Medullary Thyroid Carcinoma

MANAGEMENT OF ENDOCRINE NEOPLASMS

1055-3207 /98 $8.00 + .00

MEDULLARY THYROID CARCINOMA Genetic Advances, Treatment Recommendations, and the Approach to the Patient with Persistent Hypercalcitoninemia David D. Chi, MD, and Jeffrey F. Moley, MD

Medullary thyroid carcinoma (MTC) comprises 3% to 9% of all thyroid cancers and arises from the thyroid C cells. These parafollicular cells are neural crest derivatives and are considered to be part of the amine precursor uptake and decarboxylation (APUD) group of neuroendocrine cells. The C cells comprise only 1% of the total thyroid mass and are dispersed throughout the gland, with the highest concentration in the upper poles. The C cells are so named because of their unique ability to secrete the hormone calcitonin. Although calcitonin has been shown to be integral in calcium homeostasis in other vertebrate species, its role in humans is unclear. C cells are capable of secreting other hormones, including carcinoembryonic antigen (CEA), histaminase, neuron-specific enolase, calcitonin gene-related peptide, somatostatin, thyroglobulin, thyrotropin stimulating hormone, adrenocortical stimulating hormone, gastrin-related peptide, serotonin, chromogranin, and substance P. 30

PATHOLOGIC FEATURES

In the beginning of this century, MTC was described in the German literature as a malignant goiter with amyloid stroma. 36 For decades, From the Section of Endocrine and Oncologic Surgery (JFM), Division of General Surgery, Department of Surgery (DDC, JFM), Washington University School of Medicine, Saint Louis, Missouri SURGICAL ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 7 •NUMBER 4 •OCTOBER 1998

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MTC was considered to be a variant of anaplastic thyroid carcinoma and it was not recognized as a unique entity until 1959 when Hazard et al first described its characteristics. 32 The tumor is well demarcated, firm, graywhite, and gritty. Microscopically, there are uniform polygonal cells with finely granular eosinophilic cytoplasm with central nuclei. Varying numbers of spindle cells are present in nearly all tumors. The presence of amyloid is considered to be a distinctive feature of MTC, although it may not be found in all cases. The amyloid differs from that of other tumors in that it is formed from calcitonin or procalcitonin molecules. In sporadic tumors, approximately 68% are solitary, and 32% are bilateral or multifocal. In familial forms, 94% are bilateral or multifocal, and 6% are solitary. Histologically, the tumors can be classified according to dominant histologic patterns. These groups include the classic, amyloid-rich, insular, trabecular, and epithelial variants. Classic variants are most common (48.9%), followed by the amyloid-rich variants (38.3%). 71 C cell hyperplasia is associated with MTC, particularly in the familial forms. It has been suggested that C cell hyperplasia is a precursor in the malignant transformation to MTC. 6, 31 ,104

THE MEN 2 SYNDROMES

Approximately 20% of cases of MTC occur as part of the hereditary multiple endocrine neoplasia type 2 (MEN 2) syndromes (MEN 2A, MEN 2B, and familial non-MEN medullary thyroid carcinoma (FMTC)). All patients with MTC should be queried about any family history of thyroid, parathyroid, and hypertension disorders. The MEN 2 syndromes are inherited in a typical Mendelian autosomal dominant fashion . There is nearly complete penetrance but variable expression of the syndromes; essentially all persons who inherit the disease allele develop MTC, but other features of the diseases may not necessarily be present. In MEN 2A, approximately 42% of affected patients develop pheochromocytomas, whereas hyperparathyroidism is present in about 35%.34In MEN 2B, about half develop pheochromocytomas, and all individuals develop neural gangliomas, particularly in the mucosa of the digestive tract and conjunctiva. Although the disease is inherited as an autosomal dominant trait, in about half of the patients the disease arises de novo. 13,39 FMTC is characterized by the development of MTC without any other endocrinopathies.22 The clinical presentations of sporadic MTC, MEN 2A, MEN 2B, and FMTC differ considerably (Table 1). Sporadic MTC is unilateral in the majority of cases, whereas MTC in the MEN 2 syndromes tend to be bilateral and multifocal. The tumors in FMTC are usually indolent and appear later in life, whereas MTC in MEN 2B is extremely aggressive, with gross evidence of cancer present in children as young as 6 months of age. These patients often die of widespread metastatic MTC at an early age, underscoring the importance of early diagnosis and treatment in this particular population. 58, 87 The virulence of sporadic MTC can vary and

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Table 1. COMPARISON OF CLINICAL FEATURES OF SPORADIC MTC, MEN 2A, MEN 2B, AND FMTC Disease

Sporadic MEN2A

MEN 2B

FMTC

Endocrinopathies

Age of Diagnosis

MTC variable 1st to 3rd decade MTC Pheochromocytomas Hyperparathyroidism 1st decade MTC Pheochromocytomas Mucosal ganglioneuromas Marfanoid habitus MTC 5th to 6th decade

Virulence of MTC

Histopathology

+++ ++

Unifocal Multifocal Bilateral

++ ++

Multifocal Bilateral

+

Multifocal Bilateral

MTG = Medul lary thyroid carcinoma; MEN = Multi ple endocrine neoplasia; FMTC = Familial nonMEN medullary thyroid carcinoma; + -7 + + + + = Least aggressive to most aggressive.

the disease can present in any age group, although the peak incidence is in the sixth decade of life. 71

PATTERNS OF METASTASES

Most cases of sporadic MTC and index cases of hereditary MTC are diagnosed as a neck mass on physical examination. The incidence of cervical metastases at presentation varies from 25% to 63% depending on the reported series. 16, 32, 67, 68 , 103 MTC spreads to the perithyroidal and peritracheal lymph nodes in the central compartment of the neck, which extends between the trachea to the carotid sheath, and from the hyoid bone to the innominate vein. Within this compartment, spread is commonly bilateral. Further lymphatic spread can also occur to lateral neck compartments, including jugular and supraclavicular nodes. The regional lymph nodes have been classified by some authors into seven levels 2o,s4, 77 (Fig. 1). Often, lymph nodes in the upper mediastinum may be involved because of the caudal drainage of the thyroid gland. Spread to the lower tracheobronchial lymph nodes is generally considered to be equivalent to remote metastasis. 10 It is not uncommon for primary or metastatic MTC to involve adjacent structures by direct invasion or compression. Structures most commonly affected include the trachea, recurrent laryngeal nerve, jugular veins, and carotid arteries. Invasion of such structures may result in stridor, upper airway obstruction, hoarseness, dysphagia, and bleeding or arterial stenosis or occlusion. Hematogenous metastatic spread is considered a late event in tumor progression, but distant metastases have also been described at early tumor stages. 28 When distant spread is present, it most often occurs in the liver, lungs, and bone. In a study from a Swedish registry, it was noted that MTC patients with a palpable mass in the neck had distant metastatic disease in 20% of cases, regardless of heritability. 4 Furthermore, occult

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Lateral Compartment

Central Compartment Level VI

[ pretracheal and paratracheal (----;'"""'.::"'" ~ --E-

l l

inferior spinal accessory transverse cervical

Level IV

Level V (posterior triangle)

Mediastinum infraclavicular anterior mediastinal (thyrnic)

