1. Multiple Endocrine Neoplasia Type 1

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1 1. Multiple Endocrine Neoplasia Type 1 Rajesh V. Thakker, MD, FRCP, FRCPath, FMedSci Clinical Features and Diagnosis Multiple endocrine neoplasia type 1 (MEN1), which is inherited as an autosomal dominant disorder, is characterized by the combined occurrence of tumors of the parathyroid glands, pancreatic islet cells, and anterior pituitary gland ( 1, 2 ). Some patients may also develop adrenal cortical tumors, foregut carcinoid tumors, facial angiofibromas, collagenomas, and lipomas ( 3, 4 ). Parathyroid tumors are the first manifestation of MEN1 in more than 85% of patients, and in the remaining less than 15% of patients, the first manifestation may be an insulinoma or prolactinoma ( 4 7 ). Pancreatic islet cell neuroendocrine tumors (NETs), which consist of gastrinomas that are associated with excessive gastric acid production and severe peptic ulceration (Zollinger-Ellison syndrome [ZES]), insulinomas, pancreatic polypeptidomas, glucagonomas, vasoactive intestinal polypeptidomas and nonfunctioning (ie, nonsecreting) tumors occur in about 40% of patients. Anterior pituitary NETs, which consist of prolactinomas, somatotrophinomas, corticotrophinomas, or nonfunctioning adenomas, occur in about 30% of patients ( 4 7 ). Combinations of these affect ed glands and their pathologic features (for example, hyperplasia or single or multiple adenomas of the parathyroid glands) have been reported to differ in members of the same family ( 4 7 ) and even between identical twins ( 8, 9 ). The incidence of MEN1 has been estimated from randomly chosen postmortem studies to be 0.25% and to be 1% to 18% among patients with primary hyperparathyroidism, 16% to 38% among patients with gastrinomas, and less than 3% among patients with pituitary tumors ( 2 ). The disorder affects all age groups, with a reported age range of 5 to 81 years, with clinical and biochemical manifestations of the disorder having developed in 80% and more than 98% of patients, respectively, by the fifth decade ( 4 7 ). Acknowledgment: I am grateful to the Medical Research Council (UK) for support and to Mrs. Tracey Walker for typing the manuscript. Translational Endocrinology & Metabolism, Volume 2, Number 4,

2 The clinical manifestations of MEN1 associated tumors and their diagnoses are generally related to their products of secretion ( Table 1-1 ). A diagnosis of MEN1 may be established in an individual by 1 of 3 criteria ( Figure 1-1 ). Thus, MEN1 may be clinically diagnosed in an individual on the basis of the occurrence of 2 or more MEN1 associated endocrine tumors ( Figure 1-1 ) ( 7, 10 ). In addition, a diagnosis of familial MEN1 is established for an individual who has the occurrence of 1 of the MEN1 associated tumors and is a first-degree relative of a patient with a clinical diagnosis of MEN1. Finally, a genetic diagnosis of MEN1 is made on identification of a germline MEN1 mutation in an individual who may be asymptomatic and has not yet developed any of the serum biochemical or radiological abnormalities indicative of tumor development ( Figure 1-1 ) ( 10 ). Current Treatments and Emerging Therapies In the absence of treatment, endocrine tumors have been observed to be associated with an earlier mortality in patients with MEN1 ( 11, 12 ). Thus, patients with MEN1 have a decreased life expectancy, with a 50% probability of death by the age of 50 years, and 50% to 70% of patients with MEN1 will die as a result of a malignant tumor process or sequelae of the disease ( 11, 12 ). Although the prognosis of patients with MEN1 has improved considerably following the introduction of acid-suppressive TABLE 1-1. Summary of Biochemical and Radiological Screening Guidelines in Individuals at High Risk of Developing MEN1 Tumor Age to Begin (Years) Biochemical Test (Annually) Parathyroid 8 Calcium, PTH None Gastrinoma 20 Gastrin (±gastric acid output, ±secretinstimulated gastrin) Imaging Test (Every 3 Years) None Insulinoma 5 Fasting glucose, insulin None Other enteropancreatic 20 Chromogranin A, glucagon, proinsulin Anterior pituitary 5 Prolactin, IGF-1 MRI Foregut carcinoid 20 None CT CT, MRI, or 111 In-DTPA octreotide scan Abbreviations: CT, computed tomography; IGF-1, insulin growth factor-1; MRI, magnetic resonance imaging PTH, parathyroid hormone. [Reproduced with permission from Brandi et al. ( 7 ) The Endocrine Society] 14 Translational Endocrinology & Metabolism: Neoplasia Update

3 BASIS FOR MEN1 DIAGNOSIS CLINICAL FAMILIAL GENETIC A patient with 2 or more MEN1- associated tumours A patient with 1 MEN1- associated tumour and a first degree relative with MEN1 An individual who has an MEN1 mutation but does not have clinical or biochemical manifestations of MEN1 i.e. a mutant gene carrier FIG 1-1. Basis for a diagnosis of MEN1 in individuals. A diagnosis of MEN1 based on clinical and familial criteria may be confounded by the occurrence of phenocopies. [Reproduced with permission from Turner et al. (10) John Wiley & Sons] therapies for the treatment of gastrinoma and the ZES, it is nevertheless noteworthy that a significant proportion of individuals with MEN1 still die prematurely of MEN1 related malignancies ( 12 ). For example, studies of MEN1 related mortality have demonstrated that 28% to 46% of deaths are directly related to MEN1, most commonly as a result of malignant pancreatic NETs and foregut carcinoids ( 12, 13 ). In addition, these deaths occur at a significantly younger age than those occurring in unaffected individuals ( 11, 12 ). Furthermore, a multicenter study from France and Belgium has suggested that 70% of individuals with MEN1 currently die of causes directly related to MEN1 ( 14 ). In particular, malignant pancreatic NETs and thymic carcinoid tumors were associated with a marked increased in risk of death (hazard ratio >3, P <.005) ( 14 ). Thus, such studies highlight the shift in MEN1 associated mortality that has occurred from gastrinoma associated with ZES, to other MEN1 associated malignant tumors. The treatment for each type of MEN1 associated endocrine tumor is generally similar to that occurring in non MEN1 patients. However, the treatment outcomes of MEN1 associated tumors are not as successful as those in non MEN1 patients, for 2 main reasons. First, the MEN1 Multiple Endocrine Neoplasia Type 1 15

