- Email: [email protected]
Familial medullary thyroid carcinoma: Presymptomatic diagnosis and management in children
F
Familial medullary thyroid carcinoma: Presymptomatic diagnosis and management in children
Rubina A. Heptulla, MD, Robert P. Schwartz, MD, Allen E. Bale, MD, Stuart Flynn, MD, and Myron Genel, MD
Two kindreds with familial medullary thyroid carcinoma (MTC) are described in which affected family members had variable clinical and pathologic manifestations. Genetic testing in 2 children from one kindred revealed a mutation in exon 10, codon 618 (TGC to AGC) in the extracellular cysteine-rich region of the RET gene. In this kindred an 11-year-old had microscopic evidence of MTC; however, a 17-year-old had no evidence of pathology on thyroidectomy. In a second kindred a rare mutation in exon 14, codon 804 (GTG to TTG) of the intracellular tyrosine kinase region of the RET gene was detected. In this kindred MTC has occurred in the 4th to 5th decades of life, with a clinical spectrum in mutation-positive family members ranging from no disease and C-cell hyperplasia to carcinoma with lymph node metastasis; a 7-year-old with the mutation and a normal response to provocative testing was also identified. Management recommendations in children from families with clearly defined familial MTC may be individualized to reflect emerging genotype-phenotype correlations. (J Pediatr 1999;135:327-31)
Medullary thyroid carcinoma arises in the C cells of the thyroid gland. Familial forms of MTC are inherited as autosomal dominant diseases, and 3 variants have been recognized: multiple endocrine neoplasia 2A, MEN 2B,
and familial medullary thyroid carcinoma. MEN 2A is defined by the presence of MTC, pheochromocytoma, and hyperparathyroidism. In addition to these abnormalities, MEN 2B is characterized by an unusual facial ap-
From the Departments of Pediatrics, Genetics, and Pathology, Yale University School of Medicine, New Haven, Connecticut; and the Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina.
Supported in part by the Yale Children’s Clinical Research Center, Grant M01-RR008022, General Clinical Research Centers Program, National Center for Research Resources, National Institutes of Health. Dr Heptulla is a recipient of a postdoctoral fellowship award from the Juvenile Diabetes Foundation International. Submitted for publication Oct 26, 1998; revisions received Mar 4, 1999, and May 14, 1999; accepted May 26, 1999. Reprint requests: Myron Genel, MD, Department of Pediatrics, PO Box 208000, Yale University School of Medicine, New Haven, CT 06520-8000. Copyright © 1999 by Mosby, Inc. 0022-3476/99/$8.00 + 0 9/21/100341
pearance, marfanoid habitus, ganglioneuromas of the intestinal tract, and mucosal neuromas. FMTC is characterized exclusively by the presence of MTC.1 See related article, p. 390. In 1993 germline point mutations were identified in the RET gene, which segregated with the disease phenotype in MEN 2A and FMTC.2,3 Specific RET mutations generally correlate with particular phenotypes. Thus mutations of codon 634 typically lead to full MEN 2A expression in affected families. Mutations of codons 768 and 804 were subsequently found in families with MTC without accompanying hyperparathyroidism or pheochromocytoma. MEN 2B is caused by specific mutations in codons 918 and 883 of the RET gene.4 A schematic summary of these recognized mutations is provided in Fig 1. Genetic testing for these mutations provides unequivocal determination of carrier status and has significantly altered the management of familial disease.5,6 FMTC Familial medullary thyroid carcinoma MEN Multiple endocrine neoplasia MTC Medullary thyroid carcinoma
Recommendations for presymptomatic mutation carriers of MEN 2A include total thyroidectomy before the age of 5 years and provocative testing for calcitonin, beginning at the age of 1 year.7,8 However, definitive recommendations for families with FMTC 327
HEPTULLA ET AL
THE JOURNAL OF PEDIATRICS SEPTEMBER 1999 more common MEN 2A are applicable to FMTC and whether genotype-phenotype correlations should influence recommendations for management.
METHODS
Fig 1. RET gene with the 3 domains. Mutations in the cysteine-rich region of the extracellular domain and the intracellular tyrosine kinase region are associated with MEN and FMTC syndromes. Mutations identified in the 2 families described are indicated by an asterisk.
Fig 2. Pedigree of kindred 1, showing family members with the mutation in exon 10, codon 618. Family members who tested negative for the mutation are indicated by an n within the gender-specific symbol; family members who are potential carriers but were unavailable for testing are indicated by a question mark; and family members through marriage, presumed to be unaffected, are indicated by empty symbols.