Level VII

Figure 1. The anatomic landmarks and lymph node compartments in the neck and upper mediastinum encountered in surgical reinterventions in medullarythyroid carcinoma. The central compartment is delimited inferiorly by the innominate vein, superiorly by the hyoid bone, laterally by the carotid sheaths, and dorsally by the prevertebral fascia. It comprises lymphatic and soft tissues around the esophagus and pretracheal and paratracheal lymph nodes that drain the thyroid bed (level VI). The submandibular nodal group (level I) is subsumed in the central compartment by some classifications. The lateral compartments span the area between the carotid sheath, the stemocleidomastoid muscle and the trapezius muscle. The inferior border is defined by the subclavian vein, and the hypoglossal nerve determines the superior boundary. The lymph node chain adjacent to the jugular vein is divided cranially to caudally in superior jugular nodes (level II} , midjugular nodes (level Ill}, and inferior jugular nodes (level IV). Lymph nodes situated in the posterior triangle between the dorsolateral sternocleidomastoid muscle, the trapezius muscle and the subclavian vein are classified as level V nodes. Mediastinal lymphatic tissue is referred to as level VII lymph nodes. (From Musholt TJ, Maley JF: Management of recurrent medullary thyroid carcinoma after total thyroidectomy. Prob Gen Surg 14:89-110, 1997; with permission .)

remote micrometastases are most likely the cause of most cases of persistent hypercalcitoninemia after extensive lymph node dissection. 90

GENETIC ADVANCES

In 1987 the gene for MEN 2A was localized to the pericentromeric region of chromosome 10 (lOql 1.2). 78 Subsequent reports demonstrated that MEN 2B and FMTC mapped to the same region. 42 •59 Genetic linkage and physical mapping studies further narrowed the critical region for MEN 2A to a 480 kb segment. 27•49 The RET proto-oncogene is included within this critical region, which made this an obvious candidate gene for

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the MEN 2 diseases. The RET proto-oncogene is a member of the receptor tyrosine kinase gene family and was originally found to be a dominant transforming gene activated by the replacement of the 5' region with a portion of a zinc finger-like gene in T-cell leukemia cell lines.86 This transmembrane oncogene basically consists of three domains: a cysteine-rich extracellular receptor domain, a hydrophobic transmembrane domain, and an intracellular tyrosine kinase catalytic domain. RET consists of at least 20 exons,41 and is expressed as five major mRNA species.84, 85 Point mutations associated with MEN 2A and FMTC were first identified in exons 10 and 11 of RET, which encode the juxtamembrane portion of the extracellular receptor domain. 17, 53 These mutations result in a nonconservative substitution of one of five cysteine residues (codons 609, 611, 618, 620, and 634). Mutations associated with FMTC have also been described at codons 768 (Glu to Asp) in exon 13 and 804 (Val to Leu or Met) in exon 14 in the intracellular domain. 8, 21 The mutations have been proposed to result in "gain-of-function" in the MEN 2 syndromes with increased intrinsic tyrosine kinase activity or alterations of substrate recognition, and hence, transforming capability.46 The RET proto-oncogene mutation associated with MEN 2B is characterized by allelic homogeneity with nearly each individual sharing the identical mutation in exon 16. 12, 33 In these cases, a methionine is changed to threonine (ATG to ACG) at codon 918. This codon is positioned within the tyrosine kinase catalytic core of the intracellular domain and probably participates in the formation of the putative substrate recognition region. 12 Mutations in the RET proto-oncogene have been associated with sporadic MTC. 8, 17 , 21 , 37, 45 Most commonly, these mutations involve codon 918, the codon mutated in MEN 2B. Mutations have also been found in other regions of the extracellular and intracellular domains. Missense, deletions, and insertion mutations have been described. Although the exact role of the RET gene product is unclear, evidence suggests that it is important in the embryonic development of the enteric nervous system and the kidneys. 76 Glial derived growth factor (GDNF) has recently been implicated as a ligand to the receptor domain of the RET gene product. 52, 65, 73 GDNF is a 32 kDa protein dimer that was first purified from glial cell lines and is a potent neurotrophic survival factor for motor neurons. There is compelling evidence that GDNF transduces a signal in RET. 19,3s, 9i, 92 A glycophosphatidylinositol (GPI)-linked protein called glialderived neurotrophic factor receptor-alpha (GDNFRa) is a cofactor in the signaling heterodimeric complex with RET. Current evidence suggests that GDNF binds directly to GDNFRa and indirectly with RET (Fig. 2). DIAGNOSIS

The prognosis of MTC is associated with disease stage at the time of diagnosis. Numerous studies of patients with MEN 2A who are treated for MTC have demonstrated a direct correlation between early diagnosis and cure of the disease.43, 69, 88, 96, 98, 101 Clearly, early diagnosis and treatment are of significant importance.

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GDNF GDNFRa

GPI RET

Extracellular

Exons 10 and 11 ~(MEN2A,FMTC

/

Intracellular

mutations)

(FMTC mutations)

~ Exon 16

(MEN 2B mutation)

Figure 2. A possible RET gene product, GDNFRa, and GDNF complex. The RET gene product is divided into the intracellular, transmembrane, and extracellular domains. The GDNFRa is believed to be anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage. Note that GDNF is indirectly bound to RET by way of GNDFRa, and dimerization of the RET molecules. Locations of exons containing mutations described for MEN 2A, MEN 2B, and FMTC are shown.

History and Physical Examination

More than 50% of patients with MEN 2B have unaffected parents, and the diagnosis is not usually made until a mass is discovered in the patient's neck. Occasionally, the diagnosis is made earlier by an astute clinician, who notes the characteristic phenotype. MEN 2B patients have a "marfanoid" habitus with long axial skeletal features, soft-tissue hypergnathism of the mid-face, and hyperflexible joints. They also typically have neuromata of the lips, conjunctiva, and gastrointestinal tract. Patients with MEN 2A and FMTC have a completely normal outward appearance. In these patients, the diagnosis of MTC has been made through screening efforts (elevated plasma calcitonin level in the basal or stimulated state) undertaken because of other affected family members, or by detection of a thyroid nodule on physical examination. Signs and symptoms of pheochromocytoma may be present in patients with MEN 2A and 2B. All patients with sporadic MTC and most index cases of MEN 2A and FMTC present with a thyroid nodule. Cervical lymph node involvement is present in over half of patients. Respiratory complaints, hoarseness, and dysphagia are seen in approximately 13% of patients. Approximately 12% of patients with sporadic MTC present with evidence of distant metastatic disease. 4

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MTC does not concentrate iodine and appears as a cold nodule on thyroid scintigraphy scans. Plain films may show areas of calcifications in the neck. Fine-needle aspirates are a sensitive means for establishing the diagnosis of MTC, especially if immunocytochemical stains for calcitonin are performed. Serum Calcitonin Screening

Thyroid C cells and MTC cells secrete calcitonin, which has been an invaluable marker for the presence of disease in screening and follow-up settings. Peripheral serum levels of this hormone are measured by radioimmunoassay. In patients with MTC, a direct correlation exists between basal plasma calcitonin levels and tumor mass 95 (Fig. 3). More than 30% of patients with MTC, however, will have normal basal levels of calcitonin. Therefore, provocative calcitonin stimulation tests are routinely performed. After an intravenous infusion of calcium gluconate (2 mg/kg over

100

10

.02

10

100

1000

Tumor Size (cm3) Figure 3. Correlation of preoperative basal plasma calcitonin levels versus size. The calcitonin levels are in pg/ml x 103 . The tumor size is in cm 3 . The solid circles and the triangles represent patients in group I (no clinical evidence of MTG and undetectable basal calcitonin levels, but stimulated with calcium and pentagastrin intravenous infusion). The open circles represent patients in group II (no clinical MTG but had elevated basal calcitonin). The squares represent patients in group Ill (clinically evident MTG) . (From Wells SA Jr, Baylin SB, Gann DS, et al: Medullary thyroid carcinoma: Relationship of method of diagnosis to pathologic staging. Ann Surg 188:377, 1978: with permission.)

