Departments of 1 Molecular Pathology, 2 Biostatistics, 3 Gynecologic Oncology, Institute of Oncology, Roentgena, Warsaw; 4 University of Medical Sciences, Pozna; Departments of 5 Pathology and 6 Obstetrics and Gynecology, Bródnowski Hospital and Second Faculty of Medicine, Medical University, Warsaw; 7 Second Gynecological Department, and 8 Department of Pathology, Medical University, Gda
sk; 9 Department of Chemotherapy, Medical University,
od
; 10 Second Department of Gynecology, Medical Academy, Wroc
aw; 11 Department of Gynecological Surgery and Oncology of Adults and Adolescents, Pomeranian Academy of Medicine, Szczecin; 12 Department of Gynecologic Oncology, Institute of Oncology, Kraków, Poland
Received 21 November 2002; revised 24 March 2003; accepted 1 April 2003
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Abstract |
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The prognostic and predictive value of cell cycle regulatory proteins in ovarian cancer has not been established. We evaluated the clinical and biological significance of P21WAF1, P27KIP1, C-MYC, TP53 and Ki67 expressions in ovarian cancer patients.
Materials and methods:
Immunohistochemical analysis was performed on 204 ovarian carcinomas of International Federation of Gynecology and Obstetrics (FIGO) stage IIB to IV treated with platinum-based chemotherapy. Multivariate analysis with Cox and logistic regression models was performed in the whole group, and in the TP53-negative and TP53-positive subgroups.
Results:
High P21WAF1 labeling index (LI) was an independent positive predictor of platinum-sensitive response (P = 0.02). Overall survival was positively influenced by P21WAF1 LI (P = 0.02) or by P21WAF1 plus P27KIP1 LI (P = 0.004) in the TP53-negative group only. Ki67 LI showed borderline association with disease-free survival (P = 0.05). Growth fraction was negatively associated with P21WAF1 and P27KIP1 indices in the TP53-negative group (P = 0.023 and 0.008, respectively), and these associations were borderline or lost in the TP53-positive group. Endometrioid and clear cell carcinomas differed from other carcinomas by having a low incidence of TP53 accumulation, a high incidence of C-MYC overexpression (70%) and a low median Ki67 LI (all with P <0.001).
Conclusions:
We have shown an independent predictive value of P21WAF1 LI in ovarian carcinoma patients. The prognostic value of P21WAF1 and P21WAF1 plus P27KIP1 LI was determined by TP53 status. A high frequency of C-MYC overexpression in endometrioid and clear cell carcinomas may suggest its role in the development of these tumor types.
Key words: Ki67, C-MYC, ovarian cancer, P27KIP1, P21WAF1, TP53
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Introduction |
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Proto-oncogene C-MYC and the tumor suppressor gene TP53 encode phosphoproteins that participate in the regulation of cellular proliferation, apoptosis and cell differentiation. C-MYC is a positive regulator of the cell cycle, while TP53 is a negative regulator. The main mechanism by which TP53 exerts its inhibitory effect on cell proliferation is induction of P21WAF1 expression, and this may be impaired by TP53 gene mutations [5]. On the other hand, some data suggest that C-MYC may antagonize P27KIP1 activity (reviewed in [6, 7]). P21WAF1 and P27KIP1 are inhibitors of cyclin-dependent kinases (cdk) and cause G1 arrest by binding to cyclincdk complexes. P21WAF1 expression can also be regulated by epigenetic silencing [8] and TP53-independent pathways [9]. In some experimental models C-MYC inhibited P21WAF1 activity [10] and the TP53-dependent induction of P21WAF1 [11].
In ovarian carcinoma cell lines P21WAF1 gene transfer enhanced the cytotoxic effect of cisplatin [12]. However, its predictive role in ovarian carcinoma has not been demonstrated. Data on the prognostic importance of P21WAF1 in ovarian carcinoma are controversial. In the majority of studies P21WAF1 had no prognostic value [2, 4, 1316], while in others, except in a study by Costa et al. [3], this has been demonstrated in univariate analyses only [1, 17]. As far as P27KIP1 is concerned, several groups have found it of prognostic significance in ovarian tumors [14, 18, 19], while others have not [2, 13]. Low levels of P27KIP1 expression were associated with chemoresistance in ovarian carcinoma patients in one study [20].