Basal and calcium-pentagastrin–stimulated calcitonin levels were measured by radioimmunoassay. Provocative stimulation consisted of administration of calcium gluconate (2 mg Ca++/kg/1 min) and/or pentagastrin (0.5 µm/kg/5 s) and measurements of calcitonin at 1, 2, 3, 4, 5, and 6 minutes. Basal values greater than 25 pg/mL and stimulated values of 150 pg/mL or greater were considered abnormal for kindred 1 (reference laboratory, Endocrine Sciences). For the second kindred, calcitonin levels were considered abnormal in female subjects when basal levels exceeded 4 pg/mL and stimulated values were greater than 40 pg/mL and in male subjects when basal levels exceeded 4 pg/mL and stimulated values were greater than 345 pg/mL (reference laboratory, Nichols Institute). Parathyroid function was assessed with total or ionized calcium levels, and intact parathormone levels were measured by a 2-site immunoradiometric assay. Evaluation for pheochromocytoma consisted of determination of serum and 24-hour urinary catecholamine levels, measured fluorometrically, and 24-hour urinary vanillylmandelic acid and metanephrine levels determined by spectrophotometric analysis. Interfollicular and intrafollicular hyperplasia of thyroid C cells was confirmed by immunohistochemical stains for calcitonin.
Kindred 1 (Fig 2) have not been formulated. In many kindreds with FMTC, characteristic pathologic changes appear to be more gradual and occur later in life.9 We recently encountered 2 kindreds with FMTC caused by germline mutations in exons 10 and 14 of the RET 328
gene. The variability of clinical presentation and age at onset of malignancies pose a management dilemma in children who are free of symptoms and carry these mutations. The findings of this study have prompted us to consider whether management guidelines for the
INDEX CASE. Patient III 3, a 37-yearold woman, was found to have a thyroid nodule at 22 years of age. Subsequent thyroidectomy revealed bilateral medullary carcinoma with lymph node metastases. At age 30 she was found to have metastatic disease of the liver.
HEPTULLA ET AL
THE JOURNAL OF PEDIATRICS VOLUME 135, NUMBER 3 She is currently doing well without chemotherapy or radiotherapy. Serum calcium and urine vanillylmandelic acid, metanephrine, epinephrine, and norepinephrine levels are normal. Patient III 3 was thought to have sporadic MTC because there was no other family history of MTC. However, her 2 sons were followed up by periodic determinations of baseline calcitonin levels.
Mutation Analysis Genetic analysis in the index case revealed a mutation in exon 10, codon 618, TGC to AGC of the RET gene. The mutation creates an abnormal Alu 1 restriction site and was confirmed by Alu 1 digestion of a polymerase chain reaction product.
Family Studies Because of this history in the index case, other family members were screened for the mutation. Patient IV 1, the 17-year-old son of the index case, had a normal baseline calcitonin level and a normal calcium pentagastrin test result. Nevertheless, because he was found to carry the mutation, he underwent a thyroidectomy. Pathologic examination revealed no abnormalities on either gross or microscopic examination. Patient IV 2, the 10-year-old son of the index case, was also evaluated because of his mother’s history. Findings on physical examination were normal. Baseline calcitonin level was <25 pg/mL (normal, 0-25 pg/mL) and peaked at 83 pg/mL (normal, 25-150 pg/mL) with pentagastrin stimulation. He had normal serum calcium levels; thyroid function test results; and urine metanephrine, vanillylmandelic acid, epinephrine, and norepinephrine levels. DNA testing demonstrated that he had the identical codon 618 mutation. A complete thyroidectomy revealed multiple foci of Ccell hyperplasia and an area of microscopic medullary carcinoma. On followup postoperative pentagastrin test, all calcitonin levels were <25 pg/mL. Patient II 3, the 62-year-old maternal grandmother, was found to be an
Fig 3. Pedigree of kindred 2, showing family members affected with the mutation in exon 14, codon 804. Family members who were tested and found not to carry the mutation are indicated by an n within the gender-specific symbol; family members who were not tested but are potential carriers of the mutation are indicated by a question mark, including family members known to have MTC; family members through marriage, presumed not to carry the mutation, and children of a family member known to be negative (patient III, 3) are indicated by open gender-specific symbols.
asymptomatic carrier of the mutation. Prophylactic thyroidectomy was performed and revealed MTC with bilateral lymph node metastasis. Family members III 1, III 2, and II 2 are free of symptoms, and test results for the mutation were negative.
Kindred 2 (Fig 3) INDEX CASE. Patient III 4, a 45-yearold woman, had a family history of MTC in her father, 2 paternal aunts, and 2 first cousins. She was followed up with annual calcium-pentagastrin stimulation testing since the age of 31 years. Previous genetic testing for mutations in exons 10 and 11 was unrevealing. Because of the family history and equivocal calcium-pentagastrin test results (basal calcitonin = 25 pg/ mL, stimulated calcitonin = 39 pg/mL), she underwent thyroidectomy. Pathologic examination demonstrated C-cell hyperplasia, but no carcinoma was detected.
