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Clinical Cancer Research Vol. 11, 3733-3742, May 15, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

The Relationship of Molecular Markers of p53 Function and Angiogenesis to Prognosis of Stage I Epithelial Ovarian Cancer

Michael J. Goodheart1, Justine M. Ritchie3, Stephen L. Rose1, John P. Fruehauf4, Barry R. De Young2 and Richard E. Buller1

Authors' Affiliations: 1 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology; Departments of 2 Pathology and 3 Biostatistics, Holden Comprehensive Cancer Center, University of Iowa Hospital and Clinics, Iowa City, Iowa; and 4 Oncotech Incorporated, Irvine, California

Requests for reprints: Michael J. Goodheart, Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, 4630JCP, Iowa City, IA 52242. Phone: 319-356-2015; Fax: 319-353-8363; E-mail: michael-goodheart{at}uiowa.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Multiple angiogenic factors may influence tumor progression and metastasis. Several are modified by the p53 gene. We sought to identify molecular markers for high-risk stage I epithelial ovarian cancers.

Experimental Design: Seventy-seven consecutive stage I epithelial ovarian cancers were evaluated for p53, CD31 microvessel density, thrombospondin-1, vascular endothelial growth factor (VEGF), p21 immunohistochemical staining, and p53 gene mutations. Molecular marker impact upon disease-specific survival, disease recurrence, and distant recurrence was evaluated with Cox regression.

Results: There were 12 deaths from disease. Twelve of the 77 tumors contained p53 mutations—10 missense and 3 null (one tumor had two mutations). Fesddration Internationale des Gynaecologistes et Obstetristes substage (IA/IB versus IC; P < 0.001) and VEGF staining (P = 0.02) were significant in bivariate models with relationship to disease-specific survival. Stage (P = 0.0004), grade (P = 0.008), histology (P = 0.0025), p53 dysfunction (positive stain and/or mutation; P = 0.048), and microvessel density (P = 0.04) were significant in bivariate models with relationship to time to recurrence. In multivariate analyses among stage IC patients, failure to receive chemotherapy and microvessel density were associated with disease-specific survival, time to recurrence, and time to distant recurrence with hazard ratios of 4.8 to 44.1.

Conclusions: The p53-dependent molecular markers of angiogenesis are of limited utility in developing a clinical strategy for postoperative management of stage I ovarian carcinoma. Microvessel density impacts survival and metastasis for high-risk stage IC disease. Adjuvant chemotherapy is necessary, but not sufficient, for cure of high-risk stage I epithelial ovarian cancers.

Key Words: p53 • molecular markers • stage I epithelial ovarian cancer • prognosis

When ovarian cancer is confined to the ovary as Fesddration Internationale des Gynaecologistes et Obstetristes (FIGO) stage I disease, actuarial survival is 70% to 90% (1). However, the clinical behavior of certain subgroups of stage I ovarian cancer can be quite aggressive. Following conventional surgical staging, many patients are treated with adjuvant chemotherapy. Exactly who to treat and when it is safe not to provide adjuvant chemotherapy remains controversial despite the recent publications of the results from the ICON1 and ACTION trials (2, 3). Being able to identify patients with early-stage disease who do not need adjuvant therapy would be of great benefit to these patients and would spare them potential morbidity and the costs associated with additional treatment modalities. Currently, treatment decisions are often based on the histologic grade of the tumor because grade has been identified in several studies as an adverse prognostic marker (4, 5).

As our knowledge of the molecular changes associated with ovarian carcinogenesis has increased dramatically in the last 10 years, it would seem reasonable to hypothesize that molecular markers may provide more useful prognostic information than can be achieved from conventional histopathologic review of ovarian cancers. In particular, multiple angiogenic factors seem to play an important role in the progression and spread of ovarian cancer (6–10). The use of molecular markers to characterize an angiogenic phenotype in ovarian cancers could be important in identifying which patients truly require additional treatment modalities or closer follow-up. For example, it has been shown that stage I invasive ovarian carcinomas have higher microvessel counts than tumors of low malignant potential (11).

The use of multiple molecular markers may be a better predictor of clinical outcome than the use of a single marker. We and others have been studying the role of the p53 tumor suppressor gene in ovarian carcinoma (12, 13). Expression of several molecular factors critical to the angiogenic phenotype are under the influence of p53 (10, 14, 15). Both vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) contain p53 response elements and are involved in the formation and inhibition of new blood vessels, respectively (14, 16).

