CANCER RESEARCI-155.6172-6180.December 15. 19951 Genetic Analysis of Benign, Low-Grade, and High-Grade Ovarian Tumors' Hiroshi Iwabuchi, Masaru Sakamoto, Hotaka Sakunaga, Yen-Ying Ma, Maria L. Carcangiu, Daniel Pinkel, Teresa L. Yang-Feng, and Joe W. Gra? Department of Laboratory Medicine, Division of Molecular C'ytometry, University of C'alifornia, San Francisco, San Francisco, @alzfornia94143-0808 (H. I., M. S., H. S., D. P., .1. W. G.J: Department of Gynecology, Sasaki Institute, Kyoundo Hospital, 1-8, Kanda-Surugadai @hiyoda-Ku,Tokyo,101 Japan [H. 1.. M. S., H. S.]; and Department of Genetics [Y-Y. M., T. L Y-F.J, and Department ofPathology [M. L C.], Yale University, New Haven, Connecticut 06510-8005 ABSTRACT abnormalities may be diagnostically important, whereas information about later abnormalities may guide therapy. Genetic changes asso Genetic abnormalities were assessed in 56 benign, low-, and high-grade ciated with progression that may be used for tumor characterization ovarian tumors using comparative genomic hybridization (CGH) and are beginning to be defined for ovarian cancer. Consistent chromo analysis ofloss of heterozygosity (LOH). In addition, 95 epithelial tumors were analyzed for microsatellite repeat instability. DNA sequence copy somal abnormalities detected by karyotyping and consistent genetic number abnormalities (CNAs) were not detected in the benign tumors, abnormalities detected in molecular analyses of ovarian cancers are and more were detected in high-grade than in low-grade cancers. Almost reviewed by Pejovic (4). Regions of frequent LOH3 are summarized no microsatellite repeat instability was detected in these cancers. CNAs by Osborne and Leech (5) and Yang-Feng et a!. (6). Some events, occurring in more than 30% of the cancers included increased copy such as atypical expression or overexpression of egf, fins, and HER number on 3q25—26and 8q24 and reduced copy number on 16q and 2/neu, have been associated with poor prognoses (7—9).However, the l7pter—q21.Another 14 CNAs occurred in more than 20% of the cancers. range of genetic abnormalities that are involved in ovarian cancer Increased copy number at 3q25—26and20q13 was the most frequent CNA progression, their frequency of occurrence, and their clinical and in low-grade tumors, and increased copy number at 8q24 occurred pref biological significance remain poorly understood. erentially in high-grade tumors. The presence of a large number of CNAs In this study, we have further defined the spectrum of genetic per tumor was significantly correlated with reduced patient survival duration. Reduced copy number on l7pter—q21was most strongly mao abnormalities associated with ovarian cancer, investigated the mech ciated with accumulation of a large number of CNAs. The overall con anisms by which the abnormalities occur, assessed their clinical cordance between LOH and reduced copy number detected by CGH was importance, and identified abnormalities that may be associated with 84%, but only 31% ofthe LOH was associated with reduced copy number cancer progression. To accomplish this, we mapped gene dosage detected using CGH. abnormalities in tumors of various grades using analysis of LOH (6, 10) and CGH (1 i). CGH was particularly useful because it allowed INTRODUCTION genome-wide mapping of regions of altered copy number (both in creases and decreases from the tumor average) in a single experiment The ovary is the fifth most common site of the cancer among without prior knowledge of the locations of regions of abnormality. American women, and ovarian cancer is the fourth leading cause of The comprehensive nature of CGH facilitated correlative analysis of cancer death ( 1). Among patients with pelvic reproductive cancer, the interactions between abnormalities and exploration of the biolog more deaths occur from ovarian cancer than from cervical and uterine ical and clinical consequences of the various abnormalities. cancers combined. The survival rate for ovarian cancer varies consid erably, depending on the stage at diagnosis. The fractions of patients surviving 5 years with ovarian cancers diagnosed as Federation In MATERIALS AND METHODS ternationale des Gynaecologistes et Obstetristes stage I (limited to the ovaries) or stage II (limited to pelvic metastases) are 0.89 and 0.57, Tumor Material. Samples from primary ovarian tumors were obtained from surgical specimens taken at Yale University Hospital, along with periph respectively. These fractions fall to 0.24 and 0.12 for patients with era! blood from the same patient. Material was promptly frozen at —70°Cuntil stage III and stage IV disease, respectively (2). The high mortality rate the time of DNA extraction. Tumor DNA and normal DNA were prepared as of ovarian cancer