TP53, BCL-2 and BAX analysis in 199 ovarian cancer patients treated with taxane-platinum regimens

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Gynecologic Oncology 112 (2009) 179 – 184 www.elsevier.com/locate/ygyno

TP53, BCL-2 and BAX analysis in 199 ovarian cancer patients treated with taxane-platinum regimens Izabela Ziółkowska-Seta a , Radosław Mądry b , Ewa Kraszewska c , Teresa Szymańska d , Agnieszka Timorek e , Alina Rembiszewska d , Jolanta Kupryjańczyk d,⁎ a

Department of Gynecologic Oncology, the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland b Chair of Gynecologic Oncology, Medical University, Lakowa 1/2, 61-848 Poznan, Poland c Department of Gastroenterology and Hepatology, Medical Center for Postgraduate Education, Roentgena 5, 02-781 Warsaw, Poland d Department of Molecular Pathology, the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland e Chair and Department of Obstetrics, Gynecology and Oncology, IInd Faculty of Medicine, Warsaw Medical University and Brodnowski Hospital, Kondratowicza 8, 03-242 Warsaw, Poland Received 18 June 2008 Available online 19 October 2008

Abstract Objective. In cell line studies, BCL-2 and BAX proteins interfere with cancer response to taxanes. This issue has not received much attention with regard to taxane-platinum (TP)-treated ovarian cancer patients. Methods. We evaluated prognostic/predictive significance of BCL-2 and BAX with regard to TP53 status. Immunohistochemical analysis was performed on 199 ovarian carcinomas FIGO stage IIB–IV treated with TP; the results were analyzed by the Cox and logistic regression models. Results. Clinicopathological parameters (residual tumor size, FIGO stage and/or tumor grade, but not patient's age) were the only or the strongest predictors of patient's outcome. Platinum highly sensitive response showed a positive association with TP53 accumulation (p = 0.045). As in our previously published analysis on platinum-cyclophosphamide-treated group, complete remission showed a borderline negative (paradoxic) association with high BAX expression in the whole group (p = 0.058) and with BCL-2 expression in the TP53(−) group (p = 0.058). Conclusion. Our results suggest that TP53, BCL-2 and BAX proteins carry some predictive potential in taxane-platinum-treated ovarian cancer patients, auxiliary to clinicopathological factors. We have confirmed on another patient group that clinical importance of BCL-2 may depend on TP53 status. © 2008 Elsevier Inc. All rights reserved. Keywords: Ovarian cancer; TP53; BCL-2; BAX; Taxane-platinum

Introduction Taxanes with cisplatin or its analogues (TP therapy) have been the standard first-line chemotherapy of ovarian cancer ⁎ Corresponding author. Department of Molecular Pathology, Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland. Fax: +48 22 5462181. E-mail addresses: [email protected] (I. Ziółkowska-Seta), [email protected] (R. Mądry), [email protected] (E. Kraszewska), [email protected] (T. Szymańska), [email protected] (A. Timorek), [email protected] (A. Rembiszewska), [email protected] (J. Kupryjańczyk). 0090-8258/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2008.09.008

patients in the last decade [1,2]. Although introduction of taxanes has improved treatment success when compared with cisplatin-cyclophosphamide therapy, still, about 20%–30% of patients do not show a response to this regimen. Molecular factors causing resistance to chemotherapy may be potential targets of molecular therapy. Taxanes exert a cytotoxic effect mainly by stabilising and inactivating microtubules responsible for the formation of mitotic spindle [3,4]. Another mechanism of taxol action (although not in all cell types) is inactivation of an apoptosis inhibitor BCL-2 protein by its phosphorylation [5,6]. According to some studies, the phosphorylation prevents BCL-2/BAX complex formation,

