[CANCER RESEARCH 56. 2167-2170. May 1. 1996] Heterogeneous Mutation of the RET Proto-oncogene Medullary Thyroid Carcinoma1 in Subpopulations of Charis Eng,2 Lois M. Mulligan, Catherine S. Healey, Carol Houghton, Andrea Frilling, Friedhelm Raue, Gerry A. Thomas, and Bruce A. J. Ponder Cancer Research Campaign Human Cancer Genetics Research Group ¡C.£.. C. S. H., C. H., B. A. J. P] anil Department of Histopathology ¡G.A. TJ, University of Cambridge. Addenbrooke's Hospital. Hills Road. Cambridge CB2 2QQ. United Kingdom; Division of Cancer Epidemiólogo and Control, Dana-Farber Cancer Institute. Department of Medicine. Han'anl Medical School. Boston. Massachusetts 02115-6084 [C.E.]; Departments of Pathology and Paediatrics, Queen's University. 20 Barrie Street, Kingston. Ontario K7L 3N6. Canada ¡LM. M.j; Universitals-Krankenhaus, Chirurgische Klinik. Universität Hamburg. Martinstrasse Medizinische Klinik und Poliklinik. Abteilung Innere Medizin l, Ruprecht-Karls-Universität. 69115 Heidelberg. Germanv ¡F.R.} ABSTRACT Mutations in the RET proto-oncogene are associated with the patho- genesis of medullary thyroid carcinoma (MTC). In an attempt to under stand this process, we examined microdissected subpopulations from MTC and multiple métastasesfrom these tumors. Approximately 80% of sporadic MTCs had at least one subpopulation with the RET codon 918 mutation, which is a mutation previously detected in sporadic MTC as a somatic mutation and in multiple endocrine neoplasia type 2B as a germline mutation. However, the distribution of this mutation was nonhomogeneous, occurring only in subpopulations in most tumors and among subsets of multiple métastases,thus implying that although the codon 918 mutation could be an early event, it is not necessarily an early or essential event in tumorigenesis. This heterogeneity suggests either that the codon 918 mutation can arise as an event in progression within a metastatic clone or within a single tumor, or that MTC can be of polyclonal origin. Of significance, one of two multiple endocrine neoplasia type 2A MTCs carried a somatic mutation at codon 918, in addition to the RET mutation present in the germline. We found no correlation between the presence of other somatic genetic events, such as loss of heterozygosity on chromo some arms Ip and 22q, and RET mutation status in the various subpopu lations of MTC. MTC,3 a neoplasm of the calcitonin-secreting thyroid C cells, may occur sporadically or as a component of the inherited cancer syn drome MEN 2(1). The majority of all three clinical subtypes of the MEN 2 syndromes are caused by germline mutations in the RET proto-oncogene (2), which encodes a receptor tyrosine kinase ex pressed predominantly in tissues and tumors of neural crest origin (3, 4). MEN 2A, which comprises MTC, pheochromocytoma, and para thyroid hyperplasia, is a consequence of a germline missense mutation in one of five cysteine codons in the cysteine-rich extracellular do main of RET (5-9). FMTC, characterized by the presence of MTC as the only phenotype in the family, is associated with mutations similar to those in MEN 2A and, rarely, with a missense mutation in codon 768 or 804 in the tyrosine kinase domain (5-7, 10, 11). MEN 2B, which is similar to MEN 2A, except for the presence of developmental abnormalities and a typical habitus, is caused by a germline mutation in codon 918, which lies in the substrate specificity pocket of the RET tyrosine kinase catalytic core (9, 12-15). Received 12/19/95; accepted 3/4/96. 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. 1 This work was supported by the Cancer Research Campaign [CRC] Dana-Farber Cancer Institute Fellowship, the Lawrence and Susan Marx Investigatorship. the Markey Charitable Trust, and the Charles A. Dana Foundation (C. E.); core and programme grants from the CRC and the CRC Gibb Fellowship (B. A. J. P.); and a Medical Research Council of Canada Operating Grant and the Kingston General Hospital Foundation (L. M. M.). 