Multiple Endocrine Neoplasia
Type 2 (RET)

Multiple endocrine neoplasia type 2 (MEN2) is a hereditary cancer syndrome characterized by occurrence of multifocal medullary thyroid cancer (MTC) in most and pheochromocytoma in about half of all patients (1,2). In addition, numerous other endocrine and non-endocrine tumors have been in observed in patients with MEN2. MEN2-associated MTC accounts for about 25-30% of all MTC and typically occurs at a much younger age than MEN2-unrelated MTC. Prevalence of MEN2 has been estimated at 1:30,000 (3).

Total thyroidectomy at a very young age has been shown to decrease occurrence or recurrence of metastatic MTC, which is the predominant cause of mortality from MEN2 (4,5). Initially, early diagnosis of MEN2 in pre-symptomatic patients was based on the results of provocative biochemical testing. After autosomal dominant gain-of-function mutations in the proto-oncogene RET were identified as the cause of MEN2 (6,7), genetic testing replaced biochemical testing as the method of choice for early diagnosis of MEN2. Genetic testing is not only more sensitive and more specific than biochemical testing (8), but also can clarify the prognosis, as the position of a mutation within RET is often correlated to the age of onset and the aggressiveness of MTC (1,2,9).

Types and Causes of MEN2

All forms of MEN2 are associated with germline gain-of-function mutations in the gene RET, which codes for a receptor tyrosine kinase believed to be involved in the regulation of such processes as cell proliferation, differentiation, and survival (3,10). MEN2-associated gain-of-function mutations in RET allow the receptor to become activated in the absence of a physiological stimulus, leading to neoplasms. Tumors can only occur in cells derived from neural crest and urigenital precursor cells, where RET is expressed.

Normal Function of RET

The gene RET codes for a receptor tyrosine kinase, a transmembrane protein with an extracellular receptor domain and an intracellular tyrosine kinase domain. The large extracellular section contains a cysteine-rich domain, a calcium binding site, and four domains with homology to cadherin, a protein involved in cell-cell adhesion. The receptor ligands, members of the glial cell-line derived neurotrophic factor (GDNF) family, bind to the receptor through a membrane-anchored “co-receptor” molecule, the GDNF receptor alpha. The ligand-co-receptor-receptor complex then dimerizes, triggering autophosphorylation of tyrosine residues in the cytoplasmic domain of the receptor through the tyrosine kinase also contained within the cytoplasmic domain. This autophosphorylation activates downstream signaling cascades, including the RAS-mediated MAP kinase pathway involved in mitogenesis and neuronal differentiation and the phosphatidylinositol-3-kinase pathway implicated in cell motility, proliferation, and survival (3,10).

Gain-of-Function Mutations in RET

Gain-of-function mutations activate the RET-encoded receptor tyrosine kinase through different mechanisms depending on where in the protein the mutation is located. Mutations affecting one of five cysteines in the extracellular domain are believed to disrupt intramolecular and promote intermolecular bonding between cysteines, leading to ligand-co-receptor independent receptor dimerization. Mutations in the cytoplasmic domain of the receptor molecule may modulate affinity for the tyrosine kinase substrate or for ATP (3,10).

All patients with MEN2 are at risk for developing medullary thyroid cancer (MTC), a neoplasm of the calcitonin-producing parafollicular or C cells within the thyroid gland. About 50% of patients are also affected with pheochromocytomas in the adrenal medulla. Occurrence of hyperparathyroidism and other manifestations may vary and is used to distinguish several different subtypes of MEN2 (see Table 1).

MEN2A, which accounts for 80% of all MEN2, is characterized by the occurrence of hyperparathyroidism in about 20% of patients. Onset of MTC is usually during childhood or adolescence, and metastases can be found in more than half of all patients by early adulthood. In rare cases, MEN2A occurs together with cutaneous lichen amyloidosis or with Hirschsprung disease (aganglionic megacolon).

FMTC (familial medullary thyroid carcinoma), the least aggressive form of hereditary MTC, can be considered a milder subtype of MEN2A with low penetrance of additional manifestations. Onset of MTC in individuals with FMTC is rarely in childhood.

MEN2B is associated with a very aggressive form of MTC that often starts developing during infancy. Metastases have been detected in patients with MEN2B during their first year of life. Individuals with MEN2B are characterized by marfanoid characteristics and the occurrence of mucosal neuromas and intestinal ganglioneuromas.

Of note, loss-of-function mutations in RET are associated with up to half of all cases of Hirschsprung disease (1).

MEN2A: MEN2A usually presents in late adolescence or early adulthood, as a neck mass or neck pain due to MTC (11). Diarrhea may also be present. Pheochromocytoma can occur uni- or bilaterally and may lead to intractable hypertension. MEN2A-related hyperparathyroidism rarely leads to clinical symptoms, but may be associated with kidney stones due to hypercalciuria (1,11).

MEN2A with CLA: In patients with this minor subtype of MEN2A, a lichenoid skin lesion surrounded by many small pruritic lesions on the upper back may be the presenting feature (2).

MEN2A with HSCR: In patients with this other minor subtype of MEN2A, chronic and worsening constipation associated with Hirschsprung disease is usually the presenting symptom during childhood (2).

FMTC: FMTC presents with symptoms of MTC, usually in late adolescence or adulthood (1,11).