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increases the functional levels of the apoptosis promoter BAX and leads to apoptosis [7]. The taxol-induced increase in the level of BAX occurs in a TP53 independent manner [8]. Several mechanisms underlying taxol resistance have been described [9–12]. Regarding the apoptosis proteins, it was observed that impaired phosphorylation and/or excessive BCL2 expression caused resistance of cancer cells to taxanes [13,14]; however, contradictory results have been published, too [15]. BAX expression was lower in ovarian cancer cells resistant to taxol than in the sensitive ones [16]; stimulation of BAX expression led to enhanced sensitivity to taxol [17–19]. Despite rich data from cell line studies, the role of BCL-2 and BAX expressions in ovarian cancer response to taxane-platinum agents has not received much attention [20–22]. A few more studies analyzed prognostic importance of BCL-2 in ovarian cancer patients treated with TP [23–25]. TP53 status is another factor determining tumor sensitivity to taxane-platinum therapy. Ovarian carcinomas with dysfunctional TP53 protein show a better response to paclitaxel or paclitaxel containing regimens than tumors with normal TP53 [8,26–29]. In addition, clinical value of BCL-2 and BAX may be different in subgroups divided according to TP53 accumulation, as it was shown in our previous analysis on platinumcyclophosphamide-treated ovarian cancer patients [30]. We aimed to verify this observation on another (validating) group of patients. Thus, the main goal of our study was to evaluate the clinical importance of BCL-2 and BAX protein expressions in the context of TP53 accumulation, in ovarian cancer patients treated with taxane-platinum agents. Materials and methods Patients and tumors The study was approved by the local ethics committee (decision ref. 49/2003). We studied archival material from 199 patients treated with standard taxane-platinum regimens (TP: paclitaxel or docetaxel [17 patients] with cisplatin or carboplatin). First-line chemotherapy consisted of 5 or 6 courses (mean 6; 95.9% of patients completed 6 courses of treatment). Only patients treated with standard protocols of chemotherapy were accepted for the study. Taxol, given in a 24-hour (135 mg/m2) or 3-hour infusion (175 mg/m2), or docetaxel (75 mg/m2) was followed by cisplatin (75 mg/m2) or carboplatin (AUC6). Follow-up time ranged from 4.7 to 88.7 months (median 29.4). The short follow-up resulted from early patient's death. All surviving patients had at least a 6-month follow-up and all but six surviving patients without recurrence had at least a 2year follow-up after completion of the chemotherapy. Table 1 gives detailed material characteristics. Selection of patients and reasons for exclusion Medical records were reviewed centrally and all tumors were uniformly reviewed histopathologically according to the World Health Organization criteria [31] (Table 1). The patients were carefully selected to meet several criteria, among them an

adequate staging procedure (see [29] for details), no chemotherapy before staging laparotomy, International Federation of Gynecologists and Obstetricians stage IIB to IV disease [32], completed first-line chemotherapy and tumor tissue from the first laparotomy available. Among reasons of exclusion were: other chemotherapeutic regimens, lack of important data (residual tumor size, CA125 estimations), patient drop-out, inadequate histopathological diagnosis (a borderline tumor, a metastasis), interval debulking or secondary cytoreduction after completion of chemotherapy. Only a small percentage of patients underwent second-look procedure. Patients with a microscopic recurrence were excluded, because they received a second-line chemotherapy and could not be compared with patients who were observed only clinically and biochemically. Evaluation of clinical response to chemotherapy Response to chemotherapy was evaluated retrospectively according to the World Health Organization response evaluation criteria [33]. The evaluation was based on data from medical records describing patient's clinical condition and CA125 levels in 3–4 week intervals. Complete remission (CR) was defined as disappearance of all clinical and biochemical symptoms of ovarian cancer evaluated after completion of first-line chemotherapy and confirmed at four weeks; this definition relates also to patients with the lack of residual disease. CR was achieved in 131 patients (66%). Within the CR group we have identified a platinum sensitive group (PS, disease-free survival longer than six months, 112 patients, 56%) and platinum highly sensitive group (PHS, disease-free survival longer than 24 months, 39 patients, 20%). Other tumors were described as platinum resistant [33] (Table 1). Immunohistochemical analysis All immunohistochemical stainings were performed on paraffin-embedded material after heat-induced epitope retrieval (HIER). We used PAb1801 monoclonal antibody (1:3000, Sigma-Genosys Cambridge, UK) for TP53 protein, anti-BAX (1:80, clone B-9, Santa Cruz Biotechnology Inc, Santa Cruz USA) and anti-BCL-2 (1:80, clone 124 Dako, Glostrup, Denmark) monoclonal antibodies. Deparaffinized sections were boiled in a citrate buffer (pH 6.0) at 700 W in a microwave; 2 × 5 min for TP53, 3 × 5 min for BCL-2, and 6 × 5 min for BAX. Non-specific tissue and endogenous peroxidase reactivity were blocked with 10% BSA and 3% H2O2, respectively. Tissue sections were incubated with primary antibodies overnight at 4 °C (anti-TP53, anti-BAX) or for 1 h at room temperature (anti-BCL-2) Biotinylated goat anti-mouse IgG (1:1500, cat. no. 816), peroxidase conjugated streptavidin (1: 500, cat. No. 309) (both from Immunotech, Marseille, France), and DAB were used as a detection system. Ovarian carcinomas with and without TP53 gene mutation were controls for TP53. Intratumoral lymphocytes and plasma cells, as well as a tonsil tissue served as controls for BCL-2 and/or BAX. Normal mouse IgG of the same subclasses and

