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Clinical Cancer Research Vol. 10, 5168-5177, August 1, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Cyclin D1, p53, and p21Waf1/Cip1 Expression Is Predictive of Poor Clinical Outcome in Serous Epithelial Ovarian Cancer

Anish Bali1, Philippa M. O’Brien1, Lyndal S. Edwards2, Robert L. Sutherland1, Neville F. Hacker3 and Susan M. Henshall1

1 Cancer Research Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales; 2 Department of Anatomical Pathology, Prince of Wales Hospital, Randwick, New South Wales; and 3 Gynaecological Cancer Centre, Royal Hospital for Women, Randwick, New South Wales, Australia


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Dysregulation of cell cycle control, in particular G1-S-phase transition, is implicated in the pathogenesis of most human cancers, including epithelial ovarian cancer (EOC). However, the prognostic significance of aberrant cell cycle gene expression in EOC remains unclear.

Experimental Design: The expression of selected genes from the pRb pathway that regulates G1-S-phase progression, including cyclin D1, p16Ink4a, cyclin E, p27Kip1, p21Waf1/Cip1, and p53, was examined in a consecutive series of 134 serous EOC using immunohistochemistry and the results correlated to disease outcome.

Results: Molecular markers predictive of reduced overall survival in univariate analysis were overexpression of cyclin D1 (P = 0.03) and p53 (P = 0.03) and reduced expression of p27Kip1 (P = 0.05) and p21Waf1/Cip1 (P = 0.02), with the latter three also being prognostic for a shorter progression-free interval. In addition, patients displaying overexpression of p53 with concurrent loss of p21Waf1/Cip1 had a significantly shorter overall (P = 0.0008) and progression-free survival (P = 0.0001). On multivariate analysis, overexpression of cyclin D1 and combined loss of p21Waf1/Cip1 in the presence of p53 overexpression were independent predictors of overall survival. Similarly, the combination of p21Waf1/Cip1 loss and p53 overexpression was independently predictive of a shorter progression-free interval. Overexpression of p53 and cyclin E and reduced expression of p27Kip1 and p21Waf1/Cip1 were significantly associated with increasing tumor grade.

Conclusions: This study confirms that dysregulation of cell cycle genes is common in EOC, and that aberrant expression of critical cell cycle regulatory proteins can predict patient outcome in serous EOC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In developed countries, ovarian cancer is the leading cause of death from gynecological malignancies. Typically, this cancer has an insidious onset, and consequently, 70% of women present with disease that has spread beyond the ovary, resulting in a high mortality rate despite optimal surgery and aggressive chemotherapy (1) . Epithelial ovarian cancer (EOC) constitutes at least five different histological subtypes, of which, serous cystadenocarcinoma is the most prevalent (60% of all EOCs). The remainder include mucinous, endometrioid, clear cell, Brenner, and mixed phenotype tumors. In addition to their distinct morphological appearance and subtle clinical differences, there is molecular evidence for heterogeneity between different EOC subtypes (2 , 3) . Various clinical and pathological features of ovarian cancer are used as predictors of clinical outcome, of which, the volume of postoperative residual disease and International Federation of Gynecologists and Obstetricians (FIGO) stage are the most important (4 , 5) .

Dysregulation of normal cell cycle control has been implicated in the pathogenesis of most human cancers. In particular, abnormal expression of regulatory proteins that control G1-S-phase transition, a critical rate-limiting step in cell cycle progression, are frequently observed. G1-S transition requires phosphorylation of the retinoblastoma protein pRb, which results in the release of the E2F family of transcription factors that in turn activate genes essential for entry into S phase (6) . Phosphorylation of pRb is initiated by cyclin D1/(CDK)4-6 complexes and completed by cyclin E/CDK2 in late G1. Alterations in cyclin and/or cyclin-dependent kinase (CDK) expression results in increased cell proliferation and are thought to contribute to malignancy. Furthermore, down-regulation or inactivation of the CDK inhibitors, including p21Waf1/Cip1, p27Kip1, and p16Ink4a, which normally cause G1 arrest by binding to cyclin-CDK complexes, are often observed in diverse human tumors, further rendering the cell susceptible to uncontrolled extracellular proliferation signals (7) . Frequently mutated in a wide range of human cancers, p53 is a negative regulator of cell cycle control, which inhibits cell cycle progression in part by activating p21Waf1/Cip1 expression, and also controls the exit of cells from the cell cycle into programmed cell death.

