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Previous Volume 331:828-835 September 29, 1994 Number 13 Next

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ABSTRACT

Background Multiple endocrine neoplasia type 2A (MEN-2A) is characterized by medullary thyroid carcinoma in combination with pheochromocytoma and sometimes parathyroid adenoma. Missense mutations in the RET proto-oncogene are associated with MEN-2A. Their detection by DNA analysis allows the identification of carriers of the gene, in whom the risk of medullary thyroid carcinoma is 100 percent. We compared the reliability of biochemical tests with that of DNA analysis in identifying carriers of the MEN2A gene.

Methods Starting in 1975, we screened 300 subjects in four large families with MEN-2A for expression of the disease, using measurements of plasma calcitonin after stimulation with pentagastrin or calcium and urinary excretion of catecholamines and catecholamine metabolites. We tested for carrier status by DNA analysis, including linkage analysis, and more recently by analysis of mutations in the RET gene.

Results Of 80 MEN2A gene carriers (in 61 of whom carrier status was proved by DNA analysis), 66 had abnormal plasma calcitonin values and medullary thyroid carcinoma. Fourteen young carriers had normal results of plasma calcitonin tests. In 8 of these 14, thyroidectomy revealed small foci of medullary thyroid carcinoma; the remaining 6 have not yet been operated on. Of the other 220 family members, 68 were found by DNA analysis not to carry the MEN2A gene. None of these 68 subjects had medullary thyroid carcinoma or pheochromocytoma; 6 had elevated plasma calcitonin concentrations and underwent thyroidectomy but had only C-cell hyperplasia.

Conclusions Unlike biochemical tests, DNA analysis permits the unambiguous identification of MEN2A gene carriers.

Medullary thyroid carcinoma originates in calcitonin-producing cells (C cells) of the thyroid gland⁸. Patients with this condition or its precursor, C-cell hyperplasia, have supranormal plasma calcitonin responses to various stimuli. In persons with MEN-2A, MEN-2B, or familial medullary thyroid carcinoma, screening for abnormal plasma calcitonin responses has permitted surgery to be performed at an early stage of the disease and has improved survival^9,10,11. In families with MEN-2A, the biochemical manifestations of medullary thyroid carcinoma generally appear between the ages of 5 and 25 years (mean, 15) and before pheochromocytoma.

In 1987, the MEN2A gene was assigned by linkage analysis to the pericentromeric region of chromosome 10; this information allowed the reliable determination of the disease-gene carrier state^12,13. In 1993, MEN-2A and familial medullary thyroid carcinoma were shown to be associated with mutations in exon 10 or 11 of the RET proto-oncogene, and specific RET mutations were detected in affected families^14,15. In 1994, a unique mutation in the RET gene was identified in 48 of 50 patients from 34 families with MEN-2B^16,17. The structural organization of the RET gene, as well as the predicted functional domains in the RET protein, is shown in Figure 1^18,19.

View larger version (26K): [in this window] [in a new window]   Figure 1. Schematic Representation of the RET Gene, Messenger RNA, and Protein. Within the exons, shown as rectangles, protein-encoding regions are indicated in black and noncoding regions in white. Depending on alternative RNA-processing events, either exon 20 or exon 21 is the last exon in RET messenger RNA (mRNA). The size of the intron between exons 16 and 17 is not known. The RET proto-oncogene encodes a transmembrane-receptor tyrosine kinase. All mutations known to be associated with MEN-2A or familial medullary thyroid carcinoma involve cysteine residues in the extracellular domain of this receptor,14,15 whereas MEN-2B is associated with a mutation of the intracellular tyrosine kinase domain16,17. The positions of the amino acid residues that are mutated in these diseases are shown. The four families with MEN-2A described here all have mutations in exon 11 that affect cysteine in codon 634. In Families A and B, Cys (cysteine) 634 is changed to Arg (arginine); in Families C and D, Cys is changed to Trp (tryptophan). The oligonucleotide primers used to sequence these mutations are shown above exons 10 and 11 in the RET gene structure. S denotes signal peptide, Cd cadherin-like domain, Cys-R cysteine-rich region, TM transmembrane domain, and TK and TK tyrosine kinase subdomains 1 and 2.