Mutation Analysis A DNA sample from the index case revealed a mutation in exon 14, codon
804 (GTG to TTG) of the RET gene. The mutation creates an abnormal N1a111 restriction site and was confirmed by enzymatic digestion.
Family Studies Subsequently, other available family members were screened for the mutation. Patient IV 4, a daughter (age, 7 years 10 months) of the index case was found to carry the identical codon 804 mutation. Subsequently, a calciumpentagastrin stimulation test attained a peak of 7 pg/mL. The family opted to defer prophylactic thyroidectomy at this time and continue with annual calcium-pentagastrin stimulation tests. Patient III 1 was tested at age 43 years because of the genetic and pathologic findings in his sister and was found to carry the identical mutation. Prophylactic thyroidectomy was performed but showed no pathologic evidence of MTC or CCH. Family members III 6, III 7, II 2, II 3, and II 4 were found to have MTC at thyroidectomy during their 4th and 5th decades of life; and family members III 2, III 3, and IV 3 tested negative for the mutation. 329
HEPTULLA ET AL
DISCUSSION DNA analysis for the detection of mutations of the RET gene has become a reliable method for the identification of subjects with hereditary MTC. From their analysis of 477 families, the international RET consortium concluded that a genotype-phenotype correlation does exist.10 Mutations in the cysteine-rich region of the RET gene codons 609, 611, 618, 620, and 634 are associated with either MEN 2A or isolated FMTC. Variability in the spectrum of pathology could be caused by modifying genes or environmental factors or could simply reflect random chance. Pheochromocytoma and/or hyperparathyroidism may also be relatively late occurrences in individuals who present with medullary carcinoma. Thus Moers et al11 reported a large kindred, initially classified as having FMTC. Subsequently, 4 family members were found to have parathyroid involvement. Before association of disease phenotype with RET mutations, genetic counseling and management relied on provocative stimulation and measurement of calcitonin in familial cases. The reliability of provocative testing varies with the sensitivity and specificity of the assay systems used, the stage of underlying disease, and the adequacy of control data.12 Thus false-negative or equivocal responses may be observed in mutation-positive individuals, as in patient IV 2 in kindred 1 and the index case, patient III 4, in kindred 2. Alternately, false-positive test results may occur in mutation-negative members of MEN 2A and FMTC kindreds; this has led to thyroidectomy in 5% to 10% of individuals at risk in whom subsequent gene analysis showed no abnormalities.13-16 The availability of RET mutation analysis thus provides a more precise means of identifying gene carriers than provocative stimulation with calcium and/or pentagastrin.17 Families with the MEN 2A syndrome have an earlier, 330
THE JOURNAL OF PEDIATRICS SEPTEMBER 1999 more aggressive form of MTC, which has led to the recommendation for thyroidectomy by 5 years of age.8 However, concern has been raised regarding the potential risks of thyroidectomy in young children, especially in inexperienced centers.6 The risk must be weighed against the risk of development of MTC, especially in families with mutations in the non-cysteine-rich region of the RET gene. The mild phenotype and low penetrance have been previously noted.18,19 As more families with mutations in these exons are identified, we should be able to modify screening programs, targeting primary thyroid disease and judiciously following up for other endocrinopathies in a cost-efficient and evidence-based manner.10 The nuances of current recommendations for treatment of mutation-positive children in families with FMTC are illustrated by the 2 kindreds described in this report. In the first kindred there is clear evidence of an early age of onset with metastatic disease evident in the index case at age 22 and microscopic disease in one of her sons at age 10. Notwithstanding the absence of pheochromocytoma and parathyroid hyperplasia as late as age 62 in one affected member of this kindred, this family shares a mutation at a region of the RET gene associated with early onset and a more aggressive presentation of MTC, and thus early prophylactic thyroidectomy remains appropriate in mutation-positive children in this family. In contrast, in the second kindred the mutation lies at a far less common intracellular site within the tyrosine kinase domain of the RET-coded transmembrane protein. There is limited experience in families with FMTC and mutations in exons 13 and 14. The late onset and relatively indolent course in this family may permit a more conservative approach, as indeed was selected. This was based on a plan to perform provocative testing with pentagastrin and/or calcium at annual intervals. Pentagastrin, however, is no longer
readily available, thus leaving calcium infusion alone, which is far less sensitive and reliable as a provocative stimulus.20 In contrast to recommendations for the more frequent RET mutations, families with these uncommon and less aggressive variants of FMTC can be treated on an individual basis, but definitive prophylactic thyroidectomy, if deferred, should be performed in childhood, ideally during the first decade of life. Original members of kindred 2 were studied and tested at the Tufts New England Medical Center under the direction of Dr Seymour Reichlin. We thank Dr Robert Gagel for providing results of the original mutation analysis of case III 3 kindred 2 and Dr Gustave Davis for sharing pathological specimens and slides from the index case of kindred 2.
