GYNECOLOGIC

ONCOLOGY

38, 473-477 (1990)

Chromosome Abnormalities in Human Epithelial Ovarian Malignancies H. H.

GALLION,

M.D.,* D. E.

POWELL,

M.D., L. W. SMITH, B.A., J. K. MORROW, PH.D., A. W. M.D.,” AND E. S. DONALDSON, M.D.*

MARTIN,

M.D.,?

J. R. VAN NAGELL,

*Division of G.vnecologic Oncology, Dcpurtment of Obstetrics und Gynwology, und Depctrtmrnt of Pathology. University c!f Kc~nttrcky Me&u1 Center, Lexington, Kentucky 40536, und tDepurtm?nt qf Pathology, University of‘Lorrisvi//e School c?f’Mrdicine, Lorrisville. Kentwhy 40208

Received November 30, 1989

Burkitt’s hereditary ties, including lymphoma, retinoblastoma, acute myeloblastic leukemia, and Wilms’ tumor [3-61. Although carcinomas represent over 80% of all malignant tumors in humans, fewer cytogenetic data are available concerning epithelial malignancies than for leukemias, lymphomas, and childhood tumors. There have been several studies of chromosomal changes in ovarian cancer, the leading cause of death from gynecologic malignancy 17-121. However, most of the reported series include data on previously treated patients or data from effusions or tumor metastases. This investigation was undertaken to determine whether consistent chromosome abnormalities are present in the tumors of untreated patients with epithelial ovarian cancer and to use this information as a basis for subsequent molecular genetic studies.

Karyotypic analysis of tumor specimens from 29 patients with untreated epithelial ovarian carcinoma was performed at the University of Kentucky Medical Center. Twenty-three of the twentynine tumors had adequate cells for analysis. Seventeen of these tumors exhibited chromosome abnormalities. Chromosome alterations were complex, with an average of seven different abnormal chromosomal patterns per tumor (range 2-14). Chromosomes 1 and 11 were the most commonly involved, being abnormal in 89 and 83% of tumors, respectively. Chromosomes 3 and 7 were also frequently abnormal. In contrast to invasive tumors, alterations in chromosomes 1 and 11 were not seen in the two tumors of borderline malignant potential. Evidence for DNA amplification of IGF2, Ha-ras-1, and c-ets was not observed. Amplification of the c-e&B-2 oncogene was present in two tumors. These findings indicate that multiple karyotypic abnormalities occur in untreated epithelial ovarian malignancies, with chromosomes 1 and 11 being the most frequently abnormal. These data also suggest that alterations of these chromosomes may be associated with the biologically aggressive behavior of frankly invasive ovarian tumors. 0 1990AcademicPress,Inc. INTRODUCTION Karyotypic analysis of tumor cells has established the presence of nonrandom chromosome changes in many

human malignancies [I]. In some instances, these chromosome alterations are well defined and have proven to be specific for a particular type of tumor. The Philadelphia chromosome was the first such chromosome abnormality to be identified [2]. This chromosome, formed by a reciprocal translocation between chromosomes 9 and 22, is now considered the hallmark of chronic myelogenous leukemia. More recently, characteristic cytogenetic changes have been identified in other malignanPresented at the annual meeting of the Society of Gynecologic Oncologists, San Francisco, CA, February 4-7, 1990.

MATERIALS

AND METHODS

From 1983to 1989fresh tumor samples for cytogenetic analysis were obtained from the primary ovarian tumors of 29 patients with epithelial ovarian cancer treated at the University of Kentucky Medical Center. No patient who had received prior chemotherapy or radiotherapy was included in this investigation. Tumors were staged according to the FIG0 operative staging system and classified according to the WHO classification system for epithelial ovarian cancer [ 13,141. Tumors were graded histologically as well differentiated, moderately differentiated, or poorly differentiated [ 151. Cytogenetics

Tumor specimens for cytogenetic analysis were obtained from the least necrotic-appearing portion of the

473 0090.8258190$1.50 Copyright D 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TABLE 1 Histologic Characteristics of Tumors Studied (N = 29) No. of tumors Cell type Serous cystadenocarcinoma Endometrioid carcinoma Mutinous carcinoma Undifferentiated carcinoma Clear cell carcinoma Grade Low malignant potential I 2 3

