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Variable expressivity of familial medullary thyroid carcinoma (FMTC) due to a RET V804M (GTG→ATG) mutation
Variable expressivity of familial medullary thyroid carcinoma (FMTC) due to a RET V804M (GTG→ATG) mutation Gerald L. Feldman, MD, PhD, Merrill W. Edmonds, MD, Peter J. Ainsworth, MBChB, PhD, Isabelle Schuffenecker, PhD, Gilbert M. Lenoir, DVM, Andrew W. Saxe, MD, Gary B. Talpos, MD, Jacquelyn Roberson, MD, Nancie Petrucelli, MS, and Charles E. Jackson, MD, Detroit, Mich, London, Ontario, Canada, and Lyon, France
Background. Multiple endocrine neoplasia type 2 (MEN 2) and familial medullary thyroid carcinoma (FMTC) are autosomal dominantly inherited cancer syndromes that predispose to C-cell hyperplasia and MTC. MEN 2A and FMTC are caused by mutations in the RET proto-oncogene. Methods. We used a multiplex polymerase chain reaction-based assay to screen exons 10, 11, 13, and 14 of RET for mutations in 2 families with FMTC. We correlated mutation status with calcitonin and pathologic studies to determine genotype-phenotype correlations. Results. We identified a mutation in codon 804 in exon 14 (GTG→ATG; V804M) in both families. An 86-year-old person who was a gene carrier and other individuals over age 70 who were suspected by pedigree analysis to be gene carriers had no overt clinical evidence of MTC. Four of 21 patients who underwent a thyroidectomy also had papillary thyroid cancer. One individual in each family had metastatic MTC at age 30 and 32 years, and all 26 people having thyroidectomies had either MTC or C-cell hyperplasia, leading us to continue to recommend prophylactic thyroidectomy for all identified patients who were gene carriers. Conclusions. Because of active MTC in younger members of these families, including metastases, we have continued to advocate thyroid surgery in mutation-positive individuals. While DNA diagnosis of gene carriers and subsequent genetic counseling was relatively straightforward, the acceptance of surgical recommendations was more difficult for some individuals. These families demonstrate that the search for RET mutations should include exons 13, 14, 15, and 16 in patients whose studies in exons 10 and 11 are negative. (Surgery 2000;128:93-8) From the Departments of Medical Genetics and Surgery, Henry Ford Hospital, the Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, and the Department of Surgery, Sinai-Grace Hospital, Detroit Medical Center, Detroit, Mich, the London Health Sciences Centre, London, Ontario, Canada, and the Laboratoire de Génétique, Hôpital Édouard Herriot, Lyon, France
MEDULLARY THYROID CARCINOMA (MTC), a rare thyroid malignant lesion, occurs either sporadically or as a familial entity. Familial MTC can occur as familial MTC alone (FMTC) or as part of multiple endocrine neoplasia type 2A or 2B (MEN 2A or MEN 2B).1-4 MEN 2A is characterized by MTC, pheochromocytoma, and parathyroid adenomas; MEN 2B is characterized by MTC, pheochromoSupported in part by a research grant from the Dykstra Foundation (Detroit, Mich). Accepted for publication March 11, 2000. Reprint requests: Gerald L. Feldman, MD, PhD, Center for Molecular Medicine and Genetics, 3216 Scott Hall, 540 E Canfield, Detroit, MI 48201. Copyright © 2000 by Mosby, Inc. 0039-6060/2000/$12.00 + 0 doi:10.1067/msy.2000.107103
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cytoma, ganglioneuromatosis, and a marfanoid habitus.5-6 FMTC, MEN 2A, and MEN 2B are all inherited in an autosomal dominant pattern with age-related penetrance and variable expressivity.2 Almost all patients with these disorders have a germline mutation in the RET proto-oncogene, located on chromosome 10 in band q11.2. Alterations at 1 of 5 cysteine codons in exons 10 and 11 of the RET proto-oncogene have been found in approximately 95% of families with MEN 2A.7-8 Approximately 97% of patients with MEN 2B have a mutation in codon 918 within exon 169; the remainder have mutations in codon 883 in exon 15.10-11 Mutations have been detected in about 85% of cases of FMTC, and these occur in the codons noted above in exons 10 and 11 and in codons 768, 790, and 791 in exon 1312-15 and codon 804 in exon 14.13,16-19 One famSURGERY 93
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ily with MTC has been reported with a point mutation in exon 1520 and a recent family has been reported with a 9-base pair duplication in exon 8.21 A codon 804 mutation (V804L) resulting in the substitution of a leucine for the normal valine (GTG→TTG) was initially reported by Bolino et al,13 and another family was recently reported by Nilsson et al.19 Three other reports identified a different codon 804 mutation (V804M), leading to the substitution of a methionine in place of valine (GTG→ATG).15-17 In the report by Fink et al,16 2 of 7 gene carriers in the family had MTC, while the remaining 5 gene carriers in the family had C-cell hyperplasia only. In the 2 other FMTC families reported from Italy with V804M, 4 individuals were affected in 3 generations in each unrelated family.17 Shannon et al reported 2 families with apparently sporadic MTC in which a germline V804M mutation was detected in the probands and in other at-risk family members.18 A statistically significant association between the presence of any mutation at a specific position (codon 634) and the presence of pheochromocytoma and hyperparathyroidism has been shown, while mutations at codons 768 and 804 were initially reported with FMTC only.9 However, 2 of the family members recently reported by Nilsson et al also had pheochromocytomas, suggesting that germline V804L mutations can be associated with MEN 2A.19 We report here 2 families with extensive FMTC with the GTG→ATG sequence change in codon 804 of the RET proto-oncogene. We present pathologic, biochemical, and molecular data on numerous family members, including several apparently asymptomatic family members over age 70, demonstrating the variability in age of expression within these families. METHODS High molecular weight genomic DNA was extracted by using a DNA extraction kit (Puregene; Gentra Systems, Inc, Minneapolis, Minn) according to the protocol provided by the manufacturer. RET exons 10, 11, and 13 were amplified simultaneously as previously reported by using a multiplex polymerase chain reaction assay followed by Mutation Detection Enhancement matrix for heteroduplex identification.22 For exon 14, DNA was polymerase chain reaction-amplified by using primers previously reported.23 Heteroduplex analysis allows the detection of sequence mismatches between 2 alleles, such as that produced by the GTG→ATG nucleotide substitution in codon 804 in these families. Specific codon 804