MEN2B: Pseudointestinal obstruction, diarrhea, or constipation due to intestinal ganglioneuromatosis may be the initial presentation in children with MEN2B. Patients also show a characteristic facies, with prominent lips with submucosal neuromas at the vermillion border and eversion of the eyelids at the upper margin due to neuromas on the mucosal surface of the eyelids. Neuromas are also apparent over the distal surface of the tongue. A marfanoid habitus with long, thin limbs and a decreased upper-to-lower body is present (1,11).

Since all three of the most common tumors in MEN2, MTC, pheochromocytoma, and tumors of the parathyroid glands, are hormonally active, diagnosis of MEN2 can be achieved through biochemical screening (1). MTC leads to increased serum levels of calcitonin. A pentagastrin or calcium stimulated rise in calcitonin can indicate early-stage MTC, but is also seen with benign C-cell hyperplasia, which occurs in 5% of the general population. Pheochromocytomas are indicated by increased levels of plasma metanephrines or high 24h-urinary levels of catecholamine or metanephrines and can be confirmed through retroperitoneal imaging with CT or MRI. Hyperparathyroidism is suggested by increased plasma levels of calcium and parathyroid hormone.

Genetic testing can detect an estimated 95% of MEN2 cases and has become the method of choice for diagnosis of MEN2. Importantly, genetic testing can detect carriers of MEN2-associated mutations in RET before they become symptomatic.

The recommended treatment for MEN2 is prophylactic thyroidectomy, with or without lymph node dissection, coupled with thyroid hormone replacement therapy. The age at surgery and the necessity for lymph node dissection may depend on the exact RET mutation, since many mutations are known to be correlated to a specific subtype of MEN2 and thus age at onset and aggressiveness of MTC (see Table 2) (1,2,9,12). Generally, surgery is recommended to be performed within the first months of life for MENB, before age 5 years for MENA, and at around age 10 to 15 years for FMTC. Recommendations for lymph node dissection are less clear.

After thyroidectomy, measurement of serum calcitonin levels in response to injection of pentagastrin or calcium can be used to screen for persistent or recurrent disease. Results should be carefully interpreted, as an increased calcitonin response may also be a sign of benign C-cell hyperplasia, which occurs in about 5% of the general population. Patients should also be monitored for signs of hypoparathyrodism, although autotransplantation of parathyroid tissue is usually performed concomitantly with thyroidectomy to guard against hypoparathyroidism due to surgery-related damage to the parathyroid glands.

In addition, MEN2 patients should be screened for pheochromocytoma by measuring catecholamine production. If there is evidence of pheochromocytoma, patients should be treated with alpha or beta blockers before undergoing adrenalectomy, since the increased catecholamine levels may trigger a hypertensive crisis during anesthesia. In pregnant women, pheochromocytomas may lead to a hypertensive crisis during labor and delivery if not treated with appropriate pharmacotherapy.

MEN2 shows autosomal dominant mode of inheritance and is associated with germline gain-of-function mutations in the gene RET. Germline mutations in RET are believed to account for all hereditary and about 6% of apparently sporadic MTC as well as approximately 5% of apparently sporadic pheochromocytomas (2,13). Somatic, not germline mutations in RET are found in 23-70% of truly sporadic MTC (10). Mutation detection in tumor tissue is therefore not suitable for diagnosis of MEN2.

In the majority of patients with MEN2A, one of five cysteine residues in the extracellular cysteine-rich domain of the RET-encoded receptor tyrosine kinase is changed to another amino acid (10). The cysteine at position 634 in particular serves as a mutation hot-spot. A cysteine to arginine mutation at position 634 is found in half of all kindreds with MEN2A (2).

MEN2B has only been associated with mutations in the intracellular domain of the receptor molecule, with a methionine to threonine mutation at position 918 accounting for 95% of all cases of MEN2B (3). This Met918Thr mutation is also the predominant somatic RET mutation detected in sporadic MTC (10).

Mutations at positions 768 and 891 and a valine to methionine change at position 804 are typically associated with FMTC.

Genetic testing is appropriate for probands with any type of MEN2 and is considered standard management for all at-risk members of families in which a MEN2- associated RET mutation has been identified. The MEN2 (RET) Evaluation can help to confirm a diagnosis of MEN2 and detect asymptomatic carriers of an MEN2-associated mutation in RET. Carrier detection and early diagnosis of MEN2 are crucial, since prophylactic thyroidectomy has been shown to increase survival. Genetic testing is more accurate in identifying carriers of MEN2-associated mutations than biochemical testing and can detect carriers before they develop any biochemical symptoms. Genetic testing can also inform about the risk of early onset MTC and guide the timing for thyroidectomy.

While most MEN2-associated mutations in RET are clustered within specific regions of the gene sequence, MEN2 may also be caused by mutations in other parts of the gene. The MEN2 (RET) Evaluation is therefore based on full-length sequencing of the RET coding region. However, since mutations in RET are also associated with Hirschsprung disease, novel mutations identified during sequencing cannot be interpreted as MEN2-associated without careful evaluation of the clinical phenotype and the family history.

How Is Genetic Testing for MEN2 Performed?

DNA for sequencing is obtained from leukocytes present in a small blood sample. The coding sequences of RET are amplified in a highly specific manner through a polymerase chain reaction (PCR), and all PCR products are fully sequenced. Sequencing results are interpreted, and a detailed result report is sent to the patient’s physician.

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