ELSEVIER
BEST PRACTICE & RESEARCH
Multiple endocrine neoplasms
Alberto Falchetti MD, PHD
Assistant Professor
Francesca Marini PhD
Research Fellow
Ettore Luzi PhD
Research Fellow
Francesco Tonelli MD
Professor Surgery Unit, Department of Clinical Physiopathology, University of Florence, 6-50139 Florence, Italy
Maria Luisa Brandt* MD, PHD
Professor
Department of Internal Medicine and Regional Centre for Hereditary Endocrine Tumours, Azienda Ospedaliera Universitaria Careggi, University of Florence, Viale Pieraccini, 6-50139 Florence, Italy
Multiple endocrine neoplasia type 1 (MEN1) and type 2 (MEN2) are rare autosomal-dominant disorders characterized by primary tumours in at least two different endocrine tissues. Both syndromes present as sporadic (a single case with two of the characteristic endocrine tumours) or familial form (an MEN case plus at least one first-degree relative showing one of the charac- teristic endocrine tumours).
MEN1 is characterized by the occurrence of parathyroid, gastro-entero-pancreatic and ante- rior pituitary tumours, but it can include various combinations of more than 20 endocrine and non-endocrine tumours. Generally, tumours in MEN1 are benign, although gastrinomas and foregut carcinoids may exhibit a malignant course.
MEN2 is characterized by medullary thyroid carcinoma (MTC), uni- or bi-lateral pheochro- mocytoma, and other tumours of different endocrine tissues. If not diagnosed precociously, MTC can be fatal.
* Corresponding author. Tel .: +39 (0)554296586. Fax +39 (0)554296585.
E-mail address: m.brandi@dmi.unifi.it (M.L. Brandt)
MEN1 develops after tissue inactivation of both MEN1 gene copies. Activating mutations of c-RET proto-oncogene causes MEN2.
Key words: hereditary endocrine tumours; clinical management of endocrine tumours; tumour suppressor gene; oncogene; genetic diagnosis.
Multiple endocrine neoplasia types 1 (MEN1) and 2 (MEN2) are rare hereditary disor- ders characterized by the presence of primary tumours in at least two different endo- crine tissues. Two different forms, sporadic and familial (more common), have been described for both syndromes. The sporadic form is represented by a single case with two of the principal MEN-related endocrine tumours. The familial form consists of an MEN case with at least one first-degree relative showing one of the characteristic endocrine tumours or a known mutation in the corresponding gene.
Both syndromes exhibit an autosomal-dominant pattern of inheritance. Therefore, each affected patient has a 50% probability of transmitting the gene defect to their progeny, independent of sex.
Although uncommon, it is important to recognize these syndromes at an early stage because the gene mutations confer a high risk of occurrence of multiple aggres- sive primary tumours at young ages. Genetic testing allows early recognition of those individuals who carry the mutation and identification of neoplastic lesions at an earlier stage, helping clinical management of MEN patients and reducing morbidity and mortality.
Appropriate genetic counselling is required for both syndromes. Prenatal testing is available for both conditions, although it is not commonly performed for MENI.
MULTIPLE ENDOCRINE NEOPLASIA TYPE 1
MEN1 (MIM 131100) is a rare (approximately one in 30 000) endocrine disorder presenting with various combinations of parathyroid adenoma, gastrinoma and prolac- tinoma. It can also include various combinations of more than 20 endocrine and non- endocrine tumours such as foregut, bronchial and thymic carcinoids, lipomas and skin tumours. Many tumours in MENI are benign, although entero-pancreatic gastrinomas and foregut carcinoids are often malignant. Approximately one-third of patients die from an MEN1-related cancer and associated malignancies.
MEN1 is an autosomal-dominant disorder with penetrance reaching 100% with age. Various clinical phenotypes are seen in affected members within the same MENI family.2
Tumour develops after inactivation of both gene copies at chromosome Ilq12-13 at tissue level. The initial lesion consists of diffuse proliferation of the affected endo- crine tissue with bilateral involvement of many paired organs and multifocal growth.
MEN1 clinical aspects and treatments
Primary hyperparathyroidism
Primary hyperparathyroidism (PHPT) is the most common endocrinopathy in MENI, affecting nearly 100% of patients by 50 years of age.3 It is often the first endocrine manifestation of MENI (90% of patients), and is recognized as early as 8 years of age in some cases.
