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Japanese Journal of Clinical Oncology Pages 42-45

Treatment of Minute Medullary Thyroid Carcinoma in Multiple Endocrine Neoplasia 2A Families First Diagnosed by DNA Analysis of RET Proto-Oncogene Mutations: a Case Report
Introduction
Case report
Discussion
Acknowledgments
References

Treatment of Minute Medullary Thyroid Carcinoma in Multiple Endocrine Neoplasia 2A Families First Diagnosed by DNA Analysis of RET Proto-Oncogene Mutations: a Case Report

Treatment of Minute Medullary Thyroid Carcinoma in Multiple Endocrine Neoplasia 2A Families First Diagnosed by DNA Analysis of RET Proto-Oncogene Mutations: a Case Report Tomoyuki Yamashita1, Masatoshi Iihara1, Joji Okamoto1, Masako Kanbe1, Yukio Ito1, Makio Kawakami2, Shin-ichi Egawa3, Ken Yamaguchi3 and Takao Obara1

Departments of 1Endocrine Surgery and 2Surgical Pathology, Tokyo Women's Medical College, Tokyo and 3Growth Factor Division, National Cancer Center Research Institute, Tokyo, Japan

Multiple endocrine neoplasia (MEN) 2A is an inherited disease characterized by the development of medullary thyroid carcinoma, pheochromocytoma and hyperparathyroidism. It has recently been shown to be associated with germ-line mutations in the RET proto-oncogene. We describe a 21-year-old man from a MEN 2A family who was found by DNA analysis to be a gene carrier of MEN 2A and then was diagnosed, using a stimulated calcitonin test, as having presymptomatic medullary thyroid carcinoma. His morbidity seems to have been cured by total thyroidectomy as postoperative calcitonin levels after stimulation are normal. It is concluded that direct genetic analysis for mutations in the RET proto-oncogene should be the diagnostic test of choice for identifying family members at risk for MEN 2A.

Key words: medullary thyroid carcinoma - multiple endocrine neoplasia (MEN) 2A - RET proto-oncogene

Introduction

Multiple endocrine neoplasia (MEN)-2A is an inherited disease characterized by the development of medullary thyroid carcinoma (MTC), pheochromocytoma and parathyroid adenoma or hyperplasia. The pattern of inheritance is autosomal dominant with a high degree of penetrance. Gene carriers in MEN 2A families have conventionally been detected by provocative calcitonin (CT) testing. Recently, it has been shown that MEN 2A is associated with germ-line mutations of the RET proto-oncogene (1,2), which is located in the pericentrometric region of chromosome 10 (3,4). This finding provides the basis for predictive testing and novel management alternatives for the members of MEN 2A families. We describe a case of minute MTCs associated with MEN 2A, which was first diagnosed by DNA analysis of the RET mutations and then confirmed by elevated CT values after provocative testing. The patient underwent a total thyroidectomy and the specimen histology revealed two minute nodules of MTC.

Case report

A 21-yr-old Japanese man whose father was known to have MTC and pheochromocytoma first visited our clinic in October 1992 when he was 18 years old for a check-up to find out whether he had MTC, pheochromocytoma or a parathyroid tumor. He had no complaints, no specific history, no hypertension and no abnormal physical findings. The values of basal plasma CT, carcinoembryonic antigen (CEA), calcium, parathyroid hormone (PTH), and urinary catecholamine metabolites were all within or close to the normal ranges (Table 1). No abnormality was diagnosed at that time and he was followed up carefully. However, his elder sister, a 21-yr-old woman, had a thyroid nodule and an elevated basal CT value, and was diagnosed as having MTC. She underwent total thyroidectomy in February 1994.