REFERENCES 1. Schimke RN. Genetic aspects of multiple endocrine neoplasms. Annu Rev Med 1984;35:25-31. 2. Mulligan LM, Kwok JBJ, Healy CS, Eldson MJ. Germline mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458-60. 3. 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;2:851-6. 4. Carlson KM, Dou S, Chi D, Scarvada N, Toshima K, Jackson CE, et al. Single missense mutation in the tyrosine kinase catalytic domains of the RET prot-oncogene is associated with multiple endocrine neoplasia type 2B. Proc Natl Acad Sci USA 1994;91:1579-83. 5. Marsh DJ, Robinson BG, Andrew S, Richardson AL, Pojer R, Schnitzler M, et al. A rapid screening method for the detection of mutations in the RET proto-oncogene in multiple endocrine neoplasia type 2A and familial medullary thyroid carcinoma families. Genomics 1994;23:477-9. 6. Lips CJM, Landsvater RM, Hoppener JWM, Geerdink RA, Blijham G, van Veen JM, 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. 7. Wells SA, Chi DD, Toshima K, Dehn-
HEPTULLA ET AL
THE JOURNAL OF PEDIATRICS VOLUME 135, NUMBER 3
8.
9.
10.
11.
er 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. Gill JR, Reyes Múgica M, Iyengar S, Kidd KK, Touloukian RJ, Smith C, et al. Early presentation of metastatic medullary carcinoma in multiple endocrine neoplasia type 2A: implications of therapy. J Pediatr 1996;129:459-64. Brunt LM, Wells SA. Advances in the diagnosis and treatment of medullary thyroid carcinoma. Surg Clin North Am 1987;67:263-79. Eng C, Clayton D, Schuffenecker I, Lenoir G, Cote G, Gagel RF, et al. The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2. International RET mutation consortium analysis. JAMA 1996; 276:1575-9. Moers AMJ, Landsvater RM, Schaap C, Jansen-Schillhorn van Veen JM, de Valk IA, Blijham GH, et al. Familial medullary thyroid carcinoma: Not a
12. 13.
14.
15.
16.
distinct entity? Genotype-phenotype correlation in a large family. Am J Med 1996;101:635-41. Gagel RF. The abnormal pentagastrin test. Clin Endocrinol 1996;44:221-2. Marsh DJ, McDowall D, Hyland VJ, Andrew SD, Schnitzler M, Gaskin EL, et al. The identification of false positive responses to the pentagastrin stimulation test in RET mutation negative members of MEN 2A families. Clin Endocrinol 1996;44:213-20. Gagel RF, Tashijian AH Jr, Cummings T, Papathanasopoulos N, Kaplan MM, DeLellis RA, et al. The clinical outcome of prospective screening for multiple endocrine neoplasia type 2A: an 18-year experience. N Engl J Med 1988;318:478-84. Lichter JB, Wu JS, Genel M, Flynn SD, Pakstis SD, Kidd JR, et al. Presymptomatic testing using DNA markers for individuals at risk for familial multiple endocrine neoplasia 2A. J Clin Endocrinol Metab 1992;74:368-73. Lips CJ, Landsvater RM, Hoppener JW, Geerdink RA, Blijham G, van Veen
17.
18.
19.
20.
JM, 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. Heshmati HM, Gharib H, Khosla S, Abu-Lebdeh HS, Lindor NM, Thibodeau SN, et al. Genetic testing in medullary thyroid carcinoma syndromes: mutation types and clinical significance. Mayo Clin Proc 1997;72: 420-36. Bolino A, Schuffenecker I, Luo Y, Seri M, Silengo M, Tocco T, et al. RET mutations in exons 13 and 14 of FMTC patients. Oncogene 1995;10:2415-9. Eng C, Smith DP, Mulligan LM, Healey CS, Zvelebil MJ, Stonehouse TJ, 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 1995;10:509-13. Wells SA, Baylin SB, Linehan WM, Farrell RE, Cox EB, Cooper CW. Provocative agents and the diagnosis of medullary carcinoma of the thyroid gland. Ann Surg 1978;188:139-41.
FELLOWSHIPS Fellowships available in pediatric subspecialties and those for general academic pediatric training are listed once a year, in January, in The Journal of Pediatrics. Each June, forms for listing fellowships available for the academic year beginning 18 months after publication are sent to the Chairman of the Department of Pediatrics at major hospitals in the United States and Canada. In addition, a copy of the application form appears in the July, August, and September issues of The Journal (please use the current form). Should you desire to list fellowships, a separate application must be made each year for each position. All applications must be returned to Mosby, Inc., by October 15 preceding the listing year to ensure publication. Additional forms will be supplied on request from the Periodical Editing Department, Mosby, Inc., 11830 Westline Industrial Drive, St. Louis, MO 63146-3318 (phone: 800-325-4177, ext. 2838, or 314-579-2838).
