest that MEN 2A and FMTC are a single genetic entity whose phenotypic manifestation is a consequence of differences in penetrance of each mutation type. So, codon 634 mutations appear to have the highest penetrance, likely because of the fact that all of the molecules mature and are on the cell surface. This results in the full manifestation of MTC, PC, and HPT (ie, classic MEN 2A). In contrast, non–codon 634 mutations have a smaller proportion of mutant receptors at the cell surface. This results in some constitutively dimerised, and hence, consitutively activated, receptors, but perhaps only enough for MTC to form, and to a lesser extent, PC. In addition to the variable penetrance of these mutations, there also seems to be tissue-specific differences in transformation threshold, such that C cells have the lowest threshold and parathyroid cells have the highest threshold. A similar explanation might solve the dilemma of gain of function mutations, C618R and C620R, that appear causative of both MEN 2A/FMTC and HSCR in a single family, indeed, in a single patient. Only a proportion of C618R- and C620R-bearing receptor molecules mature to the cell surface, and hence, are constitutively activated. If this hypothesis were correct, any non–codon 634 MEN 2A/FMTC-type mutation has some potential to be etiologic for the co-occurrence of MEN 2A/FMTC and HSCR. Although it is enough for MTC and perhaps PC to result from constitutive activation of some receptors at the cell surface, enteric ganglia appear to require a certain threshold number of "workable" receptors for normal gangliogenesis to occur or perhaps to prevent inappropriate apoptosis. However, because this is not an all or none phenomenon, we find that approximately one third of families with C618R and C620R have both MEN 2A/FMTC and HSCR, whereas the other two thirds have MEN 2A or FMTC only.119 CONCLUSION When RET was first identified as the susceptibility gene for MEN 2, it was the first time that a proto-oncogene was implicated in the etiology of an inherited cancer syndrome. This gene continued to surprise when it was also found to be a major susceptibility gene for HSCR. Nonetheless, the RET-MEN 2 story seems to be one of the first triumphs for molecular medicine. Within 6 months of the initial report that germline mutations in RET were associated with MEN 2A, clinical molecular diagnosis was available. The results of this sort of testing do not represent an academic exercise; rather, the knowledge that an individual is mutation-positive or mutation-negative can alter medical management. More importantly, it saves lives. Beyond that, the genetic and functional study of RET will yield further clues that will help diagnose, prevent, and perhaps effectively treat all neuroendocrine tumors and various neural and neuroendocrine disorders. NOTE ADDED IN PROOF
A third ligand, persephin, and another coreceptor for RET, GFR Preliminary work with a series of 49 unrelated patients with sporadic MTC shows that a rare sequence variant, S836S, within RET is overrepresented in cases (9%) with MTC compared with normal controls (3.7%).127 In addition, eight of nine assessable cases with the S836S variant also had somatic M918T in their tumors. This may represent a low penetrance allele predisposing to MTC.
ACKNOWLEDGMENTS Supported by the American Cancer Society (RPG-97-064-01VM), the Susan G. Komen Breast Cancer Foundation, the Massachusetts Department of Public Health Breast Cancer Research Program, the Harvard Nathan Shock Center of Excellence Award in the Basic Biology of Aging (1P30AG13314-02 from the National Institutes of Health), the Dana-Farber Partners Cancer Center Women's Cancer Program, a Barr Investigatorship, and the Lawrence and Susan Marx Investigatorship in Human Cancer Genetics. Oliver Gimm critically reviewed this manuscript. I would like to acknowledge Lois Mulligan, Stanislas Lyonnet, and members of my laboratory, Debbie Marsh, Oliver Gimm, and Patricia Dahia, for contributing to the work described in this review. I am grateful to Bruce Ponder and David Livingston for their continued enthusiastic support. NOTES Address correspondence to Charis Eng, MD, PhD, Human Cancer Genetics Program, Ohio State University Comprehensive Cancer Center, 420 W 12th Ave, 690C MRF, Columbus, OH 43210; Email eng-1@medctr.osu.edu. REFERENCES 1. Takahashi M, Ritz J, Cooper GM: Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell 42:581-588, 1985[Medline] 2. Takahashi M, Cooper GM: RET transforming gene encodes a fusion protein homologous to tyrosine kinases. Mol Cell Biol 3:1378-1385, 1987 3. Takahashi M, Buma Y, Iwamoto T, et al: Cloning and expression of the ret proto-oncogene encoding a receptor tyrosine kinase with two potential transmembrane domains. Oncogene 3:571-578, 1988[Medline] 4. Santoro M, Rosato R, Grieco M, et al: The ret proto-oncogene is consistently expressed in human pheochromocytomas and thyroid medullary carcinomas. Oncogene 5:1595-1598, 1990[Medline] 5. Nakamura T, Ishizaka Y, Nagao M, et al: Expression of the ret proto-oncogene product in human normal and neoplastic tissues of neural crest origin. J Pathol 172:255-260, 1994[Medline]
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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-1 (GDNF Family Receptor alpha one, also known as GDNFR-
three affected members) and incompletely documented families. More than 98% of the MEN 2A families, 95% of the MEN 2B families, and 85% of the FMTC families have germline RET mutations.