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mutations were confirmed by DNA sequencing or by restriction enzyme digestion with NlaIII for V804M. This restriction enzyme recognizes the sequence difference between GTG and ATG, creating a new NlaIII restriction site specific for that mutation. RESULTS The propositus in family 1 (Fig 1, III-2), had a thyroidectomy at age 55 after finding a basal calcitonin elevation associated with a thyroid enlargement that had been present for 20 years (Table I). The pathologic report noted multifocal MTC with nodal involvement. Her basal postoperative calcitonin levels remained elevated, although they have generally decreased, with the lowest level at 210 pg/mL being found 11 years postoperatively. After her MTC was found, her 26-year-old son (now 36) (Fig 1, IV-4) had a pentagastrin-stimulated calcitonin elevation to 196 pg/mL. Thyroidectomy revealed C-cell hyperplasia only. DNA studies of these two people were negative for mutations in exons 10, 11, and 13; but, later, a V804M mutation was identified in each at the Hôpital Édouard Herriot in Lyon. Studies of family members revealed the V804M mutation in 10 other family members (Fig 1, II-2, III-3, III-6, III-7, IV-3, IV-5, IV6, IV-7, IV-8, and IV-10). Interestingly, the propositus’ 86-year-old mother, II-2, is asymptomatic with no thyroid enlargement on physical examination and only a slightly abnormal basal calcitonin level. No goiter was reported in her Hungarian-born father, mother, or in her brother (Fig 1, I-5), who died at 89, 72, and 72, respectively. Thyroidectomies have been performed on 8 of these 10 gene carriers, and C-cell hyperplasia or MTC was reported in each, with 3 also having papillary microcarcinomas. A metastasis was found adjacent to the thyroid in one 32-year-old family member (IV-5), but her pentagastrin-stimulated calcitonin test 3 months postoperatively was normal (< 1 pg/mL). All affected people in this family have had normal serum calciums and, while catecholamine testing was not performed, none had signs or symptoms suggestive of pheochromocytomas. In family 2 (Fig 2), the propositus (III-2) was referred to the Genetics Clinic at Henry Ford Hospital because of her personal and family history of MTC. This 65-year-old woman had thyroid surgery at age 56, at which time MTC was found in the right lobe and C-cell hyperplasia and a 2-mm papillary carcinoma were found in the left lobe. After a total thyroidectomy, her calcitonin levels have remained normal (Table II). Her 39-year-old daughter (IV-2) had a total thyroidectomy at age
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Fig 1. Pedigree of family 1 with exon 14 804 GTG→ATG mutation. Solid circles or squares, Family members with MTC; striped circles or squares, patients with C-cell hyperplasia only; open circles or squares, women or men, respectively, with no documented MTC; open circles or squares with slash marks, deceased women or men, respectively, with no documented MTC; plus signs below circles or squares, family members who tested positive for the 804 mutation; minus signs, family members who tested negative for the 804 mutation. This pedigree was shown in part previously.24
Table I. Clinical findings in family 1 Patient
Age
Calcitonin level (pg/mL)*
Thyroid palpable
II-2 III-2
86 66
21 (normal < 4.6) 2480 preop, 210-1100 postop, 210 most recent
III-3
63
III-6
58
IV-3 IV-4 IV-5
40 36 32
Negative, pentagastrin stimulation test at age 58, 40→197 after mutation found at age 63 Basal calcitonin normal after mutation found –– 196 after pentagastrin preop 75→600 preop, negative stimulated postop
IV-6 IV-7
30 24
13→228 preop 40→1048 preop
No No
IV-8 IV-10
34 28
–– ––
No No
No Enlarged
No
No No No No
Pathology –– Bilateral MTC at age 55 (1.8 and 0.2 cm on left; 0.3 and 0.2 cm on right) Multicentric MTC
2-mm MTC and 0.5-mm papillary microcarcinoma MTC at age 40 C-cell hyperplasia at age 26 MTC with metastases adjacent to thyroid plus small focus of papillary carcinoma C-cell hyperplasia C-cell hyperplasia plus < 1-mm papillary carcinoma C-cell hyperplasia C-cell hyperplasia
*All assays performed by radioammunoassay in various laboratories in US and Canada with slight variability in normal ranges for each particular laboratory. Generally, < 20 pg/mL basal calcitonin is considered normal.
30, at which time metastases were found. Two years later, because of continued elevations of calcitonin, a node dissection was performed, which revealed peritracheal node metastases. Molecular studies of the RET gene performed at Henry Ford Hospital identified the V804M mutation in affected individuals III-2 and IV-2 and also in IV-3, IV-6, and V-1. Review of the family history disclosed that the propositus’ 70-year-old sister (III-4) and her 2 daughters (IV-12 and IV-13) had operations for MTC, as did her maternal aunt (II-4) and her maternal cousin (III-12). These people had been operated on before the identification of the V804M mutation in III-2. After the identification of the
V804M mutation in this family at Henry Ford Hospital, the V804M mutation was also identified in individuals II-4, II-7, III-4, III-6, III-7, III-10, III11, III-12, IV-8, IV-10, IV-11, IV-12, IV-13, and IV-16 at the Molecular Diagnostics Laboratory, London Health Sciences Centre. Individuals IV-10 and IV16 had thyroidectomies performed on the basis of mutation testing even though they previously had normal stimulated calcitonin levels (Table II). Thirteen other members of this kindred (not shown in Fig 2) tested positive for this mutation: 3 members of generation III, 6 members of generation IV, and 4 members of generation V. Review of the pedigree clearly shows that II-2, who died at age
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Fig 2. Partial pedigree of family 2 with exon 14 804 GTG→ATG mutation. Solid circles, Family members with MTC; striped circle, patient with C-cell hyperplasia only; open circles or squares, women or men, respectively, with no documented MTC; open circles or squares with slash marks, deceased women or men, respectively, with no documented MTC; plus signs below circles or squares, family members who tested positive for the 804 mutation; minus signs, family members who tested negative for the 804 mutation. This pedigree was shown in part previously.