331
Familial medullary thyroid carcinoma: Presymptomatic diagnosis and management in children
Rubina A. Heptulla, MD, Robert P. Schwartz, MD, Allen E. Bale, MD, Stuart Flynn, MD, and Myron Genel, MD
Two kindreds with familial medullary thyroid carcinoma (MTC) are described in which affected family members had variable clinical and pathologic manifestations. Genetic testing in 2 children from one kindred revealed a mutation in exon 10, codon 618 (TGC to AGC) in the extracellular cysteine-rich region of the RET gene. In this kindred an 11-year-old had microscopic evidence of MTC; however, a 17-year-old had no evidence of pathology on thyroidectomy. In a second kindred a rare mutation in exon 14, codon 804 (GTG to TTG) of the intracellular tyrosine kinase region of the RET gene was detected. In this kindred MTC has occurred in the 4th to 5th decades of life, with a clinical spectrum in mutation-positive family members ranging from no disease and C-cell hyperplasia to carcinoma with lymph node metastasis; a 7-year-old with the mutation and a normal response to provocative testing was also identified. Management recommendations in children from families with clearly defined familial MTC may be individualized to reflect emerging genotype-phenotype correlations. (J Pediatr 1999;135:327-31)
Medullary thyroid carcinoma arises in the C cells of the thyroid gland. Familial forms of MTC are inherited as autosomal dominant diseases, and 3 variants have been recognized: multiple endocrine neoplasia 2A, MEN 2B,
and familial medullary thyroid carcinoma. MEN 2A is defined by the presence of MTC, pheochromocytoma, and hyperparathyroidism. In addition to these abnormalities, MEN 2B is characterized by an unusual facial ap-
From the Departments of Pediatrics, Genetics, and Pathology, Yale University School of Medicine, New Haven, Connecticut; and the Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina.
Supported in part by the Yale Children’s Clinical Research Center, Grant M01-RR008022, General Clinical Research Centers Program, National Center for Research Resources, National Institutes of Health. Dr Heptulla is a recipient of a postdoctoral fellowship award from the Juvenile Diabetes Foundation International. Submitted for publication Oct 26, 1998; revisions received Mar 4, 1999, and May 14, 1999; accepted May 26, 1999. Reprint requests: Myron Genel, MD, Department of Pediatrics, PO Box 208000, Yale University School of Medicine, New Haven, CT 06520-8000. Copyright © 1999 by Mosby, Inc. 0022-3476/99/$8.00 + 0 9/21/100341
pearance, marfanoid habitus, ganglioneuromas of the intestinal tract, and mucosal neuromas. FMTC is characterized exclusively by the presence of MTC.1 See related article, p. 390. In 1993 germline point mutations were identified in the RET gene, which segregated with the disease phenotype in MEN 2A and FMTC.2,3 Specific RET mutations generally correlate with particular phenotypes. Thus mutations of codon 634 typically lead to full MEN 2A expression in affected families. Mutations of codons 768 and 804 were subsequently found in families with MTC without accompanying hyperparathyroidism or pheochromocytoma. MEN 2B is caused by specific mutations in codons 918 and 883 of the RET gene.4 A schematic summary of these recognized mutations is provided in Fig 1. Genetic testing for these mutations provides unequivocal determination of carrier status and has significantly altered the management of familial disease.5,6 FMTC Familial medullary thyroid carcinoma MEN Multiple endocrine neoplasia MTC Medullary thyroid carcinoma
Recommendations for presymptomatic mutation carriers of MEN 2A include total thyroidectomy before the age of 5 years and provocative testing for calcitonin, beginning at the age of 1 year.7,8 However, definitive recommendations for families with FMTC 327
HEPTULLA ET AL
THE JOURNAL OF PEDIATRICS SEPTEMBER 1999 more common MEN 2A are applicable to FMTC and whether genotype-phenotype correlations should influence recommendations for management.
METHODS
Fig 1. RET gene with the 3 domains. Mutations in the cysteine-rich region of the extracellular domain and the intracellular tyrosine kinase region are associated with MEN and FMTC syndromes. Mutations identified in the 2 families described are indicated by an asterisk.
Fig 2. Pedigree of kindred 1, showing family members with the mutation in exon 10, codon 618. Family members who tested negative for the mutation are indicated by an n within the gender-specific symbol; family members who are potential carriers but were unavailable for testing are indicated by a question mark; and family members through marriage, presumed to be unaffected, are indicated by empty symbols.