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1 minute) followed by pentagastrin (0.5 µg/kg over 5 seconds), blood samples are obtained before and at 1, 2, 3, and 5 minutes after the infusion. Peak plasma calcitonin values generally occur at 1 to 2 minutes. The plasma immunoreactive monomeric form of calcitonin is the most reliable biochemical marker for the presence of MTC. The gastric hormone pentagastrin stimulates calcitonin secretion and is widely used (with or without additional calcium infusion) as a secretagogoue in socalled provocative calcitonin stimulation tests. The development of immunoradiometric assays increases detection to less than 3 pg/ mL, and therefore significantly improves the sensitivity and specificity of the provocative calcitonin stimulation test. Evidence suggests that preoperative stimulated calcitonin levels less than 1000 pg/mL were associated with MTC confined to the thyroid gland in greater than 95% of patients with MEN 2 diseases (Table 2).96 Approximately half of the patients with stimulated calcitonin levels less than 1000 pg/ mL had only microscopic evidence of disease, whereas all patients with levels greater than 10,000 pg/mL had macroscopic evidence of disease, with approximately 17% of these having evidence of distant metastatic MTC. Clinical and pathologic studies have shown that MEN 2 patients with MTC had an earlier stage of disease and a higher cure rate if the thyroid neoplasms were diagnosed biochemically rather than clinically with a palpable thyroid mass. 95 Despite the fact that provocative calcitonin stimulation tests are highly specific and sensitive, there still are several disadvantages associated with the test. First of all, a positive stimulated calcitonin test requires sufficient tumor mass to produce elevations of plasma calcitonin levels and thus indicates that cancer has already developed. Even in the setting of thyroidectomy for small primary tumors of less than 1 mm, there are a few patients who will develop distant metastatic disease. These patients would benefit from earlier thyroidectomy. Second, the test is uncomfortable for the patients; they often complain of nausea, diaphoresis, agitation, and urinary urgency. Members of MEN 2 kindreds who are at risk for Table 2. PROGNOSTIC SIGNIFICANCE OF PREOPERATIVE STIMULATED PLASMA CALCITONIN LEVELS

Group

2 3 4

Postop CT* (%) (> 300pg/ml)

Preop CT (pg/ml)

RLNM* (%)

250 to 1000 (n = 25) 1000 to 5000 (n = 36) 5000 to 10,000 (n) = 8) >10,000

1 (4)

1 (4)

0

0

3 (8.3)

6 (16.7)

0

0

2 (25)

1 (12.5)

0

0

4 (17)

2 (8.7)

13 (57)

14 (61)

DTH (%)

DM (%)

Preop CT = preoperative stimulated plasma calcitonin level; Postop CT = postoperative stimulated plasma calcitonin level; RLNM = Regional lymph node metastases; DTH = Death. •Group 1 or 2 versus Group 4, P < 0.001 From Wells SA Jr, Baylin SB, Leight GS, et al: The importance of early diagnosis in patients with hereditary medullary thyroid carcinoma. Ann Surg 195: 595, 1982; with permission.

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inheriting the diseases are screened at early ages, usually around 5 years of age, and require annual testing until the age of 45. It is understandable, therefore, why compliance is often poor, especially among adolescents and young children. Furthermore, because the disease is inherited in an autosomal dominant fashion, 50% of at-risk patients will never develop disease and would be spared the expense and inconvenience of routine scheduled testing if a definitive genetic test were applied. Surgical Treatment

The surgical approach to medullary thyroid cancer has been influenced by several factors. First of all, the clinical course of MTC is usually more aggressive than that of differentiated thyroid cancer. Second, MTC cells do not concentrate iodine and are not sensitive to 1-131 treatment. Third, MTC is multicentric in 90% of patients with the hereditary forms of the disease, and in 20% of patients with the sporadic form. 69 Fourth, with the exception of children whose MTC is discovered as part of a genetic or biochemical screening program,44•97 more than 50% of patients who present with MTC have nodal metastases. 6• 48• 83 Lastly, the ability to measure postoperative stimulated calcitonin levels has allowed assessment of the adequacy of surgical extirpation. Total thyroidectomy is the appropriate treatment of the primary tumor, accompanied by a central node dissection. In this operation, all thyroid tissue and all nodal tissue from the level of the hyoid bone superiorly to the innominate vessels inferiorly is removed. After the parathyroid glands are identified, central nodal tissue on the anterior surface of the trachea is removed, exposing the superior surface of the innominate vein behind the sternal notch. Fatty and nodal tissue between the carotid sheaths and the trachea is removed, including paratracheal nodes along the recurrent nerves. On the right, the junction of the innominate and right carotid arteries is exposed, and on the left, nodal tissue is removed to a comparable level behind the head of the left clavicle. A systematic approach to the removal of all nodal tissue in the central neck has been reported to improve recurrence and survival rates when compared retrospectively with procedures in which only grossly involved nodes were removed.18 Controversy exists over the optimal management of the parathyroid glands. Some surgeons prefer to leave the parathyroid glands in situ, ensuring that the vascular pedicle is preserved. 18• 69 At our institution, we do not believe that an adequate thyroidectomy and node clearance can be achieved unless the parathyroids are also removed. Furthermore, the vascular supply to a parathyroid gland may be interrupted by dissection and excision of perithyroidal and central nodes. Therefore, we advocate total parathyroidectomy with autotransplantation as part of total thyroidectomy for MTC. Parathyroid glands are therefore removed and preserved in cold saline at the time of thyroidectomy for MTC. At the end of the procedure, one or two glands are sliced into 20 1 by 3 mm pieces and autotransplanted into the muscle of the sternocleidomastoid muscle in patients with sporadic MTC, FMTC, and MEN 2B, or into the nondominant forearm in patients with MEN 2A because they have a 30% chance

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of developing hyperparathyroidism.61 The remaining parathyroid tissue is viably frozen and stored. 102 The autografts generally function well within 4 to 6 weeks, at which time patients can be taken off of calcium supplementation. In a recent series of thyroidectomies performed in 13 patients with hereditary MTC identified by genetic screening, total thyroidectomy and central node dissection with parathyroidectomy and parathyroid autografting were performed in all patients. All patients were normocalcemic after stopping calcium supplementation 8 weeks postoperatively.97 In other series, the percentage of patients requiring calcium supplementation following parathyroidectomy with parathyroid autografting has ranged from 0 % to 18%.35,99, 101 The decision to perform a more extensive lateral node dissection is based on the clinical suspicion that the jugular nodes may be involved with tumor. Certainly patients with palpable adenopathy in the anterior or posterior jugular chain should undergo ipsilateral functional or modified radical neck dissection. In patients without such gross evidence of nodal involvement, the indications for removal of lateral nodes is less clear. Some authors have recommended sampling of the mid-jugular nodes, with formal neck dissection reserved for patients in whom metastatic tumor is demonstrated.69 Our approach is to perform ipsilateral neck dissection in patients who have palpable adenopathy in central, paratracheal, or jugular nodes, and in patients with large (greater than 2 cm) primary tumors. This operation entails removal of the jugular chain of nodes, which includes nodes anterior to and posterolateral to the jugular vein, from the level of the mastoid process to the subclavian vessels, and laterally to the accessory nerve. Unless invaded by tumor, the jugular vein and sternocleidomastoid muscle are left intact. Genetic Testing for MEN 2 and Related Syndromes

Discovery of the RET proto-oncogene mutations associated with the MEN 2 syndromes has allowed the detection of disease gene carriers before their stimulated calcitonin levels become elevated . In principle, genetic testing, which requires the drawing of blood for extraction of lymphocyte DNA, need only be performed once in an at-risk individual's lifetime. Stimulated calcitonin testing remains an important modality in following patients for recurrent or residual disease after thyroidectomy. To test a patient for the presence of a mutation in RET, peripheral blood is drawn and DNA is extracted from lymphocytes. Regions of the RET proto-oncogene are amplified by polymerase chain reaction (PCR) and mutations are detected by one of several techniques. These include direct DNA sequencing, analysis of restriction sites introduced or deleted by a mutation, or by gel shift analysis (denaturing gradient gel electrophoresis or single strand conformation polymorphism analysis). 9, 14, 15, 23, 44, 97 The technique used by our group at Washington University is shown in Figures 4 and 5.15

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PCR Amplification

~

Normal Allele

Mutant Allele

---Leu- QL§.-A rg -Thr---

-Leu - Arq-Arg-Thr---

_.