I. Ziółkowska-Seta et al. / Gynecologic Oncology 112 (2009) 179–184 Table 1 Patients characteristics Whole group Age Range Mean FIGO stage IIB, IIC IIIA, IIIB IIIC IV Residual tumor size 0 b2 cm including b1 cm ≥2 cm Histological type Serous Endometrioid Clear cell Undifferentiated Other Histological grade a G2 G3 G4 Response to chemotherapy Complete remission Partial remission/no change 2change b Progression

20–78 54.6 10(5%) 28(14%) 141(71%) 20(10%) 37(19%) 81 (41%) 57 (28.5%) 82 (41%)

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time analyses were performed with the multivariate Cox' proportional hazards model. Tumor response to chemotherapy was evaluated in a logistic regression model using backward selection technique, where factors non-significant at 0.1 were stepwise removed from the model. The analysis was performed in all ovarian carcinomas, and separately in TP53(−) and TP53 (+) subgroups. Associations between protein expressions and histological type, grade, FIGO stage and residual tumor size were studied by chi-square test. All tests were two-sided and the level of significance was set at 5%. All calculations were done using the STATA 6.0 program. Results Protein expressions and their associations

147(74%) 9(5%) 4(2%) 21(10%) 18(9%) 26(13%) 115(58%) 58(24%) 131(66%) 62(31%) 6(3%)

a

We did not have patients with advanced stage grade 1 carcinoma. We have combined these responses because it was not always possible to have objective measures of the disease in the retrospective study. b

concentrations as the primary antibodies served also as negative controls. Semiquantitative evaluation of immunohistochemical stainings was performed independently by two pathologists (JK, TSZ). Each person evaluated the immunohistochemical stainings three times. Discordant results were evaluated by the two pathologists together under two-head microscope. TP53 protein accumulation was described as present (more than 10% of positive cells, TP53-positive) or absent (TP53negative). According to our experience from different studies, and with the HIER method applied for the detection of TP53, 10% is an optimal threshold for separation of tumors with and without a TP53 gene missense mutation [34]. BCL-2 expression was described as: 1) negative (including focal), and 2) positive (diffuse or heterogeneous of any intensity). BAX expression was described as: 1) negative, trace or weak (further called low), 2) moderate and 3) strong (both further called high). These criteria were established in our previous study [30], according to the major expression patterns of BCL-2 (large proportion of negative tumors) and BAX (usually diffuse staining of different intensity). They also turned out to be useful clinically [30]. Statistical analysis Probability of survival and disease-free survival (DFS) was estimated using the Kaplan–Meier method. Overall and DFS