Basal and calcium-pentagastrin–stimulated calcitonin levels were measured by radioimmunoassay. Provocative stimulation consisted of administration of calcium gluconate (2 mg Ca++/kg/1 min) and/or pentagastrin (0.5 µm/kg/5 s) and measurements of calcitonin at 1, 2, 3, 4, 5, and 6 minutes. Basal values greater than 25 pg/mL and stimulated values of 150 pg/mL or greater were considered abnormal for kindred 1 (reference laboratory, Endocrine Sciences). For the second kindred, calcitonin levels were considered abnormal in female subjects when basal levels exceeded 4 pg/mL and stimulated values were greater than 40 pg/mL and in male subjects when basal levels exceeded 4 pg/mL and stimulated values were greater than 345 pg/mL (reference laboratory, Nichols Institute). Parathyroid function was assessed with total or ionized calcium levels, and intact parathormone levels were measured by a 2-site immunoradiometric assay. Evaluation for pheochromocytoma consisted of determination of serum and 24-hour urinary catecholamine levels, measured fluorometrically, and 24-hour urinary vanillylmandelic acid and metanephrine levels determined by spectrophotometric analysis. Interfollicular and intrafollicular hyperplasia of thyroid C cells was confirmed by immunohistochemical stains for calcitonin.
Kindred 1 (Fig 2) have not been formulated. In many kindreds with FMTC, characteristic pathologic changes appear to be more gradual and occur later in life.9 We recently encountered 2 kindreds with FMTC caused by germline mutations in exons 10 and 14 of the RET 328
gene. The variability of clinical presentation and age at onset of malignancies pose a management dilemma in children who are free of symptoms and carry these mutations. The findings of this study have prompted us to consider whether management guidelines for the
INDEX CASE. Patient III 3, a 37-yearold woman, was found to have a thyroid nodule at 22 years of age. Subsequent thyroidectomy revealed bilateral medullary carcinoma with lymph node metastases. At age 30 she was found to have metastatic disease of the liver.
HEPTULLA ET AL
THE JOURNAL OF PEDIATRICS VOLUME 135, NUMBER 3 She is currently doing well without chemotherapy or radiotherapy. Serum calcium and urine vanillylmandelic acid, metanephrine, epinephrine, and norepinephrine levels are normal. Patient III 3 was thought to have sporadic MTC because there was no other family history of MTC. However, her 2 sons were followed up by periodic determinations of baseline calcitonin levels.
Mutation Analysis Genetic analysis in the index case revealed a mutation in exon 10, codon 618, TGC to AGC of the RET gene. The mutation creates an abnormal Alu 1 restriction site and was confirmed by Alu 1 digestion of a polymerase chain reaction product.
Family Studies Because of this history in the index case, other family members were screened for the mutation. Patient IV 1, the 17-year-old son of the index case, had a normal baseline calcitonin level and a normal calcium pentagastrin test result. Nevertheless, because he was found to carry the mutation, he underwent a thyroidectomy. Pathologic examination revealed no abnormalities on either gross or microscopic examination. Patient IV 2, the 10-year-old son of the index case, was also evaluated because of his mother’s history. Findings on physical examination were normal. Baseline calcitonin level was <25 pg/mL (normal, 0-25 pg/mL) and peaked at 83 pg/mL (normal, 25-150 pg/mL) with pentagastrin stimulation. He had normal serum calcium levels; thyroid function test results; and urine metanephrine, vanillylmandelic acid, epinephrine, and norepinephrine levels. DNA testing demonstrated that he had the identical codon 618 mutation. A complete thyroidectomy revealed multiple foci of Ccell hyperplasia and an area of microscopic medullary carcinoma. On followup postoperative pentagastrin test, all calcitonin levels were <25 pg/mL. Patient II 3, the 62-year-old maternal grandmother, was found to be an
Fig 3. Pedigree of kindred 2, showing family members affected with the mutation in exon 14, codon 804. Family members who were tested and found not to carry the mutation are indicated by an n within the gender-specific symbol; family members who were not tested but are potential carriers of the mutation are indicated by a question mark, including family members known to have MTC; family members through marriage, presumed not to carry the mutation, and children of a family member known to be negative (patient III, 3) are indicated by open gender-specific symbols.
asymptomatic carrier of the mutation. Prophylactic thyroidectomy was performed and revealed MTC with bilateral lymph node metastasis. Family members III 1, III 2, and II 2 are free of symptoms, and test results for the mutation were negative.
Kindred 2 (Fig 3) INDEX CASE. Patient III 4, a 45-yearold woman, had a family history of MTC in her father, 2 paternal aunts, and 2 first cousins. She was followed up with annual calcium-pentagastrin stimulation testing since the age of 31 years. Previous genetic testing for mutations in exons 10 and 11 was unrevealing. Because of the family history and equivocal calcium-pentagastrin test results (basal calcitonin = 25 pg/ mL, stimulated calcitonin = 39 pg/mL), she underwent thyroidectomy. Pathologic examination demonstrated C-cell hyperplasia, but no carcinoma was detected.
