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© 2000 American Society for Clinical Oncology Expression of Apoptosis-Related Proteins Is an Independent Determinant of Patient Prognosis in Advanced Ovarian CancerFrom the Departments of Gynecologic Oncology and Pathology, Norwegian Radium Hospital, Oslo, Norway. Address reprint requests to M. Baekelandt, MD, Department of Gynecologic Oncology, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
PURPOSE: The present study was undertaken to investigate the prognostic and predictive relevance of the expression of apoptosis-related proteins Bax, Bcl-XL, and Mcl-1 in advanced ovarian cancer. PATIENTS AND METHODS: Tumor biopsies from 185 consecutive and homogeneously treated patients with stage III ovarian cancer were examined immunohistochemically for the expression of Bax, Bcl-XL and Mcl-1 proteins. Their prognostic relevance was examined in a uni- and multivariate survival analysis. RESULTS: Sixty-six percent of cancer cases expressed Bax, 62% Bcl-XL, and 53% Mcl-1. The expression of Bax correlated with tumor differentiation (P = .016) and less residual disease after surgery (P < .0001). In univariate analysis, Bax expression was associated with improved (P = .0004) prognosis and Mcl-1 expression with poorer (P = .011) prognosis. None of the factors studied was of independent prognostic significance by itself, but when Bax and Bcl-2 expression data were considered together, this combined variable was of independent prognostic significance (P = .0115), together with residual disease status (P = .0016), differentiation grade (P = .0014), and the presence of ascites (P = .0122). Patients with a long median survival (104 months) could be discriminated from those with a short one (16 months) by combining the individual patients’ expression data for p53, Bax, and Bcl-2 with their residual disease status (P < .00001). None of the factors studied was able to predict response to chemotherapy. CONCLUSION: The expression of selected apoptosis-related proteins is of independent prognostic significance and may be helpful in a molecular substaging of patients with stage III ovarian cancer.
TUMOR GROWTH IS dependent on both excess proliferation and deficient cell death. Apoptosis, or programmed cell death, being the main physiologic mechanism of cell death, is a highly conserved, precisely regulated, and mechanistically driven process, which is clearly distinct from necrosis.1 It has become clear that the damage inflicted by anticancer drugs on their proximal drug target can activate the apoptotic process and that this constitutes the predominant way of drug-induced cancer cell kill.2 A defect in the apoptotic pathway, which leads to a failure to undergo apoptosis in response to cell damage, is a major determinant of drug resistance.3,4 The principal regulators of the apoptotic process are the p53 tumor suppressor gene product and the Bcl-2 family of apoptosis regulators.5 The Bcl-2 family of proteins are critical determinants of the cellular apoptotic threshold and are usually divided in proteins with pro-apoptotic (eg, Bax, Bak, Bok, Bcl-XS) and antiapoptotic (eg, Bcl-2, Bcl-XL, Mcl-1, Bcl-W) functional activity.6-8 The members of this family can physically interact with one another, forming homo- and heterodimers, and the relative ratio of anti- versus pro-apoptotic proteins determines cellular sensitivity to apoptotic stimuli.6 The p53 gene is a direct transcriptional activator of the human Bax gene,9,10 but the presence of sufficiently elevated levels of Bax can bypass the need for functional p53 in some forms of chemotherapy-induced apoptosis.11 Bax is able to lower the cell’s apoptotic threshold, but the protein can also mediate cell death through direct effects on mitochondrial function and integrity.12 The Bcl-X gene can function as a Bcl-2–independent regulator of apoptosis.13 Because of alternative splicing, it encodes for Bcl-XL, an apoptosis suppressor, and Bcl-XS, a dominant facilitator of cell death. Like Bcl-2, Bcl-XL has the ability to form ion channels in biologic membranes and may, by regulating intracellular membrane permeability, maintain cell survival.14 The Mcl-1 protein can interact with Bax and prolong cell viability under different cytotoxic conditions.15 Despite the structural and functional homology between Mcl-1 and Bcl-2, both proteins are differentially expressed and regulated.15,16 Epithelial ovarian cancer is the leading cause of death from gynecologic cancer in the Western world. As a result of the lack of early and specific symptoms, approximately two thirds of the patients have advanced-stage (International Federation of Gynecology and Obstetrics stage III or IV) disease at presentation. Despite significant improvements in the treatment of advanced ovarian cancer, typical 5-year survival rates still are in the range of 10% to 20%.17 There is a need for a better understanding of the biology of this disease and for the identification of potential clinically relevant molecular targets for therapy. To this purpose, we examined the possible prognostic and predictive significance of the expression of Bax, Bcl-XL and Mcl-1 proteins in a large cohort of uniformly treated patients with stage III ovarian cancer. We previously demonstrated, in the same patient population, the independent prognostic significance of p53 and Bcl-2 protein expression.18 Another aim of this study was to explore whether a combination of the prognostic information given by the individual independent prognostic factors for each patient could help to distinguish patients who have a good prognosis from those with a poor outcome.