MEN1 PHPT shows similar clinical features to sporadic PHPT with a long period of asymptomatic hypercalcaemia and low morbidity. In contrast to sporadic PHPT, MEN1 PHPT develops at a younger age and has similar prevalence in females and males. The average age at onset of MENI PHPT is 20-25 years3,4; three decades earlier than that for sporadic PHPT. Unlike single adenoma of sporadic PHPT, MEN1 PHPT is associated with multiple, asymmetric parathyroid gland enlargement and all parathyroid glands are usually affected. MEN1 PHPT increases gastrin secretion from gastrinomas, precipitat- ing and/or exacerbating symptoms of Zollinger-Ellison syndrome (ZES).
Biochemical diagnosis is made by elevated serum parathyroid hormone (PTH) and calcium concentrations.
Surgery for hyperparathyroidism (HPT) in MEN1 is still the preferred treatment, but there is no general consensus regarding which technique is the optimal surgical approach. The decision about timing for parathyroid surgery usually takes the follow- ing criteria into account: severity of PHPT symptoms; concentration of circulating PTH and calcium; presence of MEN1-associated endocrinopathies, especially ZES; and pa- tient age. Persistence, late recurrence and hypoparathyroidism are all more common after surgery for MEN1 than sporadic adenoma.
Surgical approaches can consist of subtotal parathyroidectomy (surgical ablation of three parathyroid glands and part of the fourth gland) or total parathyroidectomy (all four parathyroid glands and thymic tissue are removed to avoid HPT recurrence or thymic carcinoids). Subtotal parathyroidectomy could avoid permanent hypoparathy- roidism. Late recurrence of HPT often follows successful subtotal parathyroidectomy in patients with MENI, affecting 50% of cases by 8-12 years after surgery.” Recurrence of HPT can be due to increased function of the remnant tumour or new development of a tumour in a normal remaining, ectopic or supernumerary parathyroid gland. Total parathyroidectomy with simultaneous autologous parathyroid graft, in the forearm, is a more radical approach for MEN1-associated HPT. The parathyroid gland to be trans- planted macroscopically exhibits the features closest to a normal gland, preferably lacking any nodular histopathology.
Calcium-sensing receptor agonists have been demonstrated to act directly on para- thyroid glands, decreasing PTH release6, and perhaps even decreasing parathyroid tumour growth. They may acquire an important role in the treatment of MENI HPT.
Well-differentiated endocrine tumours of the gastro-entero-pancreatic tract
The prevalence of well-differentiated endocrine tumours of the gastro-entero-pancre- atic (GEP) tract in MENI patients varies from 30% to 75%.7 GEP tumours are often clinically recognized during the fifth decade of life, but may be detected biochemically much earlier (third decade) in asymptomatic MEN1 carriers. Unlike non-familial endo- crine tumours of the upper gastrointestinal tract, tumours in MEN1 patients are usu- ally multiple and develop at a young age. MEN1-associated gastrinoma and insulinoma exhibit an average age at onset that is 10 years younger than that for sporadic gastri- noma and insulinoma3; ZES usually occurs before 40 years of age. Pancreatic adenomas are usually multiple and scattered throughout the whole pancreas; they may be numer- ous (up to 100 in some cases) and range in size from micro-adenomas slightly larger than unaffected islets to macro-adenomas larger than 0.5 cm. Gastrointestinal manifes- tations may be influenced by other endocrinopathies, mainly PHPT.7
The most common GEP functional endocrine tumours in MENI are gastrinomas (54%) and benign insulinomas (15%).8 Approximately 40% of MENI cases exhibit gas- trinomas. Most MEN1 gastrinomas are malignant, and half have metastasized before
diagnosis.’ Poor prognosis is associated with pancreatic primary gastrinomas (more aggressive than duodenal gastrinomas, as suggested by their larger size and greater risk for hepatic metastasis), liver metastases, ectopic Cushing syndrome or a very high gastrin level.
The diagnosis of gastrinoma is made by elevated serum gastrin and high gastric acid output, with or without symptoms. Confirmatory tests can include secretin-stimulated gastrin levels. A 72-h fast protocol may be helpful in the diagnosis of insulinoma that is characterized by fasting hypoglycaemia with a high plasma or serum concentration of insulin and a high plasma or serum concentration of C-peptide or pro-insulin.7,9 Somatostatin receptor scintigraphy is a proven method for pancreatic islet imaging.