 
In families with MEN-2A, the youngest generation poses an important clinical problem. Parents with medullary thyroid carcinoma are concerned about the future of their children. Biochemical screening allows tumors to be detected early, but even at this stage treatment is not always curative. Knowing that a person carries the disease gene gives an opportunity for preventive treatment. To validate DNA analysis as a reliable method for the early identification of such carriers, we compared the results of DNA analysis with those of biochemical, radiologic, and pathological examinations in four large families with MEN-2A.

Methods

Patients

Since 1974, we have repeatedly studied 300 members of four large families with MEN-2A, of whom 152 were obligate noncarriers because neither parent was affected. The studies involved a medical history, physical examination, and measurements of basal and stimulated plasma calcitonin concentrations, urinary excretion of catecholamines and catecholamine metabolites, and serum calcium concentrations. The screening examinations for family members with MEN-2A began when they were between 5 and 10 years old and were performed annually thereafter until they reached the age of 35 years, after which the examinations were performed every 3 years²⁰. The study protocol was approved by the ethics review committee at our hospital, and the study subjects or their parents gave written informed consent.

To evaluate the yield of screening, we divided the patients into three groups. Group 1 comprised subjects who were first examined in our hospital because of symptoms or signs of medullary thyroid carcinoma, pheochromocytoma, or hyperparathyroidism. Group 2 comprised first-degree relatives of subjects in group 1 who were found to have abnormal plasma calcitonin responses to stimulation at the time of the initial screening of the family. Group 3 comprised subjects who had initially negative biochemical-test results that later became positive.

Plasma Calcitonin Stimulation Tests

Plasma calcitonin was measured before and 2 and 5 minutes after the intravenous administration of pentagastrin (0.5 µg per kilogram of body weight in 2 ml of 0.9 percent sodium chloride, given over a period of 10 seconds) or calcium (2.5 mg per kilogram, given over a period of 30 seconds)^10,21,22. The test was considered positive if the peak plasma calcitonin concentration after stimulation was more than three times the basal concentration or if it was 300 ng per liter or higher. A total thyroidectomy was then performed.

Urinary Excretion of Catecholamines and Catecholamine Metabolites

Twenty-four-hour urine specimens were collected for the measurement of norepinephrine, epinephrine, total metanephrines, and vanilmandelic acid by spectrofluorometry²¹.

Radiologic Studies

Until 1990, computed tomographic scanning of the abdomen was performed whenever a pheochromocytoma was suspected clinically and whenever urinary catecholamine values were increased. Thereafter, 30 gene carriers who previously had normal blood-pressure and urinary catecholamine values underwent abdominal magnetic resonance imaging (MRI)²³. Those who had biochemical or radiologic evidence of a pheochromocytoma underwent ¹³¹I-metaiodobenzylguanidine (MIBG) scintigraphy.

Pathological Examination

Paraffin-embedded sections of the thyroid lobes were stained with use of the immunoperoxidase or immunoalkaline phosphatase technique to detect calcitonin, chromogranin, and carcinoembryonic antigen in the C cells.

Microscopical Criteria for C-Cell Hyperplasia and Medullary Thyroid Carcinoma

The diagnosis of C-cell hyperplasia was based on the presence of an increased number of diffusely scattered C cells (i.e., 7 per thyroid follicle), clusters of C cells, or 20 or more C cells per visual field at a magnification of 200 (objective, x20; eyepiece, x10; diameter of visual field, 1 mm)^8,24,25. The C cells were characterized immunohistochemically according to positivity for calcitonin, chromogranin, and carcinoembryonic antigen. Medullary thyroid carcinoma was diagnosed when nests of C cells appeared to extend beyond the basement membrane and to infiltrate and destroy thyroid follicles.

DNA Analysis

Until June 1993, we used linked genetic markers to assess the MEN2A gene carrier status of members of these families with MEN-2A²⁶. For Southern blot analysis of markers of linked restriction-fragment-length polymorphisms, we used probes from loci FNRb²⁷ and D10S34²⁸ on the centromeric side of the MEN2A locus and from loci D10S102,²⁹ RBP3,³⁰ and D10S15³¹ on the distal side.