2 To whom requests for reprints should be addressed, at D920C. Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115-6084. 1 The abbreviations used are: MTC, medullary thyroid carcinoma, MEN 2. multiple 20. German\ ¡A.F.]; and Like that of other tumors, the pathogenesis of MTC probably involves the accumulation of a somatic loss of tumor suppressor function, as manifested most frequently by LOH on chromosome arms Ip and 22q (16), and/or activation of proto-oncogenes (reviewed by Eng and Ponder; Ref. 17). Widely varying frequencies (between 23 and 66% in series with 10 or more) of sporadically occurring MTCs have been reported to have a somatic RET mutation in the MEN 2B-specific codon 918 (12, 13. 18-20), an alteration the has been hypothesized to be the initiating or one of the early events in the tumorigenesis of sporadic MTC. In contrast, two other somatic mu tations, at codons 768 (exon 13) and 883 (exon 15), are relatively rare, detected in <10% of sporadic MTCs (10, 19, 20).4 We examined the RET mutation status in MTC métastasesand subpopulations within individual tumors from sporadic and MEN 2 cases. The aims of the study were to determine whether MTCs are monoclonal with respect to RET codon 918 mutation status, and whether the patterns of other genetic events allow these to be assigned to a sequence in tumor progression. MATERIALS MTC Tumors. INTRODUCTION 52. 2000 Hamburg AND METHODS Eighty-seven MTC blocks from 33 patients, 82 from 28 sporadic cases. 2 from 2 MEN 2A cases, and 3 from 3 MEN 2B cases, were analyzed. All were obtained as paraffin-embedded tissue. MTC was considered sporadic if the patient did not have multiple primary tumors, and there was no history of a first- or second-degree relative with MTC or pheochromocytoma. Information regarding the presence of C-cell hyper plasia (a relative indicator of hereditary disease) in the thyroidectomy speci mens is incomplete and. therefore, has been omitted for clarity. The MEN 2 cases belong to families in which the diagnosis had been made based on pathology of thyroid and adrenal tumors and the presence of a germline mutation in either a MEN 2A- or MEN 2B-specific RET codon. Isolation of Genomic DNA. DNA was extracted from archival tissue as described (21. 22). After examination of a H&E-stained section, serial sections of paraffin-embedded tissue were initially divided into normal tissue and MTC. The MTC was then microdissected into further sections based on division by connective tissue septae. When septae were not present, which was rare, the MTC was randomly divided. PCR Amplification and Mutation Detection. An initial genomic amplicon encompassing exon 16 of the RET proto-oncogene was created by the PCR and the primer pair CRT 5G and CRT 5H or fRET 16 and rRET 16 (12, 13). Ten to 50 ng genomic DNA were amplified using the conditions described previously (12, 13) or according to the recommendation of the manufacturer (Red Hot Thermits icelanclictis DNA polymerase; Advanced Biotechnologies. Surrey. United Kingdom). No more than 25-30 cycles of amplification were carried out. A secondary amplification was performed with the primers t'16Rsa (22) and either CRT 5H or rRET 16 under similar conditions, except for an annealing temperature of 60°C.Similarly, amplicons encompassing AfTexons 13 and 15 were created with the primer pairs CRT 4E and CRT 4F and CRT 17S and CRT 17A, respectively (10, 23). under conditions identical to those described above, except for an annealing temperature of 55°Cfor the exon 13 primers. PCR products were purified through low-melting-point 4C. Eng el al., unpublished observations. endocrine neoplasia type 2. FMTC, familial MTC; LOH, loss of heterozygosity. 