Two years after the check-up, in December 1994, the patient visited our clinic for an analysis of germ-line mutations of the RET proto-oncogene in his blood. Genomic DNA was extracted from his peripheral blood mononuclear cells using a QIamp blood kit (Qiagen Inc., Chatsworth, CA, USA) and subjected to PCR-SSCP analysis. Genomic DNA was amplified for exons 10, 11 and 16, using 32P-labeled primers described previously (5,6), by an automatic thermal cycler (Robocycler Gradient, Stratagene, La Jolla, CA, USA). The resulting PCR products were heat- denatured and electrophoresed on 6% polyacrylamide gel containing 10% glycerol at 16°C and 18°C (7). PCR products of all samples were then subjected to direct sequence analysis using a Sequenase PCR product sequencing kit (United States Biochemical, Cleveland, OH, USA) according to the manufacturer's instructions. The DNA analysis showed the mutation of TGC (Cys) to TCC (Phe) at codon 634 in exon 11 of the RET proto-oncogene. His father and sister also showed the same RET mutation (Fig. 1).


Figure 1. PCR-SSCP products. Shifted fragments of the RET gene mutant were seen in genomic DNA from the patient and his family members. A, control; B, patient's father; C, the patient; D, patient's sister.

Table 1 Endocrinological examinations of the patient
  (Normal range
and units)		 Nov 92

 Dec 94

 Mar 95
CT (15-86 pg/ml) 41 41 32
CEA (<2.4 ng/ml) <1.0 <1.0 <1.0
Ca (8.8-10.6 mg/dl) 9.6   8.8
iPTH (23-73 pg/ml) 4   38
u-MN (<200 ng/mgCr) 248.9   191.2
u-NMN (<300 ng/mgCr) 254.0   165.3
CT, plasma calcitonin; CEA, carcinoembryonic antigen; Ca, serum calcium; iPTH, intact parathyroid hormone; u-MN, urinary metanephrine, u-NMN, urinary normetanephrine


Table 2 Elevation of patient's calcitonin level after a calcium-provocative test
  (Normal range
and units)		 Before

 1'

 15'

 30'

 60'
CT (15-86 pg/ml) 32 523 312 164 81
CEA (<2.4 ng/ml) <1 <1 <1 <1 <1
CT, plasma calcitonin; CEA, carcinoembryonic antigen

He had no abnormal physical findings, but calcium-provocative testing produced elevated plasma CT values (Table 0). Ultra- sonography of his neck showed multiple small nodules in both lobes of his thyroid, but was unable to confirm malignancy. Endocrinologic examinations did not reveal any pheochromocyotoma or parathyroid tumor (Table 0). His family tree is shown in Fig. 2.


Figure 2. The family tree showing the three members with the RET gene mutation. Recently, it has been found that other relatives have MTC or pheochromocytoma. Other members died suddenly or of hypertension. Arrow; reported case, HT; hypertension, Apo; apoplexy, MTC; medullary thyroid carcinoma, Pheo; pheochromocytoma


Figure 3. Cut surface of left thyroid lobe showing a small grayish-yellow nodule with a diameter of 3 mm in the upper third portion (arrow). Scale in cm.

In May 1995, the patient had a total thyroidectomy for suspected MTC. At surgery, there was no visible or palpable thyroid nodule, but the cut surface of the resected specimen revealed a minute grayish-yellow nodule with a diameter of 3 mm in the upper third portion of the left lobe (Fig. 3). At microscopy, another minute MTC nodule was detected near this nodule. The tumor cells in both nodules penetrated the follicular basal layers and extended into the interstitium (Figs 4 and 5), and were of C-cell origin because they stained positively with anti-calcitonin antibody (polyclonal, Dako,Glostrup, Denmark) (Fig. 6). They also stained positively for CEA (monoclonal, Mochida, Tokyo, Japan), neuron-specific enolase (NSE) (polyclonal, Immunotech, Marseille, France), and chromogranin A (monoclonal, Dako). The tumors were surrounded by C-cell hyperplasia, and many benign adenomatous nodules were seen within both thyroid lobes.


Figure 4. Microscopic appearance of minute MTC. The nodule has no capsule and tumor cells extend into the interstitium. Normal residual follicles are present inside the nodule.