The markers from loci D10S176,³² D10S141,³³ RET, and ZNF22³⁴ detect dinucleotide-repeat polymorphisms. To demonstrate these polymorphisms, we amplified 50-ng samples of DNA by the polymerase chain reaction (PCR), in which one of the oligonucleotide primers was end-labeled with ³²P-labeled ATP. The PCR products were size-fractionated on a DNA-sequencing gel that was then exposed to x-ray film for analysis.

More recently, we have identified mutations in the RET proto-oncogene by directly sequencing PCR products generated from genomic DNA isolated from peripheral-blood leukocytes with the oligonucleotide primers CRT19S (5'GCAGCATTGTTGGGGGACA3') in exon 10 and ret2190 (5'GTGGGCAAACTTGTGGTAG3') in exon 11. The amplified fragments were excised from ultra-low-melting-point agarose gels (Sigma Chemical, St. Louis). The sequences of the PCR products were determined with a pUC sequencing kit (Boehringer Mannheim, Germany) with ³⁵S-labeled dATP (specific activity, >600 Ci per millimole). The RET mutations specific for MEN-2A create additional cleavage sites for restriction enzymes. Therefore, the presence of these mutations can also be demonstrated by restriction-enzyme digestion of PCR-amplified exons 10 and 11. The resulting DNA fragments were fractionated according to size by agarose-gel electrophoresis.

Results

DNA Analysis

The MEN2A gene carrier state could be identified with a high degree of reliability by linkage analysis using flanking DNA markers in all 129 subjects studied (68 noncarriers and 61 carriers). Sequence analysis of PCR-amplified exons 10 and 11 of the RET gene revealed three different mutations of a single allele in the probands of the four families (Figure 1). Among the other family members, MEN2A gene carriers were identified by appropriate restriction-enzyme digestion of the PCR products. These results agreed with those of the linkage analysis in all instances.

In the probands of Families A and B, thymine was replaced by cytosine at position 2095 from the starting site of transcription (T2095C), resulting in the replacement of a cysteine residue by arginine at codon 634 (C634R). In the proband of Family C, cytosine was replaced by guanine at position 2097 (C2097G), resulting in the replacement of the same cysteine residue at codon 634 by tryptophan (C634W). In the proband of Family D, cytosines at positions 2097 and 2098 were both replaced by guanines (CC2097/2098GG), affecting codons 634 and 635. The cysteine at codon 634 was replaced by tryptophan and the arginine at codon 635 by glycine (C634W/R635G). The presence of all four mutations was confirmed by restriction-enzyme analysis. The C634R mutation introduces a CfoI restriction site, and the C634W/R635G mutation a BmyI restriction site. The C634W mutation was also confirmed by using a primer with one mismatch that, in combination with the C634W mutation, disrupts a BanI restriction site.

Screening for Medullary Thyroid Carcinoma

Biochemical and radiologic tests, pathological examinations, and DNA analysis resulted in the identification of 80 subjects with MEN-2A (Table 1 and Figure 2). Of 14 subjects who initially presented with symptoms (group 1), 8 are alive, 3 died of metastatic medullary thyroid carcinoma (Family A, Subject V-1, and Family B, Subjects IV-3 and V-5), 2 died of pheochromocytoma (Family C, Subjects II-6 and III-6), and 1 died of a myocardial infarction (Family C, Subject III-1).

View this table: [in this window] [in a new window]   Table 1. Clinical Results for Subjects with MEN-2A in Four Families Diagnosed by Biochemical, Radiologic, and Pathological Studies and DNA Analysis.

 

View larger version (56K): [in this window] [in a new window]   Figure 2. Pedigrees of Families A, B, C, and D with MEN-2A, Showing Family Members Who Had Medullary Thyroid Carcinoma (MTC) or Pheochromocytoma or Who Carried the MEN2A Gene as Determined by DNA Analysis. In these families, 116 subjects with MEN-2A were identified. Of these, 45 died of MEN-2A, 36 of them before 1975, when annual screening of family members began. Fourteen of the 45 died of metastatic MTC, 7 before and 7 after 1975. The youngest was a 22-year-old woman (Family B, Subject V-5). Thirty-one subjects died from the consequences of pheochromocytoma, 29 before 1975 and only 2 thereafter (Family C, Subject II-6, at the age of 75 years, and Subject III-6, at the age of 52 years); they had not yet been included in the screening program. Two subjects operated on for pheochromocytoma who had normal plasma calcitonin concentrations after C-cell stimulation are indicated by asterisks. Subjects with hyperparathyroidism are not identified in this figure. The superscript numbers refer to the sequence numbers within a generation; only the first and last numbers are given.

