Germline and Somatic Mutations in an Oncogene: RET Mutations in Inherited Medullary Thyroid Carcinoma1

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[CANCER RESEARCH 56. 1241-1243. March 15. I996|

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Debbie J. Marsh, Scott D. Andrew, Charis Eng, Diana L. Learoyd, Amanda G. Capes, Ruth Pojer, Ann-Louise Richardson, Carol Houghton, Lois M. Mulligan, Bruce A. J. Ponder, and Bruce G. Robinson2

Koi/ing Institute of Medical Research Â¡D.J. M.. S. D. A.. D. L L, A. C. C. R. P.. A-L R.. B. G. R.Â¡and Department of Endocrinology [D. L. L. B. G. R.Â¡,Royal North Shore Hospital, St Leonards. N.S.W. 2065, Australia; University of Sydney, Sydney, N.S.W., Australia Â¡D.J. M.. D. L. L, A. G. C., B. G. R.Â¡:Department of Medicine, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts [C. E.I; Cancer Research Campaign Human Cancer Genetics Research Group, University of Cambridge, Addi'nbrooke's Hospital, Bo\ 23X, Cambridge CB2 2QQ, England 1C. Â£., C. H., B. A, J. P.]; and Departments of Paediatrics and Pathologv, Queen's University, Kingston, Ontario. K7L 3N6 Canada Â¡L.M. M.Â¡

