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[Cancer Research 63, 1235-1241, March 15, 2003]
© 2003 American Association for Cancer Research

Clinical Investigations

Cyclin E Expression Is a Significant Predictor of Survival in Advanced, Suboptimally Debulked Ovarian Epithelial Cancers: A Gynecologic Oncology Group Study1

John Farley2, Leia M. Smith2, Kathleen M. Darcy, Eugene Sobel, Dennis O’Connor, Benita Henderson, Larry E. Morrison and Michael J. Birrer3

Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Tripler Army Medical Center (TAMC, HI), 96859-5000 [J. F.]; Department of Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523-1673 [L. M. S.]; Gynecologic Oncology Group Statistical and Data Center, Roswell Park Cancer Institute, Buffalo, New York 14263 [K. M. D., E. S.]; Norton Healthcare, Inc., Clinical Pathology Associates, Louisville, Kentucky 40207 [D. O.]; Vysis, Inc., Downers Grove, Illinois 60515 [L. E. M., B. H.]; and Cell and Cancer Biology Department, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Rockville, Maryland 20850 [M. J. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclin E is a key regulator of the G1-S transition. Abnormalities in cyclinE expression have been related to survival in a variety of cancers. Thisstudy evaluated the prognostic relevance of cyclin E in human ovarian cancer. Immunohistochemical expression of cyclin E was evaluated in 139 advanced, suboptimally debulked epithelial ovarian cancer specimens from patients treated on Gynecologic Oncology Group protocol 111. High cyclin E protein expression (>=40% cyclin E positive tumor cells) was seen in 62 (45%) of the advanced, suboptimally debulked ovarian cancer patients. Expression of cyclin E was not associated with age, race, stage, grade, cell type, or amount of residual disease. High verses low cyclin E expression was associated with a shorter median survival (29 ± 2 versus 35 ± 3 months) and worse overall survival (P < 0.05). Univariate and multivariate regression analyses revealed that high relative to low cyclin E was associated with a 40–50% increase in the risk of death (hazard rate, P <= 0.05). Fluorescence in situ hybridization was used in a subset of 20 cases to examine cyclin E gene amplification. Eight of 10 cases with high cyclin E expression exhibited amplification of the cyclin E gene, whereas only 1 of 10 cases with low expression displayed gene amplification (P < 0.006). High cyclin E expression was an independent poor prognostic factor for patients with advanced ovarian cancer, and it was associated with amplification of the cyclin E gene.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the United States, ovarian cancer is the fifth most common cause of cancer death among women and the leading cause of death from gynecologic malignancy (1 , 2) . It is estimated that 23,300 new cases of ovarian cancer will be diagnosed in the United States in 2002 and that 13,900 women will die from disease (1 , 2) . Ovarian cancer has been associated with a high lethality because it is most frequently diagnosed at an advanced stage when the potential for cure is often remote (1 , 2) . Despite improvements in the treatment of advanced disease, survival has not changed appreciably since the inception of platinum-based chemotherapy (1 , 2) . Thus, new and novel agents are needed for the treatment and prevention of this disease. Identification of such agents will require an understanding of ovarian cancer’s molecular origins and abnormalities.

All dividing cells proceed orderly through the cell cycle (3) . The central regulators of the cell cycle progression include the CDKs4(3) and the small inhibitory proteins called CDK inhibitors. During the G1-S transition, the cyclin E/CDK2 and cyclin D/CDK4 complexes promote progression and are each inhibited by the associated CDK inhibitor p27 (Kip1; Refs. 3, 4, 5, 6, 7, 8, 9 ). Alterations in the levels and activity of the components of these complexes regulate the G1-S transition. Cyclin E is a key player in this process and is therefore an ideal target for evaluation of altered expression in a variety of cancers. Normal cell cycle regulation of cyclin E has been found to be altered in breast cancer cells (10 , 11) . In non-small cell lung cancers, increased cyclin E expression was found to be an independent prognostic factor for survival along with tumor size, node status, and stage (12) .

To determine whether cyclin E expression has prognostic significance in ovarian cancer, we analyzed by immunohistochemical staining the expression of cyclin E in 139 suboptimally debulked AOC specimens.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Preparation and Histological Evaluation.
Archived, paraffin embedded tissue blocks were obtained from GOG participating institutions under active institutional review board approved protocols. Appropriate pathology blocks were provided from 139 cases of primary AOC from women who participated on GOG experimental medicine protocol 9404 along with treatment protocol 111 (13) . Samples were collected at the time of primary cytoreductive surgery and before initiation of first-line chemotherapy and were linked to clinical data as part of their treatment protocol; however, analyses of cyclin E expression was performed without knowledge of the clinical data. Serial 5-µm sections of the tumor were obtained from each tissue block, with the first slide being stained for H&E to confirm pathologic diagnosis and subsequent slides stained for cyclin E or control antibody. All operative and pathologic reports were reviewed to confirm histology and International Federation of Gynecology and Obstetrics stage of ovarian adenocarcinoma.