19 5 2 2 I 3 2 IO 14

sterile, fresh ovarian tumor and were immediately placed in L-15 transport medium. The tumor was then minced and rinsed with a low-thymidine medium to remove single cells for suspension harvest. The suspension was then incubated with 5-fluorouracil for 18 hr followed by thymidine. After the addition of colcemid and ethidium bromide, the culture was harvested and fixed according to standard procedure. In addition, tumor minces were incubated in collagenase II for l-2 hr to dissociate the tissue, rinsed and resuspended in supplemented media in Primaria flasks. Cells were harvested at 24-hr intervals until all flasks were depleted. Slides were made on all cultures if divisions were seen. Banded metaphases were analyzed microscopically and photographed for karyotyping. Molecular Studies High-molecular-weight genomic DNA was extracted from frozen tissue from nine of the cases included in this report. Southern blot and slot-blot analyses of these cases were performed using 32Pprobes for IGF2, c-e&BTABLE 2 Frequency of Structural Chromosome Abnormalities (N = 17) Chromosome Number of tumors Chromosome Number of tumors 1 11 3 7 5 14 12 13 2 6 8

I.5 (89) I4 (83) II (65) IO (59) 9 (53) 9 (53) 8 (47) 7 (41) 5 (29) 5 (2% 5 (29)

L?Percentages given in parentheses.

17 19 15 20 X 4 IO 16 18 21

4 (24) 3 (18) 3 (18) 3 (18) 3 (18)

2 (12) 2 (12) 2 (12) 1 (6) 1 (6)

ET AL.

2, Ha-ras-I, and c-ets (American Type Culture Collection, Rockville, MD) as well as a human actin probe (Stanford University, Palo Alto, CA) [16-181. Ha-ras-1, an oncogene of the GTP-binding protein class, is localized at 1lpi5 [19]. Insulin-like growth factor (IGF2) is not an oncogene per se, but is a growth factor localized at 11~15 [201. The oncogenes c-ets and c-e&B-2 are localized to 1lq23 and 17ql l-q12, respectively [21,22]. Signals were analyzed for gene dosage using a scanning laser densitometer. Immunochemistry Formalin-fixed paraffin-embedded tumors were cut at 4 pm, deparaffinized, and hydrated. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide/methanol and nonspecific antibody binding was blocked with 10% normal goat serum. Sections were incubated with rabbit (polyclonal) anti-c-e&B-2 (Triton at a 1: 10 dilution, then incubated with secondary antibody (biotinylated goat anti-rabbit immunoglobulin) (Vector) and with ABC (Vector) according to the manufacturer’s directions. Color was developed with diaminobenzidene and slides were dehydrated, cleared, and mounted. RESULTS The mean age of the patients studied was 61 years (range 29-82) and the mean gravidity was 3 (range O-7). Four patients had FIG0 stage I disease, five patients stage II disease, and twenty patients had stage III or IV disease. The histologic characteristics of the tumors studied are illustrated in Table 1. The most common cell type was serous cystadenocarcinoma, followed by endometrioid carcinoma, mutinous cystadenocarcinoma, undifferentiated carcinoma, and clear cell carcinoma. Three patients had tumors of borderline malignant potential. Two tumors were grade 1, ten were grade 2 and fourteen were grade 3. Twenty-three of the twenty-nine (79%) tumors had adequate cells for karyotypic analysis. Six tumors revealed normal chromosomes. The remaining 17 tumors exhibited chromosomal abnormalities. A mean of 7 different abnormal chromosomes were identified in each tumor (range 2-14). The frequency of structurally abnormal chromosomes identified in these 17 tumors is illustrated in Table 2. Chromosomes 1 and 11 were the most commonly involved, being abnormal in 89 and 83% of tumors, respectively. Chromosomes 3 (65%) and 7 (59%) were also often abnormal. Chromosomes 4, 10, 16, 18, and 21 were rarely abnormal (s 12%). Representative karyotypes are illustrated in Figs. 1 and 2. Chromosome abnormalities were identified in two of

CHROMOSOME

ABNORMALITIES

IN OVARIAN

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m 1~36

-

lp13 11 1

f

FIG. 1. Karyotype of an ovarian tumor exhibiting deletion of chromosome 19 and a recombinant marker consisting of the long arms of chromosomes II and 13.

