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Amplification and Overexpression of Mutant RET in Multiple Endocrine Neoplasia Type 2-Associated Medullary Thyroid Carcinoma

Molecular Pathogenesis Unit, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke (S.C.H., J.T.-C., S.D.P., A.O.V., P.M., I.A.L., Z.Z.), and Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Disease (C.A.K.), National Institutes of Health, Bethesda, Maryland 20892; and M. D. Anderson Cancer Center, University of Texas (R.F.G.), Houston, Texas 77030

Address all correspondence and requests for reprints to: Christian A. Koch, M.D., F.A.C.P., Department of Endocrinology/Nephrology, Uniklinik, Philipp Rosenthalstrasse 27, 04103 Leipzig, Germany. E-mail: kochc{at}exchange.nih.gov.

We have previously identified two second hit mechanisms involved in the development of multiple endocrine neoplasia type 2 (MEN 2)-associated tumors: trisomy 10 with duplication of the mutant RET allele and loss of the wild-type RET allele. However, some of the MEN 2-associated tumors investigated did not demonstrate either mechanism. Here, we studied the TT cell line derived from MEN 2-associated medullary thyroid carcinoma with a RET germline mutation in codon 634, for alternative mechanisms of tumorigenesis. Although we observed a 2:1 ratio between mutant and wild-type RET at the genomic DNA level in this cell line, fluorescence in situ hybridization analysis revealed neither trisomy 10 nor loss of the normal chromosome 10. Instead, a tandem duplication event was responsible for amplification of mutant RET. In further studies we could for the first time demonstrate that the genomic chromosome 10 abnormalities in this cell line cause an increased production of mutant RET mRNA. These findings provide evidence for a third second hit mechanism resulting in overrepresentation and overexpression of mutant RET in MEN 2-associated tumors.

S.C.H. and J.T.-C. contributed equally to this work.

Abbreviations: d, Deoxy; FISH, fluorescence in situ hybridization; MEN2, multiple endocrine neoplasia type 2; SSC, standard saline citrate; SSCP, single strand conformational polymorphism; TBE, Tris/Boric acid/EDTA.

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