Immunohistochemistry.
Detection of cyclin E was performed using a mouse monoclonal antibody, antiserum HE-111 (Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, before immunostaining, the deparaffinized tissue sections were microwaved in 0.01 M citrate buffer (pH 6.0) for a total of 15 min. The specimens were incubated overnight at 4°C with primary antibody (1:1000) or control antibody (MOPC 21, mouse IgG1 {kappa}, Sigma Company, St. Louis, MO) at a dilution of 1:1000. Detection of primary antibody was performed using the appropriate biotin-streptavidin-peroxidase detection system (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine as a substrate. Background staining was evaluated using the control antibody in place of primary antisera and found to be negligible (data not shown). Stromal cells were used as internal controls for positive staining. Immunohistochemical staining was evaluated by visual counting of the cells from at least 10 random high-powered fields (x100) with a minimum requirement of 1000 cells counted. The immunohistochemical results for cyclin E were expressed as a percentage of tumor cells that exhibited any staining using the HE-111 antibody, regardless of intensity. The reviewer (J. F.) was blinded as to stage, grade, and histology of tumor. Random specimens were selected for confirmation of analysis by a second blinded observer (L. S.).

FISH Procedure.
FISH was performed on 4–5-µm thick sections of formalin-fixed paraffin-embedded ovarian specimens mounted on glass microscope slides. Before hybridization, the slides were baked overnight at 56°C and then deparaffinized by soaking in Hemo-De Solvent and Clearing Agent (Fisher Scientific, Pittsburgh, PA) three times, for 5 min each, followed by absolute ethanol two times for 1 min each. After drying, the slides were placed in Vysis Pretreatment Solution at 80°C for 10 min, rinsed in water for 5 min, immersed in a solution of 2 mg of pepsin (2500–3000 units/mg) per ml 0.2 N HCl at 37°C for 15 min, rinsed in water for 3 min, and dehydrated in 70, 85, and 100% ethanol 1 min each. After drying, 10 µl of probe mixture was placed on the slide over the tissue, a coverslip applied, and the coverslip sealed with rubber cement. The specimen and probe were codenatured in a HYBrite oven (Vysis, Inc.) set for denaturation at 73°C for 5 min and hybridization at 37°C for 16 h. After hybridization, the rubber cement was removed and the slide soaked in 2x SSC/0.3% NP40 at room temperature for several minutes until the coverslip fell off. The slides were then washed in 2x SSC/0.3% NP40 at 73°C for 2 min and rinsed in 2x SSC/0.3% NP40 at room temperature. After drying, 10 µl of 4',6-diamidino-2-phenylindole I counterstain/antifade solution (Vysis, Inc.) were applied, the coverslip added, and the specimen stored at -20°C or viewed immediately on a fluorescence microscope. Specimens were viewed using 4',6-diamidino-2-phenylindole, SpectrumOrange, and SpectrumGreen filter sets (Vysis, Inc.). A FISH probe for the cyclin E gene (CCNE1) was prepared from a single BAC clone containing the CCNE1 gene at 19q12 and labeled with SpectrumOrange. To control for aneusomy, a probe on the opposite arm of chromosome 19 was also prepared from a single BAC clone containing the insulin receptor gene (INSR) at 19p13.2 and labeled with SpectrumGreen. For each specimen, the number of CCNE1 signals and chromosome 19 (INSR signals) were counted using a fluorescence microscope, in each of 30 randomly selected nonoverlapping nuclei. A ratio of the number of copies of the cyclin E gene/copy of chromosome 19 (ratio of CCNE1-to-INSR signals per cell) >=0.5 but <1.5 was considered nonamplified for the cyclin E gene, whereas a ratio >1.5 indicated amplification and a ratio <0.5 indicated a deletion of the cyclin E gene.