the three ovarian tumors of borderline malignancy. Neither had an identifiable abnormality of chromosome 1 or 1I. A mean of 8 (range 4-14) abnormal chromosomes were seen in the 15 invasive tumors. All tumors from patients with invasive disease had a structural abnormality of chromosome 1, and 14 of these 15 tumors had a structural abnormality of chromosome 11. DNA from frozen tissue was available in nine of the tumors included in this report. Evidence for amplification of IGF2, Ha-ras-1, and c-ets was not seen. DNA blot analysis revealed evidence of DNA amplification of the c-erbB-2 oncogene in 2 of the 9 tumors tested. A representative slot blot from 5 tumors is illustrated in Fig. 3. Immunohistochemical staining using anti-c-rrbB-2 was performed in 16 tumors. Two of these tumors exhibited strong positive staining. Two patients had both blot and immunohistochemical analysis for c-erbB-2, and both tumors were negative by these methods. DISCUSSION Recent advances in molecular and cellular biology have made it possible to study the genetic differences between cancer cells and their normal counterparts. The presence of tumor-specific chromosome translocations as well as amplifications and deletions have been documented in several human tumors, particularly leukemias and lymphomas [ 1,231. Previous studies of chromosome changes in ovarian cancer have reported a number of different complex karyotypic abnormalities, most commonly involving chromosomes 1, 3, and 6 [7-121. However, many of these reports have utilized cells from effusions or from previously treated patients. The current study is unique in that only tumor specimens from primary, untreated epithelial ovarian tumors were included. Therefore, changes seen were not induced by therapy,

FIG. 2. Karyotype of an ovarian tumor showing numerous chromosomal abnormalities as well as unidentified marker chromosomes. Note abnormalities of chromosomes I and Il.

and abnormalities occurring late in the course of disease should be less prominent. Tanaka and co-workers, for example, reported complex aberrations of multiple chromosomes in 9 ovarian malignancies [12]. In this report, chromosome 1 was abnormal in 7 tumors and chromosomes 6 and 11 were abnormal in 5 cases. Similarly, Atkin and Baker observed frequent structural abnormalities of chromosome 1, 3, and 6 in 14 ovarian cancers [9]. Chromosome 1 was the most frequently identified structurally abnormal chromosome in the current series. This finding is consistent with observations in other solid tumors, including lung, cervix, melanoma, breast, and colon cancer [24,25]. Therefore abnormalities in chromosome 1 do not appear to be site specific for ovarian cancer. The second most commonly noted karyotypic abnormality in the patients studied at this institution occurred on chromosome 11. Tumor cells contained alterations of this chromosome in 83% of patients. Although abnormalities of chromosome 11 have been previously reported, they have not been observed as frequently as in the present study [9,12]. Finally, some investigators have reported characteristic translocations or deletions inONCOGENE IGF2 Ha-m

12345 i‘ I, hi

ACTIN 12345

mm

eAB-2 c-ets FIG. 3. Representative slot blots of five ovarian tumor genomic DNAs probed with IGFZ, Ha-rrrs-I, c-e&B-2, c-cts, and human actin probe. Amplification of c-e&B-2 is evident in sample 3.

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volving chromosomes 6 and 14 in patients with papillary serous ovarian carcinoma [7,26]. More recent studies have not identified these changes [12,27]. In the present series, structural aberrations of chromosome 6 were observed in only 30% of tumors, and the specific t(6;14)(q21:24) translocation described by Wake et al. was not identified [7]. Although there has been much interest concerning the biologic potential of ovarian tumor of borderline malignancy, minimal karyotypic data on these tumors have been reported. It is of interest that neither patient from this institution with borderline epithelial ovarian cancer had an identifiable abnormality of chromosome 1 or 11. The observation that nearly all patients with invasive epithelial ovarian cancer had alterations of these chromosomes raises the possibility that genes located on chromosomes 1 and 11 may be responsible for the malignant phenotype of frankly invasive ovarian tumors. One of the most interesting aspects of chromosome analysis in tumor biology is the identification of small chromosomal abnormalities which, with gene mapping and molecular genetic techniques, may give important insights into the etiology and pathogenesis of a particular malignancy. In recent years, molecular genetic studies in human tumors have focused on the role of transforming oncogenes and, more recently, tumor suppressor genes. Studies of the N-myc proto-oncogene, frequently amplified in neuroblastomas, established a direct association between oncogene amplification and the clinical behavior of a human tumor [28]. In a subsequent investigation, Slamon and co-workers reported that amplification of the HER-2/neu (e&B-2) oncogene was superior to all other prognostic factors with the exception of positive lymph nodes in predicting relapse and survival in breast cancer patients [29]. Recent data by the same authors suggest that a similar association between amplification and overexpression of the HER-2/neu (e&B2) oncogene and prognosis may exist in patients with epithelial ovarian cancer [30]. In addition to increased gene dosage due to amplification, mutations involving oncogenes and suppressor genes are of interest in human carcinogenesis because loss of both copies of the gene may lead to the development of malignancy. For example, in familial retinoblastoma an inherited loss of one of the two Rb alleles, followed by a structural or functional loss of the remaining allele, is believed to be responsible for the high frequency of malignancies observed in some families [4]. In addition to the retinoblastoma gene, at least one suppressor gene for other human tumors has been mapped to chromosome 11 [31,32]. Investigation of abnormalities by molecular techniques in the current series was limited to DNA analysis of oncogene amplification because only archival frozen tu-

