Genetic Analysis of Benign, Low-Grade, and High-Grade Ovarian Tumors'

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 3q25â€”26and 8q24 and reduced copy number on 16q and 2/neu, have been associated with poor prognoses (7â€”9).However, the l7pterâ€”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 3q25â€”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 l7pterâ€”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 â€”70Â°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 (â€”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-03â€”76SF00098, and Sasaki DNA were mixed and hybridized to normal metaphase spreads for 3â€”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

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, 13â€”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. Quantification of the hybridization signals was type were normalized to standard length and combined statistically to show the performed with a laser scanning densitometer. A reduction of more than 50% mean and SD of the ratio. A region was considered to be significantly relative to the normal signal was recorded as allelic loss. increased in DNA sequence copy number relative to the tumor average Other loci were assessed for LOH and/or microsatellite repeat instability by when the mean of the green:red ratio was above 1.25 and significantly PCR amplification of polymorphic sequences using commercially available decreased in copy number when the mean of the green:red ratio was below primers (Research Genetics). The PCR reaction mixture consisted of TaqI 0.75. These excursions are referred to as CNAs. All CNAs were confirmed DNA polymerase, 200 ,zM dNTP, 1â€”2mM MgC12, and reaction buffer (Gene

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(42%) . (20%) (0%) 20 21 22 0/5 19 (0%) (11%) 5/13 18 1@4/14 (38%) @â€”(29%) 4/20 (20%) (13%)1/1415 1314[2/16 x Fig. 1. Chromosomal locations of 40 regions tested for LOH. The bounded regions show the areas of the genome to which the polymorphic markers map. The number of loci showing LOH divided by the number of informative loci is shown for each locus. 6173

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fl:@ I @ Fig. 2. Copy number karyotype measured using CGH for a high __k@ @râ€”@_ ::: grade ovarian cancer. Mean green:red ratios (heavy lines) Â± l@ 61@â€” @i (light lines) are shown for each chromosome. , ratios of I .25 and 0.75. Bars at the bottom of each chromosome axis show regions scored as abnormal. Heavy bars, regions of increased relative copy rt@@- number; light bars, regions of reduced relative copy number. 7 __ @@::::@@

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Amp kit). Typically, one primer was end labeled with [32P}dATP.PCR was all regions of the genome were analyzed for LOH with equal resolu carried out for 25â€”35 cycles. The exact thermal cycle profile was adjusted for tion. Thus, it is likely that sites of consistent LOH have been missed. each primer pair; usually 30 s to 1 mm at 94Â°C,30 s to 2 mm at 50â€”60Â°C,and The overall concordance between reduced copy number detected 30 s to 1 mm at 72Â°C.An aliquot of the amplified DNA was denatured and using CGH and LOH was 0.84. However, the concordance ranged separated on a 6â€”8% polyacrylamide gel in Tris-borate buffer (0.089 M Tris-borate/boric acid-0.002 MEDTA) at 200â€”300Vfor 3â€”6h,depending on from 1.0 to 0.56, depending on the locus. Only 27 of 87 loci showing the fragment size. The gel was then exposed to KOdakXAR-5 film for 4â€”6h. LOH also showed reduced DNA sequence copy number when ana When a microsatellite repeat fragment appeared to be altered in size from that lyzed by CGH. Thus, only 31% of the LOH in ovarian cancer is in the matched normal sample, the experiment was repeated at least twice. In clearly attributable to physical deletions detectable by CGH. We addition, the autoradiographic films were purposely overexposed to increase investigated the concordance between CGH and LOH in more detail confidence that the band shift was due to a change in the size of the micro for chromosomes i3q and Xp. The results of analysis at 6 loci on satellite repeat rather than to a PCR artifact. chromosome 13q for 34 tumors and analysis of 6 loci on Xp for 35 tumors are shown in Fig. 4. The fraction of loci showing both LOH RESULTS and reduced DNA sequence copy number was 0.65 for chromosome Commonly Occurring Abnormalities. CNAs were mapped using Xp but only 0.08 for chromosome 13q. CGH in 26 high-grade, 18 low-grade, and 12 benign tumors. A CGH Microsatellite Repeat Instability. Ninety-five tumors were ana copy number karyotype and CNA assignments typical of a high-grade lyzed for genetic instability by PCR amplification of 66 different tumor are shown in Fig. 2. CNAs were not detected in any of the polymorphic microsatellite repeat markers. At least one marker benign tumors. Those detected for the low- and high-grade cancers are was located on every chromosome arm, except for the short arms summarized and compared in Fig. 3 and Table 1. CNAs appearing in of the acrocentric chromosomes. The 66 markers tested were >30%of all cancersarereferredtoasclass1abnormalities. composed of 62, 2, and 2 di-, tn-, and tetranucleotide repeats, LOH versus CGH. Thirty-nine tumors were analyzed for CNAs respectively. In all, 5039 loci were tested in the paired tumor using CGH and for LOH using 40 precisely mapped polymorphic normal samples (not every tumor was tested at each marker). The markers. In total, 512 loci in the LOH study were informative, and 87 normal and tumor pairs appeared identical in all but six tests, showed LOH. These results are summarized in Table 2 and Fig. 1. Not indicating that microsatellite repeat mutations are rare in ovarian 6174