2167 Downloaded from cancerres.aacrjournals.org on February 20, 2016. © 1996 American Association for Cancer Research. agarose and RET MUTATION IN MTC SURPOPULATIONS M12345678910 eluted using the Wizard PCR preparation kit (Promega. Southampton. United Kingdom). The final exon 16 PCR products were subject to digestion with Rsa\ and fractionated on 3% agarose gels. Digestion with A.val denotes the presence of the codon 918 ATG—»ACGmissense mutation (22). Lack of digestion denotes the presence of the wild-type sequence. This technique has been validated previously against samples with known sequence data (22).4 The molecular Fig. 1. Ethidium bromide-stained 3^ agarose gel of Alni digestions of exon 15 amplicons of DNA from three MTC métastasesfrom a single individual. The presence of the codon 883 mutation causes loss of the A/uI restriction site. M. A l(X)-hp ladder marker: Lunes ÃŒ and 2. one metastasis: Lanes .f-5. second metastasis: Limes 6-8. third metastasis: Lanes 9 and 10. positive (presence of codon 883 mutation) and negative (wild-type) controls, respectively. sensitivity of this technique is such that the codon 918 mutation can be detected at a minimal dilution of I:2(X) (data not shown). As a control, these products were also subject to Sau3A\ digestion, and all were digested with this enzyme. This site is ubiquitously present within exon 16 and is not dependent on the presence or absence of the codon 918 mutation. The exon 13 and 15 amplicons were digested with Alu\. The absence of the site denotes the presence of the codon 768 and codon 883 mutations, respec tively. No subpopulation had a codon 768 mutation. I.OH Analysis. LOH was assessed using the markers DIS211 and D1S233. the markers D22S272 and D22S284. which are in proximity to the NF2 locus, as described previously (16). and D10S141 and ZNF22. markers Hanking RET (24-26). RESULTS RET Mutations in Subpopulations of MTC. To determine whether somatic RET mutations occurred in subpopulations of MTC, we carried out analyses in separate metastatic clones and in dissected subpopulations from individual tumors. Eighty-two MTC blocks from 28 patients with sporadic disease could be analyzed for the presence or absence of the somatic RET codon 918 ATG—»ACG or codon 883 GCT-»TTT mutation. Of these 28 individuals. 6 had a total of 4 primary tumors and 56 métastasesthat were available for analyses (Table 1). Two individuals had 1 primary plus 2 métastaseseach, all of which contained the codon 918 mutation (Table 1, patients A and C). Of the remaining 4 individuals, one did not have the codon 918 mutation in the primary tumor or the metastasis (Table 1. patient B). The remaining 3 individuals each had 8 or more métastases(Table 1, patients D-F). The first had 11 métastases(patient D). Of these 11.6 contained a somatic codon 918 mutation, and 5 did not. Of the 5 without the 918 mutation. 3 had the rare somatic codon 883 mutation (Fig. 1). The second patient had 32 separate métastases,and 11 contained the codon 918 mutation (patient E). The third patient had 1 primary and 8 métastases(patient F). The primary tumor did not have the codon 918 mutation, whereas 2 of these 8 métastasesdid. Well over one-half of all métastasesdid not have the codon 918 or codon 883 mutation. The remaining 22 individuals had single blocks available represent ing primary MTC or did not have metastatic disease. A histológica! section from each block was divided along connective tissue septae or randomly, if none existed; each of these divisions is operationally termed a subpopulation for purposes of this study. Three of the 22 had the codon 918 mutation in all subpopulations. i.e.. the whole tumor: 5 were shown not to have the mutation in any subpopulation: and 14 had mixed subpopulations. In this last category, within a single MTC. certain tumor subpopulations had the codon 918 mutation, whereas others did not (e.g.. Fig. 2, Lanes 14-17). In sum. of 28 patients with sporadic MTC. 80% had at least 1 subpopulation that had a somatic codon 918 mutation. In addition, of the 248 subpopulations from these 28 patients. 