Table II. Clinical findings of family 2 Patient
Age
Calcitonin level (pg/mL)*
Thyroid palpable
II-4
86
Basal: 8300, 1976; 5000, 1987; 1743, 1996
III-2
65
Preop: 110→1000 9 y postop: 0.4 (normal < 4.6)
Enlarged
III-4 III-12
72 71
Stimulated elevation at age 62 Basal: 1065
No Enlarged
IV-2
40
Elevated after pentagastrin
No
IV-10 IV-12
39 45
No No
IV-13 IV-16
40 39
Stimulated normal ages 31-35 Stimulated normal ages 35-42; although gradually increasing; elevated age 43 Stimulated normal ages 34-39; preop: 15Æ180 Stimulated normal ages 23-27
“Papillary thyroid cancer” reported at age 53, later found to be MTC after nodes removed at age 65 Thyroidectomy, age 56; right lobe: MTC, Hashimoto’s thyroiditis; left lobe: 2.0-mm papillary carcinoma, Hashimoto’s thyroiditis, and focal C-cell hyperplasia 12 × 5-mm MTC at age 63 MTC at left lobectomy, age 54; metasteses at age 55 at total thyroidectomy At age 31: Right lobe: Hashimoto’s thyroiditis; left lobe: 6-mm MTC and Hashimoto’s thyroiditis At age 32: metastatic MTC at node resection Age 39: 3-mm MTC 2-mm MTC at age 43
No No
MTC, 1 small focus only, at age 33 Age 39: C-cell hyperplasia
Nodule
Pathology
*All assays were performed by radioammunoassay in various laboratories in the US and Canada, with slight variability in the normal ranges for each particular laboratory. Generally, < 20 pg/mL basal calcitonin is generally considered normal.
84, and II-5, who died at age 86, were gene carriers, although neither had thyroid surgery. Both had affected offspring with the V804M mutation and MTC. Since the likelihood that each of their affected offspring carried V804M as a result of a new mutation, one can assume that each was a carrier of this mutation. Similarly, I-2, who died at age 85, was likely to have also carried this same mutation, since her brother’s granddaughter is a known mutation carrier (data not shown in pedigree). All affected members tested in this family have had normal
serum calciums, with negative catecholamine studies in 8 of 8 carriers tested (ages 32-77). We have collectively analyzed 65 patients (130 chromosomes) with apparently sporadic MTC for exon 14 mutations and have not identified additional patients with mutations in this codon. CONCLUSIONS The families reported here are interesting in several ways. While FMTC is known to be variable in age of onset, this mutation appears to cause a much
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wider age range than do most other RET mutations.13,16-19 Although DNA diagnosis of gene carriers and subsequent genetic counseling of the results to patients was relatively straightforward, individual decisions regarding the option of a prophylactic thyroidectomy were more difficult. The presence of active MTC, including metastases in young members(32 and 30 years old) of each family (family 1, IV5 and family 2, IV-2), suggests that surgery be done at an early age in all known mutation carriers. However, the apparent absence of MTC in older gene carriers (family 1, II-2 and II-5) caused one known mutation carrier (family 1, III-7) to be reluctant to consider surgery despite genetic counseling and even though all diagnosed gene carriers who underwent prophylactic thyroidectomies had either C-cell hyperplasia (family 1, IV-4, IV-6, IV-7, IV-8, and IV-10; family 2, IV16) or MTC (family 1, III-2, III-3, III-6, IV-3 and IV-5; family 2, II-4, III-2, III-4, III-12, IV-2, IV-10, IV-12, and IV-13). In our opinion, a less desirable but alternative option is to follow known mutation carriers carefully with stimulated calcitonin testing and operate when levels become elevated. The clinical findings in these families are similar to those observed by Fattoruso et al,17 who noted that subsequent generations seemed to show an earlier and more aggressive phenotype. Similarly, Halling et al25 recently reported a family with a codon 609 RET mutation in which a later age of onset was noted in many individuals. The presence of papillary thyroid cancer (PTC) as seen in these 2 families (Tables I, II) has infrequently been reported in patients with a germline point mutation in the RET proto-oncogene.26 The papillary carcinomas in these patients were occult, and no further treatments were necessary. A somatic rearranged form of the RET proto-oncogene (termed RET/PTC oncogene) has been detected specifically in a minority of papillary thyroid carcinomas,27 including some with karyotypic abnormalities.28 The possible increased frequency of PTC in addition to MTC in these families may be coincidental or may be causally related to the specific codon 804 mutation. Further studies are indicated in ascertaining the role of RET germline mutations versus rearrangements in PTC. These families demonstrate that the search for RET mutations should not be confined to exons 10 and 11, even though most mutations are present in those 2 exons. As additional mutations in RET are identified, it is important to study those patients in families in which a mutation has not previously been identified. In addition, discussion of the genotype-phenotype correlation must be specific not only for the mutation identified, but must also take into account the relationship between the spe-