Several studies have determined expression of these critical cell cycle regulatory proteins in EOC. Although changes in expression levels are frequently detected, there are many conflicting findings, making it difficult to delineate the role of individual genes in EOC development and progression (8) . Similarly, the prognostic significance of changes in cell cycle regulatory gene expression in EOC is unclear. The results of such studies may be confounded by several factors, including small patient sample sizes, differing therapeutic treatments between institutions that influence patient outcome, and the considerable disease heterogeneity of EOC. Indeed, it is likely that different mechanisms contribute to loss of cell cycle control in different histological subtypes of EOC (8) . Therefore, the aim of this study was to determine the expression levels of key proteins regulating G1-S-phase progression in a large consecutive series of 134 patients sourced from a single referral center, all of whom had been diagnosed with serous EOC and treated using similar surgical and adjuvant chemotherapy regimes. The prognostic significance of gene expression was determined using comprehensive clinicopathological follow-up data for each patient.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Tissue Samples.
A total of 134 patients treated by primary laparotomy between 1988 and 1998 for serous EOC was identified from the case records of the Gynaecological Cancer Centre at the Royal Hospital for Women, Sydney, Australia. The age at diagnosis, preoperative CA125 level, performance status, volume of postoperative residual disease, presence of intraoperative ascites, FIGO stage, and tumor grade were obtained retrospectively from patient records and are shown in Table 1Citation . Patient outcome was obtained from medical records and the New South Wales State Register of Births, Deaths and Marriages. Median follow-up time for the cohort was 29.8 months (2.6–135.7 months) and 2- and 5-year disease-specific survival rates were 67 and 31%, respectively. All experimental procedures were approved by the Research Ethics Committee of the South Eastern Sydney Area Health Service (00/115).


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  		Table 1 Clinical and pathological characteristics of the serous EOC patient cohort (n = 134)

 
Immunohistochemistry.
Immunohistochemistry was performed on formalin-fixed, paraffin embedded tissue samples sectioned at 4 µm and mounted on SuperFrost slides (Menzel-Glaser, Braunschweig, Germany). The tissue sections were deparaffinized in xylene (twice for 5 min each) and rehydrated through graded ethanol. Antigen retrieval was achieved by heating the samples using a microwave (p53, p21Waf1/Cip1), pressure cooker (pRb), or boiling water bath (p27Kip1, p16Ink4a, cyclin D1, and cyclin E). Endogenous peroxidase activity was quenched using 3% hydrogen peroxide (H2O2) in methanol for 5 min. The sections were then blocked with normal horse serum followed by incubation with mouse monoclonal antibodies to either p53 (clone DO-7; DAKO Corporation, Carpinteria, CA), p21Waf1/Cip1 (clone 70; Transduction Laboratories, Lexington, KY), pRb (clone G3-245; PharMingen, San Diego, CA), p27Kip1 (clone 57; Transduction Laboratories), p16Ink4a (clone 16PO4; Neomarkers, Fremont, CA), cyclin D1 (clone DCS-6; DAKO Corporation), or cyclin E (HE12; PharMingen). The Vectastain Elite Avidin-Biotin Complex kit (Vector Laboratories, Burlingame, CA) was used as the detection system and color development was obtained using 3,3-diaminobenzidine (Vector Laboratories). Counterstaining was undertaken with Whitlock’s hematoxylin, Scott’s blueing solution with or without light green before dehydration through graded ethanol and xylene. Formalin-fixed, paraffin-embedded cell lines, known for their status of the specific genes studied, were used as positive and negative controls.

Scoring.
Two independent observers (A. Bali and L. Edwards, a specialist gynecological pathologist) assessed the pattern of staining of molecular markers for each tissue sample. Standardization of scoring was achieved by comparison of scores between observers and any discrepancies were resolved by consensus. Scores were given as a percentage of positive nuclear staining within representative areas of the tumor sample. The percentage score above which staining was representative of overexpression was based on reports in the published literature. The median percentage of immunostaining, percentage values deemed as overexpression, and number of specimens displaying positive staining are shown in Table 2Citation . Representative photomicrographs of tumor tissue showing positive and negative staining for the specific antigens are presented in Fig. 1Citation .