__.

---CTG- TGC-CGC-ACG-----GAC- 8CG -GCG-TGC---

,-'

---CTG-QGC-CGC-ACG-----GAC-GCG-GCG-TGC -- -

.___

234 bp

.___

234 bp Cfo I Cleavage

~

---CTG- TGC -CGC-ACG-----GAC- 8CG-GCG-TGC--234 bp

61 bp

Normal 234 bp 173 bp

61 bp

-

Mutant

-

'

---CTG-QG C-CGC-ACG-----GAC qcG -GCG-TGC---

A

173 bp

Normal +Mutant

--

Gel Electrophoresis stained with Ethidium Bromide

Figure 4. PCR restriction endonuclease genetic test. Genomic DNA from an individual at risk is used as a template in the PCR amplification of a part of RET exon 11. The PCR product is 234 base pairs (bp) in length. The MEN 2A mutation is indicated by the underlined DNA and amino acid sequences. A Cfo I recognition site is produced by the mutation in exon 11 and is represented by bold print. Vertical arrowheads indicate the site of cleavage by Cfo I. Horizontal arrows indicate 5' to 3' direction of the DNA. The PCR products are incubated with the appropiate restriction endonuclease (Cfo I) . Because the normal gene does not possess a Cfo I cleavage site, it remains intact as a 234 bp product. The mutant gene, however, is cleaved by Cfo I into two products of 173 and 61 bp. The DNA fragments are separated by electrophoresis. Unaffected individuals have two copies of the normal gene and thus have a single uncleaved 234 bp product. Affected persons, however, have one normal gene and one mutated gene, and therefore have the uncleaved 234 bp product and the cleaved 173 and 61 bp products. (From Chi DD, Toshima K, Donis-Keller H, et al: Predictive testing for multiple endocrine neoplasia type 2A (MEN 2A) based on the detection of point mutations in the RET protooncogene. Surgery 116:124-133, 1994; with permission .)

Although genetic testing can be performed at birth, the age at which thyroidectomy should be performed is controversial. Presently, in children with MEN 2A and FMTC, we advocate performing total thyroidectomy with central lymph node dissection and total parathyroidectomy with heterotopic autotransplantation at the age of 5 years (management of the parathyroids in these children is controversial, because some experts advocate parathyroid preservation). In patients with MEN 2B, surgery should be performed during infancy, owing to the biologic aggressiveness

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Sile Standards 2602

Size 2605

2607

2608

2606

2610

2612

2613 2611

2615

2623 2603

2634

2636 2635 Standards

Figure 5. Direct analysis of the RET mutation in a portion of an MEN 2A kindred. Individuals with proven clinical diagnosis of MEN 2A are represented by the partially shaded circles or squares (individuals 2608, 2606, 2612, 2611, 2623, 2603, and 2636). Individuals at risk for the disease are individuals 2607, 2613, 2615, and 2636. The mutation in this kindred is located in exon 11 and creates a site cleaved by the restriction endonuclease Cfo I as illustrated in Figure 4. Individuals 2607, 2615, and 2636 have a single 234 bp PCR product and therefore do not have the RET mutation. Individual 2613, however, has the cleaved 173 bp fragment, and thus inherited the mutated gene from her mother. (From Chi DD, Toshima K, Donis-Keller H, et al: Predictive testing for multiple endocrine neoplasia type 2A (MEN 2A) based on the detection of point mutations in the RET protooncogene. Surgery 116:124-133, 1994; with permission.)

of MTC in this disease. We have performed total thyroidectomies with central lymph node dissection in MEN 2B children as young as 6 months old. 81 Preventative Surgery for MEN 2A Gene Carriers

In 1994, Wells and colleagues at Washington University in St. Louis reported a series of prophylactic thyroidectomies in MEN 2A and FMTC patients.97 Genetic testing was performed in 132 members of seven kindreds with MEN 2A. Of the 58 individuals at risk for the disease, 21 inherited the RET mutation. After the patients and their families discussed the test with the surgeons, nurses, and genetic counselors, 13 decided to proceed with prophylactic total thyroidectomy, central lymph node dissection, and total parathyroidectomy with heterotopic autotransplantation. The results are summarized in Table 3. Six patients had normal preoperative plasma calcitonin levels, and seven had elevated calcitonin levels. All patients had normal stimulated calcitonin levels postoperatively. The age at the time of thyroidectomy ranged from six to 21 years. The average age of patients with normal preoperative calcitonin levels was 12 years, with a range of six to 21 years. A total of 176 lymph nodes were examined in the 13 patients, and no evidence of metastasis was ob-

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Table 3. PROPHYLACTIC THYROIDECTOMY IN PATIENTS WITH MEN 2A AND FMTC BASED ON GENETIC MUTATION ANALYSIS. Number identified by genetic testing Consented for thyroidectomy Males Females Preoperative CTa Normalb Elevated 0 Postoperative CT Normal Elevated Regional lymphatic metastasis C cell disorder (MTC or cell hyperplasia) Mean age (range) Total Normal Preoperative CT Elevated Preoperative CT

21 13

9 4 6 7 13

0 0 13 13.2 years (6 to 21 years) 12 years (6 to 21 years) 15.1 years (8 to 20 years)

•CT = calcitonin 'Normal CT levels: :5300 pg/ml for males, :5 100 pg/ml for females 0 Elevated CT levels: >300 pg/ml for males, > 100 pg/ml for females From Wells SA Jr, Chi DD, Tashima K, et al: Predictive DNA testing and prophylactic thyroidectomy in patients at risk for multiple endocrine neoplasia type 2A. Ann Surg 220:237, 1994; with permission.

served. All thyroid specimens were found to have evidence of a C cell disorder, regardless of whether or not the preoperative calcitonin levels were elevated. The parathyroid grafts were all functional as evidenced by the normal calcium levels and the PTH gradient (determined by measuring serum PTH levels in the grafted arm compared with the nongrafted arm). In a similar study from The Netherlands, Lips et al identified 14 young members of families affected by MEN 2A who had normal calcitonin testing but who were found to be MEN lA gene carriers by DNA testing. 44 Thyroidectomy was done on eight of these 14 and foci of MTC were identified in all eight. At our institution, Skinner and coworkers subsequently evaluated thyroidectomy for MTC in 49 children with MEN 2A and MEN 2B. 81 In this series, 14 children had a prophylactic thyroidectomy based on genetic testing. The average age of the children at the time of surgery was 10.5 years. Postoperative calcitonin levels were all normal, and there was no evidence of recurrent MTC with a mean follow-up of 1.3 years (Fig. 6). To date, our group at Washington University has performed 62 prophylactic thyroidectomies in at-risk kindred members found to have RET gene mutations. Lymph node metastases were identified in three patients. Postoperative calcitonin levels were normal in all patients except for one six-year-old girl with MEN 2B. POSTOPERATIVE FOLLOW-UP

All patients should be followed postoperatively with provocative calcitonin stimulation testing and carcinoembryonic antigen (CEA) to look

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0.50 0.45 0.40 0.35

::J 0.30 E

Ci

0.25

0

0.20

-

.!:-

0.15 0.10 0.05 0 basal

1.5 min.

2 min.

3 min.

5 min.

