10 Multiple endocrine neoplasia

10

Multiple endocrine neoplasia R. V. THAKKER B. A. J. P O N D E R

In recent years there have been important advances in our understanding of the pathophysiological basis of endocrine tumours and their products of secretion. This is particularly true of the multiple endocrine neoplasia syndromes in which tumours of two or more endocrine glands occur in an individual. To some extent, these advances have stemmed from progress in hormone assays and in the recognition of clinical disorders which are associated with excessive hormone production. These developments have facilitated earlier diagnosis and have helped in the management of patients. Advances of a different type, those of molecular biology, have made it possible to clone the genes which encode hormones and to reveal the regulatory mechanisms controlling expression of these genes. Application of the techniques of molecular biology has also made it possible to localize mutant genes that cause abnormal growth and activity of endocrine glands, as for example in the clinical syndromes of multiple endocrine neoplasia. In this chapter, the molecular genetics of tumorogenesis and its investigation using recombinant D N A techniques will be described with reference to the multiple endocrine neoplasia syndromes and their underlying causes and mechanisms.

M U L T I P L E E N D O C R I N E NEOPLASIA S Y N D R O M E S

The occurrence of multiple tumours involving two or more endocrine glands within a single patient has previously been referred to as multiple endocrine adenopathy (MEA) (Underdahl et al, 1953) or the pluriglandular syndrome (Berdjis, 1962). However, glandular hyperplasia and malignancy may also occur in some patients and the term multiple endocrine neoplasia (MEN) was therefore proposed (Steiner et al, 1968; Wermer, 1974). There are two major forms of multiple endocrine neoplasia (Table 1) and these are referred to as type 1 and type 2. Multiple endocrine neoplasia type 1 (MEN1), which is also referred to as Wermer's syndrome, is characterized by the combined occurrence of tumours of the parathyroid glands, the pancreatic islet cells and the anterior pituitary gland (Wermer, 1954). However, multiple endocrine neoplasia type 2 (MEN2), which is also called Bailli~re's Clinical Endocrinology and Metabolism--Vol. 2, No. 4, November 1988

] 031

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R . V . THAKKER AND B. A. J. PONDER Table 1. The major forms of multiple endocrine neoplasia.

Types

Tnmour sites

Inheritance

1

Parathyroids Pancreatic islets

Autosomal dominant

2a 2b

Pituitary (anterior) ThyroidC cells Adrenal medulla Parathyroids ThyroidC cells Adrenal medulla (Associated abnormalities Mucosalneuromas Marfanoidhabitus)

Autosomaldominant Autosomaldominant

Sipple's syndrome, is characterized by the occurrence of medullary thyroid carcinoma (MTC) in association with phaeochromocytoma (Sipple, 1961) and three variants referred to as MEN2a, MEN2b and MTC-only, are recognized. In MEN2a, which is the most common variant, the development of MTC is associated with phaeochromocytoma and parathyroid overactivity. However, in MEN2b parathyroid involvement is absent and the occurrence of MTC and phaeochromocytoma is found in association with a marfanoid habitus and mucosal neuromas. In the third variant, MTC is the only manifestation of the syndrome and this variant is referred to as MTConly. All these forms of MEN may either be inherited as autosomal dominant syndromes (McKusick, 1988), or they may occur sporadically, i.e. without a family history. However, this distinction between 'sporadic' and 'familial' cases may sometimes be difficult, as in some sporadic cases the family history may be absent because the parent with the disease gene may have died before developing symptoms.

MOLECULAR GENETICS OF NEOPLASIA Genetic models of tumour development The development of tumours may be associated with mutations or inappropriate expression of specific normal cellular genes, which are referred to as oncogenes. Activation of these oncogenes leads to malignant transformation of the cells containing them and the genetic changes which cause this activation have recently been elucidated (Varmus, 1984; Friend et al, 1988). For example, chromosomal translocations affecting such oncogenes are associated with the occurrence of chronic myeloid leukaemia and Burkitt's lymphoma (Dalla-Favera et al, 1982; de Klein et al, 1982; Taub et al, 1982; Kurzrock et al, 1988). In these conditions, the mutations which lead to activation of the oncogene are dominant at the cellular level and therefore only one copy of the mutated gene is required for the phenotypic effect. Such dominantly acting oncogenes may be assayed in cell culture by first transferring them into recipient cells and then scoring the numbers of

MULTIPLE ENDOCRINE NEOPLASIA

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transformed colonies; this is referred to as the 'transfection assay'. However, in the case of the inherited cancer syndromes such as MEN1 and MEN2, it seems unlikely that the inherited susceptibility is due to such dominantly acting oncogenes. Instead, recessive mutations causing a functional loss of genes which control cell growth and differentiation seems more likely. This idea was first proposed in 1970 by de Mars, and subsequently elaborated by Knudson (de Mars, 1970; Knudson, 1971; Knudson et al, 1973). The essence of the idea is that to develop malignancy a cell must undergo at least two recessive genetic events, which are also sometimes referred to as two 'hits', and the theory is often called the 'two-hit hypothesis'. In familial cancers such as MEN, the first hit is inherited in the germ line and so is present in every cell in the body. Since this mutation is recessive it has no phenotypic expression but it does confer a predisposition to malignancy. When a second mutation causes loss of the remaining normal allele in the cell of the target tissue, the recessive mutation is expressed, resulting in malignant transformation. Occurrence of a sporadic tumour of the same type requires that both mutational events must coincide within the same somatic cell. Because this is improbable, the risk of tumour in an individual who has not inherited the first mutation is relatively much smaller. The seeming paradox that the inherited cancer syndromes are due to recessive mutations but dominantly inherited at the level of the family is explained because, in individuals who have inherited the first mutation, loss of the single remaining wild-type allele is almost certain to occur in at least one of the larger number of cells in the target tissue. This cell will be detected because it forms a tumour. As a result, almost all individuals will express the disease even though they inherited only a single copy of a recessive gene. Support for this model of neoplasia has recently come from studies of retinoblastoma (Cavenee et al, 1986; Hansen and Cavenee, 1988). Development of retinoblastoma has been shown to involve loss of both alleles at a single locus on the long arm of chromosome 13, and individuals with the hereditary form have been shown to have inherited the loss of one copy of the gene. A feature of key importance for studies of other inherited tumours is that the loss of the remaining allele, which occurs in the somatic cell and gives rise to the tumour, often involves a large-scale loss of chromosomal material (Figure 1). This represents a much larger target than the inherited mutation, which may be a small deletion or point mutation, in the search for the genetic loci involved in the development of different inherited tumours. Thus, in the case of MEN1 and MEN2, the finding of a consistent region of chromosomal loss in the tumour cells would both suggest that the genetic mechanism of tumour development was similar to retinoblastoma, and indicate a likely chromosomal location of the inherited mutation. The investigation of these genetic events and the search for the inherited cancer .genes have become possible only in the past few years as a result of advances m molecular biology which have provided cloned human DNA sequences to detect these mutations. These cloned DNA probes identify restriction fragment length polymorphisms which are inherited in a mendelian fashion and are thus useful genetic markers in linkage studies (Little et al, 1980; Orkin, 1981; White et al, 1985).

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R.V. Normal tissue

•

THAKKER A N D B. A. J. P O N D E R Tumour

•

II

M

Loss of segment containing remaining normal allele at disease locus, indicated by loss of marker allele b

a --

Marker alleles detected as RFLPs by Southern Blotting

a - -

b

- -

a ~

Loss of allele b in D N A from tumour

Figure 1. The 'two-hit' model of neoplasia and detection of chromosomalloss in tumours. A pair of chromosomesis shown from normal tissue and tumour of the same individual. M is a germ-line recessive mutation which predisposes to cancer. Mutation causing loss of the remaining normal allele at this locus in a somaticcell leads to turnout formation. (In a sporadic tumour, both mutations would occur in the same somatic cell), a, b are DNA sequence polymorphisms (RFLPs, see text) which provide genetic markers. Loss of marker allele b in DNA from tumour indicates the somatic mutational event which has also led to loss of the remaining normal allele at the disease locus. Note that the marker allele which is lost is on the chromosome which does not carry the inherited mutation and is therefore the chromosome inherited from the unaffected parent.