cific mutation and the phenotype within a particular family. For example, studies of oncogenic activation of RET by Pasini et al29 have demonstrated that the E768D and V804L mutations found in the context of FMTC appear to be less activating than MEN 2A or MEN 2B mutations. Even so, it is clear that codon 804 mutations can predispose a person to MTC at a young age. Thus, we are continuing to recommend prophylactic thyroidectomies in childhood in all patients with identified RET mutations associated with the clinical phenotype consistent with MEN 2A, MEN 2B, or FMTC. We thank Karen Dziubek and Javed Siddiqui in the Henry Ford Hospital DNA Diagnostic Laboratory and the Molecular Diagnostic Laboratory staff at London Health Sciences Centre for their technical assistance. We thank the following physicians for information on various family members: Dr Michael R. Aulicino (Department of Pathology, Sinai Hospital), Dr Maurice Budow (Ann Arbor, Mich), Dr Harold C Yang and Dr James M. Hammond (Department of Surgery and Medicine, Pennsylvania State University), Dr Charles Taylor, (Detroit, Mich), Dr Benjamin Parish (Mechanicsburg, Pa), and Dr Norman W. Thompson (Ann Arbor, Mich). REFERENCES 1. Farndon JR, Leight GS, Dilley WG, Baylin SB, Smallridge RC, Harrison TS, et al. Familial medullary thyroid cancer without associated endocrinopathies: a distinct clinical entity. Br J Surg 1986;73:278-81. 2. Jackson CE, Norum RA. Genetic mechanisms of neoplasia in MEN 2. Henry Ford Hosp Med J 1989;37:116-9. 3. Sipple JH. The association of pheochromocytoma with cancer of the thyroid gland. Am J Med 1961;31:163-6. 4. Steiner AL, Goodman AD, Powers SR. Study of a kindred with pheochromocytoma, medullary thyroid carcinoma, hyperparathyroidism and Cushing’s disease: multiple endocrine neoplasia syndrome, type 2. Medicine 1968;47:371-409. 5. Carney JA, Sizemore GW, Lovestedt SA. Mucosal ganglioneuromatosis, medullary thyroid cancer, and pheochromocytoma: multiple endocrine neoplasia, type 2b. Oral Surg Oral Med Oral Path 1976;41:739-52. 6. Schimke RN, Hartmann WH, Prout TE, Rimoin DL. Syndrome of bilateral pheochromocytoma, medullary thyroid cancer, and multiple neuromas: a possible regulatory defect in the differentiation of chromaffin tissue. N Engl J Med 1968;279:1-7. 7. Mulligan LM, Kwok JBJ, Healey CS, Elsdon MJ, Eng C, Gardner E, et al. Germ-line mutations of the RET protooncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458-60. 8. Donis-Keller H, Dou S, Chi D, Carlson TM, 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. 9. 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. JAMA 1996;276:1575-79.
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10. Smith DP, Houghton C, Ponder BAJ. Germline mutation of RET codon 883 in two cases of de novo MEN 2b. Oncogene 1997;15:1213-7. 11. Gimm O, Marsh DJ, Andrew SD, Frilling A, Dahia PLM, Mulligan LM, et al. Germline dinucleotide mutation in codon 883 of the RET proto-oncogene in multiple endocrine neoplasia type 2B without codon 918 mutation. J Clin Endocrinol Metab 1997;82:3902-4. 12. 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. 13. 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. 14. Boccia LM, Green JS, Joyce C, Eng C, Taylor SAM, Mulligan LM. Mutation of RET codon 768 is associated with the FMTC phenotype. Clin Genet 1997;51:81-5. 15. Berndt I, Reuter M, Saller B, Frank-Raue K, Groth P, Grubendorf M, et al. A new hot spot for mutations in the Ret proto-oncogenes causing familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2A. J Clin Endocrinol Metab 1998;770-4. 16. Fink M, Weinhäusel A, Niederle B, Haas OA. Distinction between sporadic and hereditary medullary thyroid carcinoma (MTC) by mutation analysis of the RET proto-oncogene. Int J Cancer (Pred Oncol) 1996;69:312-6. 17. Fattoruso O, Quadro L, Libroia A, Verga U, Lupoli G, Cascone E, et al. A GTG to ATG novel point mutation at codon 804 in exon 14 of the RET proto-oncogene in two families affected by familial medullary thyroid carcinoma. Hum Mut 1998;S167-71. 18. Shannon KE, Gimm O, Hinze R, Dralle H, Eng C. Germline V804M mutation in the RET proto-oncogene in two apparently sporadic cases of MTC presenting in the seventh decade of life. J Endocrine Genetics 1999;1:39-45. 19. Nilsson O, Tisell LE, Jansson S, Ahlman H, Gimm O, Eng C. Adrenal and extra-adrenal pheochromocytomas in a family with germline RET V804L mutation. JAMA 1999; 1587-8.
Surgery July 2000 20. Hofstra RMW, Fattoruso O, Quadro L, Wu Y, Libroia A, Verga U, et al. A novel point mutation in the intracellular domain of the RET proto-oncogene in a family with medullary thyroid carcinoma. J Clin Endocrinol Metab 1997;82:4176-8. 21. Pigny P, Bauters C, Wemeau J-L, Houcke ML, Crepin M, Caron P, et al. A novel 9-base pair duplication in RET exon 8 in familial medullary thyroid carcinoma. J Clin Endocrin Metab 1999;84:1700-4. 22. Kambouris M, Jackson CE, Feldman GL. Diagnosis of multiple endocrine neoplasia (MEN) 2A, 2B and familial medullary thyroid cancer (FMTC) by multiplex PCR and heteroduplex analyses of RET proto-oncogene mutations. Hum Mutat 1996;8:64-70. 23. Cecherini 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. 24. Jackson CE. A 50-year perspective on endocrine neoplasia: clinic to genes and back. J Intern Med 1998;243:419-23. 25. Halling KC, Bufill JA, Cotter M, Artz SA, Carpenter AB, Schaid D, et al. Age-related disease penetrance in a large medullary thyroid cancer family with a codon 609 RET gene mutation. Molec Diag 1997;2:277-86. 26. Komminoth P. The RET proto-oncogene in medullary and papillary carcinoma: molecular features, pathophysiology, and clinical implications. Virchows Arch 1997;431:1-9. 27. Grieco M, Santoro M, Berlingieri M, Melillo RM, Donghi R, Bongarzone I, et al. PTC is a rearranged form of the RET proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell 1990;60:557-63. 28. Smanik PA, Furminger TL, Mazzaferri EL, Jhiang SM. Breakpoint characterization of the RET/PTC oncogene in human papillary thyroid carcinoma. Hum Mol Genet 1995;4:2313-8. 29. Pasini A, Geneste O, Legrand P, Schlumberger M, Rossel M, Fournier L, et al. Oncogenic activation of RET by two distinct FMTC mutations affecting the tyrosine kinase domain. Oncogene 1997;15:393-402.