We have recently found that ovarian carcinomas with and without TP53 protein accumulation differ in clinical significance of apoptosis-regulating proteins [21]. The purpose of the present study was to examine the value of P21WAF1, P27KIP1, Ki67 and C-MYC proteins in predicting prognosis and response to chemotherapy in a large cohort of uniformly treated patients with advanced epithelial ovarian cancer, with respect to TP53 status.
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Materials and methods |
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Evaluation of clinical response to chemotherapy
Response to chemotherapy was evaluated retrospectively according to the World Health Organization (WHO) response evaluation criteria [23]. The evaluation was based on data from medical records describing patients clinical condition and CA 125 levels in 3- to 4-week intervals. Complete remission (CR) was defined as the disappearance of all clinical and biochemical symptoms of ovarian cancer evaluated after completion of first-line chemotherapy and confirmed at 4 weeks. Within the CR group we have distinguished a platinum-sensitive group (PS) according to criteria given by Christian and Trimble [24] [disease-free survival (DFS) longer than 6 months; 89 patients]. Thus, the other tumors [partial remission (PR), progression (P), no change (NC)], as well as the CR group with DFS shorter than 6 months were described as resistant to cisplatin [24] (Table 1).
Histopathological data
All tumors came from the first laparotomy. They were uniformly reviewed histopathologically (J. Kupryjaczyk) and classified according to the criteria of WHO [25]: 159 (78%) tumors were of the serous type, 13 (6%) were of the endometrioid type, 10 (5%) were of the clear cell type, 12 (6%) were undifferentiated and 10 were of other types. Histological grade was evaluated on a 4-grade scale according to the Broders criteria [26]: 27 tumors (13%) showed moderate differentiation (G2), 128 (63%) showed poor differentiation (G3) and 49 (24%) were mostly undifferentiated or completely undifferentiated (G4).
Immunohistochemical analysis
Immunohistochemical stainings were performed on paraffin-embedded material. We used PAb1801 monoclonal antibody (1:500; Sigma-Genosys, Cambridge, UK) for the TP53 protein, sc-6246 monoclonal antibody (1:40; Santa Cruz Biotechnology Inc., Santa Cruz, USA) for P21WAF1, NCL-P27 monclonal antibody (1:40) for P27KIP1 and NCL-cMYC monoclonal antibody (1:200) for C-MYC (both from Novocastra, UK), as well as MIB-1 monoclonal antibody (1:50; Immunotech, Marseille, France) for Ki67 antigen.
The immunohistochemical procedure has been described previously [27, 28]. Briefly, deparaffinized sections were boiled in a citrate buffer (pH 6.0) (HIER) at 700 W in a microwave: 2 x 5 min for TP53, 3 x 5 min for Ki67 and 6 x 5 min for P27KIP1; for P21WAF1 detection, the sections were boiled in the same buffer for 5 min at 120°C and 15 psi in an autoclave. P21WAF1 antigen appeared more sensitive to fixation and processing conditions than other antigens studied; it was also relatively resistant to retrieval in a microwave (35% of the cases were negative or only focally positive). C-MYC expression was detected without any antigen retrieval. Non-specific tissue and endogenous peroxidase reactivities were blocked with 10% bovine serum albumin (BSA) and 3% H2O2, respectively. Biotinylated goat anti-mouse immunoglobulin G (IgG; 1:1500, cat. no. 816), peroxidase-conjugated streptavidin (1:500, cat. no. 309) (both from Immunotech, Marseille, France), and diaminobenzioline (DAB) were used as a detection system. Tissue sections were incubated with primary antibodies for 1 h at room temperature (anti-TP53, anti-Ki67) or overnight at 4°C (anti- P27KIP1, anti-P21WAF1, anti-C-MYC). Ovarian carcinomas with and without a TP53 gene missense mutation were controls for TP53 while a human tonsil was a control for proliferation antigens. Ovary with corpus luteum and plasma cells were positive controls for C-MYC. An isotype-matched antibody from the same company directed against a cytoplasmic antigen [anti-MEK1 (clone H-8), Santa Cruz Biotechnology Inc.], was used as a negative control for anti-P21WAF1. Anti-P27KIP1 antibody was a negative control for anti-C-MYC (both of the same isotype). Normal mouse IgG of the same subclasses and concentrations as the primary antibodies served as negative controls also (all from Dako, Glostrup, Denmark).