Patients and Samples Tumor tissue was obtained at first surgery in 185 previously untreated patients with International Federation of Gynecology and Obstetrics stage III epithelial ovarian cancer. All patients were consecutively treated in the period from January 1988 to May 1993 at the Department of Gynecologic Oncology at the Norwegian Radium Hospital and were included in a multicenter trial on consolidation treatment after second-look laparotomy in stage III ovarian cancer.19 Selection and response evaluation criteria have been described previously.18 The median age of the patients was 54 years (range, 21 to 70 years). Histologic slides were reviewed by one pathologist (J.M.N.) without knowledge of the clinical data. After primary debulking surgery, treatment consisted of a standard regimen that contained cisplatin (50 mg/m2 every 4 weeks) and epirubicin (50 mg/m2 every 4 weeks). To assess response, a second-look laparotomy was performed in 149 of 185 patients. In 35 cases, no second-look laparotomy was performed because of clinically evident disease progression, and one patient withdrew her consent. All patients were followed up until death or August 1, 1999, whichever occurred first. Follow-up information was collected from the medical records, and no patients were lost for follow-up. Median follow-up time for patients still alive was 85 months. Patient characteristics are given in Table 1, in the way they were grouped for the univariate survival analysis.
Immunohistochemistry Sections from formalin-fixed, paraffin-embedded blocks were immunostained using the biotin-streptavidin-peroxidase method (Supersensitive Immunodetection System, LP000-UL, Biogenex, CA) and OptiMax Plus Automated Cell Staining System (Biogenex). Deparaffinized sections were microwaved in 10 mmol/L of citrate buffer, pH 6.0, to unmask the epitopes and treated with 1% hydrogen peroxide for 10 minutes to block endogenous peroxidase. The sections were incubated with a polyclonal Bax antiserum (cat. no. 13666E, Pharmingen, CA) diluted 1:700, a polyclonal Mcl-1 antibody (cat. no. A3534, Dako Corporation, Carpinteria, CA) diluted 1:25 (40 µg of immunoglobulin G per milliliter), and a monoclonal Bcl-XL (cat. no. 18-0217, Zymed Laboratories, Inc, South San Francisco, CA) diluted 1:25 (2 µg of immunoglobulin G2a per milliliter) for 30 minutes at room temperature. The sections were then incubated with biotin-labeled secondary antibody (1:30) and streptavidin-peroxidase (1:30) for 20 minutes each. Tissue was stained for 5 minutes with 0.05% 3'3-diaminobenzidine tetrahydrochloride freshly prepared in 0.05 M of tris(hydroxymethyl)-aminomethane buffer at pH 7.6 that contained 0.024% hydrogen peroxide and then was counterstained with hematoxylin, dehydrated, and mounted in Diatex (Becker Industrifarg, Marsta, Sweden). All the dilutions of antibody, biotin-labeled secondary antibody, and streptavidin-peroxidase were made in phosphate-buffered saline, pH 7.4, that contained 1% bovine serum albumin. All series included positive controls. Negative controls consisted of the replacement of the monoclonal antibody with mouse myeloma protein of the same subclass and concentration as the monoclonal antibody and the replacement of primary polyclonal antiserum/antibody with normal rabbit serum/antibody at the same concentration as the polyclonal antiserum/antibody. All controls gave satisfactory results. The immunohistochemical evaluation was performed by one of the authors (R.H.), unaware of the clinical data. Immunostaining was scored on a three-tiered scale for both extent (percentage of positive tumor cells = 0%, score = 0; < 5%, score = 1; 5% to 50%, score = 2; > 50%, score = 3) and intensity (absent, 0; weak, 1; moderate, 2; strong, 3). Both scores were added to give a composite score (0 to 6) for each tumor. This composite score was divided into three main groups: 0 = no immunostaining, score 0; 1 = low immunostaining, score 1 to 3; 2 = high immunostaining, score 4 to 6.