The postoperative course was uneventful and the patient is well with normal stimulated CT levels one year after the operation.

Discussion

It is clinically significant in this case that the diagnosis for MEN 2A was confirmed by DNA analysis of the RET mutations before conventional provocative CT testing. Gene carriers of MEN 2A used to be detected by provocative testing with pentagastrin or calcium. However, this method had several disadvantages (8). First, repeated testing until at least 30 years of age was often required to safely exclude the disease. Second, unpleasant side-effects produced by the pentagastrin reduced patient compliance. Third, interpretation of the results was subjective and could lead to an unnecessary operation in borderline cases. Fourth, C-cell hyperplasia is not necessarily associated with MEN 2A (9).

Historically, in 1987 Mathew et al. (3) and Simpson et al. (4) decribed the location of the gene for MEN 2A on chromosome 10 by genetic linkage studies. In 1993, Mulligan et al. (1) and Donis-Keller et al. (2) identified the gene responsible for MEN 2A and familial MTC as the RET proto-oncogene, and many mutations within exons 10 and 11 of the RET gene have been found (10). Examination for RET mutations has proved to be a precise and specific screening method despite minor technical problems. Besides providing an accurate diagnosis for gene carriers, this test also means that non-gene carriers no longer need repeated provocative testing.


Figure 5. High-power view of the nodule of MTC. This shows polygonal cells with rich cytoplasm and a solid growth pattern .


Figure 6. CT staining of MTC. CT is diffusely positive in the cytoplasm and strongly positive in the tubular lumina.

Recently, several clinical studies have been performed to investigate the effectiveness of DNA analysis for members of MEN 2A families. Wells et al. (11) reported that 21 of 58 kindred members had mutations in the RET proto-oncogene as shown by direct DNA testing; of these 21 members, 13 underwent thyroidectomy, all of whom had C-cell hyperplasia or MTC. Lips et al. (9) reported that of 80 MEN 2A gene carriers as shown by DNA analysis, 14 young carriers had normal stimulated CT levels. Eight of these 14 carriers were operated on and all revealed small foci of MTC. Decker et al. (8) reported that RET mutations occurred in 29 of 93 MEN 2A members. Of these 29, 17 underwent thyroidectomy; seven had MTC and 10 had C-cell hyperplasia. DNA analysis is an established method for detecting the gene carriers of MEN 2A and is clinically useful because MTC occurs in nearly 100% of MEN 2A patients (12). Therefore, biochemical screening for MTC can now be replaced by DNA analysis.

However, the appropriate timing of thyroidectomy for the gene carriers is controversial. Lips et al. (9) recommended that it should be performed at 12 or 13 years of age, or sooner if the screening test becomes positive, because nearly all those operated on soon after the test became positive have been free of recurrences. Utiger (13) recommended that thyroidectomy should be performed at four or five years of age because C-cell hyperplasia and MTC are identifiable by biochemical screening in children under 10 years of age.

MTC in carriers of MEN-2B is more aggressive than in carriers of MEN 2A, with local invasiveness and distant metastasis (14). We thus believe that carriers of MEN-2B should be treated with a total thyroidectomy as early as possible. However, the biological behavior of MTC in MEN 2A varies geographically. Generally MTC in patients with MEN 2A in Japan tends to be indolent compared to that in patients in Western countries (15). We are now investigating the surgical outcome of MEN 2A patients in Japan to decide whether prophylactic total thyroidectomy is appropriate for all Japanese patients.

Strictly speaking, the case described here was not in a preclinical stage of MTC, because provocative CT testing was positive (16). However, the patient is well and has probably been cured because provocative CT values have remained normal since the operation. A good result would be expected, as treatment was carried out at an early stage. We tentatively suggest that thyroidectomy should be performed when both an elevation in CT and the RET mutation have been confirmed, as in this patient.

Acknowledgments

This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare, Japan, for the second-term comprehensive 10-year strategy for cancer control.