Time Figure 6. Preoperative and postoperative stimulated serum calcitonin testing. This patient had preoperative calcitonin stimulation testing performed before surgery on December 27, 1995 (open squares). A total thyroidectomy, central lymph node dissection, and total parathyroidectomy with heterotopic autotransplantation was performed. Postoperative calcitonin stimulation testing was performed on November 30, 1995 (solid squares) . Upper limits of normal for males at each time point is represented by diamonds. Calcitonin levels were determined by radioimmunoassay (Nichols Institute, San Juan Capistrano, CA) on plasma samples before (basal), and at 1.5, 2, 3, and 5 minutes after intravenous infusion of calcium and pentagastrin.

for persistent or recurrent disease. In more than 50% of patients with MTC, the CEA levels are elevated. This marker has been found to be useful in following these patients postoperatively.3, 100 One study found that a rising CEA level in MTC patients with a stable or falling calcitonin level corresponded to dissemination of a virulent dedifferentiated tumor.11 Our recommendations for patients who have undergone prophylactic total thyroidectomy and central lymph node dissection for MTC is to perform the provocative calcitonin test postoperatively, and then at 2 and 5 years if there is no evidence of disease in the lymph nodes. Although elevated basal calcitonin levels may indicate residual disease after thyroidectomy for MTC, normal levels do not rule out residual disease89 and are therefore not sufficient to identify early tumor progression in the postoperative follow-up of MTC patients. For this reason, provocative calcitonin stimulation testing is routinely performed. The postsurgical reduction of provocative calcitonin stimulation levels indicates degree of success in eradicating the tumor and serves as a guideline for considering a patient free of disease and possibly cured. The expression "biochemically cured" has been used to describe a normocalcitoninemic MTC patient who does not demonstrate a calcitonin peak in a provocative calci-

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tonin stimulation test after surgical intervention. Because delayed reduction of plasma calcitonin has been described,26 elevated levels in the immediate postoperative period may be misleading. Furthermore, despite the widespread use of more sensitive calcitonin assays, some uncertainty still remains about normal ranges of the hormone level. Therefore, it remains unclear how to interpret borderline elevated but stable stimulated calcitonin levels. Yearly screening for pheochromocytoma (MEN 2A and MEN 2B) and hyperparathyroidism (MEN 2A) should be done. Testing for pheochromocytoma requires a 24 hour urine collection for catacholamines and metabolites, and hyperparathyroidism can be monitored by serum calcium measurements.

PERSISTENT OR RECURRENT HYPERCALCITONINEMIA

A problem that is frequently encountered in MTC patients is that of elevated calcitonin levels following primary surgery, indicating residual or recurrent MTC. In one study of patients who presented with palpable tumors, 15/18 (83%) of patients with hereditary disease and 11/20 (55%) of patients with sporadic tumors had persistent disease as indicated by elevated calcitonin levels postoperatively.6 Many patients with persistently high levels of calcitonin following thyroidectomy and node dissection continue to do well without other evidence of disease for many years. 7•56•83•94 In contrast, in a Norwegian study of 84 cases of MTC,56 it was noted that more than 50% of patients who presented with cervical node metastases eventually died of disease. The variable outcome of patients with positive lymph nodes is explained by differences in the biologic virulence of the tumor, the extent of spread at the time of treatment, and the adequacy of surgical extirpation. Clinical settings in which patients have persistent or recurrent hypercalcitoninemia after surgical treatment for MTC is most commonly caused by an inadequate surgical procedure performed at the index operation. If a patient with sporadic MTC presents with a thyroid nodule and FNA is either nondiagnostic or not performed, it is not unusual for the patient to be treated with a lobectomy or subtotal thyroidectomy. Furthermore, an extensive lymph node dissection is usually not performed. Indeed, in our experience, more than 85% of cases of persistent hypercalcitoninemia that were referred to us had what we consider an inadequate primary operation. These cases necessitate early, more extensive reoperation, including appropriate lymph node clearance. Patients who are index cases of MEN 2A, MEN 2B, and FMTC, and patients with sporadic MTC usually present with clinically evident disease. The presence of an intrathyroidal mass is associated with metastatic spread to the locoregional lymph nodes in more than half of the patients and increases the risk of persistent hypercalcitoninemia after operation in 50% to 100% of cases.6•62•83

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Preoperative Evaluation of Persistent Hypercalcitoninemia

Despite the introduction of genetic testing, new diagnostic procedures, and significant surgical efforts, the prevalence of persistent and recurrent hypercalcitoninemia after primary resection of MTC remains high. In 1986 Tisell et al90 described a novel surgical approach to persistent and recurrent hypercalcitoninemia. They termed this operation microdissection, which entails removal of all lymphatic, fatty, and connective tissue in anatomically defined compartments of the neck and mediastinum, which contain the lymphatic drainage of the thyroid. Several studies have subsequently demonstrated that this approach results in biochemical cure in 20% to 30% of patients and may prolong survival time in others.10, 16,50,51,54, 90 The indication, strategy, and goal of reoperation for MTC have to take into account the individual clinical setting. Considerations include the type of MTC, adequacy of primary tumor extirpation according to the operative report, histologic findings, and results of the preoperative staging. Careful patient evaluation is imperative for the success of a reintervention with curative intent. Determination of not only the presence of metastatic disease, but careful localization of the tumor is of paramount importance in determining whether attempts for palliation or cure will be undertaken. As a result, considerable effort is spent on correct identification of metastatic disease. On physical examination, patients may present with adenopathy of the paratracheal and jugular areas. By careful palpation, firm, nodular lymph nodes or residual thyroid tumor may be appreciated. In advanced cases, subcutaneous metastatic lesions have been described. Invasion of the aerodigestive tract may be manifested by stridor, dysphagia, dysphonia, or hemoptysis. Conventional imaging studies for MTC localization include CT scans, MR images, and ultrasound. In approximately half of all cases, micrometastases are not detected by these methods. These are defined as occult micrometastases. In these circumstances, other diagnostic modalities need to be used, such as nuclear imaging studies or invasive procedures. High resolution ultrasound was described in one study as the most sensitive and reliable method to detect recurrent disease when compared with physical examination, CT scans, selective venous catheterization, fine-needle aspiration biopsies, and combinations of these.66 Even with careful microsurgical resection, however, few patients had normalization of the provocative plasma calcitonin levels, suggesting missed, residual disease. 25 Ultrasound, CT scans, and MR images are highly specific for detecting metastatic MTC, but notably lack sensitivity, especially MR imaging, when compared with selective venous catheterization (SVC) with provocative calcitonin testing. More sensitive methods have been described, but are neither widely available or cost-effective. As a result, ultrasound, CT, or MR images are usually the first imaging studies obtained to rule

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out residual disease. Ultrasound and ultrasound guided biopsy are particularly useful to evaluate neck masses, whereas CT or MR imaging is done to look for evidence of intrathoracic or intra-abdominal spread. A number of different radiopharmaceuticals have been described to localize metastatic MTC. Thallium chloride 201 (201 Tl) and technetium 99m dimercaptosuccinic acid (99 mTc DMSA) have been shown to be useful in evaluating hypercalcitoninemic patients. 5 , 40, 60 The use of 1311-m-iodobenzylguanidine (MIBG) scintigraphy has demonstrated the ability to identify MTC, but not consistently. 80 Octreotide scans with indium-111 (111 ln) have been used to localize metastatic disease, but only have a sensitivity of approximately 57% to 67%, 2, 24 Selective venous catheterization for calcitonin in occult metastatic disease is a sensitive technique that can localize MTC to a general area of the neck Catheters are placed into the internal and external jugular veins, innominate veins, hepatic veins, and mediastinal veins, and stimulated calcitonin levels are measured and compared with peripheral levels, Metastatic disease is localized to regions of the neck drained by veins in which a step-up is noted, 1 Although the test is extremely sensitive, not all metastatic foci are detected by SVC sampling. Cervical venous drainage may have been altered by previous surgery, and the general areas drained by these veins is large, At our institution, we have found diagnostic laparoscopy to be invaluable in the evaluation of distant hepatic disease in MTC. When MTC metastasizes to the liver, the lesions may have a miliary appearance with multiple small (1 to 5 mm) white, raised lesions on the liver surface (Fig, 7), In other cases, a small lump may be noted on the surface of the liver which indicates a subcapsular lesion. These metastases are usually not detected on CT, MR imaging, or ultrasound. In our series of 44 patients, metastatic MTC lesions were demonstrated in 10 patients, nine of whom had negative CT or MR imaging.50, 93 TREATMENT OF PATIENTS WITH PERSISTENT OR RECURRENT HYPERCALCITONINEMIA Radiation Therapy