4 tumors, with the exception of pituitary NETs, are multiple, thereby making it difficult to achieve a successful surgical cure. Second, pancreatic and pituitary NETs in MEN1 patients are larger, more aggressive, and resistant to treatment. In addition, pancreatic NET metastatic disease is present in 50% of patients at presentation, and their lower cell division rate makes them less responsive to chemotherapy and radiotherapy ( 15 ). These difficulties encountered in treating MEN1 associated endocrine tumors are further illustrated by a review of the current treatments for parathyroid tumors, gastrinomas, and nonfunctioning pancreatic NETs. Treatments for Parathyroid Tumors Surgical removal of the overactive parathyroid tumors in patients with MEN1 is the definitive treatment, but it is controversial whether to perform subtotal or total parathyroidectomy and whether it should be performed at an early or late stage. Minimally invasive parathyroidectomy is not recommended ( 2, 7 ), as all 4 parathyroid glands are usually affected with multiple adenomas or hyperplasia ( 2, 5, 7 ). This histologic distinction may be difficult, however, and subtotal Minimally invasive parathyroidectomy for primary hyperparathyroidism in patients with MEN1 has been parathyroidectomy is not recommended in MEN1 associated with a high failure rate ( 7, ). patients, as all 4 parathyroid Subtotal parathyroidectomy (ie, removal glands are usually affected with of 3.5 glands) has resulted in persistent or multiple adenomas or recurrent hypercalcemia, within 10 to 12 years hyperplasia. after surgery, in 40% to 60% of patients, and in hypocalcemia requiring long-term therapy with vitamin D or its active metabolite calcitriol in 10% to 30% of patients with MEN1 ( ). These rates are markedly higher than those observed for parathyroidectomies in patients who do not have MEN1, in whom recurrent hypercalcemia occurs in 4% to 16% of patients and hypocalcemia in 1% to 8% of patients ( 18, 19 ). To avoid neck re-exploration, which is difficult, and to improve the treatment of primary hyperparathyroidism in patients with MEN1, total parathyroidectomy with autotransplantation of parathyroid tissue in the forearm has been performed ( 16, 20, 21 ). Both fresh parathyroid tissue and cryopreserved parathyroid tissue have been used for autotransplantation. The use of cryopreserved parathyroid tissue allows confirmation of hypoparathyroidism in the patient before autotransplantation, but unfortunately, only 50% of parathyroid grafts survive cryopreservation. The use of fresh parathyroid tissue for autotransplantation in the forearm 16 Translational Endocrinology & Metabolism: Neoplasia Update

5 results in viable grafts that secrete parathyroid hormone (PTH). However, the presence of functioning autotransplanted parathyroid tissue leads to recurrent hypercalcemia in more than 50% of patients with MEN1, and surgical removal of transplanted grafts has been required. Thus, management of primary hyperparathyroidism in patients with MEN1 is difficult; parathyroid surgery in these patients is associated with a higher prevalence of persistent or recurrent hypercalcemia. Total parathyroidectomy, which would prevent such recurrence, has therefore been suggested as a treatment for primary hyperparathyroidism in MEN1, with the resultant life-long hypocalcemia being treated with oral calcitriol (1,25-dihydroxyvitamin D) ( 2 ). However, the management of hypoparathyroidism can be challenging in some patients, even with the use of vitamin D and calcium replacement. One recommendation is that parathyroidectomy be reserved for symptomatic hypercalcemic patients with MEN1 and that asymptomatic hypercalcemic patients with MEN1 not have parathyroid surgery but have regular assessment for the onset of symptoms and complications, at which time total parathyroidectomy (or 3.5 gland parathyroidectomy), together with a possible transcervical, near-total thymectomy, should be undertaken ( 2, 16, 17 ). The type of surgery (ie, subtotal or total parathyroidectomy, Cinacalcet, which acts via the with or without autotransplantation of parathyroid tissue) and its timing need careful (CaSR) to decrease secretion of calcium-sensing receptor consideration, and factors such as the surgical experience, the availability of facilities patients in whom surgery for PTH, has been efficacious in for long-term regular serum calcium monitoring, the accessibility of calcitriol (or possible or had failed. hyperparathyroidism was not vitamin D analogues), and the preferences of the patient should be taken into account. Medical treatment with the calcimimetic Cinacalcet, which acts via the calcium-sensing receptor (CaSR) to decrease secretion of PTH, has been efficacious in patients in whom surgery failed to treat the hyperparathyroidism or in whom surgery was not possible because of a high surgical risk due to other existing medical conditions ( 22 ). Treatment for Gastrinomas Medical treatment of patients with MEN1 and ZES is directed toward reducing basal acid output to less than 10 mmol/l, and such reduced acid output may be achieved by parietal cell H + -K + -adenosine triphosphatase inhibitors (eg, omeprazole or lansoprazole), which have proved efficacious and become the drugs of choice for gastrinomas ( 2, 23 ). Some Multiple Endocrine Neoplasia Type 1 17

6 patients may also require additional treatment with the histamine H 2 receptor antagonists cimetidine or ranitidine ( 2, 23 ). The role of surgery in the treatment of gastrinomas in patients with MEN1 is controversial ( ). The ideal treatment for a nonmetastatic gastrinoma situated in the pancreas is surgical excision of the gastrinoma. In addition, duodenal gastrinomas, which occur more frequently in patients with MEN1, have been treated successfully with surgery ( 27 ). However, in most patients with MEN1, gastrinomas are frequently multiple or extrapancreatic, and with the exception of duodenal gastrinomas, surgery has often not been successful ( 25 ). For example, the results of 1 study revealed that only 16% of patients with MEN1 were free of disease immediately after surgery, and at 5 years, this number had decreased to 6%; the respective outcomes in patients without MEN1 were better, at 45% and 40% ( 28 ). Given these findings, most physicians and about 50% of surgeons recommended a nonsurgical management for gastrinomas in MEN1 ( 2, 7 ). The use of tumor localization studies involving ultrasonography, endoscopic ultrasonography, computed tomography (CT), nuclear magnetic resonance imaging (MRI), selective abdominal angiography, selective arterial secretagogue (eg, with secretin or calcium gluconate) with hepatic venous gastrin sampling, or somatostatin receptor scintigraphy are >2 cm has been Surgery for gastrinomas that has demonstrated that these techniques help recommended, as the to improve the surgical success rate ( 29 ). disease-related survival in Surgery for gastrinomas that are >2 to 2.5 cm these patients was reported to has been recommended, as the diseaserelated survival in these patients was reported surgery. be improved following to be improved following surgery ( 30 ). Total gastrectomy is rarely undertaken now and would perhaps be considered only for persistently noncompliant patients. Treatment of disseminated gastrinomas is difficult, and chemotherapy with streptozotocin and 5-fluorouracil; hormonal therapy with octreotide or lanreotide, which are human somatostatin analogues; hepatic artery embolization; administration of human leukocyte interferon, and removal of all resectable tumor have all been successful occasionally ( 2 ). Clinical Management of Nonfunctioning Pancreatic NETs Nonfunctioning pancreatic NETs have been reported to occur in asymptomatic patients who are <15 years of age ( 13 ). The identification of nonfunctioning pancreatic NETs is of clinical importance for the following reasons. Firstly, malignant pancreatic NETs are now reported to be 18 Translational Endocrinology & Metabolism: Neoplasia Update

7 the most common cause of death in individuals with MEN1 ( ). Secondly, nonfunctioning tumors are increasingly recognized, with recent studies indicating that these tumors are the most common enteropancreatic NET associated with MEN1 and that they are associated with a worse prognosis than other functioning tumors, including insulinoma and gastrinoma ( 31, 32 ). Finally, the absence of both a clinical syndrome and specific biochemical abnormalities may result in a delayed diagnosis of nonfunctioning pancreatic NETs in the absence of radiological assessment ( 15 ). The optimum screening method and its timing interval remain to be established. For example, comparison of imaging modalities for the detection of pancreatic NETs has demonstrated that endoscopic ultrasound is likely to represent the most sensitive method of detecting small pancreatic tumors, while somatostatin receptor scintography is the most reliable method for detecting metastatic disease ( 31 ). However, the ability to undertake regular endoscopic ultrasound for screening will depend on availability of local resources and will likely represent an increased expense. Furthermore, the clinical significance of small pancreatic tumors (eg, <1 cm) in asymptomatic individuals is yet to be fully evaluated, as most centers advocate surgery for tumors >2 cm ( 33 ). Indeed, the management of nonfunctioning pancreatic NETs in the asymptomatic patient is controversial ( 7, 13, 15 ). Thus, 1 recommendation is to undertake surgery irrespective of tumor size once an unequivocal diagnosis is made, Malignant pancreatic NETs are and another recommendation is to undertake surgery if the imaged tumor is 1 cm. common cause of death in not reported to be the most Yet another recommendation is to undertake surgery only if the tumor is larger than MEN1 patients. 2 cm or is growing ( 24 ). Pancreatoduodenal surgery may be successful in removing the tumors in 80% of patients, but more than 40% of patients will develop complications that include diabetes mellitus, frequent steatorrhoea, early and late dumping syndromes, and other gastrointestinal symptoms ( 26 ). However, between 47% and 60% of patients were alive at a follow-up period of 5 years or more, and global quality of life (QOL) assessments in the surgically treated patients were similar to those in the reference population ( 25, 26 ). These studies suggest that early diagnosis and surgical removal of pancreatic NETs in MEN1 patients prolongs survival, without significant comprise in patient-perceived QOL ( 25, 26 ). When considering these recommendations, it is important to consider that occult metastatic disease (ie, tumors not detected by imaging investigations) is likely to be present in a substantial proportion of these patients at the time of presentation ( 15 ). Multiple Endocrine Neoplasia Type 1 19