In some cancers, including melanoma, breast, bladder, and prostate, the intricate relationship between mutant p53 and TSP-1 has already been established (17–20). Recently, in prostate cancer, it was shown that mutant p53 was inversely related to TSP-1 expression as measured by image analysis. In prostate cancers with p53 gene mutations, a decrease in TSP-1 was correlated with an increase in angiogenesis (20). This exemplifies the importance of wild-type p53 gene function in the prevention of uncontrolled vessel growth via up-regulated TSP-1 expression.

Other studies have shown that VEGF expression is an important prognostic and survival factor for patients with a variety of solid tumors including breast (21), thyroid (22), and ovarian cancer (23, 24). Sequenced tumor p53 mutations together with VEGF protein levels are predictive of both overall breast cancer survival as well as relapse-free survival (21). Breast cancers containing p53 null mutations seem to have concomitant up-regulated VEGF expression (21). In early-stage ovarian carcinomas, Paley et al. (23) showed that elevated levels of VEGF mRNA expression were associated with a 5-fold lower median disease-free survival when compared with tumors with lower levels of VEGF expression.

Our group has previously shown the importance of the p53 tumor suppressor gene and its relationship to ovarian cancer survival (7–10, 25). Whereas both p53 immunohistochemistry and/or sequencing have been used to predict survival in ovarian cancer (7, 26–28), conflicting results are often obtained unless sequence data is incorporated into the analyses.

Likewise, p21 expression alone is a prognostic factor in ovarian cancer (10) and has been studied in combination with other stains (28, 29). P21 expression also carries prognostic significance in rectal (30) and breast cancers (31). The use of microvessel density is of prognostic significance in ovarian cancer (6) and has been correlated with stage, grade, and p53 mutation type (9).

For the present study, we sought to identify a group of patients with stage I epithelial ovarian cancer that were at high risk for adverse outcome by classifying multiple factors related to the angiogenic genotype. This allows us to test the hypothesis that the angiogenic genotype facilitates identification of patients that would benefit from adjuvant therapy.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
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Seventy-seven consecutive stage I epithelial ovarian cancer patients seen either initially or in consultation at Holden Comprehensive Cancer Center of University of Iowa Hospitals and Clinics from January 1, 1988, to December 31, 1999, were identified. All patients with peritoneal cancers, fallopian tube cancers, or tumors with low malignant potential were excluded. This project was carried out with the approval of the Institutional Human Subjects Protection Review Board. Tumor procurement, DNA isolation, and p53 gene sequencing techniques are described in our previous publications (7, 8, 10), as have the basic techniques for p53 immunohistochemical analysis (7). There are 38 patients with stage I disease included in this paper that were previously analyzed (7). Sequenced p53 mutations were characterized as null (frameshift or nonsense) and missense. No distinction was made between expressed wild-type p53 sequence and potentially unexpressed wild-type sequences. P53 dysfunction includes tumors with a positive p53 stain and/or sequenced mutation.

All immunohistochemistry slides were read by a single pathologist (B.R. De Young), who was blinded to the clinical outcome and p53 sequence data. Staining for microvessel density was done with mouse monoclonal CD31 antibody (DAKO Corporation, Carpinteria, CA) using the labeled streptavidin-biotin method. Briefly, sections were baked at 56°C for 60 minutes, deparaffinized in xylene, rehydrated in graded alcohols, and rinsed. For heat-induced epitope retrieval, sections were placed in 0.01 mol/L citrate buffer (pH 6.0) and heated in a microwave oven for cycles of 2 to 5 minutes each on high power. The slides were then cooled for 20 minutes, whereas endogenous peroxidase activity was blocked using hydrogen peroxide in distilled water. Slides were coated with CD31 antisera at a 1:20 dilution (DAKO), incubated for 30 minutes at room temperature, and then rinsed. Reactivity was detected using mouse immuglobulin G (vector) as the secondary antibody with an avidin-biotin technique and 3',3'-diaminobenzidine as the chromogen. Slides were then counterstained with Mayer's hematoxylin for 1 minute. Negative control slides were prepared by substituting mouse immunoglobulin. Sections of normal human tonsil were included with each group of CD31 slides stained and processed as above and served as the control. All rinses were done with PBS (pH 7.4) with 0.048% Brig 35 solution. The microvessel density (microvessels/high-power field) was determined by the study pathologist who counted and averaged the vessels from three different areas of tumor with the highest apparent vessel density using x200 magnification (32).