Glucagonomas, vasoactive intestinal peptide (VIP)-omas, growth hormone releasing factor (GRF)-omas, and somatostatinomas have also been described in MENI. º How- ever, non-functional endocrine tumours, difficult to diagnose by both biochemical and im- aging tests, are the most prevalent GEP endocrine neoplasms in MENI, with more than half being pancreatic polypeptide (PP)-omas.8 Even enterochromaffin-like (ECL)-omas are included in the well-differentiated endocrine tumours of the GEP tract. They are com- mon in MEN1 and are usually recognized incidentally during gastric endoscopy for ZES.
Treatment of gastrinomas can consist of both drug administration and surgical in- tervention. Gastric acid hypersecretion can be controlled using histamine H2-receptor antagonists or H+, K+-ATPase inhibitors. The surgical approach to be taken for gas- trinomas in MEN1 is controversial as successful surgical outcome is rare. Some au- thors only recommend surgery in cases of precise localization of gastrinoma, or in the presence of particularly aggressive familial gastrinomas , while other authors would perform surgery in all cases. 12
Surgery is usually indicated for insulinoma for the following reasons: hypoglycaemia is not usually easily controlled by drugs; during pancreatic surgery, one or more inci- dental macroscopic lesions are found; hypoglycaemic syndrome is cured by resection of pancreatic macroscopic lesions; and malignant degeneration is prevented. Although there is no general consensus, intra-operative monitoring of glucose and insulin may be helpful to verify removal of the tumour.
In other functioning neuroendocrine tumours (glucagonoma, VIP-oma, PP-oma and somatostatinoma) and non-functioning tumours, the localization of the tumours is eas- ier because they are generally represented by large neoplasms. Pancreatic surgery for asymptomatic patients can be decided when the size of the lesion approaches 2 cm. Patients with systemic metastases are usually excluded from surgery.
The long-acting somatostatin analogue octreotide is considered to be the drug of choice for controlling hormone secretion under conditions such as glucagonoma and VIP-oma.
Anterior pituitary adenomas
The occurrence of anterior pituitary tumours in MENI ranges between 10% and 60%.9 Between 65% and 85% of pituitary tumours in MENI are macro-adenomas9, associated with diffuse or nodular hyperplasia of extratumoural pituitary cells, compared with only 42% in common tumours.13 MEN I pituitary tumours are only multiple and invasive in 10- 15% of cases. They often secrete prolactin (PRL) or growth hormone (GH). The frequency of MENI plurihormonal tumours is greater than that of sporadic pituitary tu- mours. 4 The mean age at the time of diagnosis of MENI pituitary tumours is approxi- mately 40 years3, similar to that for sporadic pituitary tumours.”> Symptoms of MENI pituitary neoplasms depend on both tumour volume and hormonal secretion.
PRL-oma is the most common pituitary tumour in MENI, followed by GH-oma. 4 Rou- tine surveillance consists of circulating PRL measurement under basal conditions at 0 and 60 min. Magnetic resonance imaging (MRI) is the preferred method for pituitary imaging.
In general, treatment (medical, radiation or surgical) of MEN1 pituitary tumours is the same as that for sporadic pituitary tumours. Dopamine agonists are the preferred treatment for PRL-secreting micro-adenomas, whereas somatostatin analogues are the medical therapy of choice for the treatment of GH-secreting tumours.
Conversely, in non-secreting pituitary adenomas, surgery is the treatment of choice; treatment with dopamine agonists or somatostatin analogues may shrink the adenoma before surgery in 5-15% of cases.
Carcinoids
GEP tract, thymic and bronchial carcinoids form an integral part of MENI. GEP MENI carcinoids are all localized in the foregut 6 and rarely oversecrete amine or peptide hormones. The thymic carcinoids in MENI tend to be aggressive. 7 MENI thymic car- cinoid is more common in males, and cigarette smoking appears to be associated with a higher risk. 8 MENI bronchial carcinoids mainly occur in females. Most bronchial car- cinoids are benign, although they have the potential for local mass effect, metastases and recurrence after resection.
Computed tomography (CT) or MRI of the chest are recommended for early diag- nosis of thymic or bronchial carcinoids. Type II gastric ECL cell carcinoids are often recognized at gastric endoscopy, incidental to evaluation for ZES.