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  		Table 2 Immunohistochemical analysis of cell cycle gene expression in serous EOC

 


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  		Fig. 1. Immunohistochemistry on primary tumor tissue. A, p53 overexpression in a poorly differentiated serous EOC. B, p53-negative staining in a poorly differentiated (G3) serous EOC. C, p21Waf1/Cip1 expression >10% in a moderately differentiated (G2) serous EOC. D, p21Waf1/Cip1-negative staining in a moderately differentiated (G2) serous EOC. E, cyclin D1 overexpression in a poorly differentiated (G3) serous EOC. F, cyclin D1-negative staining in a poorly differentiated (G3) serous EOC. G, high p27Kip1 expression in a poorly differentiated (G3) serous EOC. H, low p27Kip1 expression in a poorly differentiated (G3) serous EOC. Magnification, x40.

 
Survival Analysis.
Survival analysis was performed using a Kaplan-Meier analysis and Cox proportional hazards model. Predictive variables of disease outcome on univariate analysis were incorporated into multivariate analysis to identify factors that predict clinical outcome independently of other clinicopathological or molecular parameters. The progression-free interval was calculated from the date of primary laparotomy to the date of disease progression as specified by a rise in CA125 or radiological or surgical evidence of relapse. The length of overall survival (OS) was defined from the date of primary laparotomy to the date of patient death or to the date of last follow-up. The relationship between gene expression and the clinical and pathological parameters was determined using {chi}2 and Fisher’s exact test where appropriate. P ≤ 0.05 was accepted as statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Correlation between Patterns of Gene Expression and Clinicopathological Parameters.
Increased expression of p53 (P = 0.03) and cyclin E (P = 0.03) and reduced levels of p21Waf1/Cip1 (P = 0.002) and p27Kip1 (P = 0.04) were all significantly associated with increasing tumor grade (Table 3)Citation . FIGO stage, volume of postoperative residual disease, and presence of ascites did not correlate with the expression pattern of any of the proteins studied.


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  		Table 3 Association of gene expression and clinicopathological parameters in serous EOC

 
The expression of p53 appeared to be negatively correlated with that of p21Waf1/Cip1 (Table 3)Citation . Of the cancers demonstrating p53 expression of <10%, 40% displayed p21Waf1/Cip1 expression > 10% in contrast to those with p53 overexpression in which only 14% demonstrated a concurrent increase in p21Waf1/Cip1 levels (P = 0.007). An inverse relationship between p53 and p16Ink4a expression (P = 0.004) was also observed. Of the p53-positive tumors, 37% demonstrated p16Ink4a expression, compared with 62% expressing p16Ink4a in the p53-negative cancers. We also determined a weak positive correlation between overexpression of p53 and cyclin E expression. Sixty-seven percent of p53-positive tumors were also positive for cyclin E expression in contrast to p53-negative tumors where 50% were cyclin E positive (P = 0.05).

The expression of p27Kip1 was positively correlated with that of p21Waf1/Cip1 (P = 0.006). In 55% of tumors that demonstrated p21Waf1/Cip1 expression, >10% also showed increased levels of p27Kip1, as compared with p21Waf1/Cip1-negative cancers (<10%), in which increased p27Kip1 staining was observed in only 28% of cases. We also determined a negative correlation between p27Kip1 and cyclin D1 expression. Of the p27Kip1-positive tumors, 9% demonstrated cyclin D1 expression, whereas 24% of the p27Kip1-negative cancers were positive for cyclin D1 expression (P = 0.03). No other significant associations between gene expression were observed.