8 Emerging Therapies for Pancreatic NETs Studies of pancreatic NETs have revealed that they may express the tyrosine kinase receptor (TKRs), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) ( 34, 35 ). Additionally, they have insulin-like growth factor-mediated autocrine activation of the mammalian target of rapacycin (mtor) signaling pathway, which is a serine-threonine kinase that stimulates cell growth proliferation and angiogenesis. Two studies have recently reported on the efficacy of treatments with a TKR and mtor inhibitor ( 34, 35 ). Treatment of patients with advanced, well-differentiated pancreatic NETs with sunitimib malate, which inhibits TKRs, lead to an increase in overall survival and a doubling in progression-free survival when compared with patients receiving a placebo (11.4 months versus 5.5 months, P <.001) ( 34 ). Treatment of patients with advanced, low-grade, or intermediate-grade pancreatic NETs with everolimus, which is an inhibitor of mtor, also lead to a doubling in the median progression-free survival when compared with patients treated with a placebo (11.0 months versus 4.6 months, P <.001) ( 35 ). These 2 studies mainly included non MEN1 patients; for example, in the sunitimib study, which had 171 patients, there were only 2 MEN1 patients, and neither was in the treatment arm ( 34 ). In the everolimus study, which had 410 patients, details of the presence or absence of MEN1 were not provided ( 35 ). Nevertheless, these 2 studies represent major advances in the treatment of pancreatic NETs in non MEN1 patients, and it seems highly plausible that their results can be extrapolated to MEN1 patients with pancreatic NETs. Genetics and Molecular and Cellular Mechanisms MEN1 Gene, Mutations, and Polymorphisms The MEN1 gene is located on chromosome 11q13 and consists of 10 exons and encodes a 610-amino acid protein, Menin ( 36, 37 ) ( Figure 1-2 ). The inheritance of a germline MEN1 mutation predisposes an individual to developing a tumor that arises following a somatic mutation, which may be a point mutation or a deletion, leading to loss of heterozygosity (LOH) in the tumor DNA, consistent with the Knudson 2-hit hypothesis and a tumor suppressor role of Menin ( 5, 38 ). Thus, the non tumor cells of a patient will be heterozygous in having the wild-type (normal) and mutant alleles of the MEN1 gene, while the tumor cells, which will have LOH, will be homozygous for the mutant MEN1 alleles. MEN1 is 20 Translational Endocrinology & Metabolism: Neoplasia Update

9 inherited as an autosomal-dominant disorder, but a nonfamilial (ie, sporadic) form may have developed in 8% to 14% of patients with MEN1, and molecular genetic studies have confirmed the occurrence of de novo mutations of the MEN1 gene in 10% of patients with MEN1 ( 2 ). Mutations of the MEN1 gene (see Figure 1-2 ) have been characterized, and 1336 mutations (1133 germline and 203 somatic mutations) have been reported in the first decade following the identification of the gene ( 3 ). The 1133 germline mutations of the MEN1 gene, which consist of 459 different mutations are scattered throughout the entire 1,830-bp coding region and splice sites of the MEN1 gene ( 3 ) (see Figure 1-2 ). Most (75%) of the MEN1 germline mutations are inactivating and are consistent with those expected in a tumor suppressor gene ( 3 ). Approximately 23% are nonsense mutations; around 41% are frameshift deletions or insertions. Six percent are in-frame deletions or insertions, 9% are splice site mutations, and 20% are missense mutations. One percent are whole or partial gene deletions ( 3 ). However, some of the germline mutations have been observed to occur several times in unrelated families (see Figure 1-2 ). Mutations at 9 sites in the MEN1 gene accounted for over 20% of all the germline mutations ( Figure 1-2 ). More than 10% of the MEN1 germline mutations arise de novo and may be transmitted to subsequent generations ( 3 ). It also is important to note that between 5% and 10% of patients with MEN1 may not harbor germline mutations in the coding region of the MEN1 gene ( 3 ), and that these individuals may have gene deletions or mutations in the promoter or untranslated regions, which remain to be investigated ( 3 ). One study showed that 33% of patients who do not have germline mutations within the coding region have large deletions involving complete exons ( 39 ). Such abnormalities will not be easily detected by DNA sequence analysis. The germline MEN1 mutations are typically detected in leukocyte (or other non-tumor) cells that are heterozygous for the wild type and mutant MEN1 alleles, consistent with an autosomal dominant inheritance of the disorder. However, studies of an Italian kindred have reported on the possible existence of 2 affected individuals who may be homozygous for MEN1 mutant alleles ( 40 ). The 2 individuals affected with MEN1 were children of parents who both had MEN1, and apart from infertility had MEN1 phenotypes that were similar to those observed in other MEN1 patients ( 40 ). Haplotype analysis using polymorphic loci from chromosome 11q13 revealed that these 2 children had inherited the affected haplotype from each parent, thereby raising the possibility that they were either homozygous ( 40 ) or compound heterozygous for mutant MEN1 alleles. An alternative explanation may be that 1 of the parents does not Multiple Endocrine Neoplasia Type 1 21

10 A I II III IV V VI VII IX VIII Mutation frequency 5% ab c d ef gh i j k l m no p qrs t u v w x 0% ATG TGA B Menin G1 G2 G3 G4 G5 NLS1 NLSa NLS2 C Menin interacting proteins: JunD NF- B Smad3 Pem NM23H1 RPA2 NMHC II-A FANCD2 msin3a HDAC1 ASK CHEST1 FIG 1-2. Schematic representation of the genomic organization of the MEN1 gene, its encoded protein (Menin), and regions that interact with other proteins. (A) The human MEN1 gene consists of 10 exons that span more than 9 kb of genomic DNA and encode a 610-amino acid protein. The 1.83-kb coding region (indicated by shaded region) is organized into 9 exons (exons 2-10) and 8 introns (indicated by a line but not to scale). The sizes of the exons (boxes) range from 41 to 1297 bp, and the sizes of the introns range from 80 to 1564 bp. The start (ATG) and stop (TGA) codons in exons 2 and 10, respectively, are indicated. Exon 1, the 5 part of exon 2, and the 3 part of exon 10 are untranslated (indicated by open boxes). The promoter region is located within a few 100 base pairs (bp) upstream of exon 2. The sites of the 9 germline mutations (I to IX) that occur with a frequency >1.5% are shown, and their respective frequencies (scale shown on the right) are indicated by the vertical lines above the gene. These germline mutations, which collectively represent 20.6% of all reported germline mutations, are: I- c.249_252delgtct; II- c.292c>t; III- c.358_360delaag; IV4- c.628_631delacag; V- c.784-9g>a; VI- c. 1243C>T; VII- c.1378c>t; VIII- c.1546delc; and IX- c.1546_1547insc. The locations of the 24 polymorphisms (a-x) are illustrated. (B) Menin has 3 nuclear localization signals (NLSs) at codons (NLS1), (NLSa), and (NLS2), indicated by closed boxes, and 5 putative guanosine triphosphatase sites (G1-G5), indicated by closed bars. (C) Menin regions that have been implicated in the binding to different interacting proteins are indicated by open boxes. These are JunD (codons 1-40, , ); continued 22 Translational Endocrinology & Metabolism: Neoplasia Update