GENETIC ANALYSIS OF OVARIAN CANCERS

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Fig. 3. Summary of CNAs found during analysis of 44 ovarian cancers. Lines to the left of each chromosome ideogram show regions of reduced relative copy number, and lines to the right show regions of increased relative copy number. . data for high-grade tumors; - , data for low-grade tumors. Each line shows a CNA found in one tumor. CNAs were not scored near the chromosome centromeres because CGH measurements are not reliable in these regions.

Table I Su@nman of I8 cancersHighcommon C'NAs foundin 44 osarian tumors, respectively. Table I shows that most of the common genetic gradeCNA gradeLow abnormalities occurred both in low- and high-grade tumors, but the frequencies in low-grade tumors were typically less than 20%. The (%)+and regionNo.Frequency (%)No.Frequency exceptions were increased copy number abnormalities at 3q26 and 1p22â€”31831211+ 1q25â€”3172716+ 20q13, which occurred in 28 and 22%, respectively, of low-grade 2q32â€”3372700+ tumors. Increased copy number abnormality at 8q24 occurred prefer 3q25â€”261350528+5p62316+ entially in high-grade tumors (i.e., an incidence of 58% of high-grade

6p22â€”2562300+7q22â€”3162300â€” tumors versus 11% of low-grade tumors). Correlations with Survival Duration. Studies of node-negative 8p2lâ€”2372700+8q24IS58211+llql4â€”2283116+l2pl2623317+ breast cancers have suggested that patients with tumors with many CNAs have a shorter survival duration than patients whose tumors have few abnormalities (14). This is consistent with the hypothesis 13q3lâ€”346232IIâ€”16q1038317â€” that tumors accumulate CNAs because they are genetically unstable,

l7pter-q211246317+ and that unstable tumors are able to progress most rapidly. We tested 18q12â€”2272716â€”19727317+20q13519422â€”Xp7272II this possibility for ovarian cancers by assessing survival duration for patients with >10 and <5 CNAs/tumor and with and without one or more class 1 abnormalities. The survival durations were shorter for

cancers. Autoradiographs showing the six microsatellite repeat Table 2 Comparison of analysesfor512 of LOH and reduced relative DNA copy number mutations are shown in Fig. 5. cancersThese loci in 39 ovarian asnot data include 5 cases with LOH and increased relative copy number (scored Correlation with Grade. The change in CNA frequency and lo decreased).Decreased cation with grade was assessed by measuring CGH copy number innumberin copy Not decreased karyotypes for benign, low-, and high-grade tumors. Fig. 6a shows lociLOH copy number Total that the total number of CNAs clearly increases with grade. Specifi 87No 27 60 cally, the average total numbers of CNAs per tumor (Â± 1 u) were 425TotalLOB 20 405 0.0 Â±0,5.4 Â±7,and 11.2Â±10for benign,low-,andhigh-grade regions 47 465 512 6175

GENETIC ANALYSIS OF OVARIAN CANCERS

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Fig. 4. Comparison of results obtained using CGH and analysis of LOB on chromosomes l3q (a, 34 tumors) and Xp (b, 35 tumors). Solid lines, regions of reduced relative copy number detected using CGH; gray lines, regions of increased copy number detected using CGH; 0, regions with no LOH; â€¢,regions with LOH. Loci tested for LOH are shown in the lower right portions of the panels.

