[CANCER RESEARCH 56. 1241-1243. March 15. I996| Advances in Brief Germline and Somatic Mutations in an Oncogene: RET Mutations in Inherited Medullary Thyroid Carcinoma1 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 ¡D.J. M.. S. D. A.. D. L L, A. C. C. R. P.. A-L R.. B. G. R.¡and Department of Endocrinology [D. L. L. B. G. R.¡,Royal North Shore Hospital, St Leonards. N.S.W. 2065, Australia; University of Sydney, Sydney, N.S.W., Australia ¡D.J. M.. D. L. L, A. G. C., B. G. R.¡: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. £., C. H., B. A, J. P.]; and Departments of Paediatrics and Pathologv, Queen's University, Kingston, Ontario. K7L 3N6 Canada ¡L.M. M.¡ Abstract Inherited cancer syndromes predispose an individual to the develop ment of specific tumors. Somatic and germline mutations in the same tumor suppressor gene, as described in Knudson's two-mutation model, are well recognized. Inherited mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, predispose individuals to the multiple endocrine neoplasia type 2 (MEN 2) cancer syndromes. The major component tumor of these syndromes is medullary thyroid carci noma (MTC). To date, somatic mutations in RET have not been identified in tumors from individuals with MEN 2, although they have been well documented in sporadic MEN 2-related tumors. We have identified, among 16 MEN 2 cases with well-defined RET germline mutations, a 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 MTC). We suggest that the presence of a somatic mutation, in addition to the preexisting germline mutation in hereditary MTCs, may contribute to tumorigenesis in vivo. Introduction MTC1 is a malignancy of the thyroid C-cells and is the character istic component tumor of the inherited cancer syndromes MEN types 2A and 2B and FMTC. Germline mutations in the RET proto-onco gene, which encodes a receptor tyrosine kinase, cause MEN 2A, MEN 2B, and FMTC (1-9). The majority of germline mutations that have been found in MEN 2A and FMTC families lie within exons 10 and 11 of RET at codons 609, 611,618, 620, and 634 and result in the substitution of a cysteine by an alternative amino acid (1, 2). A single mutation at codon 918 causing the substitution of a methionine (ATG) with a threonine (ACG) is associated with MEN 2B and sporadic MTC (3-5, 10-12). Somatic mutations in RET codons 768 and 883 have also been described in sporadic MTC at very low frequencies (7, 8, 13). Studies using transient transfection and other methods have shown that RET codon 634 missense mutations result in increased receptor dimerization independent of ligand binding, and the codon Received 12/13/95; accepted 1/31/96. 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 18 U.S.C. Section 1734 solely to indicate this fact. 1This work has been supported by a number of grants and scholarships: Mary Jo Reeve 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 (CRC), CRC Dana-Farber Cancer Institute Fellowship. Lawrence and Susan Marx Inves- tigatorship in Cancer Genetics in the Division of Cancer Epidemiology and Control, 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 Life Fellowship (to B. A. J. P.). 2 To whom requests for reprints should be addressed, at Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, N.S.W. 2065. Australia. Phone: +61 2 9926 7267: Fax: +612 9926 8523. The abbreviations used are: MTC, medullary thyroid carcinoma; MEN, multiple endocrine neoplasia; FMTC. familial MTC; CCH. C-cell hyperplasia. 918 mutation leads to altered substrate specificity (6, 9, 14). Both mutations increase receptor tyrosine kinase activity (6, 9). Inherited cancer syndromes are generally caused by germline mu tations in tumor suppressor genes (15). Activating mutations in the RET proto-oncogene in the germline of MEN 2 and FMTC individuals represent an exception to this rule. According to Knudson's two- mutation model originally invoked for tumor suppressor genes (16), a primary mutation in a tumor suppressor gene occurs in the germline, with a secondary mutation occurring in somatic tissue within the same gene on the other alÃ-eleleading to tumorigenesis (17, 18). It has been thought that this double mutation is not necessary in the case of oncogenes since a single activating mutation in RET has been shown to transform cells (6, 9). However, although the RET germline muta tion is obviously present from birth in an affected individual, the factors that determine how CCH, the presumed precursor to hereditary 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 the preexisting germline mutation in hereditary MTCs, may contribute to tumorigenesis in vivo. Materials and Methods Patients. Thyroid tissue, from 16 members of MEN 2A or FMTC families, in which germline RET mutations had been identified previously (21-23), was studied. Patient 3. aged 11 years, was identified as having a high risk of inheriting the MEN 2A phenotype by linkage analysis, prior to the advent of direct mutation detection, and thyroidectomy was performed (24). CCH was diagnosed in the thyroid specimen from this patient. All other patients were adult and had MTCs that varied in extent from 8 mm (patient 1) to widespread metastatic disease in the remaining patients, with the exception of patients 6 and 16. Mutation Detection. DNA was extracted from 5 paraffin-embedded and 11 frozen thyroid tissue specimens by methods described previously (25, 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 primary amplification were used as templates in a secondary PCR, which used a modified forward primer, to generate fragments specifically de signed to be cleaved by the restriction endonuclease Rsal (New England Biolabs, Inc., Beverly, MA) only in the presence of the codon 918ATG —¿Â» ACG mutation (28). Ten MTCs, whose codon 918 mutation status has been reported previously as negative (patients 6-8 and 10-16, Table 1; Ref. 12), were also re-analyzed with this sensitive primer-mediated technique. Sam ples were screened for mutations at codons 768 and 883 (7, 8, 13) by PCR amplification with CRT4E and CRT4F (exon 13), and CRT17B and CRT17G or CRT17S and CRT17A (exon 15), respectively (27), and digestion with the restriction endonuclease Alul (New England Biolabs, Inc.). The presence of each of the mutations causes loss of an Alul site. All results were independently replicated in separate laboratories. 1241 Research. on October 17, 2015. © 1996 American Association for Cancer cancerres.aacrjournals.org Downloaded from