Mutation Analysis A DNA sample from the index case revealed a mutation in exon 14, codon
804 (GTG to TTG) of the RET gene. The mutation creates an abnormal N1a111 restriction site and was confirmed by enzymatic digestion.
Family Studies Subsequently, other available family members were screened for the mutation. Patient IV 4, a daughter (age, 7 years 10 months) of the index case was found to carry the identical codon 804 mutation. Subsequently, a calciumpentagastrin stimulation test attained a peak of 7 pg/mL. The family opted to defer prophylactic thyroidectomy at this time and continue with annual calcium-pentagastrin stimulation tests. Patient III 1 was tested at age 43 years because of the genetic and pathologic findings in his sister and was found to carry the identical mutation. Prophylactic thyroidectomy was performed but showed no pathologic evidence of MTC or CCH. Family members III 6, III 7, II 2, II 3, and II 4 were found to have MTC at thyroidectomy during their 4th and 5th decades of life; and family members III 2, III 3, and IV 3 tested negative for the mutation. 329
HEPTULLA ET AL
DISCUSSION DNA analysis for the detection of mutations of the RET gene has become a reliable method for the identification of subjects with hereditary MTC. From their analysis of 477 families, the international RET consortium concluded that a genotype-phenotype correlation does exist.10 Mutations in the cysteine-rich region of the RET gene codons 609, 611, 618, 620, and 634 are associated with either MEN 2A or isolated FMTC. Variability in the spectrum of pathology could be caused by modifying genes or environmental factors or could simply reflect random chance. Pheochromocytoma and/or hyperparathyroidism may also be relatively late occurrences in individuals who present with medullary carcinoma. Thus Moers et al11 reported a large kindred, initially classified as having FMTC. Subsequently, 4 family members were found to have parathyroid involvement. Before association of disease phenotype with RET mutations, genetic counseling and management relied on provocative stimulation and measurement of calcitonin in familial cases. The reliability of provocative testing varies with the sensitivity and specificity of the assay systems used, the stage of underlying disease, and the adequacy of control data.12 Thus false-negative or equivocal responses may be observed in mutation-positive individuals, as in patient IV 2 in kindred 1 and the index case, patient III 4, in kindred 2. Alternately, false-positive test results may occur in mutation-negative members of MEN 2A and FMTC kindreds; this has led to thyroidectomy in 5% to 10% of individuals at risk in whom subsequent gene analysis showed no abnormalities.13-16 The availability of RET mutation analysis thus provides a more precise means of identifying gene carriers than provocative stimulation with calcium and/or pentagastrin.17 Families with the MEN 2A syndrome have an earlier, 330
THE JOURNAL OF PEDIATRICS SEPTEMBER 1999 more aggressive form of MTC, which has led to the recommendation for thyroidectomy by 5 years of age.8 However, concern has been raised regarding the potential risks of thyroidectomy in young children, especially in inexperienced centers.6 The risk must be weighed against the risk of development of MTC, especially in families with mutations in the non-cysteine-rich region of the RET gene. The mild phenotype and low penetrance have been previously noted.18,19 As more families with mutations in these exons are identified, we should be able to modify screening programs, targeting primary thyroid disease and judiciously following up for other endocrinopathies in a cost-efficient and evidence-based manner.10 The nuances of current recommendations for treatment of mutation-positive children in families with FMTC are illustrated by the 2 kindreds described in this report. In the first kindred there is clear evidence of an early age of onset with metastatic disease evident in the index case at age 22 and microscopic disease in one of her sons at age 10. Notwithstanding the absence of pheochromocytoma and parathyroid hyperplasia as late as age 62 in one affected member of this kindred, this family shares a mutation at a region of the RET gene associated with early onset and a more aggressive presentation of MTC, and thus early prophylactic thyroidectomy remains appropriate in mutation-positive children in this family. In contrast, in the second kindred the mutation lies at a far less common intracellular site within the tyrosine kinase domain of the RET-coded transmembrane protein. There is limited experience in families with FMTC and mutations in exons 13 and 14. The late onset and relatively indolent course in this family may permit a more conservative approach, as indeed was selected. This was based on a plan to perform provocative testing with pentagastrin and/or calcium at annual intervals. Pentagastrin, however, is no longer
readily available, thus leaving calcium infusion alone, which is far less sensitive and reliable as a provocative stimulus.20 In contrast to recommendations for the more frequent RET mutations, families with these uncommon and less aggressive variants of FMTC can be treated on an individual basis, but definitive prophylactic thyroidectomy, if deferred, should be performed in childhood, ideally during the first decade of life. Original members of kindred 2 were studied and tested at the Tufts New England Medical Center under the direction of Dr Seymour Reichlin. We thank Dr Robert Gagel for providing results of the original mutation analysis of case III 3 kindred 2 and Dr Gustave Davis for sharing pathological specimens and slides from the index case of kindred 2.