Radioactive iodine (1311) has not been found to be helpful in the treatment of patients with metastatic MTC because the C cells do not take up iodine. Nonetheless, several authors have used 1311 to treat MTC on the assumption that uptake by the follicular cells would expose the MTC cells to high doses of radiation based on proximity, There are, however, no data to support the use of 131 1 in the treatment of MTC. 70 The use of external beam radiation has been reported with variable results. Although several authors have advocated the use of external beam radiation therapy, these studies were retrospective and were done on small numbers of patients, 6s, 7o, 79, 32 Other studies have not supported the use of radiation therapy in MTC. In one retrospective study of 202 patients, patients who underwent external beam radiation were found to

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Figure 7. A, CT scan of liver in 29-year-old woman with MEN 2A and elevated calcitonin levels after total thyroidectomy and bilateral neck dissections. No radiographic evidence of hepatic metastases is present. B, Laparoscopic video image of superior surface of right lobe of liver from the same patient. Arrows indicate two 3-mm nodules on the surface of the liver that were biopsied and found to be metastatic MTG. (From Tung WS, Vesley TM, Moley JF: Laparoscopic detection of hepatic metastases in patients with residual or recurrent medullary thyroid cancer. Surgery 118:1024-1030, 1995; with permission) .

have worse outcomes than those who did not. 72 In a retrospective study from a French cooperative group, the investigators studied the effects of external beam radiation postoperatively to patients who were treated for MTC. Of the 59 patients who were followed (mean 5.4 years), 18 developed clinically evident recurrent disease, and many developed local complications, primarily invasion into the aerodigestive tract. 55

Chemotherapy

Data for the use of chemotherapeutic agents in the treatment of MTC is limited to advanced or disseminated disease. Single agent treatment with doxorubicin wa·s shown to provide complete response in 3 of 5 patients in an early trial, 29 but subsequent studies have failed to confirm these results. Combination chemotherapy with doxorubicin, cisplatin, and vindesine resulted in one partial remission and three minor responses out

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of 10 patients treated. 74 Other combinations of chemotherapeutic regimens reported in the literature include dacarbazine and 5-fluorouracil (5-FU) cyclophosphamide, vincristine, and dacarbazine, 5 FU and streptozocin. None of these combinations have been shown to be of significant benefit. 63, 64, 75, 105

Cervical Reoperation

Several groups have reported their experience with reoperation for persistent or recurrent MTC in the neck. 7•10•2o,so,si,57•9 o. 94 A significant reduction in stimulated calcitonin levels following reoperation was reported in many patients, and normalization of calcitonin levels was noted in some. We reported two series of cervical reoperations for medullary thyroid carcinoma: from 1990 to 1993,51 and from 1993 to 1996.50 In our first reported series of such reoperations, we described experience with 37 operations in 32 patients. 51 The patients had previously undergone total thyroidectomy and most of the patients also had previous lymph node dissections. All patients had elevated stimulated calcitonin levels. Localization studies, including selected venous catheterization, CT scanning, and physical examination were successful in localizing tumor in half the cases. Operative morbidity was low and there were no deaths. In 28 of the 35 operations, discharge from the hospital occurred 2 to 5 days postoperatively. In nine cases (group 1), calcitonin was reduced to undetectable levels following reoperation. In 13 cases (group 2), postoperative calcitonin levels were decreased by 40% or more. In 10 cases (group 3), postoperative calcitonin levels were not improved. Patient sex, disease, number of nodes previously resected, preoperative calcitonin levels, and preoperative localization study results were not significantly different between the three groups and therefore unlikely to predict outcome for reoperation. Previously resected tumors from patients in group 3, however, were more likely to have demonstrated invasive features (invasion of adjacent structures, extranodal or extracapsular spread) than tumors from patients in groups 1 and 2 (P < 0.05, Fisher's exact test). We concluded that reoperation with meticulous removal of residual nodal and tumor tissue in patients with persistent postoperative hypercalcitoninemia, resulted in normalization of calcitonin levels in 28% of patients, and a decrease in calcitonin levels by 40% or more in another 42% of patients. The results also suggested that determination of the degree of invasiveness of the primary tumor may help in selecting patients likely to benefit from reoperative surgery for recurrent medullary thyroid cancer. We sought to improve our results through better selection of patients likely to benefit from reoperation. 50 We achieved this by applying systematic metastatic work-up including routine CT or MR imaging of the neck, chest, and abdomen, SVC in selected patients, and by institution of routine staging laparoscopy, described earlier.

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One hundred and fifteen patients with persistent elevation of calcitonin after primary surgery for MTC were evaluated. Ten patients were found to have liver metastases by laparoscopy and did not undergo cervical re-exploration. Seven patients had palliative cervical operations and 45 patients had cervical re-exploration with curative intent. In the seven patients who had palliative cervical operations, one patient had persistent postoperative hypocalcemia. There were no other complications in that group. In the 45 patients who underwent reoperation with curative intent, there were no postoperative deaths and no transfusions were required. Complications included thoracic duct leak that occurred in four patients (8.9%), and 4% hypocalcemia (follow-up 3 months and 2 years). There were no permanent nerve injuries. Of the 45 patients who underwent reoperation with curative intent, the mean decrease in postoperative stimulated calcitonin level was 73.1% (Fig. 8). In 22 of 45 patients (48%), the postoperative stimulated calcitonin level dropped more than 90% compared with the preoperative value. Of these 45 patients, 17 (38%) had postoperative stimulated calcitonin levels that were within the normal range (group 1), and six (13%) had no significant decrease in stimulated calcitonin levels (group 3). The remaining patients had a greater than 35% reduction in stimulated calcitonin levels (group 2). As in our earlier series, tumor invasiveness was the only pa-

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Patients Figure 8. Postoperative change in peak stimulated calcitonin levels. The shaded bars indicate the postoperative stimulated calcitonin levels of 45 patients who underwent the curative cervical re-exploration and dissection. The postoperative calcitonin level is expressed as a percentage of the preoperative calcitonin level. One hundred percent indicates no change in calcitonin level , 10% indicates that the stimulated calcitonin level fell by 90%. *Postoperative levels were higher than were preoperative levels. (From Maley JF, Dilley WG, DeBenedetti MK: Improved results of cervical reoperation for medullary thyroid carcinoma. Ann Surg 255:734-743, 1997; with permission.)