Background. Multiple endocrine neoplasia type 2 (MEN 2) and familial medullary thyroid carcinoma (FMTC) are autosomal dominantly inherited cancer syndromes that predispose to C-cell hyperplasia and MTC. MEN 2A and FMTC are caused by mutations in the RET proto-oncogene. Methods. We used a multiplex polymerase chain reaction-based assay to screen exons 10, 11, 13, and 14 of RET for mutations in 2 families with FMTC. We correlated mutation status with calcitonin and pathologic studies to determine genotype-phenotype correlations. Results. We identified a mutation in codon 804 in exon 14 (GTG→ATG; V804M) in both families. An 86-year-old person who was a gene carrier and other individuals over age 70 who were suspected by pedigree analysis to be gene carriers had no overt clinical evidence of MTC. Four of 21 patients who underwent a thyroidectomy also had papillary thyroid cancer. One individual in each family had metastatic MTC at age 30 and 32 years, and all 26 people having thyroidectomies had either MTC or C-cell hyperplasia, leading us to continue to recommend prophylactic thyroidectomy for all identified patients who were gene carriers. Conclusions. Because of active MTC in younger members of these families, including metastases, we have continued to advocate thyroid surgery in mutation-positive individuals. While DNA diagnosis of gene carriers and subsequent genetic counseling was relatively straightforward, the acceptance of surgical recommendations was more difficult for some individuals. These families demonstrate that the search for RET mutations should include exons 13, 14, 15, and 16 in patients whose studies in exons 10 and 11 are negative. (Surgery 2000;128:93-8) From the Departments of Medical Genetics and Surgery, Henry Ford Hospital, the Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, and the Department of Surgery, Sinai-Grace Hospital, Detroit Medical Center, Detroit, Mich, the London Health Sciences Centre, London, Ontario, Canada, and the Laboratoire de Génétique, Hôpital Édouard Herriot, Lyon, France
MEDULLARY THYROID CARCINOMA (MTC), a rare thyroid malignant lesion, occurs either sporadically or as a familial entity. Familial MTC can occur as familial MTC alone (FMTC) or as part of multiple endocrine neoplasia type 2A or 2B (MEN 2A or MEN 2B).1-4 MEN 2A is characterized by MTC, pheochromocytoma, and parathyroid adenomas; MEN 2B is characterized by MTC, pheochromoSupported in part by a research grant from the Dykstra Foundation (Detroit, Mich). Accepted for publication March 11, 2000. Reprint requests: Gerald L. Feldman, MD, PhD, Center for Molecular Medicine and Genetics, 3216 Scott Hall, 540 E Canfield, Detroit, MI 48201. Copyright © 2000 by Mosby, Inc. 0039-6060/2000/$12.00 + 0 doi:10.1067/msy.2000.107103
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cytoma, ganglioneuromatosis, and a marfanoid habitus.5-6 FMTC, MEN 2A, and MEN 2B are all inherited in an autosomal dominant pattern with age-related penetrance and variable expressivity.2 Almost all patients with these disorders have a germline mutation in the RET proto-oncogene, located on chromosome 10 in band q11.2. Alterations at 1 of 5 cysteine codons in exons 10 and 11 of the RET proto-oncogene have been found in approximately 95% of families with MEN 2A.7-8 Approximately 97% of patients with MEN 2B have a mutation in codon 918 within exon 169; the remainder have mutations in codon 883 in exon 15.10-11 Mutations have been detected in about 85% of cases of FMTC, and these occur in the codons noted above in exons 10 and 11 and in codons 768, 790, and 791 in exon 1312-15 and codon 804 in exon 14.13,16-19 One famSURGERY 93
94 Feldman et al
ily with MTC has been reported with a point mutation in exon 1520 and a recent family has been reported with a 9-base pair duplication in exon 8.21 A codon 804 mutation (V804L) resulting in the substitution of a leucine for the normal valine (GTG→TTG) was initially reported by Bolino et al,13 and another family was recently reported by Nilsson et al.19 Three other reports identified a different codon 804 mutation (V804M), leading to the substitution of a methionine in place of valine (GTG→ATG).15-17 In the report by Fink et al,16 2 of 7 gene carriers in the family had MTC, while the remaining 5 gene carriers in the family had C-cell hyperplasia only. In the 2 other FMTC families reported from Italy with V804M, 4 individuals were affected in 3 generations in each unrelated family.17 Shannon et al reported 2 families with apparently sporadic MTC in which a germline V804M mutation was detected in the probands and in other at-risk family members.18 A statistically significant association between the presence of any mutation at a specific position (codon 634) and the presence of pheochromocytoma and hyperparathyroidism has been shown, while mutations at codons 768 and 804 were initially reported with FMTC only.9 However, 2 of the family members recently reported by Nilsson et al also had pheochromocytomas, suggesting that germline V804L mutations can be associated with MEN 2A.19 We report here 2 families with extensive FMTC with the GTG→ATG sequence change in codon 804 of the RET proto-oncogene. We present pathologic, biochemical, and molecular data on numerous family members, including several apparently asymptomatic family members over age 70, demonstrating the variability in age of expression within these families. METHODS High molecular weight genomic DNA was extracted by using a DNA extraction kit (Puregene; Gentra Systems, Inc, Minneapolis, Minn) according to the protocol provided by the manufacturer. RET exons 10, 11, and 13 were amplified simultaneously as previously reported by using a multiplex polymerase chain reaction assay followed by Mutation Detection Enhancement matrix for heteroduplex identification.22 For exon 14, DNA was polymerase chain reaction-amplified by using primers previously reported.23 Heteroduplex analysis allows the detection of sequence mismatches between 2 alleles, such as that produced by the GTG→ATG nucleotide substitution in codon 804 in these families. Specific codon 804
Surgery July 2000