For the p21 staining, an immunoperoxidase reaction was done using the labeled streptavidin-biotin plus method as above. The only modifications were the p21 antisera was used at a 1:10 dilution (DAKO) for a 1-hour incubation at room temperature before the rinse. A positive stain was reported when nuclear staining was detected by the study pathologist (B.R. De Young) in ≥2% of the stained cells (10).

VEGF staining was also carried out using the labeled streptavidin-biotin method. Unmasking was done with 10 mmol Tris-HCl (pH 10.0) buffer in the microwave for 2- to 5-minute cycles on high power. VEGF (NeoMarkers, Lab Vision Corporation, Fremont, CA) antisera was used at a dilution of 1:50 for overnight incubation at 4°C before the rinse. After a second antibody was applied in the avidin-biotin technique, slides were counterstained with Harris hematoxylin without acid for 1 minute. VEGF slides were assigned an immunohistochemical score based upon intensity and percent of specific tumor cell staining. The intensity was scored as follows: negative, <5% tumor cells display staining; 1+, when the intensity of staining is mild; 2+, moderate; 3+, intense and equal to the positive controls; 4+, when intensity is greater than the positive controls. Immunohistochemical score was the percent positive cells multiplied by [intensity + 1] (32).

In collaboration with Oncotech Incorporated (Irving, CA), computer-assisted image analysis was done to quantitate TSP-1 staining. A CAS 2000 two-color system (Becton Dickinson, San Jose, CA) attached to a microcomputer was used for digital image processing. Image channels were used to identify and enhance one immunohistochemical stain each. One channel identified all components stained with methyl green (nuclear components) and the other channel identified all components that were stained brown with diaminobenzidine. The results were displayed as absorbance units. A negative control was included with each specimen for subtraction of background staining.

Failure-time methods were used for disease-specific survival, time to recurrence (at any site), or time to distant recurrence. Patients who were alive at last follow-up were censored on that date. Censoring for time to recurrence was done for those alive without recurrence of disease. Local recurrences were considered censored observations in time to distant recurrence analyses.

Cox proportional hazards regression was used in bivariate and multivariate analyses to identify independent prognostic factors with P < 0.10 and to estimate hazard ratios (HR; ref. 33). Groups were compared using the likelihood ratio test. Ninety-five percent confidence intervals (95% CI) for the HRs were based on the normal approximation. Exploratory statistical methods were used to determine the optimal cutoff value for VEGF, CD31, and TSP-1 in bivariate analyses. The exploratory analyses involved identifying the deciles of the VEGF, CD31, or TSP-1 distribution and then examining the HRs estimated for each of the nine possible dichotomizations. The dichotomization that yielded the largest HR was selected as the optimal cutoff value to be used in multivariate analyses. The limitations of this method have been previously discussed (34). This was also done among analyses involving stage IC patients except that the terciles rather than the deciles of the distributions were used.

The backward stepwise selection procedure with significance set to P < 0.10 was used to identify independent prognostic factors in multivariate analyses. Time to event curves were estimated by the Kaplan-Meier method (35). All statistical analyses were done using SAS version 8.0 (36). Survival curves were created using S-Plus 2000 (37).


   Results
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 Materials and Methods
 Results
Discussion
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The clinicopathologic data for the entire cohort is presented in Table 1 and a detailed summary of the histology, p53 mutational status, and immunohistochemistry is presented in Table 2. The mean patient age was 50 years (range 21-85). Stage IB cancers were distinctly uncommon at 6%, whereas stage IA tumors represented more than half of the study population. Tumor grade was approximately equally distributed between grades 1, 2, and 3. All histologic types of epithelial ovarian cancer were represented. Mucinous and endometrioid histology together accounted for 56% of the tumor histologies. Five patients were diagnosed with dual primaries. In addition to ovarian carcinoma, one patient had early microinvasive breast cancer, one patient had stage IA uterine adenocarcinoma, and three patients had stage IB uterine adenocarcinoma. Overall, slightly more than half (52%) of the patients received adjuvant chemotherapy. Twelve patients have died from disease, whereas four died from other causes.