patients with tumors with more CNAs and with one or more class 1 was high. Therefore, this stratification requires validation in a larger abnormality, as illustrated in Fig. 7, a and b. However, the difference study. was statistically significant only between patients with and without Correlations between Abnormalities Detected Using CGH. one or more class 1 abnormality (Cox-Mantel; P < 0.05; n = 40). Table 3 shows that there are numerous statistically significant corre One important identifier of specific genetic events that contribute to lations between the common CNAs (especially between the class 1 disease progression is correlation between those events and indicators abnormalities). That is, many of the frequently occurring abnormali of clinical outcome. Correlative studies of the 18 most common ties seem to occur together in the same tumor. In addition, the class 1 abnormalities detected using CGH in this study identified reduced abnormalities are associated with a high number of CNAs per tumor. relative copy number on 16q as one event associated with reduced For example, the average number of CNAs per tumor for tumors with survival duration (Fig. 7c). However, the association is not strong, and one or more class 1 abnormalities is 15.0 Â±7.8, whereas that for the number of cases analyzed was small. In addition, because abnor tumors without any class 1 abnormalities is 0.6 Â±0.9 (Mann malities at 18 different regions of the genome were tested for corre Whitney; P < 0.01). The total number of CNAs is highest for tumors lation with survival, the possibility of finding a correlation by chance with reduced copy number involving l7pterâ€”q2l (19.1 Â±6.2). Fig. 6,

TN T N TN TN T N

Fig. 5. Autoradiographs showing microsatellite repeat mutations detected at D5S433 (Lane 1), 1.365283 (Lane 2), D7S496 (Lane 3), D10S197 (Lane 4), D115901 (Lane 5) AND VWF (Lane 6). T, tumor DNA; N, normal lymphocyte DNA; arrows, mutant alleles.

1 2 3 4

6176

GENETIC ANALYSIS OF OVARIAN CANCERS

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I 8

High grade -Lowgrade Benign

@@@@ 35. - 3q24@:q2ÃgaIn 8q24gaIn 16qlou 17p-q21loss(b) @@@ 30 c::1

@ UI c:D 4 @@ z 25 C.) Fig. 6. a, distribution of the total number of CNAs 20T T V in benign, low-grade, and high-grade ovarian tumors. b, distribution of the class I abnormalities in high @@ grade ovarian cancers. c, distribution of class 1 ab 15 normalities in low-grade ovarian cancers. 0, does not carry the specified abnormality; â€¢,does carry the 10 â€¢ S specified abnormality. @ I 5 S O-9E@-E3E3@ @ Highgrade

.@ 10 0 @ I- S 8 8 5.

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b and c, shows the association between the total number of CNAs and gene dosage. Many, however, may have accumulated by chance the various class 1 abnormalities for the low- and high-grade tumors. during clonal evolution of genetically unstable tumors. The CNAs that do contribute to progression in ovarian cancer are likely to be those that occur frequently in ovarian and other cancers and/or that DISCUSSION correlate with clinical outcome or biological behavior. The class 1 Candidate Oncogenes and Tumor Suppressor Genes. This abnormalities are of particular interest because of their high prey study of 56 benign, low-grade, and high-grade ovarian tumors alence and because they have all been observed at high frequency using CGH and analysis of LOH showed 18 regions of abnormality in one or more other tumor types. These regions contain several that occurred in more than 20% of ovarian cancers. Some of these known genes that may contribute to development or progression in may contribute to cancer progression through activation of onco ovarian cancer when differentially expressed. For example, re genes, inactivation of tumor suppressor genes, or change in relative duced copy number on l7pterâ€”q21 may contribute to disregulation 6177

GENETIC ANALYSIS OF OVARIAN CANCERS

1.0 phate kinase NM23 (20). Reduced copy number on l6q may lull! (a) @ .L...l..L..il I facilitate inactivation of the cell adhesion molecule E-cadherin .L JJ.J CNAs <5 (21) and the cell adhesion regulator CMAR (22). Increased copy number on 8q24 may lead to overexpression of the transcription factor CMYC (23), the SRC family tyrosine kinase LYN (24), the 0.5 DNA-binding protein MYBLI , and the serine-threonine kinase I j Ii CNAs>10 MOS (25), whereas increasedcopy number at 3q25â€”26mayaffect expression of EVIl (26) and the tyrosine kinase receptor RYK (27). Of course, the regions defined by CGH typically extend over 0 several megabases and contain many genes of unknown function. Thus, definitive identification of the genes affected by these and 0 1.0 J..J.L_.JLUL; LI! J....I.J.L.IL I 1._I (b) other less frequent CNAs will require that these regions of abnor @t No Class 1 ab a â€˜IL II...! mality be defined more precisely.