mutations were confirmed by DNA sequencing or by restriction enzyme digestion with NlaIII for V804M. This restriction enzyme recognizes the sequence difference between GTG and ATG, creating a new NlaIII restriction site specific for that mutation. RESULTS The propositus in family 1 (Fig 1, III-2), had a thyroidectomy at age 55 after finding a basal calcitonin elevation associated with a thyroid enlargement that had been present for 20 years (Table I). The pathologic report noted multifocal MTC with nodal involvement. Her basal postoperative calcitonin levels remained elevated, although they have generally decreased, with the lowest level at 210 pg/mL being found 11 years postoperatively. After her MTC was found, her 26-year-old son (now 36) (Fig 1, IV-4) had a pentagastrin-stimulated calcitonin elevation to 196 pg/mL. Thyroidectomy revealed C-cell hyperplasia only. DNA studies of these two people were negative for mutations in exons 10, 11, and 13; but, later, a V804M mutation was identified in each at the Hôpital Édouard Herriot in Lyon. Studies of family members revealed the V804M mutation in 10 other family members (Fig 1, II-2, III-3, III-6, III-7, IV-3, IV-5, IV6, IV-7, IV-8, and IV-10). Interestingly, the propositus’ 86-year-old mother, II-2, is asymptomatic with no thyroid enlargement on physical examination and only a slightly abnormal basal calcitonin level. No goiter was reported in her Hungarian-born father, mother, or in her brother (Fig 1, I-5), who died at 89, 72, and 72, respectively. Thyroidectomies have been performed on 8 of these 10 gene carriers, and C-cell hyperplasia or MTC was reported in each, with 3 also having papillary microcarcinomas. A metastasis was found adjacent to the thyroid in one 32-year-old family member (IV-5), but her pentagastrin-stimulated calcitonin test 3 months postoperatively was normal (< 1 pg/mL). All affected people in this family have had normal serum calciums and, while catecholamine testing was not performed, none had signs or symptoms suggestive of pheochromocytomas. In family 2 (Fig 2), the propositus (III-2) was referred to the Genetics Clinic at Henry Ford Hospital because of her personal and family history of MTC. This 65-year-old woman had thyroid surgery at age 56, at which time MTC was found in the right lobe and C-cell hyperplasia and a 2-mm papillary carcinoma were found in the left lobe. After a total thyroidectomy, her calcitonin levels have remained normal (Table II). Her 39-year-old daughter (IV-2) had a total thyroidectomy at age
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Fig 1. Pedigree of family 1 with exon 14 804 GTG→ATG mutation. Solid circles or squares, Family members with MTC; striped circles or squares, patients with C-cell hyperplasia only; open circles or squares, women or men, respectively, with no documented MTC; open circles or squares with slash marks, deceased women or men, respectively, with no documented MTC; plus signs below circles or squares, family members who tested positive for the 804 mutation; minus signs, family members who tested negative for the 804 mutation. This pedigree was shown in part previously.24
Table I. Clinical findings in family 1 Patient
Age
Calcitonin level (pg/mL)*
Thyroid palpable
II-2 III-2
86 66
21 (normal < 4.6) 2480 preop, 210-1100 postop, 210 most recent
III-3
63
III-6
58
IV-3 IV-4 IV-5
40 36 32
Negative, pentagastrin stimulation test at age 58, 40→197 after mutation found at age 63 Basal calcitonin normal after mutation found –– 196 after pentagastrin preop 75→600 preop, negative stimulated postop
IV-6 IV-7
30 24
13→228 preop 40→1048 preop
No No
IV-8 IV-10
34 28
–– ––
No No
No Enlarged
No
No No No No
Pathology –– Bilateral MTC at age 55 (1.8 and 0.2 cm on left; 0.3 and 0.2 cm on right) Multicentric MTC
2-mm MTC and 0.5-mm papillary microcarcinoma MTC at age 40 C-cell hyperplasia at age 26 MTC with metastases adjacent to thyroid plus small focus of papillary carcinoma C-cell hyperplasia C-cell hyperplasia plus < 1-mm papillary carcinoma C-cell hyperplasia C-cell hyperplasia
*All assays performed by radioammunoassay in various laboratories in US and Canada with slight variability in normal ranges for each particular laboratory. Generally, < 20 pg/mL basal calcitonin is considered normal.
30, at which time metastases were found. Two years later, because of continued elevations of calcitonin, a node dissection was performed, which revealed peritracheal node metastases. Molecular studies of the RET gene performed at Henry Ford Hospital identified the V804M mutation in affected individuals III-2 and IV-2 and also in IV-3, IV-6, and V-1. Review of the family history disclosed that the propositus’ 70-year-old sister (III-4) and her 2 daughters (IV-12 and IV-13) had operations for MTC, as did her maternal aunt (II-4) and her maternal cousin (III-12). These people had been operated on before the identification of the V804M mutation in III-2. After the identification of the
V804M mutation in this family at Henry Ford Hospital, the V804M mutation was also identified in individuals II-4, II-7, III-4, III-6, III-7, III-10, III11, III-12, IV-8, IV-10, IV-11, IV-12, IV-13, and IV-16 at the Molecular Diagnostics Laboratory, London Health Sciences Centre. Individuals IV-10 and IV16 had thyroidectomies performed on the basis of mutation testing even though they previously had normal stimulated calcitonin levels (Table II). Thirteen other members of this kindred (not shown in Fig 2) tested positive for this mutation: 3 members of generation III, 6 members of generation IV, and 4 members of generation V. Review of the pedigree clearly shows that II-2, who died at age
96 Feldman et al
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Fig 2. Partial pedigree of family 2 with exon 14 804 GTG→ATG mutation. Solid circles, Family members with MTC; striped circle, patient with C-cell hyperplasia only; open circles or squares, women or men, respectively, with no documented MTC; open circles or squares with slash marks, deceased women or men, respectively, with no documented MTC; plus signs below circles or squares, family members who tested positive for the 804 mutation; minus signs, family members who tested negative for the 804 mutation. This pedigree was shown in part previously.