11 FIG 1-2. (Continued) nuclear factor-kappa B (NF- κ B) (codons ); Smad3 (codons , ); placenta and embryonic expression (Pem) (codons ); NM23H1 (codons 1-486); a subunit of replication protein A (RPA2) (codons 1-40, ); non-muscle myosin II-A heavy chain (NMHC II-A) (codons ); FANCD2 (codons ); msin3a (codons ); histone deacetylase-1 (HDAC1) (codons ); activator of S-phase kinase (codons ) and CHES1 (codons ). The regions of Menin that interact with glial fibrillary acidic protein, vimentin, Smad 1/5, Runx2, MLL-histone methyltransferase complex and estrogen receptor-alpha remain to be determined. [Reproduced with permission from Lemos and Thakker ( 3 ) John Wiley & Sons] have MEN1, but represents a phenocopy ( 10 ). However, mutational analysis of the MEN1 gene to confirm these possibilities has not been reported from these children and their family. It would be important to undertake this mutational analysis in these children, as germline homozygosity for MEN1 loss in mouse models has been reported to result in death in early embryonic life ( 41, 42 ); thus if homozygosity (or compound heterozygosity) for mutant MEN1 alleles is demonstrated in these children, it would highlight important differences between MEN1 in humans and the mouse model. Twenty-four different polymorphisms (12 in the coding region [10 synonymous and 2 non-synonymous], 9 in the introns, and 3 in the untranslated regions) of the MEN1 gene have been reported (see Figure 1-1 and Table 1-2 ) ( 3 ). It is important to recognize the occurrence of these polymorphisms, as they need to be distinguished from mutations when performing analysis for genetic diagnosis, and also because they may occasionally help in segregation analysis in families in whom a MEN1 mutation has not been identified. More than 90% of tumors from MEN1 patients have LOH, and this has generally been taken as evidence that the MEN1 gene acts as a tumor suppressor gene, consistent with Knudson s 2-hit hypothesis ( 3, 5 ). However, this LOH represents only 1 mechanism by which the second hit may occur, with the other mechanisms being intragenic deletions and point mutations. MEN1 tumors (eg, parathyroid tumors, insulinoma, and lipoma) that do not have LOH have been shown to harbor different somatic and germline mutations of the MEN1 gene, and this is consistent with the Knudson 2-hit hypothesis ( 38 ). Correlations between MEN1 mutations and clinical manifestations of the disorder appear to be absent. For example, a detailed study of 5 unrelated families with the same 4-bp deletion in codons 210 and 211 revealed a wide range of MEN1 associated tumors ( 1, 3, 43 ); all affected family Multiple Endocrine Neoplasia Type 1 23

12 TABLE 1-2. MEN1 Associated Tumors in 5 Unrelated Families with a 4-bp Deletion at Codons 210 and 211 Tumor Family Parathyroid Gastrinoma Insulinoma + Glucagonoma + Prolactinoma Carcinoid + + presence of tumors; absence of tumors. [Reproduced with permission from Thakker ( 1 ) The Endocrine Society] members had parathyroid tumors, but members of families 1, 3, 4, and 5 had gastrinomas, whereas members of family 2 had insulinomas. In addition, prolactinomas occurred in members of families 2, 3, 4, and 5 but not in family 1, which was affected with carcinoid tumors ( 1 ). Another study of 7 unrelated families with the same g a novel acceptor splice site mutation in intron 4 revealed a similarly wide range of MEN1 associated tumors and a lack of genotype/phenotype correlation ( 44 ). MEN1 Mutations in Sporadic Non MEN1 Endocrine Tumors Parathyroid, pancreatic islet cell, and anterior pituitary tumors may occur either as part of MEN1 or more commonly as sporadic, nonfamilial tumors ( 2 ). Tumors from patients with MEN1 have been observed to harbor the germline mutation together with a somatic LOH involving chromosome 11q13 ( 5 ), or point mutations, as expected from Knudson s model ( 38 ) and the proposed role of the MEN1 gene as a tumor suppressor. However, LOH involving chromosome 11q13, which is the location of MEN1, has also been observed in 5% to 50% of sporadic endocrine tumors, thus implicating the MEN1 gene in the etiology of these tumors ( 2, 3 ). From 1997 to 2007, 203 somatic MEN1 mutations were reported, and these occurred in several different endocrine tumors ( 3 ). Thus, these have been detected in 18% of sporadic parathyroid tumors (total number, n = 452), 38% of gastrinomas (n = 105), 14% of insulinomas (n = 43), 57% of vasoactive intestinal polypeptidomas (n = 7), 16% of nonfunctioning pancreatic tumors (n = 32), 60% of glucagonomas (n = 5), 2.0% of adrenal cortical tumors (n = 83), 35% of bronchial carcinoid tumors (n = 26), 24 Translational Endocrinology & Metabolism: Neoplasia Update

13 3.5% of anterior pituitary adenomas (n = 167), 10% of angiofibromas (n = 19), and 28% of lipomas (n = 8) ( 3 ). These somatic mutations are scattered throughout the 1830-bp coding region (see Figure 1-2 ), and 18% are nonsense mutations; 40% are frameshift deletions or insertions. Six percent are in-frame deletions or insertions, 7% are splice-site mutations, and 29% are missense mutations ( 3 ). A comparison of the locations of the somatic and germline mutations revealed a higher frequency (39% somatic versus 23% germline; P <.001) of somatic mutations in exon 2, but the significance of this observation in the context of the Knudson 2-hit hypothesis remains to be elucidated ( 3 ). The tumors harboring a somatic MEN1 mutation had chromosome 11q13 LOH as the other genetic abnormality, consistent with Knudson s hypothesis. A recent study, which determined the exomic sequence of protein coding genes in sporadic pancreatic NETs, reported that 44% of these tumors had somatic inactivating MEN1 mutations. Forty-three percent had mutations of the death domain associated protein (DAXX) and alpha thalassemia mental retardation syndrome, X-linked (ATRX) that encodes subunits of a transcription/chromatin remodeling complex, and 15% had mutations involving genes (PTEN, TSC2, and PIK3CA) in the mtor pathway ( 45 ). Furthermore, mutations of MEN1, DAXX/ATRX, or the combination of both MEN1 and DAXX/ATRX were associated with prolonged survival relative to those patients whose tumors lacked these mutations, such that 100% of patients with pancreatic NETs harboring MEN1 and/or DAXX/ ATRX mutations had a 10-year survival, whereas >60% of patients whose pancreatic NETs did not have these mutations died within 5 years ( 45 ). The results of this study have 2 important potential clinical implications: first, that MEN1, DAXX, and ATRX mutational analysis of pancreatic NETs may help in predicting survival; and second, mutational analysis of genes in the mtor pathway may help to stratify those patients for treatment with mtor inhibitors, such as everolimus ( 35, 45 ). MEN1 Mutations in Hereditary Endocrine Disorders The role of the MEN1 gene in the etiology of other inherited endocrine disorders, in which either parathyroid or pituitary tumors occur as an isolated endocrinopathy, has been investigated by mutational analysis ( 2 ). MEN1 mutations have been reported in 42 families with isolated hyperparathyroidism (FIHP), and 38% of these have been missense mutations; fewer than 31% have been nonsense or frame-shift mutations, which would result in a truncated and likely inactivated protein ( 3, 46 ). The mutations associated with FIHP are also scattered throughout the Multiple Endocrine Neoplasia Type 1 25