Restriction fragment length polymorphisms (RFLPs) R F L P s are the result of variations in the primary D N A sequence of individuals, and may be due to either single base changes, or deletions or additions or translocations. These changes in D N A sequence occur frequently (approximately once in every 250 base pairs) in the non-coding regions, do not usually affect gene function and are often at a distance away from the disease gene (Cooper and Schmidt, 1984). These polymorphisms may however lead to the presence or absence of a cleavage site for a restriction enzyme. Restriction enzymes are derived from micro-organisms and found to cleave D N A in a sequence-specific manner (Roberts, 1983). For example, the enzyme Hind III, which originated from Haemophilus influenzae, will only cleave if the sequence AAGCT-Y is present, and then it will cleave specifically ( $ ) between the two adenine (A) residues, i.e. A $ A G C T T . A D N A polymorphism such as a single base change in this sequence would result in loss of an enzyme cleavage site and this would be revealed as an R F L P as follows. A restriction enzyme is used to cleave human leukocyte D N A and the resulting D N A fragments are separated according to size by agarose gel electophoresis, the smaller size fragments migrating furthest away from the cathode (Figure 2). The D N A fragments are then transferred by Southern

1035

MULTIPLE ENDOCRINE NEOPLASIA

.To ® E

~

Venesect Extract DNA Digestwith restriction endonuclease

ILHI

0

Transfer DNA to membrane Hybridize with 32p_labelled probe

(9 Gel etectrophoresis

Autoradiograph

Figure 2. Schematic representation of Southern blotting to identify restriction fragment length polymorphisms (RFLPs). Human leukocyte DNA is digested with a restriction enzyme and the fragments separated by agarose gel electrophoresis and transferred to a membrane, which is hybridized with a radiolabelled probe and the RFLPs are revealed at autoradiography. From Thakker et al (1988).

blotting (Southern, 1975) to a nylon membrane. Digested fragments of single-stranded human DNA are thus immobilized according to size (i.e. fragment length) on the membrane, which is next hybridized with a singlestranded, radiolabelled DNA probe. The labelled DNA probe will anneal to any fragments which have a complementary sequence and these restricted fragments of varying lengths are revealed by autoradiography. The exact number and size of RFLPs will vary from individual to individual in relation to the number of recognition sites for the restriction enzyme, as shown in Figure 3. In this example, the DNA sequence of individual (1) has three restriction enzyme cleavage sites and, following digestion, fragments of two sizes will result. One fragment size will be 5 kilobases (kb) in length and the other fragment size will be 10 kb in length. The labelled DNA probe will hybridize only to the 5kb fragments, which contain a complementary sequence, and autoradiography will therefore only reveal one band, the RFLP, at 5 kb. However, in individual (2) there has been a loss of one restriction enzyme cleavage site, due to a change in the DNA sequence, and following digestion only restriction fragments of 15 kb in size will result. A single 15 kb RFLP is observed at autoradiography. The heterozygous individual (3) who has one chromosome with three cleavage sites and another with two cleavage sites, will reveal two RFLPs at autoradiography, one at 15 kb and another at 5 kb. Alleles can be designated to these RFLPs; for example, individual (1) who has the smaller RFLP is designated as having allele 'aa', individual (2) who has the larger RFLP is designated 'AA', and the heterozygous individual (3) with both the large and smaller RFLPs is 'Aa'. The use of RFLPs to identify the chromosomal rearrangements and mutations which occur specifically in a patient's tumour cells and not his normal cells, for example leukocytes, is illustrated in Figure 1. Mutations or deletions in tumour cells which lead to a loss of a restriction enzyme site are detected by the absence of an RFLP. This difference in the RFLPs between

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R. V. THAKKER AND B. A. J. PONDER

Individual

Homozygous 1

F DNA sequence

,3£ O

Q

Autoradiograph

Homozygous

Heterozygous

(2)

(3)

iiii 1131! -15kb

~ Allele

aa

AA

-5kb Aa

Sequence complementary to DNA probe m Radiolabelled probe DNA sequence • Cleavagesite for restriction enzyme RFLP revealed on autoradiograph

Figure 3. Schematic representation of restriction fragment length polymorphisms (RFLPs) resulting from variations in the number of restriction enzyme cleavage sites. Paired chromosomes (autosomes) with DNA sequences are shown for each individual.

the tumour and normal cells from an individual patient is referred to as a loss of alleles or loss of heterozygosity. In addition, RFLPs can also be used as genetic markers for linkage studies of affected families to localize a disease gene (Thakker and O'Riordan, 1988).

Linkage analysis RFLPs are inherited in a mendelian manner and their inheritance can be fOllowed together with a disease in an affected family. The consistent inheritance of an R F L P allele with the disease indicates that the two genetic loci are close together, i.e. linked. Genes that are far apart do not consistently co-segregate but show recombination because of crossing over during meiosis. By studying recombination events in family studies, the distance between two genes and the probability that they are linked can be ascertained (Morton, 1955; Ott, 1974). The distance between two genes is expressed as the recombination fraction (0), which is equal to the number of recombinants divided by the total number of offspring resulting from informative meioses within a family. The value of 0 can range from 0 to 0.5. A value of zero indicates that the genes are very closely linked, while a value of 0.5 indicates that the genes are far apart. The probability that the two loci are linked at these distances is expressed as a ' L O D score' which is log10 of the odds ratio favouring linkage. The odds ratio favouring linkage is defined as the likelihood that two loci are linked at a specified recombination (0) versus the likelihood that the two loci are not

MULTIPLE ENDOCRINE NEOPLASIA

1037

linked. A LOD score of + 3, which indicates a probability in favour of linkage of 1000 to 1, establishes linkage between two loci, and a LOD score of - 2, indicating a probability against linkage of 100 to 1, is taken to exclude linkage between two loci. LOD scores are usually evaluated over a range of 0, thereby enabling the genetic distance and the maximum (or peak) probability favouring linkage between two loci to be ascertained. This is illustrated for the results in MEN2 in Figure 9 (see below). Computer programs are available to facilitate the calculation of two point LOD scores from genetic marker data obtained from large pedigrees (Lathrop and Lalouel, 1984; Lathrop et al, 1984) and these also enable the results from different families to be combined. A fuller description of linkage analysis in families with inherited metabolic disorders is provided by Thakker and O'Riordan (1988). MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 Patients with multiple endocrine neoplasia type 1 are characterized by the occurrence of tumours of the parathyroid glands, the pancreatic islet cells and the anterior pituitary gland. The disease may arise sporadically or be inherited as an autosomal dominant condition. The first occurrence of multiple endocrine tumours was described in a patient with acromegaly at autopsy, which revealed the presence of an anterior pituitary tumour and enlarged parathyroid glands (Erdheim, 1903). Pancreatic islet cell tumours were subsequently observed in association with parathyroid and pituitary tumours (Cushing and Davidoff, 1927; Lloyd, 1929), and a familial occurrence was suggested by the finding of these tumours in two sisters (Rossier and Dressier, 1939). Further case reports revealing the triad of parathyroid, pancreatic islet cell and anterior pituitary tumours in individual patients led to the recognition of a unifying disorder (Underdahl et al, 1953), and a familial basis was demonstrated by documenting its occurrence in a father and daughter of one family (Moldawer, 1953; Moldawer et al, 1954) and in a father and four daughters of another family (Wermer, 1954). An autosomal dominant mode of inheritance was proposed (Wermer, 1954) and established by further family studies which demonstrated inheritance of the syndrome in five generations with equal frequency in males and females (Ballard et al, 1964). It was proposed that these tumours, which occurred in several different endocrine glands, had a common neuroectodermal origin (Pearse, 1968, 1969) within cells that were capable of amine precursor uptake and decarboxylation (APUD). However, the parathyroids which are involved in the majority of MEN1 patients were found to have cytochemical characteristics which differed from A P U D cells, and the relationship between MEN1 and the A P U D system needs to be further defined (Le Douarin, 1982). Clinical findings, biochemical abnormalities and treatment The incidence of MEN1 has been estimated from randomly chosen post-

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R . V . THAKKER AND B. A. J, PONDER

mortem studies to be 0.25% (Berdjis, 1962; Lips et al, 1984). The disorder affects all age groups, with a reported age range of 5-81 years (Ballard et al, 1964; Gelston et al, 1982) and 80% of patients have presented by the fifth decade (Shepherd, 1985). The clinical manifestations of MEN1 are related to the sites of tumours and to their products of secretion. In addition to the parathyroid, pancreatic and pituitary turnouts, which constitute the major components of MEN1, adrenal cortical, carcinoid and lipomatous tumours have also been described. Parathyroid tumours