Surgery remains the primary treatment option for bronchial and thymic carcinoid tumours. Thymic carcinoid recurred in all MENI patients followed for more than I year after resection of the tumour.”Prophylactic thymectomy should be considered at the time of neck surgery for PHPT in males with MEN1, particularly those who are smokers or have relatives with thymic carcinoids. Bronchial carcinoid has to be removed using a pulmonary procedure that is as conservative as possible. The sur- gical approach to type II gastric ECL cell carcinoids in MENI is controversial because of the multiplicity of gastric primary lesions, and the uncertainties regarding the prospects of long-term cure as a result of partial or total gastrectomy. The treat- ment protocol for MEN1 type II gastric ECL cell carcinoids should, therefore, be lowering of gastrin levels, intensified endoscopic surveillance therapy, and gastrec- tomy on appearance of macrolesions. Somatostatin analogues have been used suc- cessfully in the treatment of type Il gastric carcinoids in three MENI patients2º, showing a decrease in serum gastrin and tumour regression in all patients. Although long-acting somatostatin analogues can control the secretory hyperfunction associ- ated with carcinoid syndrome, the risk for malignant progression of the tumour remains unchanged.
Tumours of the adrenocortical glands and pheochromocytoma
Primary adrenocortical neoplasms are common in MENI (20-40%) and they are usu- ally bilateral, hyperplastic, non-functional and with an indolent clinical course.3 The adrenal cortex tumours are occasionally associated with primary hypercortisolism, resulting from pituitary oversecretion of adrenocorticotrophic hormone (ACTH); hyperaldosteronism is much rarer. Pheochromocytoma (PHEO), always unilateral, affects <1% of MEN1 patients9 , but it is appropriate to diagnose and treat PHEO prior to surgery to avoid potentially lethal blood pressure peaks during surgery.
Non-endocrine tumours
Various tumours of cutaneous, visceral adipose tissue and skin have been described in MEN1 such as lipomas, angiofibromas, collagenomas and leiomyomas. They are mainly benign neoplasms.
Lipomas in MENI (incidence 20-30%)21 are often multiple and may occur viscerally. Lesions, often multicentric, may be small or large and cosmetically disturbing. They do not usually recur after removal.
Multiple facial angiofibromas were observed in 40-90% of MENI patients, with half of the cases having five or more.22 Angiofibromas are benign tumours comprised of blood vessels and connective tissue, consisting of acneiform papules that do not re- gress and may extend across the vermillion border of the lips.
Collagenomas of the trunk are commonly found in MENI patients21, consisting of multiple, skin-coloured, sometimes hypopigmented, cutaneous nodules, symmetrically arranged on the trunk, neck and upper limbs. They are typically asymptomatic, round- ish and firm-elastic, ranging from a few millimetres to several centimetres in size.
Leiomyomas of the oesophagus, uterus or rectum can occur in MEN1.
MEN1 gene and its protein
The gene responsible for MENI, MENI, maps at Ilq12-13.23 The proposed model for tumourigenesis followed Knudson’s ‘two hit’ hypothesis for tumour suppressor genes.24 An affected MENI copy is inherited from the affected parent, while the wild-type copy, from the healthy parent, is eliminated at somatic level.
The MENI gene consists of 10 exons encoding a 610-amino acid protein, menin, which is widely expressed from an early stage in development.
Approximately 700 different somatic and germline mutations have been identi- fied1,25, scattered throughout the entire coding region. Most MEN1 mutations are in- activating: 60% of mutations are frameshift and 25% are non-sense, both predicting a protein truncation. The remaining 15-20% are mis-sense mutations that potentially alter the interaction with one or more menin partners and/or favour a rapid degra- dation of menin. No MENI mutations have been correlated with a clinical phenotype.26,27
DNA tests for MENI fail to detect mutations in 5-30% of MENI patients. This may be because the mutation consists of a large deletion, with loss of the whole gene or exon.26,27 Deletions or other gross re-arrangements represent 1-3% of MENI germ- line mutations and can be detected by Southern blot analysis or other gene dosage procedures.28 Alternatively, undiscovered mutations may be within the untested parts of the gene, such as introns or regulatory regions. Thus, although no MEN1 gene mu- tation is found in some MEN1 index cases, this does not exclude its involvement.
Mutational analysis of MEN1-associated endocrine tumours revealed that most tu- mours from MENI patients harbour MENI germline mutations together with a so- matic loss of heterozygosity involving chromosome Ilq13.29
Analysis of the menin amino acid sequence did not reveal homologies to any other proteins, sequence motifs, signal peptides or consensus nuclear localization signals, and thus the putative function of menin could not be predicted.30
Menin is located primarily in the nucleus; two independent nuclear localization sig- nals (NLS) have been identified in the C-terminal portion of the protein.3º The nuclear localization of menin suggests that it may act in the regulation of transcription, DNA replication or cell cycle.
Menin partners include several nuclear and cytoplasmic transcriptional regulatory, cytoskeletal, DNA processing and repair proteins.3 To date, none of the menin part- ners or menin pathways has been shown to be critical in MEN1 tumourigenesis.