ET AL.

mor tissue not suitable for RNA analysis was available from nine patients. Insulin-like growth factor (IGF2), Haras-1, and c-ets were selected because of their location on chromosome 11 [19-211. The oncogene c-e&B-2, located on chromosome 17, was studied because of recent reports of amplification of this oncogene in epithelial ovarian cancers [30]. Evidence for amplification of c-ets, Ha-vas-1, and IGF2 was not observed. However, amplification of the c-e&B-2 oncogene was seen in 2 of the 9 tumors. Moreover, immunohistochemical staining for the protein product of c-e&B-2 indicated overexpression in 2 of 16 tumors. The findings of the present investigation indicate that chromosome aberrations in ovarian cancer are complex, with chromosome 1 and 11 being the most commonly involved. These data would also suggest that significant karyotypic differences may exist between borderline and frankly invasive epithelial ovarian tumors. Karyotypic analysis of large numbers of borderline and invasive tumors may provide insight into the pathogenesis of this biologically aggressive malignancy. Prospective studies utilizing restriction fragment length polymorphisms to provide molecular evidence of chromosome wide alterations and a search for increases in gene expression at the RNA level are needed. REFERENCES 1. DeVita, V. T., Jr., Hellman, S., and Rosenberg, S. A. Cancer principles and practice in oncology, J. B. Lippincott, Philadelphia, 2nd ed., pp. 73-77 (1985). 2. Rowley, J. D. A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining, Nature (London) 243, 290 (1973). 3. Zech, L., Haglund, U., and Nilsson, K. Characteristic chromosomal abnormalities in biopsies and lymphoid-cell lines from patients with Burkitt and non-Burkitt lymphomas, Znt. J. Cancer 17, 47-56 (1976). 4. Benedict, W. F., Fung, Y. T., and Murphree, A. L. The gene responsible for the development of retinoblastoma and osteosarcoma, Cancer 62, 1691-1694 (1988). 5. Rowley, J. D. Identification of a translocation with quinacrine fluorescence in a patient with leukemia, Ann. Genet. 16, 109-112 (1973). 6. Koufos, A., Hanse, M. F., Lampkin, B. C., Workman, M. L., Copeland, N. G., Jenkins, N. A., and Cavaness, W. K. Loss of alleles at loci on human chromosome 11 during genesis of Wilms’ tumour, Nafure (London) 309, 170-172 (1984). 7. Wake, N., Hreshchyshyn, M. M., Piver, S. M., Matsue, S., and Sandbert, A. A. Specific cytogenetic changes in ovarian cancer involving chromosomes 6 and 14, Cancer Res. 40, 4512-4518 (1980). 8. Panani, A., and Ferti-Passantonopoulou, A. Common marker chromosomes in ovarian cancer, Cancer Genet. Cytogenet. 16, 65-71 (1985). 9. Atkin, N. B., and Baker, M. Abnormal chromosomes including

CHROMOSOME ABNORMALITIES small metacentrics in 14 ovarian cancers, Cancer Genet. Cytogene?. 26, 355-361 (1987).