Statistical Analysis
Immunohistochemistry The number of positively stained tumor cells, the staining intensity, and their combined score were assessed to determine which was the most appropriate. The number of positively stained tumor cells was found to provide the most clinically relevant information. The intensity of cytoplasmic staining closely correlated with the number of cells staining positively (Pearson R = 0.714; P < .0001) and contributed no additional information. Fourteen normal ovaries were examined as internal controls. Cytoplasmic Bax staining in the epithelium was found in five (36%) of 14 specimens: four (29%) had less than 5%, and one (7%) had between 5% and 50% of the cells stained. All normal ovaries were negative for Bcl-XL, whereas in seven of 14 (50%), Mcl-1 cytoplasmic staining was found in less than 5% of the epithelial cells. Of ovarian cancer cases, 122 (66%) of 185 were positive for Bax expression (Fig 1 A): 14 (7.5%) with less than 5% of tumor cells staining, 60 (32.5%) with between 5% and 50%, and 48 (26%) with more than 50% of cells showing cytoplasmic immunoreactivity. Thirty-nine (21%) cases stained weakly, 60 (32.5%) moderately, and 23 (12.5%) strongly. One hundred fifteen (62%) of the specimens showed cytoplasmic expression of the Bcl-XL protein (Fig 1B): 35 (19%) in less than 5%, 61 (33%) between 5% and 50%, and 19 (10%) in more than 50% of the tumor cells. Staining intensity was weak in 42 (22.5%) cases, moderate in 62 (33.5%), and strong in 11 (6%). Mcl-1 expression was detected in 98 (53%) of cancer cases (Fig 1C): 26 (14%) had less than 5% of cells staining, 55 (30%) between 5% and 50%, and 17 (9%) more than 50% of cells. Thirty-eight (20.5%) cases stained weakly, 52 (28%) moderately, and eight (4.5%) strongly.
Clinicopathologic Correlations Bax protein expression correlated with a better differentiation of the tumor tissue (Pearson R = 0.177; P = .016) and smaller amounts of residual disease after surgery (Pearson R = 0.286; P < .0001). Bax expression did not correlate with p53 protein expression (Pearson R = 0.028; P = .706) or with Mcl-1 expression (Pearson R = 0.031; P = .672). In contrast, a correlation was found between Bax and Bcl-XL protein expression (Pearson R = 0.263; P = .001) and between Bcl-XL and Mcl-1 expression (Pearson R = 0.164; P = .026), whereas no correlation was demonstrated between the expression of antiapoptotic proteins Mcl-1 and Bcl-2 (Pearson R = 0.037; P = .62). Also, there were no significant correlations between the expression of Bcl-XL or Mcl-1 and any of the clinicopathologic determinants of prognosis. To investigate the possible predictive value of these proteins, correlations were examined between their expression and second-look laparotomy-assessed response status. Neither Bax (Pearson R = 0.068; P = .373), nor Bcl-XL (Pearson R = 0.020; P = .791), nor Mcl-1 (Pearson R = 0.016; P = .831) expression was related to response status after four courses of chemotherapy.