 
During the first biochemical screening in the 52 subjects with no symptoms, 39 had abnormal results (group 2). Currently, only 15 of these subjects have normal test results. Among the remaining 24 subjects, all of whom had increased plasma calcitonin concentrations after thyroidectomy, 4 died of metastatic medullary thyroid carcinoma (Family A, Subjects IV-6, IV-13, V-2, and V-39) and 2 died of other causes (Family A, Subject IV-22, and Family D, Subject III-5).

Thirteen subjects (group 3) had initially normal results of plasma calcitonin stimulation tests that later became positive. Most of these 13 subjects had moderate increases in plasma calcitonin after stimulation for several years before the test became positive by our definition. The age of subjects at the time of conversion to a positive test result and at the onset of progressive disease was not predictable. After total thyroidectomy, nearly all subjects in group 3 remained disease-free; only one (Family B, Subject V-9, who once missed an annual examination) had recurrent disease. The results of the plasma calcitonin stimulation tests performed annually after thyroidectomy were normal in 14, 38, and 92 percent of the subjects in groups 1, 2, and 3, respectively.

Fourteen family members who were MEN2A gene carriers had negative or equivocal stimulation-test results. Eight of them, who were between 4 and 18 years of age, underwent total thyroidectomy on the basis of the DNA analysis, including screening for family-specific RET mutations (Family A, Subjects VI-23, VI-25, and VI-26, and Family B, Subjects VI-4, VI-7, VI-8, VI-9, and VI-10). The other six MEN2A gene carriers were scheduled for surgery at this writing (Family A, Subjects VI-4, VI-5, and VI-16; Family B, Subject V-14; and Family D, Subjects V-9 and V-13) (Table 1 and Figure 2).

Pathological Findings in Thyroid Glands of Asymptomatic MEN2A Gene Carriers

Sections of thyroid tissue from the eight subjects who underwent total thyroidectomy on the basis of the DNA analysis alone revealed C-cell hyperplasia (Figure 3A) and scattered, generally small, irregular foci of medullary thyroid carcinoma (Table 2 and Figure 3B, Figure 3C, Figure 3D, and Figure 3E). Destruction of thyroid follicular structures by invading clusters of tumor cells was seen in all specimens (Figure 3B, Figure 3C, Figure 3D, and Figure 3E). The carcinomas were composed of medium-sized round, oval, or spindle-shaped cells containing abundant eosinophilic cytoplasm and vesicular nuclei with prominent nucleoli. There was mild nuclear pleomorphism, but mitoses were very rare. Highly vascular stroma was seen within the nests of tumor cells, and some contained foci of calcification. No amyloid was demonstrable by Congo-red staining in any sections. The tumor cells stained with antibodies to calcitonin, carcinoembryonic antigen (Figure 3B, Figure 3D, and Figure 3E), and chromogranin (Figure 3A). Areas of thyroid tissue adjacent to the nests of tumor cells contained more C cells that stained for calcitonin than were found in regions free of tumor.

View larger version (280K): [in this window] [in a new window]   Figure 3. Immunohistochemical Staining of Thyroid Tissue Removed because the Subject Carried the MEN2A Gene or Had Positive Results of Calcitonin Stimulation Tests. Panel A shows chromogranin-positive C-cell hyperplasia with small nodular proliferations in the specimen from Subject VI-25 in Family A (immunoalkaline phosphatase, x200). Panel B shows calcitonin-positive C-cell hyperplasia and C-cell cluster formation invading and destroying follicular structures in the thyroid gland of Subject VI-9 in Family B (immunoperoxidase, x275). Panel C shows a section of the thyroid gland of Subject VI-9 in Family B, with calcium deposits (arrows) in a small focus of medullary carcinoma (hematoxylin and eosin, x275). Panel D shows a calcitonin-positive medullary thyroid carcinoma in which the destruction of preexisting follicular boundaries can be seen (arrows), along with a solid proliferation of atypical C cells (Family B, Subject VI-10) (immunoperoxidase, x27). Panel E shows calcitonin-positive C cells that form part of a large focus of medullary thyroid carcinoma, invading thyroid connective tissue and a small nerve (arrows) (Family B, Subject VI-9) (immunoalkaline phosphatase, x67). Panel F shows calcitonin-positive C-cell hyperplasia in a section of the thyroid from a noncarrier of the MEN2A gene (Family C, Subject IV-6). The boundaries of the thyroid follicles are intact (immunoperoxidase, x400).