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rameter that correlated with failure to reduce postoperative calcitonin levels to the normal range (P < 0.05, Fisher's exact test). In group 1 patients, review of pathology from the primary operation did not reveal invasiveness in any case (0/17). In group 2 and 3 patients, invasiveness was identified in 8/28 cases. Of the 17 patients who had normal postoperative stimulated calcitonin levels, 1 year follow-up levels are available for 12 patients. In 10 of the 12 patients the levels remained normal. Two patients had recurrent elevation of calcitonin levels that has persisted after a 2-year follow-up. In five patients with normal calcitonin levels at 1 year, the stimulated calcitonin level remained normal at 2-year follow-up . These results indicate an improvement in outcome following reoperation for persistent or recurrent MTC. In our most recent series (1992 to 1996), 38% (17 I 45) of patients had normal postoperative stimulated calcitonin levels, compared with 28% (9 /32) in the first series. In the most recent series, only 13% (6/ 45) of patients had no decrease in calcitonin levels following reoperation, compared with 31 % (10/32) in the first series (P = 0.07, Fisher's exact test). This improvement has mainly occurred through better preoperative selection of patients and the institution of routine laparoscopic liver examination preoperatively, which identified metastases in 10 patients, nine of whom had normal CT or MR imaging of the liver and who would otherwise have undergone neck reoperation with curative intent. CONCLUSION

Recent advances in the understanding of MTC at the clinical, cellular, and molecular levels have led to dramatic changes in the management of the disease. After the introduction of microsurgical dissection for persistent hypercalcitoninemia, further developments, including more accurate localization and staging studies, have led to improved outcome and better patient selection for surgery. Although we still await long-term analysis for these data, early results are encouraging. The identification of mutations in the RET proto-oncogene associated with MEN 2A, MEN 2B, FMTC, and some cases of sporadic MTC has become the cornerstone in our understanding and management of patients with these diseases. Prophylactic thyroidectomy based on direct mutation analysis seems to be curative in MEN 2A and FMTC patients when they are screened at a young age. Again, longer follow-up is required to fully evaluate the efficacy of predictive genetic testing. Predictive genetic testing is also becoming available for other diseases. For instance, genetic analysis programs have been established for hereditary forms of colon cancer (such as familial adenomatous polyposis and hereditary nonpolyposis colon cancer) and familial forms of breast cancer. In these diseases, however, genetic DNA testing serves a more useful role in selective screening for individuals determined to be at high risk. As the genetic basis of more diseases are discovered, the usefulness of molecular

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genetics for diagnosis and therapy will be more commonplace and its role in prophylactic interventions will be better defined. Even with all the improvements and recent discoveries, there still remain many questions regarding MTC. As the understanding of the RET proto-oncogene and its interactions with GDNF and other ligands, and as the mechanism for intracellular interactions becomes elucidated, we are hopeful that newer and novel forms of diagnosis and therapy will become available. References l. Abdelmoumene N, Schlumberger M, Gardet P, et al: Selective venous sampling cath-

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

eterisation for localization of persisting medullary thyroid carcinoma. Br J Cancer 69:1141-1144, 1994 Baudin E, Lumbroso J, Shlumberger M, et al: Comparison of octreotide scintigraphy and conventional imaging in medullary thyroid carcinoma. J Nucl Med 37:912-916, 1996 Becker KL, Liva OL, Snider RH, et al: The surgical implications of hypercalcitoninemia. Surgery, Gynecology, and Obstetrics 154:897, 1982 Bergholm U, Adami HO, Bergstrom R, et al: Clinical characteristics in sporadic and familial medullary thyroid carcinoma: A nationwide study of 249 patients in Sweden from 1959 through 1981. Cancer 63:1196, 1989 Bigsby RJ, Lepp EK, Litwin DE, et al: Technetium 99m pentavalent dimercaptosuccinic acid and thallium 201 in detecting recurrent medullary carcinoma of the thyroid. Can J Surg 35:388-392, 1992 Block MA, Jackson CE, Greenwald KA, et al: Clinical characteristics distinguishing hereditary from sporadic medullary thyroid carcinoma: Treatment implications. Arch Surg 115:142, 1980 Block MA, Jackson CE, Tshjian AH Jr: Management of occult medullary thyroid carcinoma: Evidenced only by serum calcitonin level elevations after apparently adequate neck operations. Arch Surg 113:368, 1978 Bolino A, Schuffenecker I, Luo Y, et al: RET mutations in exons 13 and 14 of FMTC patients. Oncogene 10:2415, 1995 Borst MJ, VanCamp JM, Peacock ML, et al: Mutational analysis of multiple endocrine neoplasia type 2A associated with Hirschsprung's disease. Surgery 117:386-391; 1995 Buhr HJ, Kallinowski F, Raue F, et al: Microsurgical neck dissection for occultly metastasizing medullary thyroid carcinoma. Three-year results. Cancer 72:3685, 1993 Busnardo B, Girelli ME, Simioni N, et al: Nonparallel patterns of calcitonin and carcinoembryonic antigen levels in the follow-up of medullary thyroid carcinoma. Cancer 53:278, 1984 Carlson KM, Dou S, Chi D, et al: Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B. Proc Natl Acad Sci US A 91:1579, 1994 Carney JA, Go VLW, Sizemore GW, et al: Alimentary tract ganglioneuromatosis: A major component of the syndrome of multiple endocrine neoplasia type 2b. N Engl J Med 295:1287, 1976 Chi DD, Dou S, Carlson K, et al: A rapid presymptomatic genetic test for the detection of mutations in the RET proto-oncogene associated with multiple endocrine neoplasia type 2A (MEN 2A). Surgical Forum 45:510, 1994 Chi DD, Toshima K, Donis-Keller H, et al: Predictive testing for multiple endocrine neoplasia type 2A (MEN 2A) based on the detection of point mutations in the RET protooncogene. Surgery 116:124-133, 1994 Chong GC, Beahrs OH, Sizemore GW, et al: Medullary carcinoma of the thyroid gland. Cancer 35:695, 1975 Donis-Keller H, Dou S, Chi D, et al: Mutations in the RET protooncogene associated with MEN 2A and FMTC. Hum Mol Genet 2:851, 1993