Statistical Methods.
Biomarker and clinical data were analyzed using SPSS version 10.0 (SPSS, Inc., Chicago, IL). SigmaPlot 2000 (SPSS, Inc.) was used for graphing. All biomarker data used for the statistical analyses reflected immunohistochemical results expressed as a percentage of tumor cells expressing any staining with the antiserum against cyclin E regardless of intensity. The hypothesis of independence between biomarker expression and a categorical covariate such as race, initial performance status, amount of residual disease after primary debulking surgery, or type of chemotherapy was tested using Fisher’s exact test (14) . The association between biomarker expression and a variable with more than two categories such as tumor cell type was estimated using Pearson’s {chi}2 test (14) . Ranked covariates such as patient age, tumor stage, and histological grade were evaluated using the Kruskal-Wallis nonparametric rank test (15) . All tests were two-sided and the level of significance was set at 0.05.

Overall survival time was calculated as the time in months from enrollment in GOG 111 to death for noncensored events or to the date of last contact for censored events when the woman was still alive. Estimates of the cumulative proportion surviving were calculated using the Kaplan-Meier method, and the log rank test was used to assess the null hypothesis of equality in survival distributions among patient subgroups (16 , 17) . RR (hazard ratio) was estimated using Cox proportional hazard regression analyses (18) . All covariates were evaluated for their association with survival using the Wald Test (19) . Two methods were used to adjust for appropriate clinical characteristics (19 , 20) . The all-inclusive enter method adjusted for patient age, race, initial performance status, tumor stage, histological grade, tumor cell type, amount of residual disease after primary debulking surgery, and/or type of chemotherapy. The forward stepwise inclusion method with variable entry set at 0.1 was also used to assess the independent prognostic relevance of cyclin E.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increased Cyclin E Expression Is Prognostic of a Poor Outcome in AOCs.
The specimens used for this study were obtained at the time of primary cytoreductive surgery and before the initiation of frontline chemotherapy, from women participating in GOG protocol no. 111. This prospective randomized Phase III trial evaluated the use of cisplatin and cyclophosphamide compared with cisplatin and taxol for the treatment of women with suboptimally debulked primary advanced epithelial ovarian cancer. As shown in Table 1Citation , of 139 cases, 62% were stage III and 38% were stage IV ovarian cancers. In addition, 4% were well differentiated, 44% were moderately differentiated, and 52% were poorly differentiated tumors. The distribution by tumor cell type was: 72% serous; 9% endometrioid; 7% mixed epithelial; 4% mucinous; 2% clear cell; and 7% other. Only 59% of the cases had clinically measurable residual disease after primary cytoreductive surgery. Fifty percent of the women were randomized to receive six courses of cisplatin (75 mg/m2) and cyclophosphamide (750 mg/m2), whereas the other 50% were randomized to receive six courses of cisplatin (75 mg/m2) and taxol (135 mg/m2).


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  		Table 1 The association between cyclin E expression and clinical characteristics in women with suboptimally debulked advanced epithelial ovarian cancer

 
Immunohistochemical analysis of cyclin E expression in the 139 cases of AOC from women with suboptimal disease revealed that the mean percentage of cyclin E positive staining tumor cells was 40%. High cyclin E staining (>40% cells staining positive) was observed in 62 (45%) suboptimally debulked AOC patients (Table 1)Citation . High cyclin E protein expression was not correlated with clinical prognostic factors such as age, race, performance status, tumor stage, histological grade, cell type, amount of residual disease, or type of chemotherapy (Table 1)Citation . However, suboptimally debulked AOC patients with high cyclin E staining had a significantly shorter median survival of 29.2 ± 2.2 versus 35.4 ± 2.9 months, respectively, and worse overall survival (Fig. 1Citation ; P = 0.045). Univariate Cox proportional hazard analysis found that high cyclin E expression was associated with a 40% increase in the RR of death (hazard ratio = 1.4, 95% CI: 1.0–2.1, P = 0.05). A multivariate model was then used to adjust for the potential influence of confounding factors. High cyclin E expression was shown to be a significant, independent predictor of poor survival among women with suboptimal advanced stage ovarian cancer (Table 2)Citation . Specifically, the estimated RR of death in AOC patients with high cyclin E expression was 1.4 times greater than that estimated for AOC patients with low cyclin E expression (hazard ratio = 1.4, 95% CI: 1.0–2.1, P = 0.05) after adjusting for age, race, performance status, stage, grade, cell type, residual disease, and therapy. A forward-stepwise selection method was used as an alternative approach for the multivariate Cox regression analysis. High cyclin E expression was associated with a 50% increase in the hazard ratio (hazard ratio = 1.5, CI: 1.1–2.2, P = 0.02) after adjusting for race, tumor cell type, and type of therapy, the only variables that satisfied the inclusion entry set at P of 0.1.