Table II. Clinical findings of family 2 Patient
Age
Calcitonin level (pg/mL)*
Thyroid palpable
II-4
86
Basal: 8300, 1976; 5000, 1987; 1743, 1996
III-2
65
Preop: 110→1000 9 y postop: 0.4 (normal < 4.6)
Enlarged
III-4 III-12
72 71
Stimulated elevation at age 62 Basal: 1065
No Enlarged
IV-2
40
Elevated after pentagastrin
No
IV-10 IV-12
39 45
No No
IV-13 IV-16
40 39
Stimulated normal ages 31-35 Stimulated normal ages 35-42; although gradually increasing; elevated age 43 Stimulated normal ages 34-39; preop: 15Æ180 Stimulated normal ages 23-27
“Papillary thyroid cancer” reported at age 53, later found to be MTC after nodes removed at age 65 Thyroidectomy, age 56; right lobe: MTC, Hashimoto’s thyroiditis; left lobe: 2.0-mm papillary carcinoma, Hashimoto’s thyroiditis, and focal C-cell hyperplasia 12 × 5-mm MTC at age 63 MTC at left lobectomy, age 54; metasteses at age 55 at total thyroidectomy At age 31: Right lobe: Hashimoto’s thyroiditis; left lobe: 6-mm MTC and Hashimoto’s thyroiditis At age 32: metastatic MTC at node resection Age 39: 3-mm MTC 2-mm MTC at age 43
No No
MTC, 1 small focus only, at age 33 Age 39: C-cell hyperplasia
Nodule
Pathology
*All assays were performed by radioammunoassay in various laboratories in the US and Canada, with slight variability in the normal ranges for each particular laboratory. Generally, < 20 pg/mL basal calcitonin is generally considered normal.
84, and II-5, who died at age 86, were gene carriers, although neither had thyroid surgery. Both had affected offspring with the V804M mutation and MTC. Since the likelihood that each of their affected offspring carried V804M as a result of a new mutation, one can assume that each was a carrier of this mutation. Similarly, I-2, who died at age 85, was likely to have also carried this same mutation, since her brother’s granddaughter is a known mutation carrier (data not shown in pedigree). All affected members tested in this family have had normal
serum calciums, with negative catecholamine studies in 8 of 8 carriers tested (ages 32-77). We have collectively analyzed 65 patients (130 chromosomes) with apparently sporadic MTC for exon 14 mutations and have not identified additional patients with mutations in this codon. CONCLUSIONS The families reported here are interesting in several ways. While FMTC is known to be variable in age of onset, this mutation appears to cause a much
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wider age range than do most other RET mutations.13,16-19 Although DNA diagnosis of gene carriers and subsequent genetic counseling of the results to patients was relatively straightforward, individual decisions regarding the option of a prophylactic thyroidectomy were more difficult. The presence of active MTC, including metastases in young members(32 and 30 years old) of each family (family 1, IV5 and family 2, IV-2), suggests that surgery be done at an early age in all known mutation carriers. However, the apparent absence of MTC in older gene carriers (family 1, II-2 and II-5) caused one known mutation carrier (family 1, III-7) to be reluctant to consider surgery despite genetic counseling and even though all diagnosed gene carriers who underwent prophylactic thyroidectomies had either C-cell hyperplasia (family 1, IV-4, IV-6, IV-7, IV-8, and IV-10; family 2, IV16) or MTC (family 1, III-2, III-3, III-6, IV-3 and IV-5; family 2, II-4, III-2, III-4, III-12, IV-2, IV-10, IV-12, and IV-13). In our opinion, a less desirable but alternative option is to follow known mutation carriers carefully with stimulated calcitonin testing and operate when levels become elevated. The clinical findings in these families are similar to those observed by Fattoruso et al,17 who noted that subsequent generations seemed to show an earlier and more aggressive phenotype. Similarly, Halling et al25 recently reported a family with a codon 609 RET mutation in which a later age of onset was noted in many individuals. The presence of papillary thyroid cancer (PTC) as seen in these 2 families (Tables I, II) has infrequently been reported in patients with a germline point mutation in the RET proto-oncogene.26 The papillary carcinomas in these patients were occult, and no further treatments were necessary. A somatic rearranged form of the RET proto-oncogene (termed RET/PTC oncogene) has been detected specifically in a minority of papillary thyroid carcinomas,27 including some with karyotypic abnormalities.28 The possible increased frequency of PTC in addition to MTC in these families may be coincidental or may be causally related to the specific codon 804 mutation. Further studies are indicated in ascertaining the role of RET germline mutations versus rearrangements in PTC. These families demonstrate that the search for RET mutations should not be confined to exons 10 and 11, even though most mutations are present in those 2 exons. As additional mutations in RET are identified, it is important to study those patients in families in which a mutation has not previously been identified. In addition, discussion of the genotype-phenotype correlation must be specific not only for the mutation identified, but must also take into account the relationship between the spe-