Survival Analysis.
On univariate analysis, clinicopathological determinants of reduced OS and progression-free interval included the presence of ascites, a volume of residual disease ≥ 2 cm, advanced FIGO stage (III/IV), increasing tumor grade (grade 2/3), and poor performance status (≥1; Table 4Citation ). Molecular markers predictive of reduced OS on univariate analyses were overexpression of p53 (P = 0.03), loss of p21Waf1/Cip1 (P = 0.02), increased levels of cyclin D1 (P = 0.03), and reduced expression of p27Kip1 (P = 0.05; Table 4Citation ). Aberrant expression of p53 (P = 0.004), p21Waf1/Cip1 (P = 0.01), and p27Kip1 (P = 0.05) were also significant determinants of reduced progression-free interval. The simultaneous assessment of p53 and p21Waf1/Cip1 expression revealed that patients whose tumors displayed p53 overexpression with concurrent loss of p21Waf1/Cip1 had a poorer prognosis for progression-free interval (P = 0.0001) and OS (P = 0.0008) in comparison to patient tumors with other combinations of expression of these two proteins.


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  		Table 4 Univariate analysis associating clinicopathological parameters and gene expression to clinical outcome in serous EOC.

 
In a multivariate analysis using a Cox proportional hazards model incorporating residual disease, presence or absence of ascites, and patient performance status, the volume of residual disease remained the most important determinant of progression-free interval and OS (Table 5)Citation , similar to other cohorts (4 , 5) . The presence of ascites and performance status became insignificant in both multivariate models. Tumor grade and clinical stage were not included for purposes of multivariate analysis. Consistent with population-based studies of serous ovarian cancer, which show that only ~15% of these cancers are classified as stage I/II (9) , only 14 of 134 tumors (10.4%) in this cohort were classified as stage I/II, and thus, the numbers were too small to be included in a multivariate analysis. Similarly, only seven patients were classified as having well-differentiated tumors (G1). Moreover, we found no survival advantage for patients with moderately differentiated tumors (G2, n = 64) as compared with poorly differentiated ones (G3, n = 63; Table 4Citation ), reflected when only those patients with G2 or G3 tumors were included in a multivariate model of progression-free interval (hazard ratio 0.90, 95% confidence interval 0.58–1.39, P = 0.64) or OS (hazard ratio 0.79, 95% confidence interval 0.48–1.29, P = 0.34) along with residual disease, ascites, and performance status.


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  		Table 5 Multivariate Cox regression analysis of progression-free survival and OS of patients with serous EOC

 
Overexpression of p53 and low p21Waf1/Cip1 expression remained significant when incorporated into the multivariate analysis (data not shown) and when combined had a stronger effect at predicting OS (P = 0.0006) and progression-free interval (P = 0.0003; Fig. 2Citation and Table 5Citation ). Overexpression of cyclin D1 (P = 0.038) was also identified as an independent predictor of OS when incorporated into the multivariate model. Hence, cyclin D1 and combined p53 and p21Waf1/Cip1 expression are stronger predictors of patient outcome than tumor classification as moderately (G2) or poorly differentiated (G3) tumors, commonly used as prognostic indicators in the clinic. Overexpression of p27Kip1 did not retain its significance when considered together with residual disease, presence of ascites, and patient performance status (data not shown).



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  		Fig. 2. Kaplan-Meier curves and log-rank P values for 5-year progression-free interval (PFI), and OS according to cyclin D1 expression (A); p53 expression (B); p21Waf1/Cip1 expression (C); and combined p53 and p21Waf1/Cip1 expression (D). Numbers in brackets following parameter stratification indicate the number of patients in each group.

 
Stratification of p53 Expression and Survival.
The staining pattern of p53 across the sample set revealed a bimodal distribution (Fig. 3)Citation . p53 expression of <5% was observed in 55 (41%) tumors, >50% in 53 (40%) tumors, with the remainder (n = 26, 19%) being equally distributed between 5 and 50%. Only two (8%) patients that had p53 expression of between 5 and 50% were alive 5 years after initial diagnosis, in contrast to 23 patients (42%) who were still alive at 5 years after diagnosis in whom p53 expression was <5% (P < 0.0001). The 5-year survival rate of patients with p53 expression > 50% was 36%, which is similar to those with <5% expression. A similar association between expression of p53 and progression-free interval was also demonstrated (P < 0.0001). Kaplan-Meier survival curves of progression-free interval and OS and log-rank P values are shown in Fig. 3Citation .



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  		Fig. 3. A, distribution of p53 expression in serous EOC patient cohort. B, Kaplan-Meier curves and log-rank P values for 5-year progression-free interval (PFI), and (C) 5-year OS, according to p53 stratification. Patients were assigned three subgroups according to their percentage of p53 nuclear staining: p53 < 5% [n = 55 (41%)]; p53 > 5 < 51% [n = 26, (19%)]; p53 > 50% [n = 53 (40%)].