38% had the codon 918 mutation, whereas 62% did not. Interestingly, one MTC from 1 of the 2 MEN 2A patients with the germline mutation in codon 634 TGC—»CGCalso had a somatic codon 918 mutation (Fig. 2, Lanes 9-13). As a positive control, the MEN 2B MTCs were shown to have the codon 918 mutation in every subpopulation of MTC and in areas of normal thyroid tissue, as expected (Fig. 2. Lanes 18-24). As negative controls, available nonneoplastic thyroid tissues within some of the sections from non-MEN 2B patients were shown not to have codon 918 mutations in all subpopulations. To determine whether the nonuniform presence of the codon 918 mutation within subpopulations of a single tumor or within some métas tases from a single individual was due to subsequent loss of one or the other RET alÃ-ele.LOH analysis was performed with markers closely flanking the RET locus. No LOH was observed (data not shown). LOH 1p and 22q. LOH at Ip and 22q has been shown to occur with some frequency in both familial and sporadic MTCs (16). Thus, we determined whether the RET mutation status in each MTC subpopulation was related to LOH at these loci. Of the 274 MTC subpopulations that were analyzed for mutation status and LOH. 105 did not have LOH at either Ip or 22q; 26 were uninformative at one or the other locus (i.e., homozygous or PCR failure); and 143 had LOH at either Ip or 22q (Table 2). Within any single tumor, LOH was not clonal, but certain subpopulations within that tumor exhibited loss, whereas others did not (Fig. 3). Two hundred forty-eight tumor subpopulations were informative for codon 918 and codon 883 mu tation status and the presence or absence of LOH of Ip or 22q markers. Overall, there was no significant correlation between the presence or absence of the RET codon 918 mutation and LOH at Ip or 22q markers. The RET codon 918 mutation status and LOH at Ip and 22q were further examined in each of the three individuals with 9 or more métas tases each. For this part of the analysis, each of the métastaseswas divided into subpopulations. The first patient (Table 1, patient F), with 9 métastases,could not be analyzed, because seven of these métastases were uninformative (i.e., homozygous or had PCR failure) at either all Ip or 22q markers or both markers. The second (Table 1. patient D) with 11 Table 1 Number of MTC métastaseswith RET mutation in si.\ individuals No. of métastases" Primary PatientAliCI)EFPrimary Mutation +21)26II2883 MTC111NA''NAIWith mutation1 Mutation +000300918- and 883-0202216 (918 mutation+)01NANA0Total2:211328918 + . positive: —¿negative. . ' NA. primary tumor not available. 2168 Downloaded from cancerres.aacrjournals.org on February 20, 2016. © 1996 American Association for Cancer Research. RET MUTATION Ml 234 5678 910111213 IN MTC SUBPOPULATIONS M141516 17 181920 2122 2324 25 Fig. 2. Ethidium bromide-stained 3% agarose gel after electrophoretic separation of Rial digestions of modified exon loamplicons (see "Materials and Methods"! of subpopulations of sporadic MTC. Digestion occurs only in the presence of the codon 918 mutation and modified primer sequence. M, A 100-bp ladder; Lanes 1-5, MTC subpopulations belonging to a single sporadic case; Lanes 6-8. MTC subpopulations belonging to another sporadic case; Lanes 9-13, single MEN 2A MTC divided into five subpopulations: Lanes 14-17. single sporadic MTC divided imo four subpopulations; Lanes 18-20. 