PROGNOSTIC VALUE OF CODON 918 (ATG3 ACG) RET PROTO-ONCOGENE MUTATIONS IN SPORADIC MEDULLARY THYROID CARCINOMA Tobias SCHILLING 1 * , Julia B¨ URCK 1 , Hans-Peter SINN 2 , Andreas CLEMENS 1 , Herwart F. OTTO 2 , Wolfgang H¨ OPPNER 4 , Christian HERFARTH 3 , Reinhard ZIEGLER 1 , Manfred SCHWAB 5 and Friedhelm RAUE 6 , 1 Department of Internal Medicine I, University of Heidelberg, Heidelberg, Germany 2 Department of Pathology, University of Heidelberg, Heidelberg, Germany 3 Department of Surgery, University of Heidelberg, Heidelberg, Germany 4 Institute for Hormone and Fertility Research, Hamburg, Germany 5 Department of Cytogenetics, German Cancer Research Center, Heidelberg, Germany 6 Endocrine Practice, Heidelberg, Germany We have determined the frequency of 918 RET proto- oncogene mutations (ATG3 ACG) in primary MTC tumors and metastases and correlated the presence or absence of this mutation with the clinical outcome of patients suffering from sporadic medullary thyroid carcinoma (MTC). A total of 197 samples, consisting of both primary tumors and lymph node metastases from 34 patients with sporadic MTC, were collected for PCR analysis of the RET 918 mutation. In 75 of the samples (38%), codon 918 (ATG3 ACG) mutations could be detected. The mutations showed a heterogeneous distri- bution: 21/34 patients (62%) had mutations in at least 1 tu- mor sample, and in 13 patients (38%) the mutation was present in all examined samples. Patients were considered 918mt when at least 1 tumor sample showed the RET 918 mutation. These 918mt and 918 wild-type (918wt) patients did not differ significantly concerning sex, age at diagnosis, TNM stage at diagnosis, number of examined tumor samples or follow-up time. However, 918mt patients showed more aggressive development of distant metastases during fol- low-up (p  0.032, Fisher’s exact test) with decreased me- tastases-free survival (p < 0.005, log-rank test). Further- more, 918mt patients had a significantly lower survival rate than 918wt patients (p  0.048, log-rank test). These data show that the RET codon 918 mutation has a prognostic impact on patients with sporadic MTC which may influence follow-up treatment. © 2001 Wiley-Liss, Inc. Key words: medullary thyroid cancer; multiple endocrine neoplasia; germline mutation; prognosis; RET proto-oncogene Specific germline mutations in the RET proto-oncogene result in 4 different syndromes: Hirschsprung’s disease, familial medullary thyroid carcinoma (FMTC), multiple endocrine neoplasia (MEN) type 2A and MEN type 2B. 1–3 The mutation responsible for MEN 2B is a point mutation in exon 16, codon 918 of RET (ATG3 ACG), resulting in the substitution of methionine by thre- onine. In addition to the germline mutations, there are somatic mutations of RET in sporadic carcinomas, which include re-ar- rangements associated with papillary thyroid carcinoma 4–7 and point mutations associated with pheochromocytoma 8 –10 and spo- radic medullary thyroid carcinoma (MTC). 11–16 The somatic point mutation most frequently seen in MTC (mean incidence 42%) is identical to the germline mutation identified in MEN 2B at codon 918 of RET. Other known germline point mutations of RET at codons 883, 768, 634 and 611 and a somatic 12 bp in-frame deletion in exon 15 are considerably more infrequent. 11,17–27 It has been suggested that the codon 918 mutation alters substrate spec- ificity, resulting in autophosphorylation and activation of RET. 28 –30 Thus, the codon 918 mutation of RET may play a role in the pathogenesis of sporadic MTC. Indeed, in the 2 studies performed so far, an association of RET codon 918 mutation with tumor recurrence was found. 17,20 How- ever, 1 study did not find any impact of RET codon 918 mutation on the clinical outcome of patients. 23 While the 2 studies examined mutations exclusively in the primary tumor, we analyzed RET codon 918 mutations in both primary MTC tumors and metastases in a retrospective study, with a follow-up of up to 20 years. The presence or absence of the mutation was correlated with the clinical outcome of the patients. MATERIAL AND METHODS Patients and tumor specimens Thirty-four patients with sporadic MTC were selected for our study. Patients were considered as having sporadic MTC if there was no family history of any MEN 2–related disease. No patient showed the characteristic phenotype of MEN 2B. Patients were treated by radical thyroidectomy (if not performed already in an external institution) and cervical lymph node dissection at the Department of Surgery, University of Heidelberg. In most cases, only lymph node metastases were available for examination since primary tumor resection (radical thyroidectomy) had been per- formed in an external referring institution. Since heterogeneous mutation of RET in subpopulations of MTC has been demonstrated, 16 several tumor samples, mostly lymph node metastases, were collected from each patient (Table I). From 31 patients, 2 to 26 tumor samples were obtained; from 3 patients, only 1 specimen was available. In the original histological evaluation, all specimens were judged to contain tumor tissue. This histological diagnosis of MTC was re-established by reviewing the original tissue sections from the primary tumor and cervical lymph node metastases again and applying the WHO criteria for classi- fication of thyroid malignancies. To confirm that the patients had no MEN 2-specific mutation in the germline, we examined blood samples, which were available in 23 cases. DNA extraction, PCR and mutation detection From peripheral blood leukocytes, germline DNA was isolated and the RET proto-oncogene was screened for MEN 2-specific mutations in exons 10 and 11 and in exons 13 and 16, as previously Grant sponsor: Forschungsf¨ orderung der Medizinischen Fakult¨ at der Universit¨ at Heidelberg; Grant number: 153/95; Grant sponsor: Tumorzen- trum Heidelberg/Mannheim; Grant number: FSP III./I.2. This work is the thesis of Julia B¨ urck. *Correspondence to: Department of Internal Medicine I, University of Heidelberg, Bergheimerstrasse 58, 69115 Heidelberg, Germany. Fax: +49 6221 56 4101. E-mail: Tobias_Schilling@med.uni-heidelberg.de Received 15 August 2000; Revised 9 November 2000; Accepted 20 November 2000 Published online 18 January 2001 Int. J. Cancer (Pred. Oncol.): 95, 62– 66 (2001) © 2001 Wiley-Liss, Inc. 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