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The past 5 years have seen a number of studies reporting conflicting results on the prognostic value of aberrant cell cycle gene expression in determining the survival of patients with EOC. One reason for this discrepancy is the considerable disease heterogeneity of EOC because different alterations in cell cycle regulatory genes are likely to underlie the varied presentations of EOC. Therefore, we confined our study to patients diagnosed with a single histological subtype, serous ovarian cancer, the most frequently diagnosed form of EOC. In addition, the patients were recruited from a single referral institution, which is reflected in the high proportion of advanced FIGO stage III/IV tumors in our cohort and is consistent with the frequent late-stage presentation of women with serous EOC. Nevertheless, examination of traditional clinicopathological variables confirms that this cohort behaves in accordance with other published series of EOC with regard to disease-specific and progression-free survival (4 , 5) . Therefore, we examined the prognostic value of selected cell cycle regulatory genes in this cohort of 134 patients.

The formation of cyclin D1/CDK4–6 complexes is an early critical step regulating G1-S-phase progression, and abnormally high levels of cyclin D1 are found in many tumor types. Although mutation or amplification of the cyclin D1 gene is rare, increased levels of mRNA and protein have been reported in 14–59% of invasive ovarian cancers (10, 11, 12, 13, 14) . In our cohort of serous EOC, 19% of tumors demonstrated >10% nuclear accumulation of cyclin D1, consistent with these studies. In addition, we found that overexpression of cyclin D1 is an independent prognostic indicator of OS in patients with serous EOC. Overexpression of cyclin D1 with or without gene amplification has been identified as an adverse prognostic biomarker in lung, pancreas, and tongue carcinoma (15, 16, 17) ; however, this is the first study of an association with OS for EOC. Only one other study has reported a significant relationship between cyclin D1 overexpression (as determined by mRNA expression) and a shorter progression-free interval, performed in a small series of 24 patients with invasive ovarian malignancies (12) . These authors also reported a significant association between cyclin D1 overexpression with serous carcinomas and with poorly differentiated tumors. With regards to the latter finding, we observed a trend but not a significant association between cyclin D1 overexpression and increasing tumor grade (likely due to the small number of well-differentiated tumors). In contrast, early studies of cyclin D1 overexpression in ovarian cancer reported significant co-segregation with well-differentiated tumors (10 , 14) . A larger study may be warranted to define the relationship between cyclin D1 expression and tumor differentiation in EOC.

The activity of the cyclin D1/CDK4-6 complex is predominantly regulated by p16Ink4a, a ubiquitous tumor suppressor gene inactivated in many human cancers (18) . Although loss of heterozygosity of p16Ink4a occurs in 30–40% of ovarian tumors, mutations in the remaining allele are rare (19) . In addition, promoter methylation of p16Ink4a also appears to be uncommon (20 , 21) . Regardless of the mechanism, down-regulation of p16Ink4a is often observed in EOC (21, 22, 23) . However, it appears that levels of p16Ink4a may vary between histological subtypes. In particular, low p16Ink4a expression may be more common in mucinous and endometrioid EOC, and overexpression of p16Ink4a is more common in high-grade serous cancers (24) . Our results fit with the latter observation, with 55% of serous cancers expressing p16Ink4a in >15% of tumor cells.

Loss of p16Ink4a has been associated with poor clinical outcome in several cancers, including head and neck and prostate cancer (17 , 25) . There is no evidence to suggest that aberrant expression of p16Ink4a is associated with clinicopathological parameters or patient outcome in ovarian cancer, and our results would support these findings (19 , 22) . However, we did observe a significant negative correlation between the expression of p16Ink4a and p53. p53 expression is indirectly regulated by a second tumor suppressor gene encoded at the INK4A/ARF locus, p14ARF. There is thought to be extensive cross-talk between the p16Ink4a/Rb and the p14ARF/p53 pathways, therefore, raising the possibility that levels of p16Ink4a and p53 may be coregulated (18) .