Primary hyperparathyroidism is an important feature of MEN1 and occurs in more than 95% of all MEN1 patients (Majewski and Wilson, 1979; Eberle and Grun, 1981; Marx et al, 1982). Patients may present with asymptomatic hypercalcaemia, or nephrolithiasis, or osteitis fibrosa cystica or vague symptoms associated with hypercalcaemia, for example polyuria, polydipsia, constipation, malaise or occasionally with peptic ulcers. Biochemical investigations reveal hypercalcaemia in association with raised circulating parathyroid hormone (PTH) concentrations. Surgical removal of the abnormally overactive parathyroid tumours is the definitive treatment. However, all four parathyroid glands are usually affected with multiple adenomas or hyperplasia (Croisier et al, 1971a; Lamers et al, 1979), although this histological distinction may be difficult (Black and Utley, 1968), and both sub-total parathyroidectomy and total parathyroidectomy with autotransplantation of fresh or frozen parathyroid tissue in the forearm (Wells et al, 1976; Saxe and Brennan, 1982) have been associated with persistent and recurrent hypercalcaemia (Rizzoli et al, 1985; Mallete et al, 1987). Regular postoperative, long-term measurements of serum calcium concentrations are therefore necessary, and at present no effective medical treatment of primary hyperparathyroidism is generally available. Pancreatic turnours

The incidence of pancreatic islet cell tumours in MEN1 patients varies from 30 to 80% in different series (Croisier et al, 1971a; Majewski and Wilson, 1979; Eberle and Grun, 1981; Marx et al, 1982). The majority of these tumours produce excessive amounts of hormone, for example gastrin, insulin, glucagon or vasoactive intestinal peptide (VIP), and are associated with distinct clinical syndromes. Gastrinoma. Zollinger and Ellison initially described two patients in whom non-B-islet cell turnouts of the pancreas were associated with recurrent peptic ulceration and marked gastric acid production (Zollinger and Ellison, 1955), and gastrin was subsequently extracted from such tumours (Gregory et al, 1960, 1967). These gastrin-secreting tumours (gastrinomas) represent over 50% of all pancreatic islet cell tumours in MEN1 (Eberle and Grun, 1981) and are the major cause of morbidity and mortality in MEN1 patients. This is due to the recurrent severe multiple peptic ulcers which may per-

MULTIPLE ENDOCRINE NEOPLASIA

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forate. Additional prominent clinical features of this Zoltinger-Ellison syndrome include diarrhoea and steatorrhoea. The diagnosis is established by demonstration of a raised fasting serum gastrin concentration in association with an increased basal gastric acid secretion (Wolfe and Jensen, 1987). Occasionally intravenous provocative tests with either secretin (2 units/kg) or calcium infusion (4 mg C a 2+ kg -1 h - 1 for 3 h) are required to distinguish patients with Zollinger-Ellison syndrome from other patients with hypergastrinaemia, as for example in antral G-cell hyperplasia. Medical treatment of patients with the Zollinger-Ellison syndrome is directed to reducing basal gastric acid output to less than 10 tool/h, and this may be achieved by large doses of the histamine Hz-receptor antagonists cimetidine and ranitidine (Deveney et al, 1983; Jensen et al, 1984). More recently the parietal cell H+-K+-ATPase inhibitor, omeprazole, has proved efficacious and may become the drug of choice for gastrinomas (McArthur et al, 1985). Surgical treatment with total gastrectomy is recommended only for persistently non-compliant patients (Wolfe and Jensen, 1987). However, the ideal treatment for non-metastatic gastrinoma is surgical excision of the gastrinoma. Prior turnout localization studies should be undertaken by ultrasonography, computerized tomography, nuclear magnetic resonance imaging, selective abdominal angiography or venous sampling techniques, as gastrinomas are frequently multiple or extra-pancreatic. Treatment of disseminated gastrinomas is difficult and chemotherapy with streptozotocin and 5-fluorouracil (Moertel et al, 1980), hormonal therapy with somatostatin analogue SMS201-995 (Kvols et al, 1987), hepatic artery embolization (Carrasco et al, 1983), administration of human leukocyte interferon (Erickson et al, 1986), and removal of all resectable tumour (Norton et al, 1986) have all occasionally been successful. Insulinoma. [3-islet cell tumours secreting insulin (insulinomas) represent one-third of all pancreatic tumours in MEN1 patients (Crosier et al, 1971a). These insulinomas occur with gastrinomas in 10% of MEN1 patients (Crosier et al, 1971b; Peurifoy et al, 1979) and the two tumours may arise at different times. Patients with an insulinoma present with hypoglycaemic symptoms which develop after a fast or exertion and improve after glucose intake. Biochemical investigations reveal raised plasma insulin concentrations in association with hypoglycaemia. Circulating concentrations of Cpeptide and proinsulin, which are also raised, may be useful in establishing the diagnosis, as may an insulin suppression test (Turner and Johnson, 1973). Medical treatment, which consists of frequent carbohydrate feeds and diazoxide, is not always successful and surgery may be required. Most insulinomas are multiple and small, and preoperative localization with computed tomography scanning, coeliac axis angiography and pre-perioperative percutaneous transhepatic portal venous sampling is difficult and success rates have varied (Daggett et al, 1981). Surgical treatment, which ranges from enucleation of a single tumour to a distal pancreatectomy or partial pancreatectomy, may result in considerable improvement. Chemotherapy, which consists of streptozotocin or somatostatin, is used for metastatic disease (Broder and Carter, 1974).

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THAKKER AND B. A. J. P O N D E R

Glucagonoma. Glucagonomas, which are a-islet cell, glucagon-secreting pancreatic tumours, have been reported in only five MEN1 patients (Croiser et al, 1971b; Croughs et al, 1972; Tiengo et al, 1976; Leclerc et al, 1977; Stacpoole et al, 1981). The characteristic clinical manifestations of a skin rash (necrolytic migratory erythyema), weight loss, anaemia and stomatitis may be absent and the presence of the turnout is indicated only by glucose intolerance and hyperglucagonaemia. The tail of the pancreas is the most frequent site for glucagonomas and surgical removal of these is the treatment of choice. However, treatment may be difficult as 50% of patients have metastases at the time of diagnosis (Stacpoole et al, 1981). Medical treatment of these with somatostatin or its long-acting analogue, or with streptozotocin or dimethyltri-azenolmidazole carboxamide (DTC) has been successful in some patients (Strauss et al, 1979; Marynick et al, 1980). Vipoma. Patients with vipomas, which are vasoactive intestinal peptide (VIP) secreting pancreatic turnouts, develop watery diarrhoea, hypokalaemia and achlorhydria. The clinical syndrome has been referred to as the Verner-Morrison syndrome (Verner and Morrison, 1958), the W D H A syndrome (Marks et al, 1967) and the vipoma syndrome (Bloom et al, 1973). Vipomas have been reported in only a few MEN1 patients (Verner and Morrison, 1958; Brown and Crile, 1964; Burkhardt, 1976; Long et al, 1979) and the diagnosis is established by documenting a markedly raised plasma VIP concentration. Surgical management of vipomas, which are mostly located in the tail of the pancreas, has been curative. However, in patients with unresectable tumour, treatment with streptozotocin (Kahn et al, 1975), long-acting somatostatin analogue (Long et al, 1979), corticosteroids (Kingham et al, 1978), indomethacin (Jaffe et al, 1977), metoclopramide (Long et al, 1981), and lithium carbonate (Pandol et al, 1980) has proved beneficial. PPoma. Turnouts secreting pancreatic polypeptide (PP) are called PPomas, and are found in a large number of patients with MEN1 (Friesen et al, 1980; Gelston et al, 1982). No pathophysiological sequelae of excessive PP secretion are apparent and the clinical significance of PP is unknown, although the use of serum PP measurements has been suggested for the detection of pancreatic tumours in MEN1 patients (Lamers and Diemel, 1982; Skogseid et al, 1987). Pituitary tumours The incidence of pituitary tumours in MEN1 patients varies from 15 to 90% in different series (Croisier et at, 1971a; Majewski and Wilson, 1979; Eberle and Grun, 1981; Marx et al, 1982). The clinical manifestations depend upon the size of the pituitary tumour and its product of secretion. Enlarging pituitary tumours may compress adjacent structures such as the optic chiasm or normal pituitary tissue and cause bitemporal hemianopia or hypopituitarism. The tumour size and extension are radiologically assessed by computed tomography scanning and nuclear magnetic resonance imaging.