@I. @i I.' Reduced Copy Number and LOH. We analyzed 39 tumors for CNAs using CGH and for LOH using 40 precisely mapped polymor phic markers to assess the extent to which LOH might be caused by physical deletions detected using CGH. The overall concordance Class 1 ab between LOH and reduced copy number determined using CGH was 0.84. However, this strong concordance was driven by the large 101 number of regions showing no CNA or LOH. The concordance was @@ j_J.L..JLU ______only 0.31 in regions showing LOH. Thus, only this fraction of the 1.0 LOH in ovarian cancer is clearly attributable to physical deletions detected using CGH. This may explain why the spectrum of aberra tions defined by analyses of LOH (5, 6) is different from that defined

@ ! : LI_I by analysis of CGH. In addition, the concordance varied across the @ 0.5 Nolossl6q genome. The concordance between LOH and CGH appears high in t 11 regions in which LOH and reduced copy number occur as a result of Loss 16q extended physical deletions that are reliably detected using CGH. Fig. 4a shows that this occurs on Xp. The concordance between LOH and CGH is lower when the regions of LOH are interspersed with regions 0 1 2 3 4 5 6 that do not show LOH. Fig. 4b shows that this occurs on chromosome Time(years) 13q. Genetic Instabilityand CNA Formation.The totalnumberof Fig. 7. Kaplan-Meier curves showing survival for patients with ovarian cancer. a, CNAs may be considered a measure of the degree of genetic insta survival for patients with tumors having >10 CNA versus survival for patients with tumors having <5 CNAs. b. survival for patients with tumors having one or more class 1 bility, using the logic that unstable tumors will accumulate more abnormalities versus survival for patients with tumors having no class 1 abnormality. c, abnormalities as they proliferate more than stable tumors. The varia survival for patients with tumors having reduced copy number at l6q versus survival for patients with tumors having normal copy number at l6q. tion in the number of abnormalities per tumor is remarkable in this study, ranging from 0 to >30 for high-grade tumors and from 0 to >20 for low-grade tumors. The detection by CGH of 0 abnormalities in or inactivation of p53 ( I5) and/or a tumor suppressor gene distal to these tumors does not appear to be an artifact resulting from normal p53 (16), BRCAI (17); the GTPase-activating protein NFl (18); the cell contamination, because each tumor of this type showed LOH for retinoic acid receptor RARA (19); and/or the nucleotide diphos at least one locus tested. Genetically unstable tumors also may be

Table 3 Correlations between the CNAs found in ovarian cancers using CGH The numbers in the boxes show the significance of the correlations between the 18 most frequent CNAs. The band locations for the CNAs on the various chromosome arms are the same as those in Table I.

+ Ip + lq + 2q + 3q + 5p + 6p + 7q â€”8p + Sq + I lq + l2p + l3q â€” l6q â€” l7p, q + l8q â€” 19 + 2Oq â€”Xp +lpNAa+ lq<0.01NA+2q0.01NA+

3q<0.010.040.01NA+5p0.01NA+

6p0.02+ NA 7q0.040.030.04NAâ€” 0.03 8p<0.010.010.04NA+ 0.04 8q0.03<0.01<0.01<0.010.03<0.01NA+ <0.01 Ilq<0.010.02<0.010.01NA+ 12p0.04NA+ l3q0.030.010.010.03NAâ€”

16q0.030.020.02<0.010.02<0.01<0.01NAâ€”

l7p.q<0.01<0.01<0.01<0.01<0.010.020.02<0.010.05NA+ l8q<0.010.010.010.020.03NAâ€”

19<0.01<0.01<0.01<0.01<0.01<0.01<0.010.04NA+20q<0.01NAâ€”

Xp<0.010.020.02<0.010.020.020.010.020.03 NA

a NA, not applicable. 6178

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1995 American Association for Cancer Research. GENETIC ANALYSIS OF OVARIAN CANCERS expected to advance more rapidly than more stable tumors. This was ACKNOWLEDGMENTS observed in this study (Fig. 7). Patients whose tumors had many The authors gratefully acknowledge the continuing support of Drs. Y. CNAs survived for significantly shorter periods than patients whose Tenjin and T. Sugishita. tumors showed few CNAs. Several genetic abnormalities have already been identified in one or REFERENCES more tumors that are associated with genetic instability. These include cell cycle checkpoint genes such as p53 (28) and DNA repair genes I. Phyllis, A., Tony, 1., and Sherry, B. Cancer statistics. CA Cancer i. Clin., 45: 8â€”30, 1995. such as hMSH2 (29). However, little is known about mechanisms of 2. Nguyan, H. N., Averette, H. E., Hoskins, W., Sevin, B. 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Hiroshi Iwabuchi, Masaru Sakamoto, Hotaka Sakunaga, et al.

Cancer Res 1995;55:6172-6180.

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