The second pathway involved in cell cycle progression to S phase involves cyclin E/CDK2 complexes and is primarily regulated by the CDK inhibitors p27Kip1 and p21Waf1/Cip1. Cyclin E mRNA overexpression has been detected in 22–45% of ovarian cancers and, correspondingly, gene amplification frequently detected (26, 27, 28) . High cyclin E expression has been associated predominantly with serous and clear cell carcinomas (24 , 29) , consistent with our results where 68% of patients exhibited overexpression of cyclin E. Only one study has reported a significant association of high cyclin E expression with patient outcome (28) ; our results agree with earlier studies where such an association was not found (24 , 29) . We did, however, note an association of cyclin E expression with increasing tumor grade, not previously reported in EOC.

We also found that cyclin E levels appeared to correlate with p53 expression. Mutation of the p53 gene has been implicated in the development of >50% of all human cancers. Gene mutations and/or overexpression of p53 have been detected in 30–80% of EOC, particularly in serous EOC, the majority of which are missense mutations that closely correlate with accumulation of mutated p53 protein (30) . However, the role of p53 mutation and cellular accumulation in determining the outcome of patients with EOC is far from conclusive (31, 32, 33, 34) . In the present study of serous EOC, p53 overexpression (>10% nuclear staining) was able to predict patient outcome independent of the strongest clinicopathological prognostic markers in our cohort. A closer examination of p53 expression revealed that only 8% of patients with intermediate expression (5–50%) were alive at 5 years after diagnosis, which was significantly poorer than those with low (<5%: 23% survival) or high (>50%: 36% survival) levels of p53. A similar association was also observed for progression-free survival. The reason for this observation is unclear. First, it is possible that some of the tumors are overexpressing wild-type functional p53. Expression levels of p53 in normal cells are very low, increasing transiently in response to cellular stresses. However, levels of p53 can be stabilized by interactions with other intracellular proteins such as mdm-2, leading to positive immunostaining (35 , 36) . Second, certain p53 mutations may result in very high levels of p53 protein that may have some residual activity in regulating cellular functions (37 , 38) . Third, p53 point mutations resulting in a stop codon, frameshift, or nonsense mutations may result in altered or truncated proteins that are not detected by immunohistochemistry (39) . Ovarian cancers with p53 gene mutations, resulting in truncated proteins, develop distant metastases more rapidly than tumors with missense or no mutations, and consequently, survival in these patients is significantly shorter (40) . More comprehensive mutation and functional studies are required to decipher the role of mutant and wild-type p53 in ovarian carcinogenesis.

One of the roles of p53 in cell cycle control is regulation of expression of the CDK inhibitor p21Waf1/Cip1, an inhibitor of cyclinE/CDK2 complexes. Inactivation of p53 function leads to loss of p21Waf1/Cip1 induction and impairment of cyclin/CDK complex inhibition. Indeed, we found that 77% of serous EOC in our cohort had low or no expression of p21Waf1/Cip1, which was significantly associated with poor survival and a shorter progression-free interval, as previously demonstrated by others (41, 42, 43, 44) . In addition, we found significant loss of p21Waf1/Cip1 expression in tumors that overexpressed p53, although this did not seem to be the case in all tumors. Again, conflicting data have been reported regarding an association between p53 and p21Waf1/Cip1 expression in EOC (32 , 41 , 42) , presumably reflecting both p53-dependent and -independent mechanisms of p21Waf1/Cip1 regulation. We analyzed the prognostic relevance of combining p53 and p21Waf1/Cip1 expression and found loss of p21Waf1/Cip1 in conjunction with p53 overexpression was a stronger predictor of reduced survival and progression-free interval than either molecule alone, suggesting p53-dependent mechanisms dominate p21Waf1/Cip1 expression in serous EOC. These results are consistent with findings in other studies (41 , 45) .

p21Waf1/Cip1 also plays a functional role in cell differentiation (7) . We found a significant correlation of elevated p21Waf1/Cip1 protein with well-differentiated serous EOC compared with a marked loss of p21Waf1/Cip1 expression in moderately and poorly differentiated cancers. A larger series of serous EOC is required to confirm this result; however, a similar finding has been reported in a study incorporating EOC of varying histological subtypes (41) .