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  		Fig. 1. Kaplan-Meier survival analysis for cyclin E expression positive tumor cells in women with suboptimally debulked AOC. Low cyclin E expression represents <40% (mean) cyclin E-positive tumor cells and high cyclin E expression indicates >=40% (mean) cyclin E-positive tumor cells. The survival distribution, mean survival time in months ± the SE, and significance of the log rank test to evaluate the equality of survival distributions are provided.

 

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  		Table 2 Cox regression analyses for cyclin E expression in suboptimally debulked advanced epithelial ovarian cancer

 
Cyclin E Expression Predicts Survival in Patient Subsets.
Subset analysis among all stage III ovarian cancer patients revealed that high compared with low cyclin E expression was associated with a shorter median survival of 30 ± 4 versus 36 ± 4 months, respectively (P = 0.02; Fig. 2ACitation ). Cox regression analysis then revealed high cyclin E expression was an independent prognostic factor for women with suboptimally debulked stage III ovarian cancer (Table 2)Citation . Cyclin E expression among stage IV patients was not associated with survival (P > 0.8; Table 3Citation ). Furthermore, when serous histology, the most commonly observed ovarian adenocarcinoma in the study (72%) was analyzed, high compared with low cyclin E expression was associated with shorter median survival, 30 ± 3 versus 41 ± 7 months, respectively (P < 0.01; Fig. 2BCitation ). High expression among serous tumors was also associated with an increase in the RR of death in both univariate and multivariate Cox regression analysis (Table 2)Citation . For nonserous tumors, there was not a statistically significant difference in survival based on cyclin E expression (P > 0.6; Table 3Citation ). Subset analysis of patients with nonmeasurable disease (Fig. 3A)Citation and those receiving the treatment regimen containing cisplatin and taxol (Fig. 3B)Citation demonstrated that high cyclin E expression was associated with shorter median survival, 29 ± 2 versus 65 ± 12 (P < 0.01) and 33 ± 4 versus 43 ± 7 (P < 0.05), respectively. Cox regression analysis demonstrated that high cyclin E expression was an independent poor prognostic factor for both of these groups (Table 2)Citation . For patients with measurable disease and those treated with cisplatin and cyclophosphamide, survival time was not dependent on cyclin E expression levels (P > 0.9 and P > 0.5, respectively; Table 3Citation ).



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  		Fig. 2. Kaplan-Meier survival analysis for cyclin E expression in women with suboptimally debulked ovarian cancer by tumor stage and cell type. Low cyclin E expression represents <40% (mean) cyclin E positive tumor cells and high cyclin E expression indicates >=40% (mean) cyclin E-positive tumor cells. The survival distribution, median survival time in months ± the SE, and the significance of the log rank test to evaluate the equality of survival distributions are provided for stage III ovarian cancers (A) and advanced serous adenocarcinomas of the ovary (B).

 

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  		Table 3 Kaplan-Meier survival analysis of cyclin E expressiona in women with suboptimally debulked advanced ovarian cancer

 


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  		Fig. 3. Kaplan-Meier survival analysis for cyclin E expression in women with suboptimally debulked ovarian cancer by residual disease and treatment. Low cyclin E expression represents <40% (mean) cyclin E-positive tumor cells and high cyclin E expression indicates >=40% (mean) cyclin E-positive tumor cells. The survival distribution, median survival time in months ± the SE, and the significance of the log rank test to evaluate the equality of survival distributions are provided for patients with nonmeasurable disease (A) or who were treated with cisplatin and taxol therapy (B).

 
FISH Analysis Demonstrates Amplification of the Cyclin E Gene.
To determine the mechanism of increased cyclin E expression, we analyzed the genomic status of the cyclin E in 20 ovarian cancers (10 high- and 10 low-expressing tumors) by FISH. A FISH probe was prepared from a single BAC clone containing the CCNE1 (cyclin E) gene at 19q12 and labeled with SpectrumOrange. To control for aneusomy, a probe on the opposite arm of chromosome 19 was also prepared from a single BAC clone containing the INSR gene located at 19p13.2 and labeled with SpectrumGreen. Ratios of cyclin E to chromosome 19 (CCNE1 to INSR) signals/cell close to 1 (<1.5) were considered nonamplified for cyclin E relative to chromosome 19, whereas ratios significantly greater than 1 (>1.5) were considered amplified for cyclin E, and ratios significantly <1 represented deletion of cyclin E gene relative to the chromosome 19 copy number.