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Approximately 60% of MENl-associated pituitary turnouts secrete prolactin, 25% secrete growth hormone (GH), 3% secrete adrenocorticotrophin (ACTH), and the remainder appear to be non-functioning (Prosser et al, 1979; Farid et al, 1980; Eberle and Grun, 1981). Treatment of pituitary t umours in MEN1 patients is similar to that in non-MENi patients and consists of medical therapy or selective hypophysectomy, by the transsphenoidal approach if feasible, with radiotherapy being reserved for residual unresectable tumour. Prolactin-secreting tumours, which are called prolactinomas, may cause amenorrhoea, infertility and galactorrhoea in women and impotence in men. With the advent of radioimmunoassays, over 70% of previously reported non-secreting pituitary chromophobe adenomas Were found to be prolactinomas (Franks et al, 1977). Medical treatment with bromocriptine, which is a dopamine agonist, has been effective in decreasing tumour size, reversing visual abnormalities, decreasing serum prolactin levels, reducing galactorrhoea and restoring gonadal function (Wass et al, 1979; Vance et al, 1984) and is used in preference to surgery. GH-secreting tumours cause acromegaly, which is characterized by acral enlargement, hyperhidrosis, headaches, paraesthesiae, hypertension and diabetes mellitus. Hypophysectomy is the treatment of choice, but medical treatment with bromocriptine (Wass et al, 1977, 1979) and the long-acting somatostatin analogue (Lamberts et al, 1985) has also been reported to be effective. ACTHsecreting tumours, which cause corticosteroid excess and Cushing's disease, are rare in MEN1 patients (Eberle and Grun, 1981; Maton et al, 1986). Medical therapy with metyrapone is used prior to surgery and a selective hypophysectomy is the treatment of choice. Aminoglutethimide, ketoconazole and the adrenolytic drug mitotane (o, p ' D D D ) have also been effective in reducing the hypercortisolaemia (Child et al, 1979; Luton et al, 1979; Sonino et al, 1985). Associated tumours

Patients with MEN1 may have tumours involving glands other than the parathyroids, pancreas and pituitary. Thus carcinoid, adrenal cortical, thyroid and lipomatous tumours have been described in association with MEN1. Carcinoid tumours, which occur more frequently in patients with MEN1 (Duh et al, 1987) may be inherited as an autosomal dominant trait in association with MEN1 (Farid et al, 1980). The carcinoid tumour may be located in the bronchi (Williams and Celestin, 1962), the gastrointestinal tract (Fisher and Hicks, 1960; Snyder et al, 1972; Rode et al, 1987), the pancreas (Lee et al, 1986), or the thymus (Farid et al, 1980). Most patients are asymptomatic and do not suffer from the flushing attacks and dyspnoea associated with the carcinoid syndrome, which usually develops after the tumour has metastasized to the liver. The incidence of asymptomatic adrenal cortical turnours in MEN1 patients has been reported to be as high as 40% (Croisier et al, 1971a). The majority of these tumours are non-functioning. However, functioning adre-

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R. V. THAKKER AND B. A. J. PONDER

nal cortical tumours in MEN1 patients have been documented to cause hypercortisolaemia and Cushing's syndrome (Raker et al, 1962), and primary hyperaldosteronism, as in Conn's syndrome (Dluhy and Williams, 1969; Croughs et al, 1972; Fertig et al, 1980). Thyroid turnouts consisting of adenomas, colloid goitres and carcinomas have been reported to occur in over 25% of MEN1 patients (Croisier et al, 1971a). However, the prevalence of thyroid disorders in the general population is high and it has been suggested that the association of thyroid abnormalities in MEN1 patients may be incidental and not significant.

Biochemical screening The attempts to screen for the development of MEN1 tumours in the asymptomatic relatives of an affected individual have depended largely on measuring the serum concentrations of calcium, gastrointestinal hormones and prolactin. Parathyroid overactivity causing hypercalcaemia is invariably the first manifestation of the disorder (Benson et al, 1987) and this has become a useful and easy screening investigation. Pancreatic involvement in asymptomatic individuals has previously been detected by estimating the fasting plasma concentrations of gastrin and PP (Oberg et al, 1982). However, a stimulation test which consists of a 563 Cal mixed meal was found to be a better method of detecting pancreatic disease in individuals who had no demonstrable pancreatic turnouts by computerized tomography (Skogseid et al, 1987). An exaggerated increase in serum gastrin and/or PP proved to be a reliable early indicator for the development of pancreatic turnouts in these individuals. Some asymptomatic pituitary tumours may be detected by demonstration of hyperprolactinaemia (Farid et al, 1980). Screening in MEN1 is difficult because the age-related penetrance (i.e. the proportion of gene carriers who have manifested symptoms or signs of the disease by a given age) has not been established. The proportion of affected individuals who have been detected at a certain age by clinical symptoms or biochemical screening in different series has ranged from 11 to 18% at 20 years of age, 52 to 94% at 35 years, and 83 to 100% at 50 years (Anderson, 1970; Shepherd, 1985; Benson et al, 1987); biochemical screening, which detects asymptomatic patients, increased the proportion of affected individuals at all ages. Thus, the likelihood of wrongly attributing an 'unaffected' status to an individual who has no manifestations of the disease at the age of 35 years may be as high as i in 2 or approaching i in 20 and depends on whether clinical symptoms alone or biochemical screening methods are used to detect the disease. In order to improve this situation, further biochemical screening and systematic family studies are required to clarify the age-related penetrances for MEN1. In addition, localizing the MEN1 gene and establishing genetic markers will also facilitate the identification of gene carriers, as has been demonstrated by studies of MEN2 (see below).

Localization of the MEN1 gene Linkage studies using classical genetic markers, for example the human

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leukocyte antigens, red cell antigens (HLA), serum proteins and enzymes, were unable to localize the gene causing MEN1 (Bear et al, 1985; Bale et al, 1987). In addition, cytogenetic studies which indicated a segmental deletion of the short arm of chromosome 20 in MEN1 (Jackson et al, 1986) remain unconfirmed. Molecular genetic studies were therefore undertaken to map INS HRASI PTH CAL

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PGR

ETS1 APOA1

Figure 4. Map of human chromosome 11 showing the locations of some genes encoding hormones, clinically useful DNA probes, oncogenes and disease loci. Chromosome 11 with Giemsa bands is schematically represented and the probe and disease loci are shown juxtaposed to their regional localization, which has been ascertained by in situ hybridization, somatic cell hybrid or multipoint linkage studies: insulin (INS); harvey ras sarcoma 1 viral oncogene homologue (HRAS1); parathyroid hormone (PTH); calcitonin (CAL); tryptophan hydroxylase (TRHPH); follicle stimulating hormone, [3polypeptide (FSHB); Wilms' tumour, aniridia, genitourinary abnormalities and mental retardation complex (WAGR); catalase (CAT); coagulation factor II, prothrombin (F2); acid phosphatase 2, lysosomal (ACP2); pepsinogen (PGA); phosphorylase glycogen muscle, McArdle syndrome glycogen storage disease type V (PYGM); multiple endocrine neoplasia type I (MENI); murine mammary tumour virus integration site oncogene homologue (INT2); B cell lymphoma 1 (BCLI); progesterone receptor (PGR); avian erythroblastosis virus E26 oncogene homologue ] (ETS1), and apolipoprotein A1 (APOA1). Clusters of genetic loci whose order remains to be established are shown in square brackets.

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THAKKER A N D B. A. J. PONDER

the MEN1 gene, as a first step towards defining the genetic abnormality and its product of expression (protein). This approach, called 'reverse genetics' (Ruddle, 1984), has been successfully used to identify the genetic and protein abnormalities which lead to Duchenne muscular dystrophy (Hoffman et al, 1987; Koenig et al, 1987) and retinoblastorna (Cavenee et al, 1986; Hansen and Cavenee, 1988). The genetic mutations causing one type of MEN1 turnout (insulinomas) were identified by molecular studies in which RFLPs obtained from the patient's tumour D N A were compared to the RFLPs obtained from the same patient's leukocyte D N A (Larsson et al, 1988). A loss of RFLPs (i.e. alleles) obtained with D N A probes from the short and long arms of chromosome 11 were found to occur in two insulinomas from related MEN1 patients. D N A probes from 16 other autosomes and the X and Y chromosomes did not reveal such loss of alleles in the insulinomas. These results therefore strongly suggested that the oncogenic recessive mutations causing MEN1 were localized to chromosome 11. Confirmation of the localization of the MEN1 gene was obtained by linkage studies of four affected families, using D N A probes from chromosome 11, and linkage between the MEN1 and human muscle phosphorylase gene (PYGM) genetic loci was established, peak LOD score = 4.37, 0 =0.00. The PYGM locus, which is involved in McArdle's syndrome (glycogen storage disorder type V), had previously been localized to the pericentromeric region of the long arm of chromosome 11 by somatic cell hybrid and multipoint linkage studies (Lebo et al, 1984; Leppert et al, 1987). The results of this study, which demonstrated that the probability favouring linkage between the marker locus PYGM and the MEN1 gene was in excess of 20 000 to 1, mapped the MEN1 gene to the pericentromeric region of the long arm of chromosome 11 (Figure 4). Further studies are required in additional families to define this disease locus more closely (see section on MEN2) and to clarify the mutational events in other MEN1 tumours, for example parathyroid tumours, which are the commonest type of turnouts in MEN1 (see Note added in proof, p. 1067).