14 coding region and not clustered, a situation that is similar to that found for germline MEN1 mutations (see Figure 1-2 ). In the Burin or prolactinoma variant of MEN1, there is a high occurrence of prolactinomas and a low occurrence of gastrinomas ( ), and this is associated with nonsense MEN1 mutations (Tyr312Stop and Arg460Stop). The absence of somatotrophinomas in a MEN1 kindred from Tasmania has been associated with a splice mutation (c.446-3c g ). MEN1 Phenocopies and Mutation in Other Genes Studies have reported that 5% to 25% of patients with MEN1 may not have mutations of the MEN1 gene ( 3, 39, 43, 50 ). This variability in detecting MEN1 mutations among these studies may partly be attributable to the differences in methods used to identify the mutations; for example, most studies do not systematically examine for large gene deletions, which may be found in 33% of patients who do not have coding region mutations ( 39 ). In addition, this variability may be due to the ascertainment of the phenotype, as some studies have included nonfamilial (ie, sporadic) patients who may have developed only 2 (or fewer) endocrine tumors; the detection rate for MEN1 mutations in these patients was found to be <5% ( 50 ). Such patients with MEN1 associated tumors but without MEN1 mutations may represent phenocopies ( Figure 1-3 ), or they may have mutations involving other genes ( 3, 10 ). Phenocopy refers to the development of disease manifestations that are usually associated with mutations of a particular gene, but instead are due to another etiology ( 10, 47 ); the occurrence of phenocopies has been reported in 5% to 10% of MEN1 kindreds ( 10, 47 ). One example of such a phenocopy has been reported in a patient with hyperparathyroidism-jaw tumor (HPT-JT) syndrome (see Figure 1-3 ), which is an autosomal dominant disorder characterized by occurrence of parathyroid tumors that often have malignant features, ossifying fibromas of the jaw, and other visceral tumors ( 10, 51 ). Thus, the recognition of such phenocopies has clinical implications for a patient with HPT-JT (see Figure 1-3 ), as the patient and family members are at a higher risk of developing HPT-JT associated tumors, which include parathyroid carcinomas, renal tumors, uterine tumors, and pancreatic adenocarcinomas ( 51 ), and therefore require appropriate regular investigations to detect the development of these tumors. The involvement of another gene, CDNK1B, which encodes the 196-amino acid cyclindependent kinase (CDK) inhibitor p27 kip1, has also been reported by studies of unrelated patients, who did not have MEN1 mutations but did have MEN1 associated tumors. CDNK1B mutations have been reported in 26 Translational Endocrinology & Metabolism: Neoplasia Update

15 1.5% of these patients and their families ( ). In addition, germline mutations of the CDK inhibitors, p15, p18, and p21 may be probable causes of MEN1 in 1%, 0.5%, and 0.5% of patients, respectively ( 54 ). Finally, a mutation of the aryl hydrocarbon receptor interacting protein gene, which is also located on chromosome 11q13 and is mutated in families with isolated acromegaly, has been reported in a patient without MEN1 mutation ( 55 ). Cellular Function of MEN1 Protein (Menin) Menin is predominantly a nuclear protein in non-dividing cells, but in dividing cells, it is found in the cytoplasm. Menin has been shown to interact with several proteins that are involved in transcriptional regulation, genome stability, cell division, and proliferation ( 3 ) (see Figure 1-2 ). Thus, in transcriptional regulation, Menin has been shown to interact with activating protein-1 and the transcription factors JunD and c-jun to suppress Jun-mediated transcriptional activation; members (eg, p50, p52, and p65) of the nuclear factor-kappa B (NF- κ B) family of transcriptional regulators to repress NF- κ B mediated transcriptional activation; members of the Smad family, Smad3, and the Smad 1/5 complex to inhibit the transforming growth factor- β (TGF- β ) and the bone morphogenetic protein-2 (BMP-2) signaling pathways, respectively; Runx2, also called cbfa1, which is a common target of TGF- β and BMP-2 in differentiating osteoblasts; and the mouse placental embryonic expression gene, which encodes a homeobox-containing protein ( 3 ). Additional studies have shown that the interaction of Menin with JunD may be mediated by a histone deacetylasedependent mechanism, via recruitment of an msin3a-histone deacetylase (HDAC) complex to repress JunD transcriptional activity. The forkhead transcription factor CHES1 has also been shown to be a component of this transcriptional repressor complex and to interact with Menin in an S-phase checkpoint pathway related to DNA damage response. Menin uncouples ELK-1, JunD, and c-jun phosphorylation from mitogen-activated protein kinase activation and suppresses insulin-induced c-jun mediated transactivation in CHO-1R cells ( 3 ). A wider role in transcription regulation has also been suggested, as Menin has been shown to be an integral component of histone methyltransferase complexes that contain members from the mixed-lineage leukemia (MLL) and trithorax protein family ( 3, 56 ). These can methylate the lysine 4 residue of histone H3 (H3K4), and H3K4 trimethylation is linked to activation of transcription. Menin, by serving as a molecular adaptor, physically links the MLL histone methyl transferase with the lens Multiple Endocrine Neoplasia Type 1 27

16 A Codon number WT Gln Pro Gly... Val Amino Acid Met m His Gln Gly... Stop WT Nucleotide m ATG CAA CCA GGG... CAC CAG GGG... GTA TAG B I Family 18/ II Control WT 1 U 2 U 3 4 U N 1 N 2 N 3 S 300 bp 200 bp Control m 300 bp 200 bp C Male Female Unaffected Parathyroid tumours Jaw tumour-ossifying fibroma Prolactinoma Renal tumours-cysts and hamartoma U Uterine tumours and hysterectomy FIG 1-3. Phenocopy in an individual with 2 MEN1 associated endocrine tumors. Individual II.2 was initially attributed to have clinical MEN1 on the basis of primary hyperparathyroidism and a microprolactinoma. DNA sequence analysis of the MEN1 gene did not identify any abnormalities. However, DNA sequence analysis of the CDC73 gene, mutations of which result in the hyperparathyroidism jaw tumor (HPT-JT) syndrome, identified a deletion (c.1239dela) of 1 bp involving codon 413 (A) of exon 14 in family 18/91. The deletion is predicted to result in a frameshift with the introduction of 15 missense amino acids followed by a premature stop at codon 428. Amplification refractory mutation system polymerase chain reaction (ARMS-PCR) using DNA from the family members and separation of the products by agarose gel electrophoresis facilitated the detection of the continued 28 Translational Endocrinology & Metabolism: Neoplasia Update