TP53 positive ovarian carcinomas predominated over TP53 negative ones: 117 (59%) versus 81 (41%). BCL-2 was negative in 97 tumors (49%) and positive in 102 tumors (51%), including 32 cases with strong expression (16%). BAX expression was low (negative, trace or weak) in 78 (39%) tumors, moderate in 58 (29%) and strong in 63 (32%) tumors. BCL-2 and BAX expressions were associated with each other (p = 0.052), but not with TP53 protein accumulation. TP53 protein accumulation showed a borderline association with poor tumor differentiation/undifferentiation (p = 0.058), with serous/undifferentiated/other type carcinomas (p = 0.059) and with lower patients age (p = 0.047). Strong BAX expression was more frequent than low BAX expression in patients with complete tumor debulking (43% versus 30%), and the reverse was observed for residual tumor equal or greater than 2 cm (22% versus 50%) (p = 0.013). BCL-2 expression did not show associations with clinicopathological parameters. Clinical significance of the proteins expressions Clinical parameters were the strongest predictors of complete remission (CR, Table 2) and the only predictors of platinum sensitivity. The both clinical endpoints were associated with small RT size and with lower FIGO stage (in the whole group) (Table 2). The clinical significance of residual tumor b 2 cm was more apparent in the TP53(−) group (Table 2). Complete remission showed a borderline association with the apoptosis proteins. It was positively associated with low BAX staining in the whole group (odds ratio for CR in high BAX expressors was 0.5) and with negative BCL-2 staining in the TP53(−) group (odds ratio for CR in the BCL-2 positive group was 0.27) (Table 2). The odds of achieving platinum highly sensitive response (PHS) (CR with disease-free survival longer than 24 months) were higher for patients with TP53(+) tumors (OR 2.35, Table 2). Disease-free survival (DFS) was associated with clinicopathological parameters (FIGO stage, tumor differentiation) and not with any protein expression studied. As to the overall survival, poor tumor differentiation/undifferentiation had an adverse effect. Relative risk of death ranged from 2.3 to 4.1 in different groups, and was the highest for poorly differentiated

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Table 2 Probability of complete remission and platinum highly sensitive response (PHS—disease-free survival longer than 24 months) in the whole group of ovarian carcinomas, and in the TP53(+) and TP53(−) group (a logistic regression model) TP53(−) and (+) N = 199, 131 CR

Complete remission FIGO IV versus other Residual tumor a b2 cm versus 0 ≥ 2 cm versus 0 BAX High versus low BCL-2 (+) versus (−) Platinum high sensitivity FIGO IIIC versus IIB–IIIB IV versus IIB–IIIB Residual tumor b2 cm versus 0 ≥ 2 cm versus 0 Histological grade 3 versus 2 4 versus 2 TP53(+) versus (−)

TP53(−) N = 82, 51 CR

RR

95% C.I. for OR

p

0.2

[0.08, 0.007]

0.011

0.20 0.08

[0.05, 0.007] [0.02, 0.029]

0.017 0.000

0.50

[0.24, 1.02]

0.058

0.38 0.19

[0.15, 0.99] [0.03, 1.11]

0.049 0.065

0.29

[0.11, 0.79]

0.016 0.087

0.33 0.28 2.35

[0.11, 0.94] [0.08, 0.96] [1.01, 5.45]

0.04 0.049 0.045

TP53(+) N = 117, 80 CR

RR

95% C.I. for OR

p

RR

95% C.I. for OR

p

0.11 0.05

[0.01, 0.009] [0.006, 0.004]

0.046 0.009

0.23

[0.09, 0.005]

0.001

0.27

[0.06, 1.04]

0.058

0.29 0.15

[0.10, 0.85] [0.02, 0.95]

0.025 0.045

0.13 0.10

[0.03, 0.57] [0.01, 0.51]

0.007 0.006

0.16 0.18

[0.03, 0.77] [0.03, 0.87]

0.023 0.034

RR — relative risk; C.I. confidence interval. Only factors showing associations are presented. a tumor size b1 cm has been established as a criterion of optimal cytoreduction in the years 1992–1994 and implemented in the mid and late 1990s. An earlier criterion was b2 cm. We had to evaluate taxane-platinum group in the same way as the earlier PC/PAC group [30].

and undifferentiated tumors in the TP53(+) group (p value from 0.03 to 0.007). Discussion Our study presents some evidence of BAX and BCL-2 influence on ovarian cancer response to taxane-platinum therapy, obtained in multivariate analysis. The currently observed associations are not strong (possibly, higher group size might reveal stronger associations); however, they are nearly identical to those observed in our earlier study on platinum-cyclophosphamide-treated patients [30] (Table 3). BAX expression, in contrast to its apoptosis promoter function and in similarity to BCL-2 expression, had a negative impact on complete remission (Table 3). Paradoxic effects of apoptosis proteins on tumor chemosensitivity have been described previously, both in experimental Table 3 Odds of complete remission according to BAX and BCL-2 expression in platinum-cyclophosphamide (PC)- and taxane-platinum (TP)-treated patients Complete remission