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  		Table 1. Clinicopathologic data for stage I (n = 77) ovarian carcinomas (mean age, 50 years)

 

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  		Table 2. Histology, immunohistochemistry, and p53 mutations for stage I (n = 77) ovarian carcinomas

 
Eighteen patients (23%) have developed recurrent disease. Clinical and pathologic data on this group are summarized in Table 3. Overall, 14 of 33 patients (42%) initially diagnosed with stage IC disease developed any recurrence compared with 4 of 44 (9%) of patients with stage IA/IB disease (P ≤ 0.001). Of the 18 recurrences, 13 (72%) were classified as distant because of their location outside the pelvis: small bowel (1), liver (4), lung (3), paraaortic lymph node (4), and the abdominal wall (1). The remaining five were local recurrences. Median survival was 70.3 months following distant recurrences versus 76.3 months for local recurrences. To date, six individuals with disease recurrence remain alive with a median follow-up of 74 months; three are disease-free after retreatment at a median follow-up of 63 months from their initial diagnosis. Median survival has not been reached for the cohort as a whole. Among those still alive at last follow-up (n = 61), median follow-up was 73 months (range 24-176 months), 48 months for those dead of disease, and 84 months for those alive with disease. Ninety percent of the patients have been followed for at least 3 years.


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  		Table 3. Stage I ovarian carcinomas: recurrences (n = 18)

 
Of the 12 patients who have died of disease, 9 (75%) had developed distant recurrences. All but one had stage IC disease at presentation, all but one had grade 2 to 3 cancers, and all histologic subtypes were represented. Six patients who died had not received adjuvant chemotherapy and included (a) one patient with a dual primary: stage IC, grade 2 endometrioid ovarian histology and stage IB grade 2 endometrial carcinoma treated with adjuvant whole pelvic radiation therapy; (b) one patient with stage IC, grade 3 "adenocarcinoma not otherwise specified" histology who refused chemotherapy; (c) one patient with stage IC, grade 3 clear cell carcinoma who was not offered chemotherapy; (d) one patient with stage IA, grade 2 mucinous histology; and (e) two patients treated with i.p. 32P.

In bivariate modeling, conventional parameters shown in Table 4 that predicted recurrence (either distant or at all) included FIGO substage (IA/IB versus IC), tumor grade, and tumor histology (adenocarcinoma not otherwise specified/serous versus others). Only FIGO substage was predictive of disease-specific survival. Figure 1A shows the distant recurrence-free probability based upon FIGO substage.


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  		Table 4. The impact of clinicopathologic and molecular factors of angiogenesis on disease outcome for stage I (n = 77) ovarian carcinomas

 


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  		Fig. 1. Distant recurrence-free probability by stage of disease (A) and p53 dysfunction (B) based on the Kaplan-Meier method. A, solid line, stage IA/IB (n = 44); dashed line, stage IC (n = 33). B, solid line, absence of p53 dysfunction (n = 54); dashed line, p53 dysfunction (n = 23). Distant recurrence-free probability by chemotherapy (C) and CD31 count (D) based on the Kaplan-Meier method among stage IC patients only. C, solid line, other chemotherapy [cytoxan, platinum, or taxane; (n = 27)]; dashed line, no chemotherapy/i.p. 32P (n = 6). D, solid line, CD31 count ≤12 (n = 11); dashed line, CD31 count >12 (n = 22).

 
The p53 expression and mutation data are listed in Table 4. Thirteen mutations, including 10 missense and 3 null mutations, were sequenced from 12 tumors. P53-positive tumors were more likely to develop distant recurrence (HR, 3.4; P = 0.029). P53 mutation was not predictive of disease-specific survival or the likelihood of developing recurrent disease. By combining mutation data with immunohistochemical staining data, taken together as p53 "dysfunction," this parameter was predictive both of the likelihood of distant recurrence (HR, 4.1; P = 0.012) or any recurrence (HR, 2.6; P = 0.048), but not disease-specific survival. Figure 1B shows the distant recurrence-free probability based upon p53 dysfunction.

The results of the rest of the immunohistochemistry studies and their ability to predict either recurrent disease or survival in bivariate models are summarized at the end of Table 4. Only VEGF staining was predictive of disease-specific survival (HR, 6.5; P = 0.02). VEGF-positive staining was of borderline significance in projecting recurrence. A CD31 count >11 vessels/high-power field increased the likelihood of any recurrence (HR, 3.7; P = 0.04), but especially distant recurrence (HR, 5.6; P = 0.03). TSP staining was of borderline significance in prognosticating distant recurrence (P = 0.055).