21 and 22, and 23 and 24. three MEN 2B specimens: Lane 18. MEN 2B normal thyroid; Lanes IV and 20, MEN 2B MTC; Lanes 21 and 22, MEN 2B MTC; Lane 23. MEN 2B MTC; Lane 24, MEN 2B germline (acts as positive control also); Lane 25. negative control (wild type). métastases,had 33 subpopulations that were scored for RET codon 918 mutation status and LOH. Of the 33 subpopulations, 11 had the codon 918 mutation, and 22 did not. Of the 11 with a codon 918 mutation. 9 also had LOH at a 22q marker. Of the 22 without the mutation, 11 exhibited LOH at a 22q marker. In contrast, only 4 subpopulations had LOH at a Ip marker, and all 4 did not have a codon 918 mutation. The third individual (Table 1. Patient E) had 64 subpopulations that were scorable for mutation status and LOH. Of the 23 subpopulations that were codon 918 mutation positive, 2 and 6 had LOH at Ip and 22q, respectively. Of the 41 subpopulations without a codon 918 mutation, 8 and 6 had LOH at Ip and 22q, respectively. It does not seem that the codon 918 mutation represents an early event in tumorigenesis in the majority of MTCs examined here, despite the majority of these tumors having at least one tumor cell population with the mutation. If the codon 918 mutation were an early event, then we would expect the majority of subpopulations within individual primary MTCs to have this mutation (27), unless the alÃ-ele bearing the mutation is lost once a selective advantage has been gained, and there was no evidence of this. The observations suggest that codon 918 mutations have arisen during clonal evolution of the tumor: i.e., they arise within an established primary tumor or within a metastatic clone. An alternative, which is less likely in the case of sporadic disease, is that MTC and MTC métastasescould have a DISCUSSION polyclonal origin. Our finding that somatic codon 918 mutations seem to occur frequently and in subsets of cells within sporadic MTC By analyzing subpopulations of MTC, either several métastases suggests that they may not necessarily be associated with a strong from a single individual or different regions within a primary MTC, selective advantage. Otherwise, one might expect that cells with the we have shown that 80% of individuals with sporadic MTC have at mutation would form a larger proportion of the tumor. Indeed, one least one tumor cell subpopulation with a mutation in RET codon 918. individual had a primary tumor without the codon 918 mutation and The nonuniform distribution of codon 918 mutations within MTC subsequently developed eight métastases,two of which contained the may account for the wide range of reported frequencies of codon 918 codon 918 mutation. At least in this case, the codon 918 mutation mutations in MTCs, ranging from a low of 23% to a high of 66% in seemed to develop during clonal evolution during the metastatic series that comprise 10 or more tumors (12, 13, 18-20). Indeed, 38% process. However, in just over one-half of the metastatic subpopulaof our subpopulations had the codon 918 mutation. In light of our tions sampled, there was no codon 918 or codon 883 mutation data, it seems that the detection of codon 918 mutations may depend detected; therefore, there is no strong evidence that these events are on which region of a primary tumor or which metastasis is analyzed. frequent accompaniments of metastatic tumor progression. Conceivably, these observations could be common to other tumors MTCs are commonly slow growing, and it may be that clonal and other genes as well. evolution of the tumor occurs over a protracted time scale. The finding of the codon 918 mutation or codon 883 mutation in different meta subpo¡tulation.sNo Table 2 Somatic RET initiation