References

1. Mulligan LN, Kwok JBJ, Healy CS, Elsdon NJ, Eng C, Gardner E et al. Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458-60. MEDLINE Abstract

2. Donis-Keller H, Dou S, Chi D, Carlson KM, Toshima K, Lairmore TC et al. Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet 1993;2:851-56. MEDLINE Abstract

3. Mathew CGP, Chin KS, Easton DF, Thorpe K, Carter C, Liou GI, Fong SL et al. A linked genetic marker for multiple endocrine neoplasia type 2A on chromosome 10. Nature 1987;328:527-28. MEDLINE Abstract

4. Simpson NE, Kidd KK, Goodfellow PJ, McDermid H, Myers S, Kidd JR et al. Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by genetic linkage. Nature 1987;328:528-30. MEDLINE Abstract

5. Ceccherini I, Hofstra RMW, Luo Y, Stulp RP, Barone V, Stelwagen T et al. DNA polymorphisms and conditions for SSCP analysis of the 20 exons of the RET proto-oncogene. Oncogene 1994;9:3025-9. MEDLINE Abstract

6. Futami H, Egawa S, Tsukada T, Maruyama K, Bandoh S, Noguchi M et al. A novel somatic point mutation of the RET proto-oncogene in tumor tissues of small cell lung cancer patients. Jpn J Cancer Res 1995;86:1127-30. MEDLINE Abstract

7. Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989;5:874-9. MEDLINE Abstract

8. Decker RA, Peacock ML, Borst MJ, Sweet JD, Thompson NW. Progress in genetic screening of multiple endocrine neoplasia type 2A: Is calcitonin testing obsolete? Surgery 1995;118:257-64. MEDLINE Abstract

9. Lips CJM, Landsvater RM, Hoppener JWM, Geerdink RA, Blijham G, van Veen JMJ-S et al. Clinical screening as compared with DNA analysis in families with multiple endocrine neoplasia type 2A. N Engl J Med 1994;331:828-35. MEDLINE Abstract

10. Mulligan LM, Eng C, Healey CS, Clayton D, Kwok JB, Gardner E et al. Specific mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC. Nature Genet 1994;6:70-4. MEDLINE Abstract

11. Wells SA Jr, Chi DD, Toshima K, Dehner LP, Coffin CM, Dowton B et al. Predictive DNA testing and prophylactic thyroidectomy in patients at risk for multiple endocrine neoplasia type 2A. Ann Surg 1994;220:237-50. MEDLINE Abstract

12. 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 1993;114:1070-7. MEDLINE Abstract

13. Utiger RD. Medullary thyroid carcinoma, genes, and the prevention of cancer. N Engl J Med 1994;331:870-1. MEDLINE Abstract

14. O'Riordain DS, O'Brien T, Weaver AL, Gharib H, Hay ID, Grant CS et al. Medullary thyroid carcinoma in multiple endocrine neoplasia type 2A and 2B. Surgery 1994;116:1017-23. MEDLINE Abstract

15. Takai S, Miyauchi A, Matsumoto H, Ikeuchi T, Miki T, Kuma K et al. Multiple endocrine neoplasia type 2 syndromes in Japan. Henry Ford Hosp Med J 198432:246-50. MEDLINE Abstract

16. Pacini F, Martino E, Romei C, Ceccherini I, Basolo F, Iacconi P et al. Treatment of preclinical medullary thyroid carcinoma in MEN 2A gene carrier. Lancet 1994;344:1084-5. MEDLINE Abstract

Received May 30, 1996; accepted September 4, 1996
For reprints and all correspondence: Takao Obara, Department of Endocrine Surgery, Tokyo Women's Medical College, 8-1, Kawada-cho, Shinjuku-ku, Tokyo 162, Japan
Abbreviations: MEN, Multiple endocrine neoplasia; MTC, medullary thyroid carcinoma; CT, calcitonin; CEA, carcinoembryonic antigen; PTH, parathyroid hormone; PCR-SSCP, polymerase chain reaction-single strand confirmation polymorphism; NSE, neuron-specific enolase

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