reflects the fact that disease is usually detected late described previously (6, 10). All tumors were assigned a histological subtype (—70%of all women with common epithelial cancer have stage III or and grade. Fifty-six ovarian tumors were analyzed for LOH and copy number stage IV cancer at the time of diagnosis). Thus, clinical stage is used karyotype using CGH. The cancers analyzed included 26 grade III cancers (21 routinely for management of patient treatment. Histological grade is serous cystoadenocarcinomas, 4 endometrioid carcinomas, and 1 mixed epi also used because well-differentiated, low-grade ovarian cancers thelial carcinoma), 12 grade II cancers (7 serous cystoadenocarcinomas and 5 have better clinical prognoses than do high-grade ovarian cancers endometrioid carcinomas), and 6 grade I cancers (I mixed epithelial (3). However, improved tumor classification is needed because carcinoma, 2 serous adenocarcinomas, and 3 mucinous adenocarcinomas). patients with tumors that are identical in grade and stage may have The benign tumors were composed of 10 serous adenomas and 2 mucinous adenomas. Grade I and grade II epithelial ovarian cancers are referred to as significantly different clinical outcomes and/or responses to low-grade cancers in this study, and grade III epithelial ovarian cancers are therapy. referred to as high-grade cancers. In addition, 95 epithelial tumors (3 One approach to improving diagnosis and/or treatment is to classify benign, 9 borderline, 4 grade I, 14 grade II, and 55 grade III) were tumors according to the genetic abnormalities that they contain. Early examined for microsatellite repeat instability. All tumor samples were trimmed using histological criteria to exclude normal cells as described Received 7/19/95; accepted 10/16/95. previously (11, 12). The costs of publication of this article were defrayed in part by the payment of page CGH. CGH was performedusing DNA extractedfrom peripheralblood charges. This article must therefore be hereby marked advertisement in accordance with lymphocytes and trimmed tumor samples. Isolated tumor DNA was labeled 18 U.S.C. Section 1734 solely to indicate this fact. with biotin-i4-dATP and normal DNA was labeled with digoxigenin-ll I This work was supported by Vysis, Inc., the E. 0. Lawrence Berkeley National Laboratory/University of California, San Francisco Resource for Molecular Cytogenetics dUTP. Sixty ng of each of the labeled DNAs plus 5 @gofunlabeled Cot-l under United States Department of Energy contract DE-AC-03—76SF00098, and Sasaki DNA were mixed and hybridized to normal metaphase spreads for 3—4days. Institute Kyoundo Hospital. 2 To whom requests for reprints should be addressed, at Division of Molecular Cytometry, Department of Laboratory Medicine, MCB 230, Box 0808, University of 3The abbreviations used are: LOH, loss of heterozygosity; CGH, comparative Califomia, San Francisco, CA 94143-0808. genomic hybridization; CNA, copy number abnormality. 6172 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1995 American Association for Cancer Research. GENETIC ANALYSIS OF OVARIAN CANCERS The preparations were washed to remove unbound DNA and stained with by visual inspection of the CGH images. CNAs were not scored for the Avidin-FITC (green fluorescence) to detect biotinylated tumor DNA and with repeat-rich regions at or near the chromosome centromeres (especially anti-digoxigenin-Rhodamine (red fluorescence) to detect digoxigenin-labeled chromosomes 1, 9, 13—16,21, and 22) because CGH analyses are unreliable normal DNA. The chromosomes were counterstained with 4',6'-diamidoino in these regions. z-phenylindol for chromosome identification. Three color images from the LOH and Microsateffite Repeat Instability. Tumorswere assessed for metaphase spreads were acquired using a Quantitative Image Processing LOH at 40 loci distributed as illustrated in Fig. 1. Some loci were studied by System (QUIPS) as described earlier (13). Individual chromosomes were analysis of RFLPs. RFLP analyses were performed as described previously (6). segmented, local background was subtracted, the medial axes were defined, Briefly, high-molecular weight DNA was isolated from peripheral blood and red and green fluorescence intensity profiles were calculated by integrating lymphocytes or ovarian tumors. The DNA was digested using restriction fluorescence values across the chromosome widths along the medial axes. endonucleases, sized using agarose gel electrophoresis, transferred to a mem Approximately five different metaphases were analyzed for each hybridization. brane, and hybridized to a 32P-labeledDNA probe. The hybridized probe was Green:red fluorescence ratio profiles for four to six chromosomes of the same detected autoradiographically.