Abstract 918 mutation leads to altered substrate specificity (6, 9, 14). Both mutations increase receptor tyrosine kinase activity (6, 9). Inherited cancer syndromes predispose an individual to the develop Inherited cancer syndromes are generally caused by germline mu ment of specific tumors. Somatic and germline mutations in the same tumor suppressor gene, as described in Knudson's two-mutation model, tations in tumor suppressor genes (15). Activating mutations in the are well recognized. Inherited mutations in the RET proto-oncogene, RET proto-oncogene in the germline of MEN 2 and FMTC individuals represent an exception to this rule. According to Knudson's two- which encodes a receptor tyrosine kinase, predispose individuals to the multiple endocrine neoplasia type 2 (MEN 2) cancer syndromes. The mutation model originally invoked for tumor suppressor genes (16), a major component tumor of these syndromes is medullary thyroid carci primary mutation in a tumor suppressor gene occurs in the germline, noma (MTC). To date, somatic mutations in RET have not been identified with a secondary mutation occurring in somatic tissue within the same in tumors from individuals with MEN 2, although they have been well gene on the other alÃeleleading to tumorigenesis (17, 18). It has been documented in sporadic MEN 2-related tumors. We have identified, thought that this double mutation is not necessary in the case of among 16 MEN 2 cases with well-defined RET germline mutations, a oncogenes since a single activating mutation in RET has been shown somatic missense mutation at codon 918 of RET in 3 of 15 MTCs and in a sample with hyperplastic C-cells (the presumed precursor to hereditary to transform cells (6, 9). However, although the RET germline muta MTC). We suggest that the presence of a somatic mutation, in addition to tion is obviously present from birth in an affected individual, the the preexisting germline mutation in hereditary MTCs, may contribute to factors that determine how CCH, the presumed precursor to hereditary tumorigenesis in vivo. MTC (19, 20), or MTC develop in C-cells are yet to be elucidated. We hypothesized that the presence of a somatic mutation, in addition to Introduction the preexisting germline mutation in hereditary MTCs, may contribute MTC1 is a malignancy of the thyroid C-cells and is the character to tumorigenesis in vivo. istic component tumor of the inherited cancer syndromes MEN types 2A and 2B and FMTC. Germline mutations in the RET proto-onco Materials and Methods gene, which encodes a receptor tyrosine kinase, cause MEN 2A, MEN Patients. Thyroid tissue, from 16 members of MEN 2A or FMTC families, 2B, and FMTC (1-9). The majority of germline mutations that have in which germline RET mutations had been identified previously (21-23), was been found in MEN 2A and FMTC families lie within exons 10 and studied. Patient 3. aged 11 years, was identified as having a high risk of 11 of RET at codons 609, 611,618, 620, and 634 and result in the inheriting the MEN 2A phenotype by linkage analysis, prior to the advent of substitution of a cysteine by an alternative amino acid (1, 2). A single direct mutation detection, and thyroidectomy was performed (24). CCH was mutation at codon 918 causing the substitution of a methionine (ATG) diagnosed in the thyroid specimen from this patient. All other patients were with a threonine (ACG) is associated with MEN 2B and sporadic adult and had MTCs that varied in extent from 8 mm (patient 1) to widespread MTC (3-5, 10-12). Somatic mutations in RET codons 768 and 883 metastatic disease in the remaining patients, with the exception of patients have also been described in sporadic MTC at very low frequencies 6 and 16. (7, 8, 13). Studies using transient transfection and other methods have Mutation Detection. DNA was extracted from 5 paraffin-embedded and shown that RET codon 634 missense mutations result in increased 11 frozen thyroid tissue specimens by methods described previously (25, receptor dimerization independent of ligand binding, and the codon 26). Sequencing of PCR products was performed using the Cyclist DNA Sequencing Kit (Stratagene, La Jolla, CA). Exon 16 was amplified using PCR and the primers CRT5G and CRT5H (3, 27). PCR products from this Received 12/13/95; accepted 1/31/96. primary amplification were used as templates in a secondary PCR, which The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with used a modified forward primer, to generate fragments specifically de 18 U.S.C. Section 1734 solely to indicate this fact. signed to be cleaved by the restriction endonuclease Rsal (New England 1This work has been supported by a number of grants and scholarships: Mary Jo Reeve Biolabs, Inc., Beverly, MA) only in the presence of the codon 918ATG â€”¿Â» Fellowship (to D. L. L.): National Health and Medical Research Council biomedicai scholarship (to A. G. C.); core and program grants from the Cancer Research Campaign ACG mutation (28). Ten MTCs, whose codon 918 mutation status has been (CRC), CRC Dana-Farber Cancer Institute Fellowship. Lawrence and Susan Marx Inves- reported previously as negative (patients 6-8 and 10-16, Table 1; Ref. 12), tigatorship in Cancer Genetics in the Division of Cancer Epidemiology and Control, were also re-analyzed with this sensitive primer-mediated technique. Sam Dana-Farber Cancer Institute, and Young Scientist Award from the Markey Charitable Trust (to C. E.); Medical Research Council of Canada (to L. M. M.); and the CRC Gibb ples were screened for mutations at codons 768 and 883 (7, 8, 13) by PCR Life Fellowship (to B. A. J. P.). amplification with CRT4E and CRT4F (exon 13), and CRT17B and 2 To whom requests for reprints should be addressed, at Kolling Institute of Medical CRT17G or CRT17S and CRT17A (exon 15), respectively (27), and Research, Royal North Shore Hospital, St. Leonards, N.S.W. 2065. Australia. Phone: +61 digestion with the restriction endonuclease Alul (New England Biolabs, 2 9926 7267: Fax: +612 9926 8523. The abbreviations used are: MTC, medullary thyroid carcinoma; MEN, multiple Inc.). The presence of each of the mutations causes loss of an Alul site. All endocrine neoplasia; FMTC. familial MTC; CCH. C-cell hyperplasia. results were independently replicated in separate laboratories. 1241

Table l Germline and somatic RET mutations in MTCs of MEN 2A and FMTC origin codon 918 mutation (ATG^ACG) in exon 16 of RET. A codon 918 of somatic mutation was present in 3 of 15 MTCs and in the sample with CCH codon 918 mutation (Fig. 1; Table 1). Screening of germline DNA samples for the codon in hereditary 918 mutation, both by sequence analysis and primer-mediated muta Patienl1"23"45"h789IdII1213141516PhenotypeFMTCFMTCMENmutation618TGC->CGC620TGC-CGC634TGC->TCC634TGC-<:GC6I8TGC->CGC634TGCâ€”TTC618TGC-X3GC620TGC^CGC634TGC-KTGC634TGC-<:GC634TGC-CGC634TGC-^CGC634TGC-CGC634TGC-Â»CGC634TGC-CGC634TGC-Â»TACPresenceMTCPositivePositivePositivePositiveNegativeNegativeNegativNegativNegativNegativNegativNegativNegativNegativeNegativeNegative tion detection, was negative in all patients studied (data not shown). We also examined our tumors for the presence of two other somatic 2AMEN mutations, at codons 768 (exon 13) and 883 (exon 15), reported 2AFMTCFMTCMEN2AMEN previously to be associated with sporadic MTC and/or FMTC (7, 8, 13). Neither mutation was detected in our tumor samples (data 2AMEN not shown). 2AMEN 2AMEN Discussion 2AMEN2AMEN2AMEN2AMEN This is the first report of both germline and somatic mutations in a single dominantly acting oncogene occurring in the same tumor. 2AMEN Furthermore, the presence of a codon 918 somatic mutation in hyper- 2AGermline " Individuals from the same FMTC family. plastic C-cells suggests that in this sample, the second genetic event * CCH only. occurred early in tumorigenesis. By analogy to other receptor tyrosine kinases, wild-type RET should require ligand binding for dimeri/.ution and subsequent acti Results vation, leading to autophosphorylation and phosphorylation of spe Fifteen MEN 2 MTCs and a single thyroid sample containing cific downstream targets (29). Transient transfection experiments hyperplaslie C-eells were analyzed for the presence of the somatic have shown that MEN2A mutations at cysteine codon 634 increase