Whole group BAX high versus low

TP53(−) group BCL-2 positive versus negative

PC-treated patients [30] (N = 229) TP-treated patients (N = 199)

OR 0.53 [0.08, 033], p = 0.047 OR 0.50 [0.24, 1.02], p = 0.058

OR 0.4; [0.16, 1.01], p = 0.05 OR 0.27 [0.06, 1.04], p = 0.058

and clinical studies. BAX expression was found to be a bad prognostic factor in ovarian carcinomas, while BCL-2 expression a good one [35]. Ferlini et al. described a BCL-2 downregulation as a mechanism of paclitaxel resistance [15]. Rogers et al. suggested that microenvironmental factors contribute to a clinical effect of apoptosis proteins in vivo, while they are absent in vitro [36]. According to Blagosklonny, high BCL-2 levels may be clinically convenient, because they may indicate functionality of the apoptosis machinery [37]. In our study, the functionality might be indicated by the association between BAX and BCL-2 expressions. Studies on clinical material are non-numerous. In univariate analysis on 45 patients, Tai et al. [20] described higher rate of complete remission in high BAX expressors; in a study by Geisler et al., BCL-2 together with TP53 expression had a prognostic significance [24]. Other authors did not report any clinical significance of BCL-2 or BAX expression [21–23,25]. In a recent study with the use of oligonucleotide microarrays, BAX expression was present in a profile associated with response to chemotherapy [38]. Our result related to BAX protein is among some other that do not fit the simplistic translation of protein function to a clinical effect [39]. In addition, we confirmed the observation from platinumcyclophosphamide-treated patients [30] that the negative impact of BCL-2 expression on tumor response may be seen in the TP53-negative carcinomas only. This supports our suggestions that TP53-positive and TP53-negative ovarian carcinomas may differ in clinical significance of some proteins, possibly

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those interacting with TP53 in tumor response to chemotherapy [40]. Regarding the TP53 accumulation, it was a positive predictive factor in taxane-platinum-treated patients, however, only with respect to prolonged remission (at least 24 months). While several studies suggested an advantage of TP53 dysfunction for the effects of taxane-platinum therapy [26– 28], our study appears to be the first multivariate analysis showing the predictive value of this marker. Poor tumor differentiation/undifferentiation appears as a factor negatively influencing results of treatment with taxaneplatinum therapy, predominantly in the group with tumor TP53 accumulation. We could not find a clear explanation for this observation. The mechanisms underlying taxol resistance include mutations in the β-tubulin gene [9,10], differential expression of β-tubulin isotypes [11,12], and altered microtubule dynamics [41]. The alterations may be more pronounced in less differentiated tumors. Several studies on cell lines demonstrated differences in the expression of beta-tubulin subtypes depending on cell differentiation; a higher stability of microtubules and lower activity of taxanes in differentiated cells was also described [42,43]. Shakuto et al. observed better tumor suppression in vivo by paclitaxel in well differentiated than in moderately differentiated tumors [44]. Despite the novel findings related to apoptosis proteins, clinical parameters (residual tumor size, FIGO stage) were the only factors of overall survival, the strongest predictors of complete remission and the only predictors of platinum sensitivity. In our study, patient's age did not influence clinical endpoints. Similar results were presented by other authors [45]. Bicher et al. reported that delivered dose intensity of paclitaxel was not influenced by age [46]. These data may explain why TP therapy is much more efficient that PC therapy in older patients [29]. In summary, we have shown (for the second time on a different patient group) a tendency for a paradoxic (negative) effect of BAX expression on complete remission in ovarian cancer patients treated with taxane-platinum therapy. There was also a tendency for negative influence of BCL-2 expression on complete remission in the group without TP53 accumulation, thus, confirming our results from platinum-cyclophosphamidetreated group. Separate analysis of tumors with and without evidence of TP53 dysfunction may facilitate revealing associations between molecular factors and clinical outcome.

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Conflict of interest statement The authors have no conflicts of interest to declare.

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Acknowledgment

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This study was supported by grant no. 2P05A 068 27 from the Polish Committee for Scientific Research.

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