status and LOU in MTC static deposits in a single individual suggests that either mutation can RET RET 91 8 or 833 of informative play a role in tumor progression and is an unequivocal demonstration Mutation(%)"67(64) Mutation (%)"38 + subpopulations sampled105 of clonal heterogeneity. (36) LOH Ip or 22q The presence of a somatic codon 918 mutation in an MTC from a 46(65) 71 25 (35) LOH Ip MEN 2A patient with a germline mutation at codon 634 is notewor 19(54) 16(46) 37 LOH 22q (59)154(62) 22 22qTotal" LOH Ip and 15(41)94 37248negative.No. thy. In earlier studies, nonmicrodissected MTCs from MEN 2A and FMTC patients were examined for the presence of somatic codon 918 (38)No. +, positive; -,No. mutations, and none were found (18-20, 28). These observations had 4 5 10 11 12 13 Fig. 3. Autoradiographs demonstrating alÃ-eles of polymorphic dinucleotide repeat marker D1S211. Lanes 1-9 and 10-14. subpopu lations from two sporadic MTCs. •¿ P* 2169 Downloaded from cancerres.aacrjournals.org on February 20, 2016. © 1996 American Association for Cancer Research. 14 RET MUTATION IN MTC SUBPOPUI.ATIONS suggested that the germline RET mutation in one of the cysteine codons would be sufficient for transformation, as evidenced by in vitro transfection studies (9, 29). However, the presence of both germline and somatic mutations of RET in the single MEN 2A MTC is unlikely to be redundant; more likely, the somatic mutation adds to the effect of the germline mutation (30). Additional studies of the somatic RET mutation in MEN 2A and FMTC tumors are in progress to determine whether this is observed more frequently, especially at the subpopulation level, whether both mutations are allelic. and whether these reflect more aggressive tumors. Just as the RET codon 918 mutation is heterogeneous within MTCs. LOH of Ip and 22q markers is not homogeneous within a single tumor or among métastasesin the majority of the tumors studied. The nonhomogeneous presence of LOH is usually attributed to events related to tumor progression, either as a cause or an effect. For example, in the well-worked-out model of colon carcinoma progres sion, the paucity of 17p losses in colonie adenomas and the high frequency in frank carcinoma and métastasesare explained by the loss of a gene or genes, including the tumor suppressor gene p53, leading to tumor advancement (reviewed by Fearon and Vogelstein: Ref. 27). This model may be extended to explain our observations. In MTC. we showed that the nonhomogeneous presence of an RET mutation and/or LOH occurred even within a single tumor or a single metastasis, suggesting a less rapid overgrowth of the clones bearing these changes. The distribution of l p and 22q losses across the subpopulations in primary and metustutic tumors shows no evidence of which one could construct a sequence of these events and a codon 918 mutation in tumor progression. This opportunity to look for specific mutations in microdissected tumors has revealed that somatic mutations in RET may be more common than thought and has revealed an unsuspected degree of heterogeneity, most clearly seen in the tumor métastaseswith codon 918 mutation- and codon 883 mutation-positive clones. This under scores the need to be aware of the heterogeneity, whether in molecular diagnosis using tumor templates or in potential therapy directed at molecular targets. ACKNOWLEDGMENTS We are grateful to the clinicians who obtained MTC specimens and to Maggie Ponder. Jo Dearden, Carole Garner. Jean Miller, and Wendy M. Smith for assistance. We thank Fred Li and Sig Verselis for helpful discussions: Don Kufe. Ed Garber, and Darrin Smith for critical review of an early draft of the manuscript; and Jan Vijg for his continued support and encouragement. REFERENCES 1. Schimke. R. N. Genetic aspects of multiple endocrine neoplasia. Annu. Rev. Med.. .S3.