REFERENCES 1. Schimke RN. Genetic aspects of multiple endocrine neoplasms. Annu Rev Med 1984;35:25-31. 2. Mulligan LM, Kwok JBJ, Healy CS, Eldson MJ. Germline mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458-60. 3. 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;2:851-6. 4. Carlson KM, Dou S, Chi D, Scarvada N, Toshima K, Jackson CE, et al. Single missense mutation in the tyrosine kinase catalytic domains of the RET prot-oncogene is associated with multiple endocrine neoplasia type 2B. Proc Natl Acad Sci USA 1994;91:1579-83. 5. Marsh DJ, Robinson BG, Andrew S, Richardson AL, Pojer R, Schnitzler M, et al. A rapid screening method for the detection of mutations in the RET proto-oncogene in multiple endocrine neoplasia type 2A and familial medullary thyroid carcinoma families. Genomics 1994;23:477-9. 6. Lips CJM, Landsvater RM, Hoppener JWM, Geerdink RA, Blijham G, van Veen JM, 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. 7. Wells SA, Chi DD, Toshima K, Dehn-
HEPTULLA ET AL
THE JOURNAL OF PEDIATRICS VOLUME 135, NUMBER 3
8.
9.
10.
11.
er 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. Gill JR, Reyes Múgica M, Iyengar S, Kidd KK, Touloukian RJ, Smith C, et al. Early presentation of metastatic medullary carcinoma in multiple endocrine neoplasia type 2A: implications of therapy. J Pediatr 1996;129:459-64. Brunt LM, Wells SA. Advances in the diagnosis and treatment of medullary thyroid carcinoma. Surg Clin North Am 1987;67:263-79. Eng C, Clayton D, Schuffenecker I, Lenoir G, Cote G, Gagel RF, et al. The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2. International RET mutation consortium analysis. JAMA 1996; 276:1575-9. Moers AMJ, Landsvater RM, Schaap C, Jansen-Schillhorn van Veen JM, de Valk IA, Blijham GH, et al. Familial medullary thyroid carcinoma: Not a
12. 13.
14.
15.
16.
distinct entity? Genotype-phenotype correlation in a large family. Am J Med 1996;101:635-41. Gagel RF. The abnormal pentagastrin test. Clin Endocrinol 1996;44:221-2. Marsh DJ, McDowall D, Hyland VJ, Andrew SD, Schnitzler M, Gaskin EL, et al. The identification of false positive responses to the pentagastrin stimulation test in RET mutation negative members of MEN 2A families. Clin Endocrinol 1996;44:213-20. Gagel RF, Tashijian AH Jr, Cummings T, Papathanasopoulos N, Kaplan MM, DeLellis RA, et al. The clinical outcome of prospective screening for multiple endocrine neoplasia type 2A: an 18-year experience. N Engl J Med 1988;318:478-84. Lichter JB, Wu JS, Genel M, Flynn SD, Pakstis SD, Kidd JR, et al. Presymptomatic testing using DNA markers for individuals at risk for familial multiple endocrine neoplasia 2A. J Clin Endocrinol Metab 1992;74:368-73. Lips CJ, Landsvater RM, Hoppener JW, Geerdink RA, Blijham G, van Veen
17.
18.
19.
20.
JM, 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. Heshmati HM, Gharib H, Khosla S, Abu-Lebdeh HS, Lindor NM, Thibodeau SN, et al. Genetic testing in medullary thyroid carcinoma syndromes: mutation types and clinical significance. Mayo Clin Proc 1997;72: 420-36. Bolino A, Schuffenecker I, Luo Y, Seri M, Silengo M, Tocco T, et al. RET mutations in exons 13 and 14 of FMTC patients. Oncogene 1995;10:2415-9. Eng C, Smith DP, Mulligan LM, Healey CS, Zvelebil MJ, Stonehouse TJ, 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 1995;10:509-13. Wells SA, Baylin SB, Linehan WM, Farrell RE, Cox EB, Cooper CW. Provocative agents and the diagnosis of medullary carcinoma of the thyroid gland. Ann Surg 1978;188:139-41.
FELLOWSHIPS Fellowships available in pediatric subspecialties and those for general academic pediatric training are listed once a year, in January, in The Journal of Pediatrics. Each June, forms for listing fellowships available for the academic year beginning 18 months after publication are sent to the Chairman of the Department of Pediatrics at major hospitals in the United States and Canada. In addition, a copy of the application form appears in the July, August, and September issues of The Journal (please use the current form). Should you desire to list fellowships, a separate application must be made each year for each position. All applications must be returned to Mosby, Inc., by October 15 preceding the listing year to ensure publication. Additional forms will be supplied on request from the Periodical Editing Department, Mosby, Inc., 11830 Westline Industrial Drive, St. Louis, MO 63146-3318 (phone: 800-325-4177, ext. 2838, or 314-579-2838).
331