Aetiology of parathyroid tumours The abnormal parathyroid cell growth which results in tumours may be due to extrinsic or intrinsic factors, and several studies have been undertaken to define these. An extrinsic factor with high parathyroid mitogenic activity has recently been identified in the plasma of MEN1 patients by in vitro studies, which used bovine parathyroid cells maintained in a long-term culture system (Brandi et al, 1986). Plasma from MEN1 patients stimulated these bovine parathyroid cells to rapidly incorporate [3H]thymidine and to proliferate. This plasma mitogenic activity was markedly reduced by heat, acid and dithiothreitol treatment, indicating that the stimulatory properties may be due to a protein containing disulphide bonds. Gel filtration analysis demonstrated the mitogenic activity to be within a single peak in a region between bovine serum albumin and ovalbumin, thereby indicating that the protein has a molecular weight in the range 50000-55 000 (Brandi et al,

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MULTIPLE ENDOCRINE NEOPLASIA

1986, 1987). This mitogenic factor was demonstrated to be a distinct factor from other growth factors such as epidermal growth factor, platelet-derived growth factor, nerve growth factor, fibroblast growth factor, insulin-like growth factor I and tumour growth factor [3 and was shown not to be an autocrine product from the parathyroid glands themselves. This plasma mitogenic factor appeared to be specific for parathyroid cells and did not stimulate activity in anterior pituitary or pancreatic islet cells. More recent studies have revealed that a basic fibroblast growth factor, in contrast to its acidic counterpart, has parathyroid mitogenic activity, and this appears to act by stimulating endothelial cells (Brandi et al, 1988). The role of this extrinsic mitogenic factor and its relation to any intrinsic factors in MEN1 patients needs to be further elucidated. Intrinsic factors, such as genetic defects, have not yet been reported in MEN1 parathyroid tumours (see p. 1067). However, the molecular basis of non-MEN1 parathyroid turnouts has been investigated and a structural defect within the PTH gene itself identified. The human PTH gene has been localized to the short arm of chromosome 11 by using rodent-human hybrid cell lines (Naylor et al, 1983) and its nucleotide sequence determined (Vasicek et al, 1983). Further analysis of the organization of the pre-pro-PTH gene revealed that it has two intervening sequences which separate the gene into three functional domains (Figure 5). The intervening sequences are called introns and the gene sequences which encode the mature messenger PTH genesequence(DNA)

I

Transcription

!

Splicing Mature~mRNA

I

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I

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Translation Polypeptide I Pre- I Pro-PTH I Figure 5. The parathyroid hormone (PTH) gene contains two intervening sequences, introns ( ~ ) which interrupt the three gene sequences encoding the mature m R N A , exons (1 [). Exon A encodes the 5' non-coding region of the mRNA, exon B encodes the 'pre' sequence of the protein and exon C encodes the prohormone cleavage site, the PTH sequence and the 3' non-coding region. The complete gene is transcribed into R N A and the introns are spliced out from the m R N A precusor to yield mature m R N A . Translation of this m R N A results in the precursor, pre-pro-PTH, and processing of this yields parathyroid hormone. In parathyroid adenomas the region containing exons B and C is separated from exon A and is juxtaposed to non-PTH DNA.

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T H A K K E R A N D B. A . J. P O N D E R

R N A (mRNA) are called exons. The first exon, at the 5' end, is a regulatory domain, the second exon encodes the 'pre' sequence of the protein, and the third exon encodes the 'prohormone' cleavage site, together with the PTH coding sequence and the 3' non-coding region. Structural abnormalities within the organization of the PTH gene have been identified in non-MEN1 parathyroid adenomas (Arnold et al, 1988a). These abnormalities involve a separation of the first exon from the fragment containing the second and third exons, together with a rearrangement in which the second and third exons become juxtaposed with 'new' non-PTH DNA. Investigation of this nonPTH DNA, revealed it to be highly conserved in the human and mouse genomes, with one copy per haploid genome. Somatic cell and in situ hybridization studies have localized this new non-PTH D N A to the long arm of chromosome 11, band 11q13 (Arnold et al, 1988b), which contains the oncogenes I N T 2 and B C L I . The activation of cellular oncogenes through analogous rearrangements has been implicated in the pathogenesis of several tumours, for example Burkitt's lymphoma (Dalla-Favera et al, 1982; Taub et al, 1982) and chronic myeloid leukaemia (de Klein et al, 1982), and the role of this clonal D N A rearrangement involving the PTH gene in the development of MEN1, whose gene has been mapped to the long arm of chromosome 11 (see above), requires further study. In addition, the possible relations between these genetic defects and the parathyroid mitogenic factor will also need to be elucidated. Animal models

An animal model for MEN1 would greatly facilitate studies into the pathogenesis, gene expression and pharmacological therapies for this disorder. Two animal models with multiple endocrine tumours which have similarities to the human MEN1 disorder have been described. The first animal model resulted from the transplantation of functioning endocrine tumours in rats, and the second model resulted from the introduction of cloned recombinant D N A sequences into the germ cells of mice. The transplantation of growth hormone and prolactin secreting rat pituitary tumour tissue in syngeneic rats resulted in hypercalcaemia and raised serum concentrations of parathyroid hormone, 1,25(OH)2 vitamin D, growth hormone and prolactin (Carlson et al, 1985). Parathyroidectomy in these rats led to hypocalcaemia, thereby revealing the parathyroid dependence of the hypercalcaemia. The pathophysiological basis for these endocrine abnormalities remains unknown and it has been suggested that pr01actin or growth hormone may directly stimulate parathyroid hormone secretion (Carlson et al, 1985) and increase renal l~-hydroxylase enzyme activity (Spanos et al, 1976, 1978). Transgenic mouse model

The introduction of cloned genes into the germ cells of mammals is one of the major recent technological advances in biology (Jaenisch and Mintz, 1974; Jaenisch, 1988). The inserted cloned gene is stably transmitted from

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MULTIPLE ENDOCRINE NEOPLASIA

MICE

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Figure 6. Schematic representation of the development of transgenic mice. The arginine vasopressin (AVP) promoter gene is fused to the DNA sequence coding for the large T-antigen of simian virus 40 (SV40 T-ag) to produce a hybrid oncogene, which is microinjected into fertilized one-cell stage eggs. Surviving eggs are transferred to recipient females and transgenic mice identified amongst the litter mates for further breeding and studies.

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R. V. THAKKER AND B. A. J. PONDER

generation to generation and is referred to as a transgene. These advances have enabled animal models to be produced for human genetic diseases and thereby permitted further study of these disorders. For example, the introduction of a recombinant oncogene containing the insulin gene promoter into fertilized mouse eggs resulted in an inherited form of insulinoma in transgenic mice (Hanahan, 1985). More recently, transgenic mice with pituitary and pancreatic tumours have been reported and these may provide a model for the human disorder of MEN1 (Murphy et al, 1987). These transgenic mice developed from fertilized one-cell mouse eggs which had a hybrid oncogene introduced into them by microinjection (Figure 6). The hybrid oncogene consisted of the promoter region of the bovine arginine vasopressin gene fused to the D N A sequence coding for the large T-antigen of simian virus 40 (SV40). The eggs surviving this procedure were returned by oviduct transfer to pseudopregnant recipient female mice and the transgenic offspring in the litter were identified and bred for further studies. Tumours of the pancreatic islet cells and anterior pituitary developed in these transgenic mice. The pancreatic tumours consisted of insulin-producing cells but those of the pituitary appeared to be non-functioning. Hypercalcaemia and parathyroid tumours were not detected in preliminary studies. This intriguing transgenic mouse model may prove useful in investigating the pathogenesis of MEN1 in man. M O L E C U L A R E N D O C R I N E NEOPLASIA TYPE 2

The principal tumours in MEN2 are MTC, which arises from the parafollicular 'C' cells of the thyroid, and phaeochromocytoma of the adrenal medulla. A typical small pedigree is shown in Figure 7. The disease is due to an autosomal dominant gene. The children of affected individuals are each at 50% risk of inheriting the gene, and are therefore at high risk of tumour.