We also examined expression of the CDK inhibitor p27Kip1, which is also frequently down-regulated in human cancers, and its loss correlated with poor prognosis in several tumor types, including breast and prostate cancer (46 , 47) . Three studies support an independent prognostic role of p27Kip1 in determining the clinical outcome of patients with EOC, including serous cancer (48, 49, 50) . A further study of 185 patients reported a trend (which did not reach significance) of reduced OS in a small subgroup of patients (n = 11) whose ovarian cancers completely lacked p27Kip1 expression (51) . In our study, we found reduced p27Kip1 expression was a marker of poor clinical outcome (shorter OS and progression-free interval) in univariate analysis, but this did not retain significance as an independent prognostic factor when adjusted for other clinicopathological and molecular variables of disease outcome.

We found a significant positive association of p27Kip1 expression with both p21Waf1/Cip1 and cyclin D1 expression. Correlation of expression of p27Kip1 and p21Waf1/Cip1 fits with their similar inhibitory roles in G1 checkpoint regulation and is consistent with other reports (51) , although incongruous with the different mechanisms that regulate expression of each gene, being predominantly posttranslational and –transcriptional, respectively.

A correlation between loss of p27Kip1 expression and cyclin D1 overexpression, both of which were associated with increasing tumor grade in our cohort, is more difficult to explain. A positive correlation between cyclin D1 and p27Kip1 has been reported in several cancers, including EOC (13) . Moreover, in vitro studies have shown that embryonic fibroblasts lacking genes encoding p27Kip1 and p21Waf1/Cip1 have reduced levels of cyclin D1, which are restored by reintroduction of p27Kip1 (52) , suggesting a positive association between expression of the two genes. However, an inverse correlation between p27Kip1 and cyclin D1 expression has been reported in a mouse model of mammary carcinogenesis (53) . It is thus likely that these results reflect our incomplete understanding of the effects of p27Kip1 expression on cell cycle regulation, including the promotion of G1 progression via binding to cyclin D1/CDK complexes (7) , particularly in the presence of altered levels of cyclins and CDKs as observed in tumors.

The interaction of cell cycle regulatory proteins leads to phosphorylation and inactivation of pRb and subsequent entry of the cells into S-phase. The RB gene is a well-defined tumor suppressor and mutations have been described in a wide variety of human cancers (54) . Loss of heterozygosity at the RB locus is common in EOC; however, the majority of ovarian carcinomas exhibit pRb expression (8) . Reduced expression of pRb has been previously associated with increasing tumor grade, stage, and residual disease volume in EOC but not patient outcome (55) . In contrast, a recent study determined that high expression of pRb was an independent prognostic marker of poor OS (24) . We found that 79% of serous EOC expressed pRb (>20%) but did not find any associations with clinicopathological parameters, patient outcome, or other molecular markers.

In conclusion, this study confirms that deregulation of expression of critical cell cycle regulatory proteins, in particular cyclin D1, cyclin E, p21Waf1/Cip1 and p27Kip1, are frequent events in serous EOC. Furthermore, cyclin D1 expression and a combination of p53 and p21Waf1/Cip1 expression are independent prognostic markers of disease progression.


   ACKNOWLEDGMENTS
 
We thank Professor Donald Marsden and Dr. Greg Robertson, Consultant Gynaecological Oncologists at the Gynecological Cancer Centre, Royal Hospital for Women, and Dr. Catherine Camaris, Department of Pathology, Royal Hospital for Women for their professional assistance in carrying out this study. We also thank Patricia Vanden Bergh and Therese Malone for coordinating the clinical and tissue databases held at the Garvan Institute of Medical Research and the Royal Hospital for Women and Darren Head for assistance with immunohistochemistry.


   FOOTNOTES
 
Grant support: Gynaecological Oncology Fund of the Royal Hospital for Women, Sydney, Australia, and the R. T. Hall Trust. R. Sutherland and S. Henshall are also supported by the National Health & Medical Research Council of Australia, The Cancer Council New South Wales, and the Prostate Cancer Foundation of Australia (S. Henshall).

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.

Requests for reprints: Philippa O’Brien, Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia. Phone: 61-2-9295-8337; Fax: 61-2-9295-8321; E-mail: p.obrien{at}garvan.org.au

Received 12/16/03; revised 4/ 6/04; accepted 4/22/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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