17 FIG 1-3. (Continued) wild-type (WT) (179 bp) and mutant (m) (178 bp) alleles (B). Control primers employed in both reactions confirmed that the ARMS-PCR was amplifying correctly (upper band of 239 bp on both gels). The standard size marker (S) is shown in the form of a 100-bp ladder. The absence of this 1-bp deletion in 110 alleles from 55 unrelated normal individuals (N1 N3 are shown) indicates that c.1239dela is not a common DNA sequence polymorphism. Individuals and clinical features are represented using symbols (C). Investigations revealed the combined occurrence of parathyroid tumors and ossifying fibromas of the jaw in the sister (II.4) and father (I.1) and benign uterine tumors in all 3 sisters, indicating a diagnosis of HPT-JT syndrome. These results indicate that individual II.2, who has HPT-JT syndrome and prolactinoma, represents a MEN1 phenocopy. [Reproduced with permission from Turner et al. ( 10 ) John Wiley & Sons] epithelium-derived growth factor, which is a chromatin-associated protein implicated in the etiology of leukemia, autoimmunity, and human immunodefeciency virus-1 disease ( 57, 58 ). Thus, Menin has a critical function in the MLL complex and its regulation of multiple transcriptional pathways; for example, Menin, as a component of this MLL complex, regulates the expression of genes such as the Hox homeobox genes and the genes for CDK inhibitors, p27 and p18. Interestingly, specific deregulation of a subset of 23 Hox genes has been reported in parathyroid tumors from patients with MEN1. Menin has been shown to directly interact with the nuclear receptor for estrogen (ER α ) and to act as a coacti vator for ER α -mediated transcription, linking the activated estrogen receptor to histone H3K4 trimethylation ( 59 ). Menin has also been shown to bind to a broad range of gene promoters, independently of the histone methyltransferase complex, suggesting that Menin functions as a general transcriptional regulator that helps maintain stable gene expression, perhaps by cooperating with other, currently unknown proteins ( 60 ). Menin also directly binds to double-stranded DNA, and this is mediated by the positively charged residues in the nuclear localization signals (NLSs) in the carboxyl terminus of Menin. The NLSs appear to be necessary for Menin to repress the expression of the insulin-like growth factor binding protein-2 (IGFBP-2) gene by binding to the IGFBP-2 promoter ( 3 ). In addition, each of the NLSs has been reported to be involved in Menin-mediated induction of caspase 8 expression. The NLSs may therefore have roles in controlling gene transcription as well as targeting Menin into the nucleus. Furthermore, gene expression profile studies, using pituitary and pancreatic islet tumors obtained from MEN1 mouse models, have revealed altered expression of genes involved in transcription, cell cycle, and chromatin remodeling. A role for Menin in controlling genome stability has been proposed because of its interactions with a subunit of replication protein, which is Multiple Endocrine Neoplasia Type 1 29

18 a heterotrimeric protein required for DNA replication, recombination, and repair; and the FANCD2 protein, which is involved in DNA repair and mutations of which result in the inherited cancer-prone syndrome of Fanconi anemia. Menin also has a role in regulating cell division as it interacts with the nonmuscle myosin II-A heavy chain, which participates in mediating alterations in cytokinesis and cell shape during cell division and the glial fibrillary acidic protein and vimentin, which is involved in the intermediate filament network ( 3 ). Menin also has a role in cell cycle control as it interacts with the tumor metastases suppressor NM23H1/ nucleoside diphosphate kinase, which induces guanosine triphosphatase activity ( 3 ); and the activator of S-phase kinase (ASK), which is a component of the Cdc7/ASK kinase complex that is crucial for cell proliferation. Indeed, Menin has been shown to completely repress ASK-induced cell proliferation. The functional role of Menin as a tumor suppressor also has been investigated, and studies in human fibroblasts have revealed that Menin acts as a repressor of telomerase activity via htert (a protein component of telomerase). Furthermore, overexpression of Menin in the human endocrine pancreatic tumor cell line BON1 resulted in an inhibition of cell growth that was accompanied by upregulation of JunD expression but downregulation of δ -like protein 1/preadipocyte factor-1, proliferating cell nuclear antigen, and QM/Jif-1, which is a negative regulator of c-jun ( 3 ). These findings of growth suppression by Menin also are observed in other cell types. Thus, expression of Menin in rat sarcoma (RAS) viraltransformed NIH3T3 cells partially suppressed the RAS-mediated tumor phenotype in vitro and in vivo, and overexpression of Menin in CHO-IR cells also suppressed insulin-induced activating protein-1transactivation; this was accompanied by an inhibition of C-Fos induction at the transcriptional level. Furthermore, Menin expression in Men1 deficient mouse Leydig tumor cell lines induced cell cycle arrest and apoptosis ( 61 ). In contrast, depletion of Menin in human fibroblasts resulted in their immortalization. Thus, Menin appears to have a large number of functions through interactions with proteins, and these mediate alterations in cell proliferation. Mouse Models for MEN1 Five mouse models for MEN1 have been generated through homologous recombination (ie, knockout) of the mouse Men1 gene. One mouse knockout model for MEN1 was generated by introducing a floxed PGK-neomycin cassette into intron 2 and a third loxp site into intron 8, thereby deleting exons 3 to 8 in 1 allele ( 62 ). Heterozygous mice (+/ ), when adult (9 to 30 Translational Endocrinology & Metabolism: Neoplasia Update

19 16 months of age), developed parathyroid dysplasia, adenomas, and carcinomas; pancreatic islet cell tumors that contained insulin; anterior pituitary tumors that contained prolactin; and adrenal cortical carcinomas ( 62 ). The tumors, which had LOH at the Men1 locus, were not associated with any serum biochemical abnormalities, such as hypercalcemia or hypoglycemia, but those Men1 (+/ ) mice developing pancreatic islet cell tumors or hyperplasia were found to have elevated serum insulin concentrations ( 62 ). Another mouse knockout model has been generated by deleting exon 3 ( 63 ), and heterozygous mice (+/ ), when adults, were found to develop parathyroid adenomas and carcinomas; pancreatic islet cell tumors that consisted of insulinomas, gastrinomas, or glucagonomas; and anterior pituitary tumors that consisted of prolactinomas or somatotrophinomas ( 63 ). These Men1 (+/ ) mice also developed tumors of the thyroid, Leydig cells, ovarian stroma, and mammary glands ( 63 ). Two other mouse knockout models have also been generated by deleting either exon 2, which contains the translation start site ( 64 ), or exons 1 and 2 ( 65 ). In both of these studies, Men1 (+/ ) mice were found to develop parathyroid, pancreatic islet, and anterior pituitary tumors. In addition, a proportion of these Men1 (+/ ) mice also developed tumors of the thyroid, adrenals, and gonads ( 64, 65 ). Thus, heterozygous (+/ ) mice from these 4 different types of knockouts provide a model for the human MEN1 disease. However, in another study, heterozygous mice (+/ ) surprisingly died as embryos in late gestation, with some embryos developing omphaloceles ( 66 ). Homozygous ( / ) mice from 4 studies ( 41, 42, 66 ) have been reported to die in utero at embryonic days 11.5 to Men1 ( / ) mice were developmentally delayed and significantly smaller, and 20% of them developed craniofacial abnormalities ( 66 ). The craniofacial abnormalities have been shown to be due to dysplasia of the membranous skull bones, and this developmental pathway involves the BMP-2 signaling pathway ( 41 ). Men1 ( / ) mice also developed extensive hemorrhage and edema ( 63 ) and had abnormalities of the neural tube, heart, and liver. Thus, many Men1 ( / ) mice had a failure of the closure of the neural tube, myocardial hypotrophy with a thin intraventricular septum, and decreased hepatic cellularity, which was associated with an altered organization and enhanced apoptosis ( 41 ). In 1 study, which bred the homozygous Men1 ( / ) mice onto incipient cogenic strains, marked differences in these phenotypes of embryonic lethality and failure in neural tube closure were observed, indicating a role for genetic modifiers ( 42 ). These results from the Men1 ( / ) mice also reveal an important role for the MEN1 gene in the embryonic development of multiple organs. For example, Menin has been shown to play a critical role in cranial neural Multiple Endocrine Neoplasia Type 1 31