 

View this table: [in this window] [in a new window]   Table 2. Pathological Findings in the Thyroid Glands of Eight Asymptomatic MEN2A Gene Carriers, All of Whom Had Carcinoma, and Six Family Members with False Positive Results on Plasma Calcitonin Stimulation Tests Who Proved Not to Be Gene Carriers.

 
Screening for Pheochromocytoma

According to the results of biochemical and radiologic tests, 39 of the 80 affected subjects (49 percent) had pheochromocytomas, most of them bilateral. In rare cases, a pheochromocytoma developed earlier than medullary thyroid carcinoma (Family A, Subject VI-4, and Family B, Subject V-14).

Thirty MEN2A gene carriers in whom blood pressure and urinary excretion of catecholamines were not elevated underwent abdominal MRI. Six of them had abnormalities of one or both adrenal glands. In all six, the presence of pheochromocytoma was confirmed by MIBG scintigraphy. One or both adrenal glands were resected in five subjects; all contained pheochromocytomas. The sixth subject (Family D, Subject III-6) was asymptomatic and chose to postpone adrenalectomy.

Parathyroid Disease

Nine patients had elevated serum calcium concentrations, but in only three were the elevations persistent. Subject IV-25 in Family A had a parathyroid adenoma removed, and Subjects V-4 and V-16 in Family B had persistently elevated serum concentrations of calcium and parathyroid hormone but have not yet had surgery.

Non-MEN2A Gene Carriers with Positive Calcitonin Stimulation Tests

Six family members with MEN-2A (Family A, Subjects IV-19, IV-21, IV-26, V-48, and V-49, and Family C, Subject IV-6) who underwent total thyroidectomy on the basis of positive plasma calcitonin stimulation tests later proved not to be MEN2A gene carriers on the basis of DNA analysis.

Histologic examination of the thyroid glands in these subjects revealed only C-cell hyperplasia (Table 2). This was most often evident as focal collections of C cells interspersed among the thyroid cells and bounded by the basement membrane of the thyroid follicle (Figure 3F), but in some areas the C cells formed a ring completely surrounding the lumen of the follicle. There were numerous clusters containing up to 30 C cells in the upper poles of all the thyroid glands in these subjects. Two subjects (Family A, Subjects V-48 and V-49) had nodules containing 94 and 100 C cells, respectively.

Discussion

DNA analysis for the detection of mutations in the RET gene was a highly reliable method for the identification of subjects with MEN-2A. There have been no false positive results so far. This fact indicates a very high specificity and suggests that therapeutic decisions can be based on the results of DNA analysis, even in asymptomatic family members with negative biochemical tests for C-cell hyperplasia. The sensitivity of DNA analysis also appears high. There were no false negative test results (i.e., proved medullary thyroid carcinomas or other manifestations of MEN-2A without MEN2A-specific RET mutations) in the families we studied. These results make the test very useful as a means of excluding the possibility of genetic susceptibility to the development of the MEN-2A syndrome and therefore of assuring unaffected family members that further screening with biochemical or imaging tests can safely be abandoned.

The use of DNA analysis reveals the limitations of the plasma calcitonin stimulation test. DNA analysis can identify patients who have medullary thyroid carcinoma but who do not have positive stimulation-test results. We were surprised to find a number of young MEN2A gene carriers with medullary thyroid carcinoma (stage 1 thyroid cancer) and not just C-cell hyperplasia³⁵. On the other hand, six family members who were later proved not to be gene carriers had positive stimulation tests and therefore underwent thyroidectomy. They had only C-cell hyperplasia, which occurs in normal subjects at a frequency of about 5 percent^{24,36,37,38,39}. These results indicate that the plasma calcitonin responses to stimulation do not always distinguish C-cell hyperplasia from small carcinomas, a finding that limits the value of the stimulation test as far as detecting the presence of medullary thyroid carcinoma is concerned²⁵.

