ELSEVIER Selection of Adrenal Tumor Cells in Culture Demonstrated by Interphase Cytogenetics Carla Rosenberg, Valter A. Della-Rosa, Ana Claudia Latronico, Berenice B. Mendon a, and Angela M. Vianna-Morgante ABSTRACT: Solid tumors often consist of an admixture of cell populations with different genome consti- tutions. Karyotyping of this material is complicated by the low mitotic index. Even when chromosome studies are feasible, altered representation of the original cell populations after cell cultivation is possi- ble. We report a human adrenal carcinoma that exhibited a normal karyotype after cultivation but was shown to be highly aneuploid when investigated by fluorescence in situ hybridization (FISH) in direct preparations of uncultured cells with six different centromeric probes. The high frequencies of trisomy for the investigated chromosomes in these interphase cells indicate that most of the tumor cells were in the triploid range. Strong selection for disomic cells was detected in interphase preparations after one and two subcultures and was even stronger in the corresponding metaphase preparations. Trisomy for chromosome 15 appeared to be maintained independent of triploidy and might play a role in cultured cell survival. The number of chromosome 17 centromeres was not increased in polyploid cells, suggesting loss of this chromosome in the original cells of the tumor. INTRODUCTION DNA studies are commonly performed to characterize tumors [1-3]. Although DNA techniques have high resolution and allow investigation at the gene level, an overview of chro- mosome alterations cannot be achieved by these methods. On the other hand, chromosome alterations can be detected through karyotyping, but these cytogenetic studies depend on tumor cell cultivation, which is often difficult or even impossible. Fnllhermore, many tumors consist of admixtures of cell populations and cells grown in vitro might not be rep- resentative of the original cell composition of the tumor. Some years ago, use of fluorescence in situ hybridization (FISH) was extended to interphase and has been applied to direct preparations of uncultured tumor cells. This approach allows cytogenetic information to be obtained without cul- tivation and has been designated "interphase cytogenetics" [4]. Centromeric sequences have been especially used to char- acterize numerical aberrations in tumors [5-7]. We applied interphase cytogenetics, using six different centromeric sequences to investigate chromosome numeri- cal abnormalities in uncultured cells of a human adrenal From the Departamentos de Biologia, Instituto de Bioci~ncias, University of St)o Paulo (C. Ft., V. A. D.-B.,A. M. V.-M.), e En- docrinologia e Metabologia, Faculdade de Medicine (A. C. L., B. B. M.), Departmento de Biologia Celular e Gen~tica, State Univer- sity of Maringd (E A. D.-M.), Sdo Poulo, Brazil. Address reprint requests to: Carla Ftosenberg, Ph.D., Depar- tamento de Biologia, Universidade de Sdo Paulo; C.E 11461; CEP 05422-970, Sdo Pau/o, SP, Brazil. HHeceived November 1, 1993; accepted May 3, 1994. carcinoma. Comparison was made with the results obtained in cultured interphase and metaphase cells from the same tumor. The significance of the alterations detected by inter- phase analysis is discussed. MATERIALS AND METHODS Patient Data The patient was a 19-year-old woman with a large abdomi- nal mass at the right adrenal gland. Hormonal evaluation showed abnormally high levels of androgens, glucorticoids, and mineralocorticoids. Right nephrectomy and adrenalec- tomy were performed. The adrenal cortex tumor measured 13 cm at its largest diameter, and histologic findings were typical of carcinoma. A macroscopically homogeneous area of the tumor was excised for cytogenetic studies. Cytogenetic Studies For the uncultured cell preparations, small fragments (1-3 mm 3) of the fresh tumor were fixed in colcl methanol:ace- tic acid (3:1) followed by two changes of fixative. The frag- ments were then dissociated in several drops of 60% acetic acid for 1-10 minutes. Clean slides were placed on a warm plate (40°-45°C); one drop of the cell suspension was placed on each slide and allowed to dry. Slides were kept overnight at room temperature and were either used immediately or stored at -20°C. The culture was established from small tumor fragments in a 1:1 mixture of Dulbecco's modified Eagle's medium and F-IO nutrient mixture enriched with 15% bovine calf serum. After 7 days, cells covered the surface of a 25-cm 2 Coster Cancer Genet Cytogenet 79:36-40 (1995) © Elsevier Science Inc., 1995 655 Avenue of the Americas, New York. NY 10010 0165-4608/95/$9.50 SSDI 0165-4608(94)00079-Q