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MULTIPLE ENDOCRINE NEOPLASIA

1049

Although uncommon (there are about 20 new cases each year in the United Kingdom), MEN2 is important because screening and early diagnosis will reduce morbidity and mortality in this high-risk population. MEN2 is also of great interest from a biological viewpoint. Within the family shown in Figure 7, a single gene is conferring a greater than 10000-fold increased risk of MTC or phaeochromocytoma. Elucidation of the normal and disordered function of this gene and its relationship to the biology of the disease is likely to increase our understanding of the normal growth and development of the tissues involved, as well as of the processes of tumorigenesis. This account will focus on the genetics of MEN2 and the relationship of genetics to the biology of the disease and to clinical management.

Clinical features, biochemical screening and genetics

Clinical forms of MEN2 MTC occurs in a sporadic (non-familial) form, and in three familial forms, distinguished by the other tissues involved and the aggressiveness of the disease.

Sporadic and apparently sporadic MTC. The typical tumour of MEN2 is the MTC. A histologically similar tumour also occurs in sporadic (truly nonhereditary) form, as is the case in other inherited cancer syndromes. Truly sporadic MTC probably accounts for 75 % of all cases; the remaining 25 % of MTC are familial, and most of these occur as part of MEN2a (Table 1). It is important to appreciate that some cases of MTC which present without a family history are, even so, of the familial type (Ponder et al, 1988a): in these 'apparently sporadic' cases the family history may be absent because the parent who transmitted the gene has died before manifesting symptoms (see below). This has important implications for decisions about family screening, also discussed below.

MEN2a. In MEN2a, the typical form of MEN2, MTC is associated with phaeochromocytoma in about half the cases, and with hyperplasia or adenoma of the parathyroid in probably 10-40% of cases (Gagel et al, 1988) (the figure depends upon the energy with which it is sought). The most usual clinical presentation is with a lump in the neck but some cases present with other symptoms, including hypertension or the effects of hypercalcaemia. The mean age of onset of clinical symptoms is about 40 years (Easton et al, 1989) and of those who present clinically (rather than to screening), fewer than half die of their disease (Saad et al, 1984; Telenius-Berg et al, 1984).

MTC-only and MEN2bL Two clinically distinct variants of MEN2 are recognized. In MTC-only families, MTC is the sole manifestation. The disease generally presents late and deaths from the tumour are exceptional t MEN2a and MEN2b are agreed nomenclature for the conditions sometimes previously called MEN2 and MEN3. (2nd International Workshop on MEN2. Henry Ford Hospital Medical Journal (1987) Vol 35, no. 2 and 3).

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R. V. THAKKER AND B. A. J. PONDER

(Farndon et al, 1986). Only a small number of families has been reported, suggesting that this variant is much less common than MEN2a, but since the definition requires a fairly large pedigree, it is difficult to be certain. Many families which are classified as MEN2a have only one or two individuals with phaeochromocytoma among a much larger number with MTC, which suggests that MTC-only families might be simply one end of a spectrum. In that case, however, MTC in families with few phaeochromocytomas might be predicted to have a less aggressive course, and evidence of this is lacking (see below). The phenotype of MEN2b is clearly distinct: MTC and phaeochromocytoma are associated with physical abnormalities which include a marfanoid habitus, neuromas of the lips and conjunctivae, medullated corneal nerve fibres, and disordered autonomic ganglia in the wall of the gut and other viscera (Dyck et al, 1979; Khalil and Lorenzetti, 1980; Demos et al, 1983; Rougier et al, 1983). There is no parathyroid involvement (Carney et al, 1980). MTC in MEN2b usually presents young and is aggressive: death from metastatic disease by the mid-twenties is common.

Neuroectodermal origin The involvement of thyroid C cells, adrenal medulla, and nerve tissue in MEN2 is probably explained by the common embryological derivation of these tissues from neural ectoderm. The inherited mutation of MEN2 may affect a gene responsible for control of the normal growth of cells in this lineage. The parathyroid glands, however, are thought not to be of neuroectodermal origin but to derive from pharyngeal pouch endoderm, and their involvement in MEN2a but not MEN2b remains unexplained. Recent evidence suggests that the MEN2a, MEN2b and MTC-only phenotypes may be due to alleles at the same locus (see below).

Tumour development in MEN2 Both the thyroid and adrenal turnouts develop through a stage of multiple foci of hyperplasia (Wolfe et al, 1973; Emmertsen et al, 1983). This is characteristic of the inherited turnouts and absent in sporadic cases (though appearances resembling C cell hyperplasia are seen in a minority of otherwise normal thyroids) (Gibson et al, 1982; O'Toole et al, 1985). The hyperplasia is therefore regarded as the phenotypic consequence of the inherited mutation. However, it is not clear whether the loci of hyperplasia are the direct expression of the inherited mutation, or if a second somatic genetic event is necessary for hyperplasia to develop. This could be resolved by finding whether individual hyperplastic foci are clonal or polyclonal in origin. A clonal composition would imply the occurrence of a second, somatic, event. Eventually, one or more hyperplastic foci develop into tumours, but whereas the thyroid C cell tumours (MTC) are malignant, the adrenal medullary tumours are almost always benign.

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Biochemical screening tests Because thyroid C cells produce calcitonin, an increased C cell mass is reflected in elevated plasma calcitonin levels. This is the basis of screening in MEN2. To increase sensitivity, calcitonin measurements are made before and after a stimulus (usually i.v. calcium or pentagastrin) (Telenius-Berg et al, 1977; Wells et al, 1978) which causes the C cells to release their calcitonin stores. Individuals who have inherited the MEN2a gene will almost always be detectable by this test before they develop clinical symptoms from phaeochromocytoma or hyperparathyroidism; it is the single most useful screening test (Telenius-Berg et al, 1984; Gagel et al, 1988).

Penetrance of the MEN2a gene 'Penetrance' means the proportion of gene carriers who will have manifested signs or symptoms by a given age. The clinical penetrance of MEN2a is incomplete; only about 70% of gene carriers ever present clinically with symptoms (Figure 8a) (Ponder et al 1988b; Easton et al, 1989). The penetrance to a pentagastrin stimulation test is, however, essentially complete: over 90% of gene carriers are detectable by age 30 (Figure 8b). Without extensive prospective screening of gene carriers who have been identified as such independently using genetic markers (see below), it will not be possible to determine whether 100% of gene carriers are detectable by biochemical screening: the plateau in Figure 8b lies somewhere between 92 and 100%. Anecdotal evidence suggests, however, that conversion from a negative to a positive pentagastrin screening result after the age of 40 is rare. There is thus a 25-year range over which gene carriers develop sufficient C cell hyperplasia to be detectable; furthermore, Figure 8 indicates that at least 30% of individuals who have detectable C cell hyperplasia before age 30 never develop clinically significant MTC. What determines this variation in the progression of the disease in different individuals is unknown: it might be the chance occurrence of further steps necessary for tumour formation, or there may be modifying environmental or genetic influences. The ability to predict, the course of the disease in a gene carrier would clearly be of clinical use, and elucidating the mechanisms, if there are such, which modify the expression of inherited cancer genes is of fundamental interest. A search for consistent differences in age at onset which might suggest an experimental approach to the problem has been made by analysis of more than 200 affected individuals and 320 unaffected first degree relatives in families collected by the Cancer Research Campaign MTC Study Group (Easton et al, 1989). This has, however, failed to show significant differences in age at clinical or screening onset for MTC or phaeochromocytoma between families (although even with this large series the comparisons have low statistical power). It is unlikely, therefore, that different mutant alleles at the MEN2a locus are responsible for large differences in the course of the disease. Apart from a suggestion of earlier clinical onset of MTC in females, there was also no difference in age at clinical or screening onset of MTC or phaeochromo-

1052

R. V. THAKKER AND B. A. J. PONDER (a) 10090A

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MULTIPLE ENDOCRINE NEOPLASIA

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cytoma between males and females or in paternally or maternally transmitted gene carriers.