Early expression of the mutated RET proto-oncogene in MEN2A gene carriers apparently results in multicentric C-cell lesions. However, no causal relation between specific RET mutations and medullary thyroid carcinoma or pheochromocytoma has yet been established by functional testing of the mutated RET genes in cultured cells or animals. Additional mutations in the RET gene or in other genes may be required for tumor formation. The appearance of tumors is thus unpredictable. The discovery of particular germ-line mutations of the RET proto-oncogene cosegregating with MEN-2A, MEN-2B, and familial medullary thyroid carcinoma in specific families permits reliable DNA diagnosis and presymptomatic treatment of medullary thyroid carcinoma.

Because of the high penetrance of medullary thyroid carcinoma in families with MEN-2A, it could be argued that total thyroidectomy at a very young age is indicated. However, the risk of complications of surgery (i.e., recurrent nerve paralysis and hypoparathyroidism) in young children in these families is probably not counterbalanced by the gain that accrues from the prevention of medullary thyroid carcinoma. The majority of our patients who were operated on after their plasma calcitonin stimulation tests became positive have not had a recurrence of the disease. Therefore, in general it still seems justified to postpone surgery until the results of the stimulation test become positive or until the age of 12 to 13 years, provided that periodic examinations are conducted. This program should relieve the parents of the psychological burden of postponing surgery.

The results of this study indicate that, as compared with biochemical testing, DNA analysis is a superior method of identifying subjects at risk for the components of the MEN-2A syndrome. The identification of gene carriers by this means should allow earlier identification of subjects at risk. Conversely, the identification of persons in affected families who are not gene carriers will spare them the need for periodic screening and the anxiety that attends the knowledge that they are at risk.

Supported by a grant from the Dutch Prevention Fund, The Hague, the Netherlands.

We are indebted to Ad Alleman, Marcel Smits, Albert Struyvenberg, and Jaap Van der Sluys Veer, our former colleagues; to Nico M.A. Bax, M.D., Maarten Jansen, M.D., and Jan-Maarten Wit, M.D., of the Departments of Medicine and Surgery, Wilhelmina Children's Hospital, Utrecht; to Frederik Bosman, M.D., Bert Ooms, M.D., and Werner H. Minder, M.D., for their excellent support with pathological examinations; to Erik P. Krenning, M.D., Sabine M.P.F. de Muinck Keizer-Schrama, and Bert Vermey, M.D., for contributing data on their patients; to Herman J.M. Van Rijn, Ph.D., Joop Seelen, Ph.D., and Mimi van Loon, for determinations of urinary catecholamines and metabolites; to Marinus A. Blankenstein, Ph.D., Willem H.L. Hackeng, Ph.D., Mariette Sprong, and Jos H.H. Thijssen, Ph.D., for the immunochemical determination of calcitonin; to Bruce A.J. Ponder, for providing information on CA repeats for the DNA-linkage studies; to Anne Huybers, Angelique Nortier, and Gerda Scheffer, for clinical assistance; to Ingrid G.J. Jansen, for the artwork; and to the families and general practitioners who participated in the study, for their cooperation.

Source Information

From the Departments of Internal Medicine (C.J.M.L., R.M.L., J.W.M.H., R.A.G., G.B., J.M.J.-S.V., M.J.W., R.A.Z.), Radiology (A.P.G.G.), Surgery (T.J.M.V.V.), and Pathology (R.M.L., J.W.M.H., M.J.W., R.A.Z., T.M.V.), University Hospital Utrecht, Utrecht; the Department of Pathology, Westeinde Hospital, The Hague (M.J.H.B.); and the Clinical Genetics Center, Utrecht (F.A.B., J.B.-S., R.P.M.J., H.K.P.A.) -- all in the Netherlands.

Address reprint requests to Dr. Lips at the Department of Internal Medicine, University Hospital Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands.

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