Evaluation of immunohistochemical stainings
TP53 protein accumulation was described as present (>10% of positive cells) or absent. MIB1-positive cells were counted in the three different foci most rich in proliferating cells: 500 cells were counted in each focus (total 1500). P21WAF1 and P27KIP1 expressions were evaluated in four different randomly chosen places and 200 cells were counted in each focus (total 800). Ki67, P21WAF1 and P27KIP1 LIs were defined as a proportion of positive cells to total cells counted. We also created a combined variable, which was a sum of P21WAF1 and P27KIP1 LIs, i.e. P21WAF1 plus P27KIP1. For C-MYC expression the following staining categories were created: weak, moderate and strong.
Statistical analysis
Probability of survival and DFS were estimated using the KaplanMeier method. Overall survival and DFS time analyses were performed with multivariate Coxs proportional hazards models [29]; factors that may determine tumor response to chemotherapy were evaluated in the multivariate logistic regression model. Important factors were selected using backward selection technique, where factors not significant at 0.1 were drawn one by one out of the model. The analysis was performed in all ovarian carcinomas, and separately in the TP53() and TP53(+) subgroups.
Associations between protein expressions and clinico-pathological parameters were studied by chi-square or Fishers exact test. All tests were two-sided and the level of significance was set at 5%. Associations between numerical indices and protein expressions or clinical and immunohistochemical parameters were performed using the Students t-test or F-test. All calculations were performed using the STATA 7.0 program.
In all multivariate models the cut-off points for numerical indices were determined at median value. The combined P21WAF1 plus P27KIP1 variable has been evaluated in statistical analyses alternatively to separate P27KIP1 and P21WAF1 variables.
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Results |
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Associations between Ki67, P21WAF1 and P27KIP1
Ki67 LI showed negative association with P21WAF1 LI (P = 0.0004), P27KIP1 LI (P = 0.0021) and with combined P21WAF1 plus P27KIP1 LI value (P <0.0001). P21WAF1 LI was positively correlated with P27KIP1 LI (P <0.0001).
Analysis in the TP53() and TP53(+) subgroups revealed that associations between Ki67 LI and P21WAF1, P27KIP1 and P21WAF1 plus P27KIP1 expressions are much stronger in the TP53() group (P = 0.023, 0.008 and 0.002, respectively) than in the TP53(+) group (P = 0.05, 0.24 and 0.05, respectively), despite its larger size. In TP53(+) and TP53() subgroups, P21WAF1 was positively associated with P27KIP1 at the same level of statistical significance (P = 0.004).
TP53 associations with Ki67, P21WAF1, P27KIP1 and C-MYC
TP53(+) carcinomas had higher Ki67 LIs (range 2793, mean 65.7) than TP53() carcinomas (range 788, mean 52.0) (P <0.0001). P21WAF1 LI was lower in the TP53(+) group than in the TP53() group; however, the difference was not statistically significant (P = 0.08). Neither P27KIP1 LI nor C-MYC expression showed differences in relation to the TP53 status.
C-MYC associations with Ki67, P21WAF1 and P27KIP1
C-MYC overexpression was associated with lower Ki67 LI (P = 0.02). In accordance, P27KIP1 LI was higher in tumors with C-MYC overexpression; however, the difference was not statistically significant (P = 0.09). C-MYC overexpression did not show an association with P21WAF1 LI.
TP53, Ki67, P21WAF1, P27KIP1, C-MYC and clinicopathological parameters
Endometrioid and clear cell carcinomas differed from serous, undifferentiated and other carcinomas in having a low incidence of TP53 accumulation, a low median Ki67 LI and a high incidence of C-MYC overexpression (P = 0.0004) (Table 2). The differences between histological types were not due to differences in tumor differentiation degree, because there was no association between type and differentiation. Serous carcinomas differed from undifferentiated and other carcinomas in having a slightly lower Ki67 LI only (P = 0.027) (Table 2).
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The proteins studied did not correlate with FIGO stage. P27KIP1 LI was the only LI associated (negatively) with residual tumor size (P = 0.05).