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18. Dralle H, Damm I, Scheumann GFW, et al: Compartment-oriented microdissection of regional lymph nodes in medullary thyroid carcinoma. Surg Today 24:112, 1994 19. Durbec P, Marcos-Gutierrez CV, Kilkenny C, et al: GDNF signalling through the RET receptor tyrosine kinase. Nature 381:789, 1996 20. Ellenhorn JD, Shah JP, Brennan MF: Impact of therapeutic regional lymph node dissection for medullary carcinoma of the thyroid gland. Surgery 114:1078, 1993 21. Eng C, Smith DP, Mulligan LM, et al: A novel point mutation in the tyrosine kinase domain of the RET proto-oncogene in sporadic medullary thyroid carcinoma and in a family with FMTC. Oncogene 10:509, 1995 22. Farndon JR, Dilley WG, Baylin SB, et al: Familial medullary thyroid carcinoma without associated endocrinopathies: A distinct clinical entity. Br J Surg 73:278, 1986 23. Feldman GL, Kambouris M, Talpos GB, et al: Clinical value of direct DNA analysis of the RET proto-oncogene in families with multiple endocrine neoplasia type 2A. Surgery 116:1042-1047, 1994 24. Frank-Raue K, Bihl H, Dorr U, et al: Somatostatin receptor imaging in persistent medullary thyroid carcinoma. Clin Endocrinol (Oxf) 42:31-37, 1995 25. Frank-Raue K, Raue F, Buhr HJ, et al: Localization of occult persisting medullary thyroid carcinoma before microsurgical reoperation: High sensitivity of selective venous catheterization. Thyroid 2:113-117, 1992 26. Fugazzola L, Pinchera A, Luchetti F, et al: Disappearance rate of serum calcitonin after total thyroidectomy for medullary thyroid carcinoma. Int J Biol Markers 9:21, 1994 27. Gardner E, Papi L, Easton DR, et al: Genetic linkage studies map the multiple endocrine neoplasia type 2 to a small interval on chromosome 10. Hum Mol Genet 2:241, 1993 28. Gautvik KM, Talle K, Hager B, et al: Early liver metastases in patients with medullary carcinoma of the thyroid gland. Cancer 63:175, 1989 29. Gottlib JA, Hill OS Jr: Chemotherapy of thyroid cancer with Adriamycin. Experience with 30 patients. N Engl J Med 290:193-197, 1974 30. Grauer A, Raue F, Gagel RF: Changing concepts in the management of hereditary and sporadic medullary thyroid carcinoma. Endocrinol Metab Clin North Am, 19:613, 1990 31. Graze K, Spiter IJ, Tashjian AH, et al: Natural history of familial medullary thyroid carcinoma. Effect of a program for early diagnosis. N Engl J Med 299:980, 1978 32. Hazard JB, Hawk WA, Crile G: Medullary (solid) carcinoma of the thyroid: A clinicopathologic entity. J Clin Endocrinol Metab, 19:152, 1959 33. Hofstra RMW, Landsvater RM, Ceccherini I, et al: A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature 367:375, 1994 34. Howe JR, Norton JA, Wells SA Jr: Prevalence of pheochromocytoma and hyperparathyroidism in multiple endocrine neoplasia type 2A: Results of long-term follow-up. Surgery 114:1070, 1993 35. Jansson S, Tisell LE: Autotransplantation of diseased parathyroid glands into subcutaneous abdominal adipose tissue. Surgery 101:549-556, 1987 36. Jaquet AJ: Ein Fall von metastasierenden Amyloidtumoren (Lymphosarcoma). Virchows Archiv [Pathol Anat] 185:251, 1906 37. Jhiang SM, Fithian L, Weghorst CM, et al: RET mutation screening in MEN 2 patients and discovery of a novel mutation in a sporadic medullary thyroid carcinoma. Thyroid 6:115, 1996 38. Jing S, Wen D, Yu Y, et al: GDNF-induced activation of the Ret protein tyrosine kinase is mediated by GDNFR-a, a novel receptor for GDNF. Cell 85:1113, 1996 39. Khairi MR, Dexter RN, Burzynski NJ, et al: Mucosa! neuroma, pheochromocytoma and medullary thyroid carcinoma: Multiple endocrine neoplasia type 3. Medicine 54:89, 1975 40. Koizumi M, Taguchi H, Goto M, et al: Thallium-201 scintigraphy in the evaluation of thyroid nodules. A retrospective study of 246 cases. Ann Nucl Med 7:147-152, 1993 41. Kwok JB, Gardner E, Warner JP, et al: Structural analysis of the human ret proto-oncogene using exon trapping. Oncogene 8:2575, 1993 42. Lairmore TC, Howe JR, Korte JA, et al: Familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2B map to the same region of chromosome 10 as multiple endocrine neoplasia type 2A. Genomics 9:181, 1991 43. Leape LL, Miller HH, Graze K, et al: Total thyroidectomy for occult familial medullary carcinoma of the thyroid in children. J Pediatr Surg 11:831, 1976

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44. Lips CJM, Landsvater RM, Hoppener JWM, et al: Clinical screening as compared with DNA analysis in families with multiple endocrine neoplasia type 2A. N Engl J Med 331:828-835, 1994 45. Marsh KJ, Learoyd DL, Andrew SD, et al: Somatic mutations in the RET proto-oncogene in sporadic medullary thyroid carcinoma. Clin Endocrinol 44:249, 1996 46. Marsh DJ, Mulligan LM, Eng C: RET proto-oncogene mutations in multiple endocrine neoplasia type 2 and medullary thyroid carcinoma. Horm Res 47:168, 1997 47. Mathew CGP, Chin KS, Easton DF, et al: A linked genetic marker for multiple endocrine neoplasia type 2A on chromosome 10. Nature 328:527, 1987 48. Miller HH, Melvin KE, Gibson JM, et al: Surgical approach to early familial medullary carcinoma of the thyroid gland. Am J Surg 123:438-443, 1972 49. Mole SE, Mulligan LM, Healey CS, et al: Localization of the gene from multiple endocrine neoplasia to a 480 kb region in the chromosome band lOql 1.2. Hum Mol Genet 2:247, 1993 50. Moley JF, Dilley WG, DeBenedetti MK: Improved results of cervical reoperation for medullary thyroid carcinoma. Ann Surg 225:734-743, 1997 51. Moley JF, Wells SA Jr, Dilley WG, et al: Reoperation for recurrent or persistent medullary thyroid cancer. Surgery 114:1090-1096, 1993 52. Moore MW, Klein RD, Farinas I, et al: Renal and neurona abnormalities in mice lacking GONE Nature 382:76, 1996 53. Mulligan LM, Kwok JBJ, Healey CS, et al: Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 363:458, 1993 54. Musholt TJ, Moley JF: Management of recurrent medullary thyroid carcinoma after total thyroidectomy. Prob Gen Surg 14:89-110, 1997 55. Nguyen TD, Chassard JL, Lagarde P, et al: Results of postoperative radiation therapy in medullary carcinoma of the thyroid: A retrospective study by the French Federation of Cancer Institutes-the Radiotherapy Cooperative Group. Radiother Oncol23:1-5, 1992 56. Normann T, Gautvik KM, Johannessen JV, et al: Medullary carcinoma of the thyroid in Norway. Acta Endocrinologica 83:71-85, 1976 57. Norton JA, Doppman JL, Brennan MF: Localization and resection of clinically inapparent medullary carcinoma of the thyroid. Surgery 87:616-622, 1980 58. Norton JA, Froome LC, Farrell RE, et al: Multiple endocrine neoplasia type 2b. The most aggressive form of medullary thyroid carcinoma. Surg Clin North Am 59:109, 1979 59. Norum RA, Lafreniere RG, O'Neal LW, et al: Linkage of the multiple endocrine neoplasia type 2B gene (MEN 2B) to chromosome 10 markers linked to MEN 2A. Genomics 8:313, 1990 60. Ohnishi T, Noguchi S, Murakami N, et al: Detection of recurrent thyroid cancer: MR versus thallium-201 scintigraphy. American Journal of Neuroradiology 14:1051-1057, 1993 61. Olson JA Jr, DeBenedetti MK, Baumann OS, et al: Parathyroid autotransplantation during thyroidectomy. Results of long-term follow-up. Ann Surg 223:472, 1996 62. O'Riordain OS, O'Brien T, Weaver AL, et al: Medullary thyroid carcinoma in multiple endocrine neoplasia types 2A and 2B. Surgery 116:1017-1023, 1994 63. Orlandi F, Caraci P, Berruti A, et al: Chemotherapy with dacarbazine and 5-fluorouracil in advanced medullary thyroid cancer. Ann Oncol 5:763-765, 1994 64. Petursson SR: Metastatic medullary thyroid carcinoma. Complete response to combination chemotherapy with dacarbazine and 5-fluorouracil. Cancer 62:1899-1903, 1988 65. Piche! JG, Shen L, Sheng HZ, et al: Defects in enteric innervation and kidney development in mice lacking GONE Nature 386:73, 1996 66. Raue F, Winter J, Frank-Raue K, et al: Diagnostic procedure before reoperation in patients with medullary thyroid carcinoma. Horm Metab Res Suppl 21:31-34, 1989 67. Rossi RL, Cady B, Meissner WA, et al: Nonfamilial medullary thyroid carcinoma. Am J Surg 139:554, 1980 68. Rougier P, Parmnetier C, Laplanche A, et al: Medullary thyroid carcinoma: Prognostic factors and treatment. Int J Radiat Oncol Biol Phys 9:161, 1983 69. Russell CF, van Heerden JA, Sizemore GW, et al: The surgical management of medullary thyroid carcinoma. Ann Surg 197:42, 1983 70. Saad M, Ordonez NG, Guido JJ, et al: The prognostic value of calcitonin immunostaining in medullary carcinoma of the thyroid. J Clin Endocrinol Metab 59:850-856, 1984

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