FISH analysis revealed that 8 of 10 randomly selected cases with high immunohistochemical expression of cyclin E exhibited amplification of the cyclin E gene, whereas only 1 of 10 randomly selected cases with low immunohistochemical expression of cyclin E exhibited amplification (Fig. 4Citation ; Table 4Citation ). High immunohistochemical expression of cyclin E was associated with amplification of the cyclin E gene (P < 0.006).



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  		Fig. 4. FISH analysis. Representative FISH analysis of low cyclin E protein expressor by IHC (A, specimen 11) and high cyclin E protein expressors by IHC (B, specimen 108). The BAC clone containing the CCNE1 (cyclin E) gene was labeled with spectrum orange while a BAC clone containing the INSR gene (to control for aneusomy) was labeled with spectrum green.

 

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  		Table 4 FISH analysis of cyclin E gene amplification in suboptimally debulked advanced epithelial ovarian cancers

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The primary objective for this study was to determine the potential for cyclin E expression to predict survival of patients with suboptimally debulked advanced epithelial ovarian cancer. We conducted a comprehensive analysis of cyclin E in specimens obtained from a prospective randomized trial in women with suboptimally debulked advanced epithelial ovarian cancer treated with one of two types of platinum-based first line therapies. Tumor expression of cyclin E protein was evaluated in 139 specimens using an immunohistochemistry assay. FISH analysis was then performed in a subset of cases to determine whether the mechanism of increased cyclin E protein expression may be amplification of the cyclin E gene. This study demonstrates increased expression of cyclin E protein occurring in 45% of suboptimally debulked advanced epithelial ovarian cancers tested. Multivariate analysis indicates that high cyclin E protein expression is an independent predictor of poor outcome in women with suboptimally debulked disease.

Overexpression of cyclin E has been reported in several human cancers (10, 11, 12) , and this increased expression is thought to increase activity of the cyclin E-CDK2 complex and progression through the G1-S portion of the cell cycle in the affected cancer cells. In non-small cell lung cancer, two studies have demonstrated frequent overexpression of cyclin E where it served as an independent unfavorable prognostic factor (12) . Likewise, recent studies showed that overexpression of cyclin E is a poor prognostic factor in gastric and breast cancers (10 , 11) . Enhanced expression of cyclin E protein has also been demonstrated in ovarian tumors (21, 22, 23, 24) . Unfortunately, these studies examined small numbers of ovarian cancers that varied in stage and lacked survival data (21 , 22) . The other studies in ovarian cancer examined larger numbers of tumors and showed cyclin E gene amplification and RNA overexpression in 12% of the tumors (23 , 24) . However, no analysis of cyclin E protein level was performed, and no association between cyclin E and patient survival was reported.

Our study demonstrated that high cyclin E protein expression is a poor prognostic factor for survival for patients with AOC. Multivariate modeling was used to demonstrate the independent prognostic significance of this biomarker. Subgroup analyses revealed that the prognostic relevance of cyclin E on survival appeared to be particularly evident in specific subgroups, including patients with stage III disease, serous adenocarcinoma of the ovary, and nonmeasurable disease assessed after primary cytoreductive surgery. For example, papillary serous tumors of the ovary showed an 11-month difference in median survival when cases were classified as low versus high cyclin E expression. Furthermore, high cyclin E was associated with worse survival only in the subgroup of women who received the combination of cisplatin and taxol. This may be influenced by the superior efficacy of a taxol-containing regimen compared with the cytoxan regimen (GOG 111). Conversely, cyclin E expression levels may modulate the cell’s sensitivity to taxol. Finally, high cyclin E appears to exhibit a more profound impact in patients with AOC with low volume disease (stage III).

Our finding of overexpression of cyclin E in advanced, suboptimally debulked ovarian cancer patients and its prognostic impact is important for two reasons. First, few molecular events have been convincingly identified as important prognostic factors for epithelial ovarian cancer. Although p53 (25, 26, 27) and angiogenesis (28) markers have been evaluated as prognostic markers in AOC, their impact on survival and clinical use remains unclear. Many of these studies have arrived at differing conclusions, in part, because they have evaluated small numbers of specimens (26 , 28) , heterogeneous patient populations with respect to histology (26) , stage (25, 26, 27) , and treatment (25, 26, 27, 28) . This study used a group of tumors from patients who were similarly staged and treated with a cisplatin-based combination therapy. Because these suboptimally debulked AOC cases represent a carefully characterized patient population, any marker related to survival is likely to have an underlying biological basis and be clinically relevant. Prescreening for cyclin E expression may identify those patients with enhanced sensitivity to cisplatin and taxol. In contrast, patients with high cyclin E expression may be better suited to a clinical trial evaluating the efficacy and toxicity of new agents and/or drug combinations thought to affect G1-S transition. Cyclin E-associated CDK activity could be an important molecular complex for targeted therapy. Presumably, those tumors with increased cyclin E expression are critically dependent upon the cyclin E-associated CDK activity and thus could be effectively targeted with molecular agents, which affect this complex. It is interesting to hypothesize that effective inhibition of cyclin E may enhance ovarian cancer-sensitivity to the cisplatin and taxol combination.