The first menin partner to be identified was JunD; a member of the AP1 family of transcription factors. The interaction between menin and JunD represses JunD tran- scriptional activity, and the disruption of menin-JunD interactions may be a component of MEN1-associated tumourigenesis.
Through interaction with a putative tumour metastasis suppressor nm23HI/nucle- oside diphosphate kinase, menin may reveal GTPase activity.32
Menin also interacts with the activator of S-phase kinase (ASK), which is crucial for cell proliferation, probably inhibiting ASK-induced cell proliferation.3 33
Menin interacts with glial fibrillary acidic protein (GFAP) and vimentin. In glioma cells, the menin-GFAP interaction may serve as a cytoplasmic sequestering network for menin during the S and early G2 phase of the cell cycle.34 Menin may have an in- hibitory role before the S phase starts, and it must be transferred to the cytoplasm to enable the S phase to proceed.35 Thus, the intermediate filament network could sequester menin away from the nucleus and its target genes.” 36
Menin can associate with mixed lineage leukaemia 1 and 2 (MLL1 or MLL2) pro- teins; this complex acts on the promoter of the p18 and p27 genes, resulting in growth suppression.
Menin also interacts with Smad3, an important regulator of transforming growth factor ß (TGFB) cell growth inhibitory effects. Reduced menin expression blocks the Smad3-mediated transcriptional effects of TGFR38 Impaired TGFB signalling may lead the cells towards inappropriate growth and tumour formation.
Menin can also control proliferation through the suppression of endogenous insulin-like growth factor binding protein (IGFBP-2), which inhibits cell proliferation induced by insulin-like growth factors and TGFR39 Mutations in menin NLSs compro- mise the ability of menin to repress expression of the IGFBP-2 gene.40
Menin interacts with Fanconi anaemia complementation group D2 (FANCD2) pro- tein, involved in the DNA repair pathway. The interaction between menin and FANCD2 increases DNA repair and is enhanced by Y-irradiation.41 41
Menin also interacts with the second subunit of the replication protein A complex, which is required for DNA replication, recombination and repair, and is involved in the regulation of apoptosis and gene expression. 42
In addition, menin interacts with three members of the nuclear factor KB (NFKB) family43, inhibiting NFKB-mediated transcriptional activation to repress or recruit other repressors, such as histone deacetylases.
Most menin-interacting proteins may implicate a role for menin in the regulation of gene expression, and menin may have downstream effects on critical genes regulating normal and tumour processes.
Chromatin immunoprecipitation studies revealed that menin interacts with thou- sands of sites in chromatin, mainly in promoters.44 A recent study found hundreds of menin interaction sites, and many were in the 3’ end of genes and in introns.45 A specific menin-binding sequence in DNA was not found, suggesting that many menin interactions with chromatin were indirect.
In parathyroid cells from sporadic primary and secondary HPT, menin mRNA levels were increased or not modified, whereas in MENI-associated PHPT, they were re- duced.46 TGFB is a crucial negative regulator of both parathyroid cell proliferation and PTH secretion. The loss of TGFß-dependent signalling, secondary to menin inac- tivation, may contribute to both parathyroid cell proliferation and PTH secretion.
Overexpression of menin in rat insulinoma cells inhibits their function and prolifer- ation via inhibition of the insulin gene promoter activity and an increase in cell apopto- sis. Menin expression significantly enables the inhibition of either the insulin promoter activity or glucose-dependent insulin secretion.48 48
It has been hypothesized that menin mediates its tumour suppressor action by reg- ulating histone methylation of promoters of Hox genes, p18, p27 and possibly other CDK inhibitors in pancreatic islets. 37,49
Scacheri et al44 identified the developmentally programmed transcription factor, HLXB9, that was overexpressed in islets (and not in control cells) in the absence of menin, suggesting the possibility for a specific bias for endocrine tumour formation in MEN1 due to changes in expression in distinct genes, such as HLXB9, specifically targeted by menin in endocrine tissues.