MTC DNA germline DNA

T A A C G G TC 918 A G G A A

Fig. l. Detection of the somatic codon 918ATG-Â»ACG mutation (patients I-5 and 9) and the germline codon ()2() mutation (patient 2). A, sequence analysis of MTC DNA (exon I6. codon 9l8ATG-Â»ACG> and gcrmlinc DNA (exon IO. codon 620TGCâ€”CGCl from palien! 2. ÃŸ,de 8 tection of (he codon 9I8ATGâ€”*AC(i rnulalion hy primer- mediated nuclcotidc substitution and /Ã.svÃldigestion.Ijine I, negative control for codon 918 mutation (wild-type germline); lolite 2. positive control for codon 9I8 mutation (germline MKN 2B|; Â¡Mie.*. patient I (MTC); Lane 4. patient 2 (MTC'); Urne 5. patient 3 (CCH); Lane f>.patient 4 (MTC); Lane 7, palien! 5 (MTC); Lane X. patient 9 (MTC).

140 bp

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1996 American Association for Cancer Research. OERMLINE AND SOMATIC MUTATIONS IN RET autophosphorylation and cause covalent dimerization of the RET Fusco, A.. Vecchio. G.. Matoskova, B., Kraus, M. H., and di Fiore, P. P. Activation of RET as a dominant transforming gene by germline mutations of MEN 2A and MEN receptor in the absence of ligand (6, 9). The ability of the codon 918 2B. Science (Washington DC). 267: 381-383, 1995. mutation to cause changes in RET receptor autophosphorylation and 10. Zedenius. J.. Wallin, G.. Hamberger. B., Nordenskjold, M.. Weber. G.. and Larsson, substrate specificity in the absence of covalent dimerization of the C. Somatic and MEN 2A de novo mutations identified in the RET proto-oncogene by screening of sporadic MTCs. Hum. Mol. Genet., 3: 1259-1262, 1994. RET receptor has also been demonstrated, although the level of 11. Blaugrund. J. E., Johns, M. M., Eby, Y. J., Ball, D. W.. Baylin, S. B., Hruban, R. H., phosphorylation of the mutant codon 918 monomer is less than that of and Sidransky, D. RET proto-oncogene mutations in inherited and sporadic medullary the mutant MEN 2A dimer (9, 14). A level of receptor activation thyroid cancer. Hum. Mol. Genet.. 3: 1895-1897, 1994. 12. Eng, C., Mulligan, L. M.. Smith. D. P., Healey, C. S.. Frilling, A.. Raue, F., Neumann. surpassing that achieved in the presence of either mutation alone may H. P. H.. Pfragner, R., Behmel, A., Lorenzo, M. J.. Stonehouse, T. J., Ponder, M. A., occur as a consequence of the combination of a dimer-inducing and Ponder, B. A. J. Mutation of the RET proto-oncogene in sporadic medullary thyroid carcinoma. Genes Chromosomes & Cancer. 12: 209-212. 1995. MEN2A mutation and a syntenic codon 918 mutation. Alternatively, if 13. Dou, C., Chi, D., Carlson. K. M., Moley. J. A., Wells, S. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1996 American Association for Cancer Research. Germline and Somatic Mutations in an Oncogene: RET Mutations in Inherited Medullary Thyroid Carcinoma

Debbie J. Marsh, Scott D. Andrew, Charis Eng, et al.

Cancer Res 1996;56:1241-1243.

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