20 crest development. Tissue-specific inactivation of Menin in neural crest cells, which expressed Pax3 or Wnt1, resulted in a decrease of p27kip1 expression and perinatal lethality with abnormalities of the cranial bones and ribs and cleft palate. These studies indicate that Menin is involved in osteogenesis and pathogenesis, as well as perinatal viability. Finally, studies using double knockout mice for Men1 and the CDK inhibitor p18 (ink4c) have revealed that a functional synergism between p18 and Men1 that increases phosphorylation of the retinoblastoma (Rb) protein and accelerates the occurrence of associated endocrine tumors ( 2 ). Circulating Growth Factor Tumor development in MEN1 has been reported to be associated with a circulating growth factor that is mitogenic for parathyroid cells ( 67 ) and has similarities to basic fibroblast growth factor (bfgf) ( 68 ). The parathyroid mitogenic factor that appeared to act as a tumor angiogenic factor by stimulating endothelial cells ( 69 ) was specific for parathyroid cells and did not stimulate activity in anterior pituitary or pancreatic islet cells ( 67, 70 ). However, plasma bfgf-like immunoreactivity was found to decrease after surgery for pituitary tumor and after initiation of bromocriptine therapy, thereby indicating that this plasma mitogenic factor may originate from the pituitary gland ( 69 ). These findings require further study to elucidate the specific role, if any, of this circulating growth factor in the development of MEN1 tumors. Clinical Applications of Genetic Diagnosis MEN1 Mutational Analysis MEN1 mutational analysis is helpful in clinical practice in several ways that include: (1) confirmation of the clinical diagnosis; (2) identification of family members who harbor the MEN1 mutation and require screening for tumor detection and early treatment; and (3) identification of the 50% of family members who do not harbor the familial germline MEN1 mutation and can therefore be reassured and alleviated of the burden of anxiety of developing tumors ( 2, 15 ). This latter aspect cannot be overemphasized, as it not only helps to reduce the personal cost to the individuals and their children, but also to the health services in not having to undertake unnecessary biochemical and radiological investigations (see Table 1-1 ). The current guidelines recommend that MEN1 mutational analysis should be undertaken in: 32 Translational Endocrinology & Metabolism: Neoplasia Update

21 (1) an index case with 2 or more MEN1 associated endocrine tumors (ie, parathyroid, pancreatic, or pituitary tumors); (2) asymptomatic first-degree relatives of a known MEN1 mutation carrier; and (3) a first-degree relative of a MEN1 mutation carrier expressing familial MEN1 (ie, having symptoms, signs, biochemical, or radiological evidence for 1 or more MEN1 associated tumors) ( 7, 15 ). Such mutational analysis may be undertaken in children within the first decade, because tumors have developed in some children by the age of 5 years and appropriate intervention in the form of biochemical testing or treatment or both has been considered ( 7 ). An integrated program of both mutational analysis, to identify mutant gene carriers, and biochemical screening, to detect the development of tumors ( Figure 1-4 ), is used by some centers ( 2, 15 ). Thus, a DNA test identifying an individual as a mutant gene carrier is likely to lead not to immediate medical or surgical treatment, but to earlier and more frequent biochemical and radiologic screening. In contrast, those relatives who do not harbor the MEN1 mutation will have their risk of developing MEN1 associated endocrine tumors markedly decreased from 1 in 2 for an autosomal dominant disorder to 1 in 3000, 1 in , and 1 in 1000, which are the respective risks for parathyroid, pancreatic islet cell, and anterior pituitary tumors for the general population; thus, these relatives without the MEN1 mutation will be freed from the requirement of further repeated clinical investigations ( 3, 7, 15 ). Thus, the identification of MEN1 mutations may be of help in the clinical management of patients and their families with this disorder. In addition, MEN1 mutational analysis should be considered in patients with suspicious or atypical MEN1. This would include individuals with: parathyroid adenomas before the age of 30 years or multigland parathyroid disease; gastrinoma or multiple pancreatic islet cell tumors at any age; or 2 or more MEN1 associated tumors, which are not part of the classical triad of parathyroid, pancreatic islet, and anterior pituitary tumors (eg, parathyroid tumor plus an adrenal tumor) ( 7 ). MEN1 mutational analysis in a symptomatic family member (ie, an individual already showing a clinical manifestation of MEN1) from a family with a known MEN1 mutation has been challenged as being unnecessary to establish the diagnosis of MEN1. However, phenocopies occurring in the context of familial MEN1, in which a patient with 1 MEN1 associated tumor (eg, a prolactinoma) did not have the familial mutation ( Figure 1-5 ), have been reported by 2 studies ( 10, 47 ). The clinical implications for patients with occurrence of phenocopies in the familial MEN1 context (see Figure 1-5 ) are important, as they Multiple Endocrine Neoplasia Type 1 33

22 Asymptomatic relative of MEN1 patient in whom germ-line mutation identified Test for MEN mutations Mutant carrier Non-mutant carrier Age <40 years Age >40 years Further investigations not necessary Biochemical tests: Ca++, PTH, PRL, IGF-1, CgA, fasting g-i hormones and glucose. Assess carefully for insulinomas Imaging: MRI (or CT) of PIT, PANC, ADR and for foregut CAR Biochemical tests: Ca++, PTH, PRL, IGF-1, CgA, fasting g-i hormones and glucose. Assess carefully for gastrinomas Imaging: MRI (or CT) of PIT, PANC, ADR and for foregut CAR Normal Result Abnormal Result Normal Result Abnormal Result Re-screen biochemically at 6-12 months and by imaging at months Proceed to further appropriate investigations Re-screen biochemically at 6-12 months and by imaging at months Proceed to further appropriate investigations and treatment FIG 1-4. An approach to screening in an asymptomatic relative of a patient with multiple endocrine neoplasia type 1 (MEN1). The relative should have first undergone clinical evaluation for MEN1 associated tumors to establish that the individual is asymptomatic. Relatives who are symptomatic, who should also have a test for MEN1 mutations, should proceed to appropriate investigations and management. If mutational analysis for MEN1 is not available, then this protocol could be adapted for first-degree relatives. It has been suggested that nonessential genetic testing in a child who is not old enough to make important long-term decisions be deferred. However, the finding that a child from a family with MEN1 does not have any MEN1 mutations removes the burden of repeated clinical, biochemical, and radiologic investigations and enables health resources to be more effectively directed toward those children who are MEN1 mutant gene carriers. The approaches to genetic testing and screening in MEN1 vary in different countries. Abbreviations: ADR, adrenal; CAR, carcinoid; CgA, chromogranin A; g-i, gastrointestinal hormones; IGF-1, insulin growth factor-1; MRI, magnetic resonance imaging; PANC, pancreas; PIT, pituitary; PRL, prolactin; PTH, parathyroid hormone. [Adapted with permission from Thakker ( 2 ) Elsevier 2010] 34 Translational Endocrinology & Metabolism: Neoplasia Update