Recent studies have elucidated several interactions between pituitary-specific path- ways and menin. Activin is a negative regulator of pituitary cell growth and PRL, ACTH and GH secretion.50 In addition, activin suppresses the transcription and expression of Pit-1, a pituitary transcription factor, which plays an essential role in the development and maintenance of lactotrope cells and the regulation of PRL and GH expression. The Smads pathway and menin are key regulators of activin effects, and menin is required for activin-mediated inhibition of PRL and GH expression. This effect is mediated by Pit-I gene expression and stability.” 51
Genetic testing for the MEN1 gene
Genetic testing for the MEN1 gene allows the identification of MEN1 in at-risk individ- uals even in the absence of more than one affected gland, or in first-degree relatives before the manifestation of MEN1 lesions. The test is recommended for patients who meet the clinical criteria for MEN1 and those in whom a diagnosis of MEN1 is sus- pected. The advantage of MEN1 mutation analysis is that, unlike biochemical screening, a negative test does not need to be repeated serially.
A test identifying an MEN1 mutant gene carrier may lead to earlier and more fre- quent biochemical screening for tumours. Biochemical screening may provide evidence of a neoplasm approximately 10 years earlier than clinical evidence, providing the op- portunity for earlier treatment and reducing morbidity and mortality. Annual routine surveillance (biochemical and imaging) of tumours is recommended for: asymptomatic individuals with an MEN1 mutation; individuals with clinical evidence of MEN1; and in- dividuals with an affected parent who have not undergone genetic testing. In a family with a known MEN1 mutation, identification of non-mutated gene carriers should lead to a decision for no further screening of those members. Unfortunately, the informa- tion obtained from genetic testing does not influence an immediate intervention due to the absence of a correlation between genotype and phenotype.
Mutation analysis may provide a false negative; in fact, MEN1 mutations are not detectable in 5-30% of MENI families. Sporadic MENI cases are less likely to test positive than familial cases, partly because some of these cases are caused by somatic mosaicism.52 In the event that sequence analysis fails to identify a germline mutation in an individual with typical MEN1 syndrome, deletions or other gross re-arrangements can be tested by Southern blot analysis or other gene dosage procedures.28
When the disease-causing mutation is not identified by MENI mutation analysis, haplotype analysis may be utilized in certain families. In fact, if large enough for
haplotype analysis, virtually any MENI family is likely to be positive for an Ilq13 hap- lotype shared in all affected cases.53
MULTIPLE ENDOCRINE NEOPLASIA TYPE 2
MEN2 (MIM 171400) is a rare (approximately one in 20 000) hereditary syndrome characterized by medullary thyroid carcinoma (MTC), unilateral or bilateral PHEO, and other hyperplasia and/or neoplasia of different endocrine tissues. It includes three clinical subtypes: MEN2A (approximately 90%); MEN2B (approximately 5%); and famil- ial medullary thyroid carcinoma (FMTC).
MEN2A is characterized by MTC, unilateral or bilateral PHEO (>50% of cases) and PHPT (15-30% of cases).54 MTC is generally the first manifestation of MEN2A with an age at onset between 5 and 25 years.55 Rare variants of MEN2A can be associated with paraneoplastic syndromes, such as cutaneous lichen amyloidosis or excessive produc- tion of corticotrophin, or with Hirschsprung’s disease.
MEN2B is the most aggressive of the MEN2 variants and is characterized by the earlier (usually 10 years earlier than that for MEN2A) occurrence of more aggressive MTC, PHEO (40-50% of patients), and multiple neuromas and/or diffuse ganglioneuromatosis of the gas- tro-enteric mucosa (approximately 40% of patients), responsible for abdominal distension, colic, constipation or diarrhoea. Patients with MEN2B do not develop PHPT. In some cases, MEN2B patients can manifest developmental abnormalities such as decreased upper/lower body ratio, skeletal deformations, joint laxity, marfanoid habitus and myelinated corneal nerves. Morbidity and mortality for MEN2B are greater than for MEN2A.
In FMTC, MTC is the only clinical feature and it refers to the occurrence of MTC alone in at least four affected members within the same family. In FMTC, the clinical course of MTC is more benign than that of MEN2A and MEN2B, and the prognosis is relatively good in most cases.
MEN2 clinical aspects and treatments
Medullary thyroid carcinoma
MTC occurs in all MEN2 patients, usually as the first clinical manifestation, and its se- verity generally decreases from MEN2B to MEN2A to FMTC.9 The tumoural progres- sion to MTC is extremely variable and may take several years36, with a natural tendency to local metastases (central and lateral, cervical and mediastinal lymph no- des), but bone, liver and lung lesions are not infrequent.5 57
In MEN2A patients, MTC generally occurs between 5 and 25 years of age. If un- treated, it can manifest as a neck mass or neck pain at about 15 to 20 years of age. In MEN2A patients, diarrhoea is the most common systemic symptom of untreated MTC, in association with a plasma calcitonin concentration of >10 ng/ml. In MEN2B patients, MTC is more aggressive and generally develops about a decade ear- lier. Individuals with MEN2B who do not undergo thyroidectomy at approximately I year of age are likely to develop metastatic MTC at an early age.