For multivariate analysis, the clinical, pathologic, immunohistochemical, and p53 sequencing data were modeled as predictors of disease-specific survival, time to recurrence, and time to distant metastasis. These results, for the entire cohort, are presented in the top half of Table 5. Individuals with stage IC disease were 47.8 times more likely to die from disease than those with Stage IA/B disease (95% CI, 5.3-430; P < 0.001). Failure to receive adjuvant chemotherapy was also associated with increased risk for death from disease (HR, 4.6; P = 0.01; 95% CI, 1.4-15.5). Recurrence at any site was predicted by FIGO stage (HR, 4.5; P = 0.01; 95% CI, 1.4-14.3) and adenocarcinoma not otherwise specified/serous tumor histology (HR, 2.8; P = 0.03; 95% CI, 1.1-7.4), whereas distant recurrence was predicted by FIGO stage (HR, 4.6; P = 0.02; 95% CI, 1.2-17.1) and p53 dysfunction (HR, 3.1; P = 0.05; 95% CI, 1.1-9.6). CD31 staining, VEGF score, and TSP staining were not statistically significant factors in the Cox model for either recurrence of disease or disease-specific survival when the entire stage I cohort was analyzed.


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  		Table 5. A multivariate analysis of significant factors in stage I ovarian carcinomas that determine survival and recurrence

 
Because 14 of the 18 (78%) cases of recurrent disease were found among the 33 patients initially diagnosed as having stage IC disease, a separate multivariate analysis of clinical, pathologic, and molecular factors was carried out for this high-risk subgroup. These results are presented in the lower half of Table 5. Factors significant in relationship to disease-specific survival included failure to receive adjuvant chemotherapy (HR, 10.1; P = 0.002; 95% CI, 2.4-43) and CD31 count (HR, 4.8; P = 0.04; 95% CI, 1.1-22). For the CD31 counts, a cut point of 12 vessels/high-power field among stage IC patients was more significant than the 11 vessels/high-power field determined when analyzing the entire cohort. For the time to recurrence analysis, the same clinical and pathologic parameters were identified: failure to receive adjuvant chemotherapy (HR, 22; P ≤ 0.001; 95% CI, 3.6-129) and CD31 count (HR, 14.2; P = 0.006; 95% CI, 2.1-95). For distant recurrence analysis of the stage IC group, failure to receive adjuvant chemotherapy (HR, 31.5; P = 0.004; 95% CI, 2.9-336) and CD31 count >12 microvessels/high-power field (HR, 44.1; P = 0.009; 95% CI, 2.6-762) were each significant. Figure 1C and D shows survival curves based upon delivery of chemotherapy and CD31 count, respectively, in relationship to distant recurrence-free probability.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This is a retrospective, nonrandomized study designed to examine the relationship of molecular markers of angiogenesis and clinicopathologic factors to disease recurrence and disease-specific survival from stage I epithelial ovarian carcinomas based upon the hypothesis that angiogenic factors play an important role in determining the clinical outcome of disease. The molecular markers of angiogenesis chosen for this investigation included VEGF, TSP-1, p21, p53, and microvessel density measured by CD31 immunostaining. These factors seem to interact at the molecular level (9, 10, 14, 15). Furthermore, there is a growing body of literature that suggests that these factors modify clinical outcome in several cancers (21, 22, 30, 38–40), including advanced epithelial ovarian cancer (9, 16, 26). Thus, it is rational to suspect that these factors may also contribute to the outcome of stage I ovarian cancers. Confirmation of this hypothesis could impact treatment patterns, allowing some individuals with early-stage ovarian cancer to avoid unnecessary chemotherapy and its associated short- and long-term complications.

In agreement with traditional clinical outcome studies of stage I ovarian cancer (41, 42), bivariate analysis showed that FIGO substage of disease was highly correlated with disease-specific survival (HR, 18.5; P < 0.001). As in other studies, FIGO substage, tumor grade, and histology all contributed to the likelihood of recurrence (2, 5). A unique observation from the present study is the indication that these parameters specifically contribute to distant recurrences of stage I disease. A 4- to 6-fold increased risk was suggested by the HRs.