23 can be alleviated of the anxiety of developing MEN1 associated tumors, and of their children being free from the burden of MEN1 ( 15 ). Detection of MEN1 Tumors Biochemical screening for the development of MEN1 tumors in asymptomatic members of families with MEN1 is of importance in as much as earlier diagnosis and treatment of these tumors may help reduce morbidity and mortality ( 3, 4, 7 ). The age-related penetrance (ie, the proportion of gene carriers manifesting symptoms or signs of the disease by a given age) has been ascertained ( 43 ), and the mutation appears to be nonpenetrant in those younger than 5 years. Thereafter, the mutant MEN1 gene has a high penetrance, being greater than 50% penetrant by 20 years of age and greater than 95% penetrant by 40 years ( 43 ). Screening for MEN1 tumors is difficult, because the clinical and biochemical manifestations in members of any one family are not uniformly similar ( 2, 5 ). Attempts to screen for the development of MEN1 tumors (see Table 1-1 ) in the asymptomatic relatives of an affected individual have depended largely on measuring the serum concentrations of calcium, gastrointestinal hormones (eg, gastrin), and prolactin, as well as on radiologic imaging of the abdomen and pituitary gland ( 3 ) (see Table 1-1 ). At present, it is suggested that individuals at high risk for MEN1 (ie, mutant gene carriers) undergo biochemical screening at least once per annum and also have baseline pituitary and abdominal imaging (eg, MRI or CT), which should then be repeated at 1- to 3-year intervals ( 7 ). Screening could possibly commence in childhood, because the disease has developed in some individuals by the age of 5 years; it should continue for life, because the disease has not developed in some individuals until the eighth decade ( 3, 7 ). A higher occurrence of insulinomas in patients below the age of 40 years, and higher occurrences of gastrinomas and somatotrophinomas in patients above the age of 40 years has been reported ( 4 ), and a more detailed search for these tumors in the appropriate age groups is recommended. Case 1-1 A 32-year-old man was referred with a history of recalcitrant hypercalcemia due to primary hyperparathyroidism that had not been successfully treated by partial parathyroidectomy. He had been previously well until the age of 24 years, when he presented at another center with renal colic and hamaturia. He passed several renal stones per urethra, and investigations revealed him to have hypercalcemia with Multiple Endocrine Neoplasia Type 1 35

24 A Codon number WT Amino Acid m WT Nucleotide m Ser AGC Pro Ala Pro Asp Pro Pro... Lys Pro Ala Gln Pro Arg Pro... Stop CCC GCC CCC GAC CCG CCT... AAG CCA GCC CAG CCC CGC CCC... TGA B Family 11/89 I 1 2 II III m WT 1 2 N 1 N 2 N 3 S 500 bp 400 bp C D WT m Male Female 412 bp 422 bp CCAGCCCAGC Unaffected Parathyroid tumours Pancreatic neuroendocrine tumour Prolactinoma Adrenal adenoma (non-secreting) FIG 1-5. Phenocopy in context of familial MEN1. DNA sequence analysis of individual II.2, from family 11/89, identified a 10-bp duplication (underlined) involving codons (A) in exon 2 of the MEN1 gene. This duplication is predicted to result in a frameshift with the introduction of 51 missense amino acids followed by a premature stop at codon 119. PCR amplification of DNA from the family members (B) and separation of products by agarose gel eletrophoresis facilitated the detection of the wild-type (WT) (412 bp) and mutant (m) (422 bp) alleles (C). The standard size marker (S) is shown in the form of a 100-bp ladder. The absence of this 110-bp duplication in 110 alleles from 55 unrelated normal individuals (N1-N3 are shown) indicates that duplication is not a common polymorphism. Individuals and clinical details are represented using symbols (D). The index case, continued 36 Translational Endocrinology & Metabolism: Neoplasia Update

25 FIG 1-5. (Continued) individual II.2, was affected with parathyroid tumors, a pancreatic NET, and an adrenal adenoma; individual III.2 was affected with parathyroid tumors and a prolactinoma; and individual II.4 was affected with a prolactinoma. Mutational analysis in these family members identified individuals II.2 and III.2 to be heterozygous for the m and WT alleles. However, individual II.4, with a single MEN1 associated tumor, is homozygous for the WT allele and thus does not harbor the familial germline MEN1 mutation, thereby indicating that she represents a MEN1 phenocopy. [Reproduced with permission from Turner et al. ( 10 ) John Wiley & Sons] increased plasma PTH concentrations, and hypercalciuria, consistent with a diagnosis of primary hyperparathyroidism. He was referred for surgery; a subtotal parathyroidectomy was performed, and histology revealed the presence of parathyroid hyperplasia. Over the next 8 years, he continued to have renal stones, was observed to remain hypercalcemic with raised plasma PTH concentrations, and was referred for further consultation. A detailed medical history revealed that he did not suffer from dyspepsia, ulcers, diarrhea, neuroglycopenia or impotence. However, his mother had died of pancreatic cancer, and his elder brother had recurrent peptic ulcers and hypercalcemia ( Figure 1-6 ). Physical examination revealed no abnormalities other than a neck scar and facial angiofibromas. A presumptive diagnosis of familial MEN1 was made and appropriate biochemical and radiological investigations undertaken (see Figure 1-4 and Table 1-1 ). These revealed that in addition to the primary hyperparathyroidism, he had hyperglucagonemia, and a CT scan showed the presence of a pancreatic tumor located in the tail. The recurrent primary hyperparathyroidism was treated by a total parathyroidectomy, and he was given oral calcitriol (1,25 dihydroxy vitamin D 3 ) replacement. He also had a distal pancreatectomy, and histology revealed the pancreatic tumor to immunostain for chromogranin and glucagon. He has been screened annually for the development of MEN1 associated tumors. For the past 17 years he has remained well and normocalcemic and has not suffered from renal stones or recurrence of the pancreatic NET. Following the identification of the MEN1 gene in 1997, mutational analysis was undertaken, and he and his family were found to have a 4-bp deletion involving codons 210 and 211 ( Figure 1-6 ). Comment The history of this young man is typical of a patient with MEN1 ( 2 ). The occurrence of primary hyperparathyroidism in a young individual, especially a male; the involvement of multiple parathyroid glands ; the Multiple Endocrine Neoplasia Type 1 37

26 MEN1 mutation (codon 210/211 del 4bp) A Codon number Amino acid (WT) Gln Thr Val Asn (m) Thr Met Pro Wild Type (WT) CA GTC AAT CAG A Mutant (m) CA ATG CCG B Family 8/94 I Parathyroid tumours Glucagonoma Gastrinoma II Age III ht hm N 1-3 FIG 1-6. Mutational analysis in family with MEN1, in which the proband (arrow) presented with renal stones. (A) DNA sequence analysis of the MEN1 gene revealed a 4-bp deletion (ACAG) involving codons 210 and 211 that resulted in a frameshift with incorporation of 12 missense amino acids followed by a stop codon at residue 222 (not shown). (B) Cosegregation of this frameshifting deletion, which results in a premature truncation, in the family and its absence in 110 alleles from 55 unaffected normals (N1-N3 are shown) was demonstrated by the use of gene-specific primers. The deletion results in the formation of wildtype (WT) or mutant (m) homoduplexes (hm) or heteroduplexes (ht [ie, WT/m]), which have decreased electrophoretic mobility. Individuals are represented as males (squares), females (circles), unaffected (open symbol), carrier for the mutation (dot in middle of symbol), affected with tumor (filled quadrant as shown in key), and affected but deceased with pancreatic cancer (all filled). The current ages of the individuals are indicated below each symbol. persistence of hypercalcemia following partial parathyroidectomy; and the family history, which is often inadequately ascertained by doctors, are all frequent features in patients with MEN1. The value of thorough investigations for other tumors is also illustrated by this case, as this revealed the presence of a glucagonoma, which does not present in MEN1 patients with the characteristic manifestations of a skin rash (necrolytic migratory erythemia), weight loss, and anemia, but may instead be silent and asymptomatic ( 2 ). The patient and his family had several questions related to tumor development and screening, which are discussed in detail. 38 Translational Endocrinology & Metabolism: Neoplasia Update