World J. Surg. 17, 615-621, 1993 WORLD Journal of SURGERY 9 1993 by the Soci›233 Internationale de Chirurgie Expression of a Potential Metastasis Suppressor Gene (nm23) in Thyroid Neoplasms David R. Farley, M,D., Norman L. Eberhardt, Ph.D., Clive S. Grant, M.D., Daniel J. Schaid, Ph.D., Jonathan A. van Heerden, M.D., Ian D. Hay, M.B., Ph.D., and Sundeep Khosla, M.D. Endocrine Research Unit, Division of Endocrinology and Internal Medicine, and Departments of Surgery and Oncology, Mayo Clinic, Rochester, Minnesota, U.S.A. Identification of multiple clinical and pathologic prognostic factors in differentiated thyroid cancer has permitted some degree of risk stratifi- cation. However, these clinical indices fail to distinguish potentiai intrinsic differences in tumor virulence. The nm23 gene has been identified as a potential metastasis suppressor gene that is homoiogous to nucleoside diphosphate kinases. Studies in human breast cancer bave shown a significant inverse correlation between nm23 ievels and nodal involve- ment/tumor recurrence. Given the possible clinical utility of a marker of metastatic potential in the management of thyroid carcinoma, we exam- ined 34 thyroid neoplasms and a human medullary thyroid cancer (MTC) cell line (TT) for nm23 expression. Normalized nm23 expression was assessed by Northern analysis of tumor RNA. nm23 Expression (tumor expression/TT cell expression, mean -+ SE) was 1.14 -+ 0.15" in MTCs (n = 5), 0.70 - 0.10" in follicular cancers (n = 6), 0.51 -+ 0.11 in papillary cancers (n = 19), and 0.31 -+ 0.03 in follicular adenomas (n = 4) (*p < 0.05 when compared to adenomas). Within histologic groups, we found no correlation between nm23 expression and nodal involvement or distant metastases. Our results indicate that thyroid neoplasms of different histologies express varying ievels of the nm23 transcript. AIthough nm23 expression seems diminished in metastatic breast cancer, it appears not to be the case in metastatic thyroid cancer. The nm23 gene may therefore have different roles in the evolution and metastases of different neoplasms. Patients with differentiated thyroid cancer (papillary, follicular, medullary) generally have a good long-term prognosis. There are, however, subgroups of patients with aggressive disease who develop systemic metastases and eventually succumb to their disease. Identification of several clinical (age, gender) and pathologic indices (tumor size, histologic grade, vascular or capsular invasion, nodal or distant metastases) has permitted some degree of risk stratification of these patients [1, 2]. DNA ploidy status has become an important independent prognostic indicator in thyroid cancer [3]. With increasing understanding of the biology of tumorigenesis, it is likely that certain genetic alterations in tumors may identify patients with a poor prognosis. Previous studies from our group have dem- onstrated specific alterations involving chromosomes lp and 22q in medullary thyroid cancers (MTCs) [4]. We also demon- strated cytogenetic abnormalities of chromosome 10q in papil- Offprint requests: Sundeep Khosla, Division of Endocrinology, Mayo Clinic, Rochester, MN 55905, U.S.A. lary thyroid cancers and loss of heterozygosity on chromosome 3p in follicular thyroid cancers [5]. Another group has demon- strated rearrangement of the ret oncogene (mapped to 10ql 1.2) in papillary cancers [6-8]. Ras oncogene mutations have also been demonstrated with variable frequency in benign and malignant thyroid neoplasms [9-15]. These studies have begun to define the molecular changes involved in thyroid tumor formation, but the clinical utility of these genetic changes as prognostic indicators remains unclear. The nm23 gene bas been identified as a potential "metastasis suppressor" gene that may have prognostic utility in breast cancer [16-20]. Steeg and coworkers originally identified the nm23 gene using a differential screening strategy involving subclones of a single K-1735 murine melanoma [21]. nm23 mRNA levels were markedly reduced in cell lines of high metastatic potential compared to cell lines of low metastatic potential [21]. In breast cancer, low nm23 mRNA levels were subsequently correlated with the presence of lymph node metastases at surgery [18] as well as with reduced disease-free and overall survival [19]. Other studies of infiltrating ductal breast carcinomas have also shown an association between low nm23 protein expression and reduced patient survival [20]. Given the possible clinical utility of a marker of metastatic potential in thyroid carcinoma, we examined nm23 mRNA expression in 34 resected thyroid neoplasms of different histol- ogies and a human MTC cell line (TT) [22]. We also tested for any correlations between the level of nm23 mRNA expression in these tumors and nodal involvement or distant metastases. Materials and Methods Cell Lines and Culture A human MTC cell line (TT), derived from a patient with metastatic disease [22], was used to provide control RNA (see below). TT cells were grown in monolayers (75 cm 2 flasks (Corning, Corning NY) in RPMI-1640 (Gibco Laboratories, Grand Island, NY) containing 16% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT), 2 mM glutamine,