The mechanism of high cyclin E expression appears to involve, in part, gene amplification. Gene amplification is a genetic event reflecting a molecular process critical to the survival of the tumor cells. However, the precise role increased expression of cyclin E plays in AOC biology remains to be determined. Furthermore, whether this is the only relevant gene within the amplicon on chromosome 19q12 will need to be determined. Other genes that may be located within this amplicon include SUMO-1, CD22, POLR21, PAK4, CEBP-{alpha}, and AKT2. The observation that two cases exhibited high expression of cyclin E in the absence of gene amplification indicates that other mechanisms are involved (29 , 30) .

This study reveals that increased immunohistochemical staining for cyclin E, expressed as a percentage of cyclin E positive tumor cells without consideration of staining intensity, is a significant, independent poor prognostic factor for suboptimally debulked advanced epithelial ovarian cancer. In addition, high immunohistochemical expression of cyclin E is associated with amplification of cyclin E. Additional studies are required to determine the prognostic relevance of cyclin E during earlier stages of ovarian cancer development and to compare amplification of the cyclin E gene with immunohistochemical expression of this protein as a prognostic measure of survival in women with suboptimally debulked advanced epithelial ovarian cancer.


   ACKNOWLEDGMENTS
 
We thank Robin Howard for her technical advice in the preparation of this manuscript, Caron Modeas for her assistance in formatting and editing this manuscript, and Suzanne Baskerville for coordinating the clinical data for this study. We thank Drs. Michael Teneriello and John Nash for their efforts on GOG no. 9404. We also thank Drs. William P. McGuire, William J. Hoskins, Mark F. Brady, Joan Woodward, and Martin Davidson, as well as Janet Walczak, for their contributions to GOG no. 111. We also thank Kathleen A. Donohue, Cynthia M. Westby, and Dr. Brian N. Bundy for their expert work on this study. We thank Drs. William Beck, Larry Copeland, Richard Kryscio, William McGuire, and Andrew Berchuck for their insightful comments, suggestions, and critical review of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by National Cancer Institute Grant CA 27469 to the GOG Administrative Office, Grant CA 37517 the GOG Statistical Office, and the Intramural Program of the National Cancer Institute. The following (current or former) GOG member institutions participated in this study: University of Alabama School of Medicine, Oregon Health Sciences University, Abington Memorial Hospital, University of Rochester Medical Center, Walter Reed Army Medical Center, Wayne State University, University of Southern California at Los Angeles, Colorado Gynecologic Oncology Group, P.C., University of California at Los Angeles, University of Washington, Milton S. Hershey Medical Center, Georgetown University Hospital, Wake Forest University School of Medicine, University of California Medical Center at Irvine, Stanford University Medical Center, University of Kentucky, The Cleveland Clinic Foundation, Johns Hopkins Oncology Center, Eastern Pennsylvania Gyn/Onc Center, P.C., Washington University School of Medicine, Cooper Hospital/University Medical Center, Columbus Cancer Council, University of Massachusetts Medical Center, University of Oklahoma, and Tacoma General Hospital. Back

2 These authors contributed equally to the present investigations. Back

3 To whom requests for reprints should be addressed, at National Cancer Institute Center for Cancer Research Cell and Cancer Biology Department, 9610 Medical Center Drive, Suite 300, Rockville, MD 20850-3300. Phone: (301) 402-3128, ext. 349; Fax: (301) 402-4422; E-mail: birrerm{at}bprb.nci.nih.gov Back

4 The abbreviations used are: CDK, cyclin-dependent kinase; AOC, advanced ovarian cancer; GOG, Gynecologic Oncology Group; FISH, fluorescence in situ hybridization; BAC, bacterial artificial chromosome; INSR, insulin receptor; CI, confidence interval; RR, relative risk. Back

Received 10/17/02. Accepted 1/17/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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