cific mutation and the phenotype within a particular family. For example, studies of oncogenic activation of RET by Pasini et al29 have demonstrated that the E768D and V804L mutations found in the context of FMTC appear to be less activating than MEN 2A or MEN 2B mutations. Even so, it is clear that codon 804 mutations can predispose a person to MTC at a young age. Thus, we are continuing to recommend prophylactic thyroidectomies in childhood in all patients with identified RET mutations associated with the clinical phenotype consistent with MEN 2A, MEN 2B, or FMTC. We thank Karen Dziubek and Javed Siddiqui in the Henry Ford Hospital DNA Diagnostic Laboratory and the Molecular Diagnostic Laboratory staff at London Health Sciences Centre for their technical assistance. We thank the following physicians for information on various family members: Dr Michael R. Aulicino (Department of Pathology, Sinai Hospital), Dr Maurice Budow (Ann Arbor, Mich), Dr Harold C Yang and Dr James M. Hammond (Department of Surgery and Medicine, Pennsylvania State University), Dr Charles Taylor, (Detroit, Mich), Dr Benjamin Parish (Mechanicsburg, Pa), and Dr Norman W. Thompson (Ann Arbor, Mich). REFERENCES 1. Farndon JR, Leight GS, Dilley WG, Baylin SB, Smallridge RC, Harrison TS, et al. Familial medullary thyroid cancer without associated endocrinopathies: a distinct clinical entity. Br J Surg 1986;73:278-81. 2. Jackson CE, Norum RA. Genetic mechanisms of neoplasia in MEN 2. Henry Ford Hosp Med J 1989;37:116-9. 3. Sipple JH. The association of pheochromocytoma with cancer of the thyroid gland. Am J Med 1961;31:163-6. 4. Steiner AL, Goodman AD, Powers SR. Study of a kindred with pheochromocytoma, medullary thyroid carcinoma, hyperparathyroidism and Cushing’s disease: multiple endocrine neoplasia syndrome, type 2. Medicine 1968;47:371-409. 5. Carney JA, Sizemore GW, Lovestedt SA. Mucosal ganglioneuromatosis, medullary thyroid cancer, and pheochromocytoma: multiple endocrine neoplasia, type 2b. Oral Surg Oral Med Oral Path 1976;41:739-52. 6. Schimke RN, Hartmann WH, Prout TE, Rimoin DL. Syndrome of bilateral pheochromocytoma, medullary thyroid cancer, and multiple neuromas: a possible regulatory defect in the differentiation of chromaffin tissue. N Engl J Med 1968;279:1-7. 7. Mulligan LM, Kwok JBJ, Healey CS, Elsdon MJ, Eng C, Gardner E, et al. Germ-line mutations of the RET protooncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458-60. 8. Donis-Keller H, Dou S, Chi D, Carlson TM, 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. 9. 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. JAMA 1996;276:1575-79.
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10. Smith DP, Houghton C, Ponder BAJ. Germline mutation of RET codon 883 in two cases of de novo MEN 2b. Oncogene 1997;15:1213-7. 11. Gimm O, Marsh DJ, Andrew SD, Frilling A, Dahia PLM, Mulligan LM, et al. Germline dinucleotide mutation in codon 883 of the RET proto-oncogene in multiple endocrine neoplasia type 2B without codon 918 mutation. J Clin Endocrinol Metab 1997;82:3902-4. 12. 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. 13. 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. 14. Boccia LM, Green JS, Joyce C, Eng C, Taylor SAM, Mulligan LM. Mutation of RET codon 768 is associated with the FMTC phenotype. Clin Genet 1997;51:81-5. 15. Berndt I, Reuter M, Saller B, Frank-Raue K, Groth P, Grubendorf M, et al. A new hot spot for mutations in the Ret proto-oncogenes causing familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2A. J Clin Endocrinol Metab 1998;770-4. 16. Fink M, Weinhäusel A, Niederle B, Haas OA. Distinction between sporadic and hereditary medullary thyroid carcinoma (MTC) by mutation analysis of the RET proto-oncogene. Int J Cancer (Pred Oncol) 1996;69:312-6. 17. Fattoruso O, Quadro L, Libroia A, Verga U, Lupoli G, Cascone E, et al. A GTG to ATG novel point mutation at codon 804 in exon 14 of the RET proto-oncogene in two families affected by familial medullary thyroid carcinoma. Hum Mut 1998;S167-71. 18. Shannon KE, Gimm O, Hinze R, Dralle H, Eng C. Germline V804M mutation in the RET proto-oncogene in two apparently sporadic cases of MTC presenting in the seventh decade of life. J Endocrine Genetics 1999;1:39-45. 19. Nilsson O, Tisell LE, Jansson S, Ahlman H, Gimm O, Eng C. Adrenal and extra-adrenal pheochromocytomas in a family with germline RET V804L mutation. JAMA 1999; 1587-8.
Surgery July 2000 20. Hofstra RMW, Fattoruso O, Quadro L, Wu Y, Libroia A, Verga U, et al. A novel point mutation in the intracellular domain of the RET proto-oncogene in a family with medullary thyroid carcinoma. J Clin Endocrinol Metab 1997;82:4176-8. 21. Pigny P, Bauters C, Wemeau J-L, Houcke ML, Crepin M, Caron P, et al. A novel 9-base pair duplication in RET exon 8 in familial medullary thyroid carcinoma. J Clin Endocrin Metab 1999;84:1700-4. 22. Kambouris M, Jackson CE, Feldman GL. Diagnosis of multiple endocrine neoplasia (MEN) 2A, 2B and familial medullary thyroid cancer (FMTC) by multiplex PCR and heteroduplex analyses of RET proto-oncogene mutations. Hum Mutat 1996;8:64-70. 23. Cecherini 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. 24. Jackson CE. A 50-year perspective on endocrine neoplasia: clinic to genes and back. J Intern Med 1998;243:419-23. 25. Halling KC, Bufill JA, Cotter M, Artz SA, Carpenter AB, Schaid D, et al. Age-related disease penetrance in a large medullary thyroid cancer family with a codon 609 RET gene mutation. Molec Diag 1997;2:277-86. 26. Komminoth P. The RET proto-oncogene in medullary and papillary carcinoma: molecular features, pathophysiology, and clinical implications. Virchows Arch 1997;431:1-9. 27. Grieco M, Santoro M, Berlingieri M, Melillo RM, Donghi R, Bongarzone I, et al. PTC is a rearranged form of the RET proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell 1990;60:557-63. 28. Smanik PA, Furminger TL, Mazzaferri EL, Jhiang SM. Breakpoint characterization of the RET/PTC oncogene in human papillary thyroid carcinoma. Hum Mol Genet 1995;4:2313-8. 29. Pasini A, Geneste O, Legrand P, Schlumberger M, Rossel M, Fournier L, et al. Oncogenic activation of RET by two distinct FMTC mutations affecting the tyrosine kinase domain. Oncogene 1997;15:393-402.