IO. Sheer, D., Sheppard, D. M., Gorman, P. A., Ward, B., Whelan, R. D., and Hill, B. T. Cytogenetic analysis of four human ovarian carinoma cell lines, Cancer Genet. Cytogenet. 26, 339-349 (1987). II. Smith, A., Roberts, C., van Haaften-Day, C., den Dulk, G., Russell, P., and Tattersall, M. H. Cytogenetic findings in cell lines derived from four ovarian carcinomas, Cancer Genet. Cytogenet. 24, 231-242 (1987). 12. Tanaka, K., Boice, C. R., and Testa, J. R. Chromosome aberrations in nine patients with ovarian cancer, Cancer Gene?. Cytogenet. 43, l-14 (1989). 13. Annual report on gynecologic cancer, FIGO, Stockholm, p. I6 (1976). 14. Serov, S. F., Scully, R. E., and Sobin, L. H. International his-

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15. Hendrikson. M. R., and Kempson, R. L. Surgical pathology of the uterine corpus, in Major problems in pathology (J. L. Bennington, Ed.), W. B. Saunders, Philadelphia, p. I2 (1980). 16. Gross-Bellard, M.. Oudet P., and Chambon, P. Isolation of highmolecular-weight DNA mammalian cells, Eur. J. Biochem. 36, 3238 (1973). 17. Southern, E. Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Viol. 98, 503-517 (1975). 18. Kafatos, F. C., Jones, C. W., and Effratiadis, A. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure, Nucl. Acids Res. 7, 1541-1552 (1979). 19. Human Gene Mapping 8. 8th International Workshop on Human Gene Mapping. Cytogenet. Cell Gene?. 40, l-4 (1985). 20. Bell, G. I.. Gerhard, D. S., Fong, N. M., and Sanchez-Pescador, R. Isolation of the human-like growth factor genes: Insulin-like growth factor II and insulin genes are contiguous. Proc. Nat/. Acad. Sci. USA 82, 6450-6454 (1985). 21. de Taisne, C., Gegonne, A., and Stehelin, D. Chromosomal lo-

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calization of the human proto-oncogene c-ets, Nature (London) 310, 581-583 (1984). Fukushige, S., Matsubara, K., Yoshida, M., Sasaki, M., Suzuki, T., Semba, K., Toyoshima, K., and Yamamoto. Localization of a novel v-erbB-related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line, Mol. Cell. Biol. 6, 955-958 (1986). Rowley, .I. D. Human oncogene locations and chromosome aberrations, Nature (London) 301, 290-291 (1983). Kovacs, G. Abnormalities of chromosome No. I in human solid malignant tumours, Inf. J. Cancer 21, 688-694 (1978). Rowely, J. D. Mapping of human chromosomal regions related to neoplasia: Evidence from chromosomes I and 17, Proc. Nat/. Acad. Sci. USA 74, 5729-5733 (1977). Trent, J. M., and Salmon, S. E. Karyotypic analysis of human ovarian carcinoma cells cloned in agar, Amer. J. Hum. Genet. 31, 112A (1979). Whang-Peng, J., Knutsen, T., Douglass, E. C., Chu, E., Ozols, R. F., Hogan, W. M., and Young, R. C. Cytogenetic studies in ovarian cancer, Cancer Genet. Cytogenef. 11, 91-106 (1984). Seeger, R. C., Brodeur, G. M., Sather, H., Dalton, A., Siegel, S., and Wong, K. Associated of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas, N. Engl. /. Med. 313, 1111 (1985). Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., and McGuire, W. L. Human breast cancer: Correlation of relapse and survival with amplification of the HER-Z/neu oncogene, Science 235, 177 (1987). Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, .I., Ullrich, A., and Press, M. F. Studies of the HER-2/neu proto-oncogene in human and breast cancer, Science 244, 707 (1989). Ali, I. U., Lidereau, R., Theillet, C., and Callahan, R. Reduction of homozygosity of genes in chromosome 11 in human breast neoplasia, Science 221, 185-188 (1987). Misra, B. C., and Srivatsan. E. S. Localization of HeLaceIl tumorsuppressor gene to the long arm of chromosome 11, Amer. J. Hum. Genet. 45, 565-577 (1989).

Chromosome abnormalities in human epithelial ovarian malignancies.

Karyotypic analysis of tumor specimens from 29 patients with untreated epithelial ovarian carcinoma was performed at the University of Kentucky Medica...
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