Analysis of Survival The results of the univariate survival analysis are given in Table 1. The presence of Bax was significantly correlated with a good outcome (P = .0004) (Fig 2), whereas Mcl-1 expression was related to a poorer prognosis (P = .011) (Fig 3). All variables that reached significance in the univariate survival analysis were incorporated in a multivariate analysis, with disease-related survival used as end point. In this model, only residual disease status (P < .00001), differentiation grade (P = .001), and the presence of ascites (P = .037) retained independent prognostic significance. Similar results were obtained when progression-free survival was used as an end point (data not shown).
Because patient prognosis most likely is not dependent on the expression of a single apoptosis-related protein but on an interplay between the different factors, the previously determined Bcl-2 expression status18 was combined with the Bax expression status for each patient. Patients whose tumors expressed both Bcl-2 and Bax had a median survival of 57 months, whereas patients whose tumors lacked the expression of at least one of these apoptosis-related proteins had a median survival of only 21 months (P < .00001) (Fig 4). When a new multivariate analysis was performed that incorporated this new variable, the combined Bcl-2/Bax expression status was of independent prognostic significance (P = .0115) (Table 2). To see if the expression of Mcl-1 contributes similar information as Bcl-2, the coexpression of Bax and Mcl-1 was examined for its prognostic significance. For the patients with tumors expressing Bax but lacking Mcl-1, a median survival of 46 months was observed, whereas patients with tumors that either lacked Bax protein expression or that expressed Mcl-1 had a median survival of 21 months (P = .0001). However, the combined expression of Bax and Mcl-1 was not of independent prognostic significance in a multivariate analysis (P = .35).
To maximize the prognostic information obtained from the principal prognostic variables identified in this patient cohort, we constructed a new variable in which the expression status of p53, Bcl-2, and Bax were combined with the residual disease status for each patient. As a result, three distinct prognostic groups were identified. Group I was characterized by small residual disease, the absence of p53 overexpression, and the presence of both Bcl-2 and Bax expression. Group II had a favorable status for at least one but not all of the variables, whereas group III was characterized by large-volume residual disease in combination with the presence of p53 overexpression and the absence of Bcl-2 and Bax expression. Median survival was 104 months in group I and 27 months in group II, but only 16 months in group III (P < .00001) (Fig 5). In contrast, if only residual disease status is taken into account, a group with small-volume disease and a median survival of 53.5 months could be discriminated from a group with large-volume disease and a median survival of 19.5 months.
To our knowledge, this is the first study to examine the possible prognostic significance of Bcl-XL and Mcl-1 expression in advanced ovarian carcinoma. Although expression patterns of Bax in ovarian cancer have been reported previously,22-24 only one study was found in which a multivariate analysis of its prognostic significance was performed.22 In accordance with Marone et al,24 we found higher expression levels of Bax, Bcl-XL, and Mcl-1 in carcinoma specimens compared with normal ovaries. The level of Bax expression in our study cohort (66%) is comparable to the one reported by Tai et al,22 although these authors used a positivity cutoff level of 50% of tumor cells staining. The small numbers of patients studied in some25,26 and the different techniques used in other24 studies on Bcl-XL and Mcl-1 in ovarian cancer make it hardly possible to compare the results of different studies. In the present patient cohort, no correlations could be found between the expression of these proteins and known clinicopathologic prognostic variables. The lack of correlation between Bax and p53 expression suggests that, in addition to p53, other, possibly posttranscriptional or posttranslational factors, regulate Bax expression.22,27 There is ample in vitro and in vivo evidence for the role of apoptosis-related proteins in drug resistance.4 Reduced expression of Bax has been related to lower response rates in chemotherapy-treated breast28 and ovarian22 cancer, and adenoviral gene transfer of Bax resulted in selective cytotoxicity of ovarian carcinoma cells in a nude mouse model.29 Bcl-XL expression has been associated with a multidrug resistance phenotype in cell-line studies30 and in clinical acute myeloblastic leukemia,31 whereas higher levels of Mcl-1 also seem to confer resistance in hematologic malignancies.32 With