New mutations in MEN2a New mutations to the MEN2a phenotype are probably uncommon. The reasons for thinking so are: (1) there have been no reported properly documented cases; (2) the reproductive disadvantage of MEN2a appears to be slight, so that only a low frequency of new mutations would be needed to maintain the gene pool; and (3) the incomplete penetrance of the gene can account for the majority of cases in which a small family is traced back to apparently unaffected ancestors (Ponder et al, 1988b; Telenius-Berg et al, 1984; Easton et al, 1989).

Molecular genetics of MEN2 Mapping the location of the MEN2a gene The gene for MEN2a has been mapped by genetic linkage to chromosome 10, near the centromere (Mathew et al, 1987; Simpson et al, 1987). The first clue to the location of the gene came from an 'exclusion map', made by combining all the data available from different laboratories at the stage when about one-third of the genome had been tested without a clearly positive result: this gave a very strong indication that the gene lay on chromosome 10 (Mathew et al, 1987). The first positive linkage result localized the MEN2a gene to within a large region around the retinolbinding protein locus (RBP3) including perhaps 30 million base pairs of DNA or roughly 20% of chromosome 10 (Figure 9). A great deal of work is needed to move from this to the precise identification and cloning of the gene. At the time of writing, this is still in progress. The first step is to identify a large set of DNA markers (DNA probes which show polymorphism and can be used in gene mapping, see above) which span the region of interest. A detailed genetic map is constructed by analysing the pattern of inheritance of each of these markers in relation to each of the others in the large set of 'reference' families which has been compiled for world-wide use in human gene mapping. These families do not have any genetic disease, but have been chosen simply for their size and suitability for genetic linkage. Because the same set of families is used by all investigators, each new DNA marker can rapidly be mapped against the data accumulated from all previous markers. The genetic map constructed in this way is based on frequencies of genetic recombination between markers. It must also be 'anchored' to the physical structure of the chromosome, by localizing selected DNA markers either by in situ hybridization to chromosome preparations, or by scoring their presence or absence in human-rodent somatic cell hybrids which contain different defined fragments of human chromosome 10. Finally, the pattern of inheritance of the DNA marker is also analysed in MEN2 families, and in this way the MEN2 gene is located on the genetic map (for review, see White et al, 1985).

1054

R . V . THAKKER AND B. A. J. PONDER Maximum

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Figure 9. Linkage between the loci for MEN2a and the retinol-binding protein (RBP3) on chromosome 10 in two families. Family S showed no recombinants: the most likely genetic distance between the loci is therefore zero and the LOD score is at a maximum at 0 = 0. Family D showed one recombinant in 15 meioses scored. Very close linkage of the loci in this family is therefore likely, and the most probable distance between them is expressed as a recombination fraction of 1/15 or 6.7% (0 = 0.067). The data from the two families can be added, to give the combined LOD score shown. Whereas neither family alone gives the LOD score of + 3 necessary to establish linkage, the combined data do. The combined LOD score reaches a maximum of 3.89 at 0 = 0.04, which represents the most probable distance of MEN2a from RBP, but because this estimate is based on the occurrence of a single recombinant, the confidence limits are wide. (These are usually expressed as the '1 LOD interval': the recombination fraction at a LOD score 1 unit either side of the maximum.)

A s u m m a r y of the current results from the laboratory of one of the authors ( B . A . J . P . ) , in collaboration with D r Y. N a k a m u r a , is shown in Table 2 (Ponder et al, 1989; N a k a m u r a et al, 1989). The most p r o b a b l e position of the M E N 2 locus is between the markers RBP3 and 14.34 which are on the long and short arms respectively on c h r o m o s o m e 10. This is 60-fold m o r e likely than the next most likely position for the gene, distal to RBP3 on the long arm. The physical distance between RBP3 and 14.34 is difficult to estimate because it includes the centromere: it m a y be several million base pairs of D N A . T h e r e is thus some way to go before the M E N 2 a gene is identified and cloned. Already, however, Table 2 shows that there are three D N A m a r k e r s within 5% recombination of the M E N 2 a locus, which suggests that inheritance of these markers can be used to predict with an accuracy of 95% or better which individuals in a family have or have not inherited the M E N 2 a gene. T h e application of D N A markers to genetic counselling and managem e n t of M E N 2 a families is discussed in m o r e detail below.

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Table 2. Linkage between the MEN2a locus and markers on chromosome 10. Marker TB14.34 RBP3 MCK2 TBQ16 TB10.163

Maximum LOD score

Most probable recombination distance*

3.61 18.10 19.48 5.63 5.48

0.07 0.024 0.011 0.029 0.12

* Assuming equal recombination frequency in males and females.

Are all MEN2 families the result of inherited mutations at the same locus? So far, over 30 MEN2a families have been examined for linkage to chromosome 10 markers world-wide, and none has been reported to show evidence against linkage. It is probable, therefore, that all MEN2a is due to mutations at this locus, but there remains a possibility that a second predisposing gene will eventually be found in some families. Preliminary evidence (Jackson et al, 1988; Noll et al, 1988) indicates that the MEN2b and MTC-only phenotypes also show linkage to the same chromosome 10 markers as MEN2a, suggesting that each may be due to different mutant alleles at the same locus. Genetic events in tumour development in MEN2 The nature of the inherited mutation. It is still not clear whether turnout development in MEN2 conforms to the genetic mechanism defined for retinoblastoma (and suggested for MEN1 and for several others of the inherited cancer syndromes), which requires loss of function mutations of both alleles at the disease locus (see above). If it were so, one would expect that DNA from tumours from cases of familial and sporadic MTC and phaeochromocytoma would show losses of marker alleles on chromosome 10 in the region of the MEN2 locus, as has been found for chromosome 11 in tumours related to MEN1. Using DNA markers on both the long and short arms of chromosome 10, however, we have found only one example of allele loss (involving the entire chromosome) in 42 tumours from which informative data were obtained (Ponder et al, 1989). Two interpretations are possible. Possibly MEN2 does share the same genetic mechanism as retinoblastoma and MEN1, but the event which leads to the loss of the second allele in the tumour cells is almost always localized to the MEN2 gene itself or a limited chromosomal region, and so has escaped detection by the markers we have used. In retinoblastoma, some of the events which lead to allele loss involve mitotic recombination in somatic cells (Cavenee et al, 1983). This requires a chromosomal exchange between the disease gene and the centromere. Because the MEN2 gene is apparently very close to the centromere, such events would be rare. Large scale deletions, which would also be detected as allele losses with existing probes, might be selected against if there were another gene, close to the MEN2 locus, hemizygous loss of which caused reduced viability of the cell. The occurrence of at least one case with complete chromosome 10 loss, and the frequent occurrence of

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THAKKER A N D B. A. J. PONDER

chromosome 10 losses in gliomas (Bigner et al, 1988) (also a cell type of neuroectodermal origin) argues against this explanation. The alternative is that the MEN2 mutation is not a recessive loss of function mutation, but is truly dominant at the cellular level, in which case loss of the corresponding allele in turnout cells would not be expected. The question will only be resolved by analysis with more closely linked probes, possibly not until there are probes for the MEN2 gene itself. Genetic events subsequent to the inherited mutation. These might include oncogene activation, gene amplification, or losses of recessive 'turnout suppressor genes' (Ponder, 1988) at loci other than the inherited mutation. The only oncogenes so far to be expressed in neoplastic but not normal C cells are n-myc and (in one case only) c-myc (Boultwood et al, 1988). There are no reports of gene amplification, nor of consistent chromosomal abnormalities in tumour cells. Consistent allele losses have, however, been found on chromosomes lp and 22 in MTC and phaeochromocytoma (Takai et al, 1987; Ponder et al, 1989); 10 of 28 informative tumours so far reported showing losses on lp and 5 of 24 on 22. Over 200 comparisons covering 12 other chromosomes showed losses in only three cases, which suggests that the chromosome lp and 22 losses are indeed significant. So far, the chromosome lp losses have not been localized to a chromosomal region: it is possible that loss of the entire short arm occurs, with formation of an isochromosome lq (Tanaka et al, 1987). Both the chromosome lp and 22 losses are found in primary thyroid and adrenal turnouts, so their presence is not directly related to malignancy (the adrenal tumours are benign) nor to metastasis (because the changes are present in the majority of cells in the primary MTC). It is not known whether they are present at the stage of C cell hyperplasia.