-25-31, 1984. 2. Mulligan. L. M.. Marsh. D. J.. Robinson. B. G.. Schuffenecker. I.. Zedenius. J., Lips, C. J. M.. Gagel. R. F.. Takai. S-I.. Noll. W. W.. Fink. M., Raue. F.. Lacroix. A., Thibodeau. S. N.. Frilling. A.. Ponder. B. A. J.. and Eng. C. Genotype-phenotype correlation in MEN 2: repon of the International RET Mutation Consortium. J. Intern. Med., 238: 343-346. 1995. 3. Santoro. M.. Rosato. R.. Grieco. M.. Berlingieri. M. T., Luca-Colucci D'Amato. G., de Franciscis. V., and Fusco, A. The rei proto-oncogene is consistently expressed in human pheochromocytomas and thyroid medullary carcinomas. Oncogene. 5: 15951598, 1991). 4. Nakamura. T.. Ishi/aka. Y.. Nagao, M.. Hará.M., and Ishikawa. T. Expression of the rei proto-oncogene product in human normal and neoplastic tissues of neural crest origin. J. Pathol.. 172: 255-260. 1994. 5. Mulligan, L. M.. Kwok. J. B. J., Healey, C. S., Elsdon, M. J., Eng. C., Gardner, E., Love. D. R.. Mole. S. E.. Moore. J. K.. Papi, L., Ponder, M. A., Telenius. H.. Tunnacliffe, A., and Ponder. B. A. J. Germline mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature (Lond.), 363: 458-460. 1993. 6. Donis-Keller. H.. Dou. S., Chi. D.. Carlson. K. M.. Toshima. K.. Lairmore. T. C., Howe. J. R.. Moley, J. F.. Goodfellow. P.. and Wells. S. A. Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum. Mol. Genet.. 2: 851-856. 1993. 7. Mulligan. L. M.. Eng. C.. Healey, C. S.. Clayton, D., Kwok. J. B. J.. Gardner. E., Ponder. M. A., Frilling. A.. Jackson. C. E.. Lehnen. H.. Neumann, H. P. H.. Thibodeau, S. N., and Ponder, B. A. J. Specific mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC. Nat. Genet., 6: 70-74, 1994. 8. Mulligan. L. M., Eng, C.. Healey. C. S.. Ponder, M. A.. Feldman. G. L.. Li, P.. Jackson, C. E., and Ponder, B. A. J. A de nom mutation of the RET proto-oncogene in a patient with MEN 2A. Hum. Mol. Genet.. 3: 1007-1008. 1994. 9. Santoro, M., Carlomagno, F., Romano, A., Bottaro. D. P.. Dathan, N. A., Grieco, M., 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 2B. Science (Washington DC). 267: 381-383. 1995. 10. Eng, C., Smith. D. P.. Mulligan. L. M., Healey, C. S., Zvelebil, M. J.. Stonehouse. T. J., Ponder. M. A., Jackson. C. E.. Waterfield. M. D.. and Ponder. B. A. J. 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. 10: 509-513, 1995. 11. Bolino. A.. Schuffenecker, I., Luo, Y.. Seri. M.. Silengo. M., Tocco. T., Chabrier, G., Houdent. C.. Murât,A.. Schlumoerger. M.. Toumiaire. J.. Lenoir. G. M.. and Romeo. G. RET mutations in exons 13 and 14 of FMTC patients. Oncogene. 10: 2415-2419. 1995. 12. Hofstra, R. M. W., Landsvater. R. M., Ceccherini. !.. Stülp.R. P.. Steelwagen. T., Luo. Y., Pasini. B.. Hoppener. J. W. M., Ploos van Amstel, H. K., Romeo. G.. Lips. C. J. M., and Buys. C. H. C. M. A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature (Lond.), 367: 375-376, 1994. 13. Eng. C.. Smith. D. P.. Mulligan, L. M.. Nagai. M. A.. Healey. C. S.. Ponder. M. A., Gardner. E., Scheumann, G. F. W.. Jackson, C. E., Tunnacliffe, A., and Ponder, B. A. J. Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours. Hum. Mol. Genet., 3: 237-241, 1994. 14. Carlson. K. M.. Dou. S.. Chi, D.. Scavarda. N.. Toshima, K.. Jackson. C. E.. Wells. S. A.. Goodfellow. P. J.. and Donis-Keller, H. Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B. Proc. Nati. Acad. Sci. USA, 91: 1579-1583, 1994. 15. Songyang, Z., Carraway, K. L., Ill, Eck. M. J., Harrison, S. C., Feldman. R. A.. Mohammadi. M.. Schlessinger. J., Hubbard. S. R.. Smith, D. P., Eng. C.. Lorenzo, M. J., Ponder. B. A. J.. Mayer. B. J., and Cantley. L. C. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature (Lond.), 373: 536-539, 1995. 