Overall survival, disease-free survival and tumor response to chemotherapy
Overall survival (OS) in the whole group was positively influenced by lower patients age, lower FIGO stage, lower residual tumor size, and not by any immunohistochemical parameter studied. Analysis of the TP53() group revealed an independent prognostic value of P21WAF1 LI, and alternatively included P21WAF1 plus P27KIP1 LI (Figure 2, KaplanMeier). These associations have not been confirmed in the TP53(+) group despite its larger size (Table 3).
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Platinum-sensitive response (CR with DFS longer then 6 months) was associated with small residual tumor size, lower FIGO stage and high P21WAF1 LI. Predictive value of P21WAF1 LI has been confirmed in the TP53(+) group only, at a lower level of significance (Table 4). CR status was associated with small residual tumor size only (P <0.001). However, when residual tumor size was divided into five categories (0 versus <1 versus 2 versus 25 versus >5 cm) instead of two (
2 versus >2 cm), we observed a positive impact of high P21WAF1 LI on both CR and platinum sensitivity.
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Discussion |
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P21WAF1 predictive value has been demonstrated in all patients and confirmed in the TP53(+) group only at the lower level of significance, which might have been caused by the smaller size of the subgroup; this could also explain why this association has not been found in the TP53() group. On the other hand, TP53 status might determine P21WAF1 and P21WAF1 plus P27KIP1 prognostic value, since it has been observed in the TP53() group only, and not in all patients nor in the large TP53(+) group. We have previously observed the differential clinical importance of BCL-2 and BAX proteins depending on TP53 status [21]. TP53 is a pleiotropic protein with complex interactions at gene and protein levels, including regulation of transcriptional activation of P21WAF1, BCL-2 and BAX genes [5, 31]. These functions are largely lost by mutant TP53, which results in profound alterations of cellular processes, such as signaling of DNA damage and its repair, cell cycle arrest and apoptosis. Possibly, decreased P21WAF1 expression may demonstrate its clinical significance in tumors with wild-type TP53, but as a biological change may be too subtle to show an influence in the TP53(+) group. Our results confirm our previous observations that separate evaluation of TP53(+) and TP53() subgroups may help to identify molecular markers of clinical importance in ovarian cancer.
Some studies suggested that P21WAF1 has an influence on clinical end points in ovarian cancer, but generally it has not been confirmed by multivariate analyses [1, 3, 17]. Costa et al. [3] reported P21WAF1 as a factor of overall survival in a group stratified for clinical stage and nuclear grade. Some authors reported that a variable of combined P21WAF1 and TP53 may be a marker of prognosis [1, 32] and such findings may be somehow related to our result obtained for the TP53() group. Our P21WAF1 counts are much higher (median 78%) than those of other authors (median 13%) who reported 26% to 57% of negative tumors (sometimes with negative normal tissues) [13, 32]. Thus, the difference may also be caused by the different antibody used and the more efficient antigen retrieval applied in our study, which enabled us to demonstrate P21WAF1 expression in normal tissues also. Royuela et al. studied prostatic carcinomas with the use of the same antibody, and they found similarly high P21WAF1 indices (mean 85%) [33].
In our analysis P21WAF1 and P27KIP1 expressions showed stronger associations with growth fraction in the TP53() than in the TP53(+) ovarian carcinomas. This finding is unexpected, particularly in relation to P27KIP1, and may suggest some cooperation between P27KIP1 and TP53 or P21WAF1 in cell cycle regulation. Furthermore, in our study P21WAF1 and P27KIP1 expressions were positively associated (reported also by Baekelandt et al. [2]) and their cumulative value (defined by a sum of P21WAF1 and P27KIP1 indices) showed stronger association with overall survival than either protein alone. We have found one study showing that P21WAF1 may upregulate P27KIP1 protein by inhibiting its phosphorylation, which prevents P27KIP1 ubiquitination and destruction [34].
Data on C-MYC expression and/or amplification in ovarian carcinomas are scarce and its clinical significance has not been proven [3538]. We have found that C-MYC was associated with better tumor differentiation, higher P27KIP1 expression and lower growth fraction, which seems paradoxical in the context of its biological function. However, some authors have also reported higher levels of C-MYC in better differentiated breast and gastric cancers [39, 40]. In our study, there was a striking association of C-MYC overexpression with endometrioid and clear cell carcinomas, which suggests a role of C-MYC in the development of these carcinoma types and should be the subject of further analysis.
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Acknowledgements |
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Footnotes |
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