At the molecular level, high levels of VEGF staining increased the risk of death from disease by a factor of 6.5. This result was consistent with the findings of Paley et al. (23) who utilized autoradiography to determine that higher levels of VEGF mRNA in early-stage ovarian cancer were associated with poor clinical outcome. However, the cut point determined in the present study, an immunohistochemistry score >400, does not seem to be clinically useful because only five tumors expressed VEGF at this level. Furthermore, in multivariate analysis, VEGF expression was not identified as an independent prognostic factor. These findings are in contrast to the importance of VEGF expression as a prognostic factor for localized papillary thyroid carcinoma (22) or curatively resected ductal carcinoma of the pancreas (43).

p53 dysfunction (sequenced mutation and/or positive stain) failed to predict disease-specific survival for stage I ovarian cancers. This observation is in conflict with the findings of Skirnisdottir et al. (27), who showed that positive p53 staining was associated with poor survival in early-stage epithelial ovarian carcinoma. However, ~20% of the "early" ovarian cancers studied by these authors were stage II in contrast to the present study limited exclusively to stage I disease. Moreover, the current results are in agreement with our previously reported observation that p53 dysfunction did not correlate with disease-specific survival in advanced stage ovarian cancer (7). We did discover, however, that p53 dysfunction was associated with a propensity to develop recurrent disease and in particular a predilection for distant metastasis (HR, 4.1; P = 0.012; Table 4). P53 dysfunction also remained significant in multivariate modeling of the time to distant recurrence. This novel observation, in conjunction with our previous observations pertaining to p53 dysfunction in ovarian cancer, suggests a role for p53 early in the metastatic process. Further support of the hypothesis is obtained from the observation that micrometastatic p53-positive cells can be found in the lymph nodes of otherwise apparently early-stage ovarian cancers and that this finding correlates with poor prognosis (44).

Microvessel density is predictive of recurrence of small (≤5 cm) hepatocellular carcinomas (45) and other early cancers such as breast (46), node-negative colon cancer (47), and testicular cancer (48). We (9) and others (6) have previously reported that increasing microvessel density, determined by CD31 counts, compromises survival of mixed-stage cohorts of epithelial ovarian cancer patients. In the present study, restricted to stage I ovarian cancers, CD31 counts were reflective of the likelihood of recurrent disease, but not disease-specific survival, in the bivariate models. When only stage IC cancers were studied, CD31 counts modified both disease-specific survival (HR, 4.8; P = 0.04) and the probability of recurrence (HR, 14.2; P = 0.006; Table 5). The largest impact, however, was on the probability of developing distant metastasis (HR, 44.1; P = 0.009). These findings clearly show the importance of tumor vascularity in developing a metastatic phenotype, particularly among stage IC patients, and suggest that the identification of high-risk markers could be an important indicator of patients who would benefit from adjuvant chemotherapy and should be further developed.

The recently published results of the ICON1 and ACTION trials suggest that the delivery of adjuvant chemotherapy following surgery for all stage I epithelial ovarian cancer prolongs disease-free and actuarial survival (2, 3). Indeed, failure to receive adjuvant chemotherapy in the present study also negatively impacted survival for the entire cohort, especially the stage IC subset. We were also able to show that recurrent stage IC disease was more likely in the absence of adjuvant chemotherapy. But it is also important to realize that 11 of the 18 patients who developed recurrent disease did so despite receiving platinum-based chemotherapy. Furthermore, six of these patients also received a taxane. Thus, even if we identify those at highest risk for recurrence, current therapy is inadequate to provide uniform cure. This observation should be added to those of Young (49) who would caution against blanket adjuvant chemotherapy treatment of all individuals with stage I epithelial ovarian cancer.

Clearly, the molecular environment of aggressive early-stage cancers differs from their less aggressive counterparts. This study has identified a limited association between p53 dysfunction and distant recurrence of stage I ovarian cancers. Evaluation of the expression levels of several downstream p53 response genes that impact angiogenesis or cell cycle regulation including p21, VEGF, and TSP-1 did not provide additional prognostic information. However, grossly microvessel count, as determined from CD31 expression, was uniquely identified in multivariate analysis as an adverse prognostic risk factor for stage IC disease. Further studies of early ovarian cancers utilizing techniques for the simultaneous study of thousands of genes (50) may prove more efficient in characterizing true high-risk stage I disease as well as identifying specific new therapeutic targets. We anxiously await the fruition of such studies so that more individualized therapy can become the norm rather than the current blanket therapeutic approaches that clearly treat more individuals than necessary yet fail to cure others whose tumors are resistant to conventional treatment.


   Acknowledgments
 
We thank Melanie Hatterman-Zogg, Linda Sanders, Lisa Blake, and Matthew Buller for research assistance.


   Footnotes
 
Grant support: National Cancer Institute grant R21-CA 84121 (R.E. Buller).

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.

Note: R.E. Buller is currently at GlaxoSmithKline, Oncology Medicine Development Center, 1250 South Collegeville Road, Collegeville, PA 19426.

Received 1/20/05; revised 2/23/05; accepted 3/ 1/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
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