16. Mulligan, L. M., Gardner. E.. Smith, B. A., Mathew, C. G. P.. and Ponder. B. A. J. Genetic events in tumor initiation and progression in multiple endocrine neoplasia. Genes Chromosomes & Cancer. 6: 166-177, 1993. 17. Eng, C., and Ponder. B. A. J. The role of gene mutations in the genesis of familial cancers. FASEB J., 7: 910-919. 1993. 18. Zedenius. J.. Wallin, G.. Hambcrger, B.. Nordenskjöld, M.. Weber, G.. and Larsson, 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. 19. Eng. C.. Mulligan. L. M.. Smith. D. P.. Healey, C. S.. Frilling, A.. Raue, F.. Neumann, H. P H., Pfragner. R.. Behmel. A., Lorenzo. M. J., Stonehouse, T. J.. Ponder, M. A., and Ponder. B. A. J. Mutation of the RET proto-oncogene in sporadic medullary thyroid carcinoma. Genes Chromosomes & Cancer. 12: 209-212, 1995. 20. Komminoth, P.. Kunz. E. K.. Matias-Guiu. X., Hiort. O., Christensen, 0.. Colomer. A.. Roth. J.. and Heitz, P. U. Analysis of RET proto-oncogene point mutations distinguishes heritable from nonheritable medullary thyroid carcinomas. Cancer (Phila.), 76: 479-489, 1995. 21. Wright. D. K.. and Manos, M. Sample preparation from paraffin-embedded tissues. In: M. A. Innis. D. H. Gelfand. J. J. Sninsky, and T. J. White (eds.). PCR Protocols. A Guide to Methods and Application, pp. 153-158. San Diego: Harcoun Brace Jovanovich Publications. 1990. 22. Marsh. D. J.. Learoyd. D. L., Andrew. S. D.. Krishnan, L., Pojer, R.. Richardson, A-L., Delbridge, L., Eng, C., and Robinson, B. G. Somatic mutations in the RET proto oncogene in sporadic medullary thyroid carcinoma. Clin. Endocrino!. (Oxf.), 44: 249257, 1996. 23. Kwok, J. B. J., Gardner, F... Warner, J. P., Ponder. B. A. J.. and Mulligan, L. M. Structural analysis of the human rei proto-oncogene by exon trapping. Oncogene, 8: 2572-2582, 1993. 24. Gyapay, G., Morissette, J., Vignai, A., Dib. C., Fizames. C.. Millasseau, P., Marc, S.. Bernardi. G.. Lalhrop. M., and Weissenbach. J. The 1993-94 Généthon human genetic linkage map. Nat. Genet., 7: 246-339. 1994. 25. Love. D. R.. Gardner, E.. and Ponder. B. A. J. A polymorphic dinucleotide repeat at DIOS14I locus. Hum. Mol. Genet., 2: 491, 1993. 26. Love, D. R., Gardner. E.. and Ponder. B. A. J. A polymorphic dinucleolide repeat at the ZNF22 locus. Hum. Mol. Genet.. 2: 491. 1993. 27. Fearon, E. R., and Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell. 61: 759-767, 1992. 28. Blaugrund. J. E., Johns. M. M., Eby. Y. J., Ball. D. W.. Baylin. S. B.. Hniban, R. H., and Sidransky. D. RET proto-oncogene mutations in inherited and sporadic medullary thyroid cancer. Hum. Mol. Genet.. 3: 1895-1897, 1994. 29. Borrello, M. G.. Smith. D. P.. Pasini. B., Bongarzone. 1., Greco. A., Lorenzo, M. J., Arighi, E.. Miranda. C.. Eng. C.. Alberti, L.. Bocciardi. R. P. M.. Scopsi. L.. Romeo. G.. Ponder. B. A. J., and Pierotti, M. A. RET activation by germline MEN2A and MEN2B mutations. Oncogene, in press, 1996. 30. Marsh, D. J., Andrew. S. D., Eng, C., Learoyd. D. L., Capes, A. G.. Pojer, R., Richardson, A-L., Houghton, C., Mulligan. L. M., Ponder. B. A. J., and Robinson. B. G. Germline and somatic mutations in an oncogene: RET mutations in inherited medullary thyroid carcinoma. Cancer Res., 56: 1241-1243, 1996. 2170 Downloaded from cancerres.aacrjournals.org on February 20, 2016. © 1996 American Association for Cancer Research. Heterogeneous Mutation of the RET Proto-oncogene in Subpopulations of Medullary Thyroid Carcinoma Charis Eng, Lois M. Mulligan, Catherine S. Healey, et al. Cancer Res 1996;56:2167-2170. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/56/9/2167 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, contact the AACR Publications Department at permissions@aacr.org. Downloaded from cancerres.aacrjournals.org on February 20, 2016. © 1996 American Association for Cancer Research.