MTC generally correlates with increased circulating levels of calcitonin. Therefore, specific tests to evaluate calcitonin secretion, by stimulation with pentagastrin or cal- cium, lead to earlier detection of MTC. Ultrasonography, CT or MRI can be used to localize tumour extension and possible distant metastases.
MTC (in both MEN2A and MEN2B patients) is treated by total thyroidectomy with lymph node dissection of at least the central compartment. Ideally, it should be performed
before the age of possible progression of malignancy.38 Tissue biopsy and evaluation of se- rum calcitonin level help in making a pre-operative diagnosis of MTC. An elevated calcito- nin serum level after surgery can be a sign of persistent, recurrent or generalized MTC.
Pheochromocytoma
PHEO in MEN2 (approximately 50% of MEN2A and MEN2B cases) is almost always benign, but tends to be bilateral in 50-80% of cases. Generally, PHEO is the first clin- ical manifestation of the disease in 25% of cases, manifests after MTC in 40% of cases, and MTC and PHEO are diagnosed at the same time in 35% of cases.56 PHEO may account for hypertension, palpitations, nervousness, episodic headache, sweating and blanching of the skin due to excessive synthesis of catecholamines. Prior to any surgical intervention, the presence of a functioning PHEO should be excluded by appropriate biochemical analysis to avoid lethal blood pressure peaks during surgery. Adrenal gland function can be assessed easily by 24-h measurement of urinary excre- tion of catecholamines and their metabolites; this screening is recommended on an annual basis. CT and MRI are used to localize the tumour.
PHEO is treated by surgical laparoscopy excision. Life-long follow-up after surgery is strongly recommended. Long-term drug treatment with a and ß adrenergic blockers should only be considered in those patients in whom the tumour is unresectable.
Primary hyperparathyroidism
PHTP occurs in 20-30% of MEN2A patients, and is usually asymptomatic.’ The diag- nosis is made by elevated serum PTH and calcium concentrations. MEN2 PHPT is treated by subtotal parathyroidectomy or total parathyroidectomy with autotransplan- tation of normal fresh or cryopreserved tissue in the forearm. All individuals who have undergone partial or total parathyroidectomy with autotransplantation need to be monitored for possible recurrences.
c-RET proto-oncogene and its protein
Activating mutations of c-RET proto-oncogene determine MEN2.59,60 It encodes a membrane receptor tyrosine kinase protein called RET. RET protein contains an ex- tracellular portion containing four cadherin-like domains, a calcium-binding site, a cys- teine-rich domain, a single-pass transmembrane domain, and an intracellular portion containing two distinct tyrosine kinase domains and several autophosphorylation sites. RET phosphorylated tyrosines serve as docking sites for intracellular signalling proteins.
Approximately 98% of MEN2 patients bear germline mutations in exons 10, 11, 13, 14, 15 and 16 of c-RET proto-oncogene.’ Very recently, mutations in exon 8 have been found in an FMTC family.6 Mutations of the cysteine-rich domain and the tyrosine ki- nase domains result in constitutive tyrosine kinase activation. MEN2 exhibits a strong genotype-phenotype correlation. Mis-sense mutations of one of six extracellular cys- teines are responsible for approximately 93-98% of cases of MEN2A and approxi- mately 80-96% of cases of FMTC. Eighty-five percent of MEN2A patients have a cysteine 634 mutation, particularly C634R, strictly associated with the occurrence of PHEO and/or HPT.62 Conversely, in FMTC patients, mutations are equally distrib- uted among all six cysteines. FMTC and rare cases of MEN2A have also been associ- ated with mis-sense mutations in the intracellular domain of RET. In contrast, most
MEN2B cases are caused by mis-sense mutations in the intracellular tyrosine kinase receptor domains of RET. More than 95% of MEN2B patients have the M918T muta- tion at exon 16, while approximately 5% of MEN2B patients have the A883F substitu- tion at exon 15. Recently, other infrequent mis-sense mutations at exons 14, 15 and 16 were found in MEN2B patients. 65
The oncogenic mechanisms of different RET mutations seem to be dependent on the site of the amino acid change. Cysteine substitution in MEN2A and FMTC, partic- ularly at cysteine 634, seems to prevent the formation of intermolecular disulphide bonds enabling ligand-independent receptor dimerization, resulting in constitutive ki- nase activation independently by ligand. In contrast, most mutations in MEN2B affect one intracellular tyrosine kinase domain. RET phosphorylated tyrosines interact with the docking protein FSR2 causing downstream activation of the mitogen-activated pro- tein kinase (MAPK) signalling cascade.64 Thus, mutations at this level may determinate an alteration of the regulation of MAPK pathways on cell survival and proliferation.65