Animal models of MEN2

An animal model might greatly speed the identification of the MEN2 gene and the elucidation of its biology. Unfortunately, no proven model is available. Certain inbred strains of rat show a high incidence of spontaneous endocrine tumours, which include MTC and phaeochromocytoma, but also pituitary tumours (Sass et al, 1975; DeLellis et al, 1979, 1986). Breeding studies to prove that the turnout susceptibility is the result of a single dominant gene have not been reported; if this can be established, it will be necessary to show that the gene which is involved lies within a region of chromosomal homology with the MEN2 gene in man. The combination of MTC and phaeochromocytoma is also common in a breed of American cattle (Ljungberg and Nilsson, 1985): the bulls but not the cows develop MTC, which suggests that calcium balance might be important. Methods of induction of C cell tumours in mice by combination of radioiodine and dietary supplements of vitamin D or calcium have been described (Thurston and Williams, 1982) but their relevance to the pathogenesis of tumours in MEN2 is unclear.

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Family screening using biochemical and DNA markers Estimation o f individual risks

Early diagnosis by screening will reduce morbidity and mortality from MEN2 (Telenius-Berg et al, 1984; Gagel et al, 1988). Biochemical screening by pentagastrin test, however, does involve a commitment to repeated tests from the age of 5 to at least 30 years; the test is mildly unpleasant, and false positive results do occur. The decision to screen is therefore not trivial It is usually based on the estimated risk to the individual, derived from the family history. Unfortunately, because of the incomplete penetrance of the MEN2a gene, the family history may be seriously misleading (Ponder et al, 1988a). Thus, in the family shown in Figure 7, if the affected individual 111-5 had presented in isolation she could easily have been mistaken for a truly sporadic case because neither parent has evident disease. Conversely, having identified 111-1 as a familial case by history, one could easily decide to omit the branch descended from II-3 from screening if III-5 had yet to present, because the grandparent I1-2 at the head of the branch is unaffected. The probability that an individual in a known MEN2 family, or a relative of an apparently sporadic case, has inherited the MEN2 gene must therefore be calculated taking into account the effect of the age-related penetrance on the probability that one or more close relatives may be unaffected gene carriers. For example, the calculation of the probability that II-2 in Figure 7 is a gene carrier is as follows:

1. 2.

Prior probability (genetic risk at birth) Conditional probability that II-2 would be apparently unaffected at age 64 (from Figure 7)

(1) x (2) 3.

Using Bayes' theorem, the probability that II-2 is a gene carrier

11-2 is a gene carrier

11-2 is not a gene carrier

0.5

0.5

0.45

1

0.23

0.5

=

0.23 0.23 + 0.5 32%

A full description with further examples, including the problem of the apparently sporadic case, is given in Ponder et al (1988b). The use o f D N A markers

A DNA marker for the defective MEN2 gene would clearly resolve the problem of risk estimation because those who had inherited or not inherited the gene, including apparently sporadic cases, could be unequivocally identified. It is likely that this will become possible within the next 5 years but at present identification of the MEN2 gene relies on the co-inheritance of linked DNA markers (RFLPs, see above) such as those listed in Table 2.

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R. V. THAKKER AND B. A. J. PONDER

aa[---]i I

I

aa[--']l

Ill

b

I

I

I

I

?

(32

D,

?

Figure 10. Predicted inheritance of the M E N 2 a gene by genetic linkage. I-2 and II-2 are proven cases of M E N 2 a . a, b are alleles at a m a r k e r locus, for example R B P 3 (see Figure 9). II-2 must have inherited an allele a from her unaffected father, and so her b allele is on the c h r o m o s o m e inherited from her m o t h e r which carries the M E N 2 a gene. H e r children III-1 and III-2 m u s t inherit an a from their unaffected father; if they inherit also an a from their m o t h e r (to be genotype aa) they will probably not have inherited the M E N 2 gene, whereas if they inherit the b allele, they probably will also have inherited the M E N 2 gene because it is close to b on the same chromosome.

The analysis is made on DNA from a single blood sample. The principles are illustrated in Figure 10: in this ideal case, the results in generation I and II show that marker allele b is linked to the MEN2 gene, and inheritance of this allele by a child in generation III predicts co-inheritance of the MEN2 gene with an accuracy which is determined by the probability of recombination between the marker locus and MEN2. For the closely linked marker RBP3, the probability of recombination is currently estimated at 2%, giving an accuracy of 98%; the 95% upper confidence limit on this estimate is 7%, giving a lower limit of accuracy of 93%. Simultaneous analysis of markers lying either side of the MEN2 gene can, in principle, provide even greater accuracy: co-inheritance of two such 'flanking' markers implies that the segment of DNA between them, including the MEN2 gene, has also been inherited, unless recombination has occurred simultaneously between each of the markers and the MEN2 locus. For the markers 14.34 and RBP3, at 7 and 2.4% recombination respectively, the likelihood of this (assuming each recombination to be an independent event) would be 0.07 × 0.024 = 0.0017, or 0.17%, giving an accuracy of prediction of > 99.8%. The ability to offer such genetic prediction in a given family will depend on the family structure and availability of blood samples from key individuals, and on the pattern of marker alleles being such that the inheritance of the chromosome carrying the MEN2 gene can be determined; if individual II-2 in Figure 10 had been homozygous aa, for example, prediction based on inheritance of this marker allele in her children would clearly be impossible. The minimum require-

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ment, of course, is two individuals of known disease status from whom the linkage of the marker allele to the MEN2 locus can be determined; prediction is not possible in a family with less than this minimum structure. Risk calculations are not always straightforward; clinicians wishing to use DNA markers in the management of an MEN2 family are strongly advised to seek the help of a clinical geneticist. When the probable MEN2 gene status of an individual has been determined, clinical decisions remain. Those with a low probability of being gene carriers can probably be excluded from screening, but the level of certainty which is required is a clinical judgement. Combined use of DNA and pentagastrin tests may be helpful; for example, 2.5% residual risk from a 'negative' DNA result with the RBP3 marker becomes a 0.5% risk when combined with a normal pentagastrin result at age 20. On the other hand, individuals predicted to be gene carriers clearly require regular and careful screening. Because of the wide range of age at clinical onset of MTC, however, it is unlikely that immediate thyroidectomy could be justified in the absence of biochemical evidence of C cell hyperplasia, unless compliance with screening is poor or there is anxiety resulting from a family history of aggressive disease.

Prospects The identification and cloning of the MEN2a gene is likely to be achieved within the next few years, and it will also become clear whether the MEN2a, MEN2b and MTC-only phenotypes are due to alleles at the same locus. Identification of individuals with the gene will become straightforward, but it is unlikely that this will solve the next question of clinical importance, which is the prediction of the likely natural history of the disease in a given individual. Cloning the MEN2 gene will open the way to the interesting biological questions of its normal function, and how altered function leads to disease. We may hope also to understand the reasons for the differences between MEN2a and MEN2b phenotypes, and for the benign behaviour of phaeochromocytomas contrasted with the malignant behaviour of MTC. CONCLUSION Clinical genetic and epidemiological studies have improved our understanding of the clinical varieties of MEN syndromes, their presentation, age at onset, diagnosis and patterns of occurrence within families. This information has already had an impact in terms of better screening and earlier diagnosis in MEN2 families, and the same is about to happen in MEN1. The location of the gene for MEN1 and MEN2 to chromosome 11 and 10 respectively is a major step forward which holds the promise that the genes themselves will be identified and cloned in the foreseeable future. This will refine family screening still further, allowing simple distinction of truly sporadic from familial cases, and it will be the starting point for a thorough understanding of the pathogenesis of these tumour syndromes.

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Acknowledgements R.V.T. is supported by the Medical Research Council. B.A.J.P. is supported by the Cancer Research Campaign. Much of the material in the section on MEN2 is based on data collected by the Cancer Research Campaign Medullary Thyroid Group. In particular we would like to acknowledge the collaboration of Dr M. Telenius-Berg, Dr S. Reichlin, Dr H. Hansen, Dr C. Buys, Dr G. Lenoir and their colleagues, and of Dr C. Mathew, Mr D. G. Easton and Mrs M. Ponder.

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Note added in proof Recent studies of parathyroid tumours from patients with M E N 1 have revealed abnormalities which are consistent with a two stage recessive mutation model involving the whole of c h r o m o s o m e 11. In addition, the gene causing M E N 1 has b e e n m a p p e d to the long arm region of chromosome 11, referred to as 11q13. This location was demonstrated by establishing linkage with the oncogene INT2, which has previously been localised to this region and is known to encode a protein of the fibroblast growth factor family. Thakker RV, Bouloux P, Wooding C et al (1989) Parathyroid tumours in multiple endocrine neoplasia type 1 are associated with chromosome 11 allele loss. New England Journal of Medicine (in press).