response assessed surgically after four courses of chemotherapy, we could not confirm a correlation between expression of any of the proteins studied and response to chemotherapy, a result in agreement with the findings by Marone et al.24 There are several possible explanations for this. First, the role of the different apoptosis-related proteins may well depend on the actual type of chemotherapy used. There are indications that Bax22 and Bcl-XL33 have a more selective ability to influence the cytotoxicity of paclitaxel compared with the drugs used in the present study. Also, given the highly dynamic nature of the regulation of the Bcl-2 family of proteins, the immunohistochemical determination of a limited number of them at a single point in time might not be the best way to evaluate their effect on drug cytotoxicity.3 It is most likely the dynamic interplay of the different proteins that will determine the apoptotic threshold in individual cells. Lastly, the multifactorial nature of drug resistance makes it unlikely that any single factor will determine sensitivity in the clinical setting.2 Tai et al22 showed that Bax expression was of prognostic significance for disease-free but not overall survival. In univariate survival analysis, we found both Bax (P = .0004) and Mcl-1 (P = .011) to be of prognostic significance, but neither factor was retained in a multivariate analysis. In a previous study, we demonstrated a similar situation for Bcl-2.18 Because it is most likely the interaction of the different proteins that determines the cell’s fate3,4,7 and because Bcl-2 and Bax are two of the main antagonistic players in the apoptotic process, we combined, for each patient, the expression data for Bcl-2 and Bax. In doing so, we identified two clearly distinct prognostic groups in which patients with tumors that expressed both proteins had a significantly better outcome (P < .00001) than those whose tumors did not. This combined variable retained independent prognostic significance in a new multivariate analysis (P = .0115). The survival advantage provided by the combined expression of both a pro- and an antiapoptotic protein may seem counterintuitive and may be related not only to their effect on the apoptotic threshold, but also to their effect on the cell cycle34 or interactions with other proteins.3 Others have also observed an association between an improved prognosis and increased expression of Bcl-223,35,36 and Bax22 in patients with ovarian cancer. Advanced ovarian cancer still carries a poor prognosis despite improvements in both surgery and chemotherapy. To identify possible relevant molecular targets for therapy,37 an increased insight into the biology of this disease and the identification of the factors that determine patient prognosis are necessary. Knowledge of the factors that have an independent influence on prognosis is also crucial in the development and interpretation of prospective randomized trials in which patients are stratified according to these prognostic determinants. We have shown previously that p53 expression is an independent prognostic variable in this patient group.18 To further discriminate patients with a good prognosis from those with a bad one, we combined, for each patient, the expression data for p53, Bcl-2, and Bax with the residual disease status. This allowed us to identify three subgroups of patients with a clearly distinct prognosis (P < .00001) and to increase the discriminating power of the individual prognostic factors. It should be borne in mind that these groups were identified retrospectively, but our results illustrate that we may be coming closer to a combined clinical and molecular substaging of patients within single, classical disease stages. In conclusion, although the expression of Bcl-XL did not seem to influence prognosis in advanced ovarian cancer, Bax and Mcl-1 expression were of significance in univariate survival analysis only. In contrast, the combined prognostic information contained in Bax and Bcl-2 expression was found to be of independent prognostic significance in a large, well-characterized, and homogeneously treated group of patients with stage III ovarian cancer. None of the factors studied here was of use in predicting response to cisplatin-, but not paclitaxel-, containing combination chemotherapy.
Supported in part by grants from the Norwegian Cancer Society, Oslo, and the Martin Flatners Legacy, Gjerdrum, Norway. We thank Anne Gyrid Helland, Ellen Hellesylt, Mette Ingrud, and Liv Inger Håseth for technical assistance, and Patricia Spaenhoven for assistance in data collection.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 or Fisher’s exact test, whichever was appropriate, and correlations were examined according to Pearson. Disease-related and progression-free survival rates were calculated using the method of Kaplan and Meier.