Micro-array expression analysis of PHEO and MTC from patients with MEN2A and MEN2B showed different gene expression patterns, possibly explaining the greater ag- gressiveness of MEN2B.66
Genetic testing for the c-RET proto-oncogene
Early identification of gene mutation carriers is important because of the development of multiple aggressive primary tumours at very young ages, and because of the ten- dency for MTC (mostly in MEN2B) to metastasize at an early stage. Genetic screening of c-RET mutations can be performed easily on a blood sample at any age, and permits the reduction of morbidity and mortality for MEN2. In fact, early recognition of mu- tant gene carriers makes it possible to prevent and cure MTC by performing prophy- lactic thyroidectomy before malignant progression and metastasis, and reduces the risk of an unsuspected PHEO. Moreover, genotype-phenotype correlation in MEN2 assists in the clinical management of patients. In fact, each specific c-RET mutation strictly cor- relates with the aggressiveness of MTC and is associated with a peculiar clinical phe- notype. The risk of MTC can be stratified into three categories according to the different mutations of c-RET9:
· children with codon 883, 918 or 922 mutations have the highest risk of aggressive MTC, and they should undergo total thyroidectomy, with central node dissection, within the first 6 months of life;
. children with codon 611, 618, 620 or 634 mutations have a high risk of MTC, and total thyroidectomy, with or without central node dissection, should be performed before 5 years of age; and
· children with codon 609, 768, 790, 791, 804 or 891 mutations have a less aggressive and slowly growing MTC and may be operated on at a later stage (between 5 and 10 years of age).
For all three groups, a more aggressive neck dissection should be performed if there is evidence of lateral lymph node involvement.67 For individuals bearing other known c-RET mutations, no specific recommendations can be made at this time as there is insufficient experience.
Genetic information can also be useful to assess the risk for developing PHEO. In- dividuals with codon 609, 611, 618, 620, 630, 634, 790, V804L, 883, 918 or 922
mutations should be biochemically screened each year for PHEO. Conversely, the de- velopment of PHEO is unlikely in patients with codon 768 and V804M mutations.68
SUMMARY
MEN1 and MEN2 are rare autosomal-dominant hereditary cancer syndromes that ex- press a variety of endocrine and non-endocrine tumours. Early recognition of these syndromes is important to diagnose the occurrence of multiple primary tumours at young ages.
Discovery of the genes responsible for MEN1 and MEN2 in 1997 and 1993, respec- tively, opened a new era in the early recognition and clinical management of affected and at-risk individuals. The increasing knowledge on the molecular and clinical patho- physiology of these syndromes, together with the availability of gene mutation testing, greatly reduced the morbidity and mortality of MENI and MEN2.
Individuals with a disease-causing mutation, those known to have MENI or MEN2, and those with an affected parent should undergo a surveillance clinical protocol. It has been proposed that these individuals should receive routine screening (biochemical and imaging) from early childhood and continuing for life. º Biochemical screening should be repeated each year; it can detect onset of the disease approximately 10 years before symptoms develop, thereby providing an opportunity for earlier treatment.
Regarding imaging, a syndrome-specific programme of tumour imaging should be performed approximately every 3 years according to international guidelines.”
Nevertheless, more studies on the intricate molecular pathway networks of menin and RET are necessary to clearly elucidate the molecular mechanisms underlying MEN- associated tumourigenesis.
Research agenda
· a deeper comprehension of either menin or RET pathophysiology is mandatory to design future selected therapeutical strategies
· the development of target-oriented (e.g. menin and RET proteins) agents in the treatment of MEN patients will contribute to reduce MEN-related morbidity
· the promising development of tyrosine kinase inhibitors targeting RET protein is encouraging, although their efficacy and safety has to be assessed adequately in multicentric clinical trials
Practice points
· individuals with a mutation of MENI or RET should undergo clinical surveillance
· biochemical screening may detect the onset of disease approximately 10 years before symptoms develop
· early recognition of MEN-affected or at-risk subjects is critical for successful precocious treatment
ACKNOWLEDGEMENTS
This paper was supported by the ‘Fondazione Ente Cassa di Risparmio di Firenze’ (to MLB).
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