Differences in clinical importance of Bcl-2 in breast cancer according to hormone receptors status or adjuvant endocrine therapy

Identification of specific therapeutic targets in cancer tissues is essential to select the most appropriate anti-cancer drugs. In the case of breast cancer, determining estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression has been routine practice for years. Endocrine therapy is considered for patients with hormone receptor-positive (ER-positive and/or PR-positive) tumors, whereas trastuzumab is given to those with HER2-positive tumors. For patients with so-called triple-negative tumors, which are ER-negative/PR-negative/HER2-negative, chemotherapy is the only available treatment. New targets are need to increase treatment options for breast cancer patients, especially with tumors lacking conventional therapeutic targets.

Bcl-2 protein, coded by the bcl-2 gene [1], plays an anti-apoptotic role and inhibits cell death [2], resulting in prolonged cell survival [3]. Bcl-2 is overexpressed in many cancers and contributes to tumor initiation, progression and resistance to therapy [1, 4–8]. There is increasing evidence to suggest that Bcl-2 targeting therapy may be an effective treatment for many cancers [9–15].

Bcl-2 is frequently expressed in normal breast epithelial cells and breast cancer cells, and is known to be upregulated by estrogen [16, 17]. Bcl-2 expression in breast cancer has been reported to positively correlate with differentiated markers or favorable prognostic factors such as ER/PR expression, HER2 negativity, slow proliferation, small tumor size, and so on [18]. Many studies have examined the clinical importance of Bcl-2 expression in breast cancer [19, 20]. In most studies, it has been concluded that Bcl-2 expression predicts a favorable clinical outcome [18, 21–29]. Taking the therapeutic protocol into consideration, Bcl-2 has been reported to be an independent predictor of clinical outcome in patients treated with endocrine therapy [23, 25, 26], but not in those given only local-regional treatment [18, 23, 24, 30]. A favorable clinical outcome in Bcl-2-positive cases is surprising considering the anti-apoptotic nature of Bcl-2; however, correlation of Bcl-2 with differentiated markers seems to be at least partly responsible for these results. Correlation of Bcl-2 expression with ER/PR expression, and endocrine therapy frequently given to patients with hormone receptor-positive tumors, may obscure the independent role of Bcl-2 [31]. In order to elucidate the independent clinicopathological role of Bcl-2 in breast cancer, Bcl-2 expression was assessed immunohistochemically and compared with other clinicopathological factors and with clinical outcome in 1081 breast cancer cases with a long follow-up period. Separate analysis was performed on 634 cases without any adjuvant therapy and 477 cases with adjuvant tamoxifen monotherapy.

The clinicopathological role of Bcl-2 in breast cancer was systematically investigated in 634 cases without any adjuvant therapy and 447 cases with tamoxifen monotherapy using full sections from routinely processed archival materials as used in the clinical setting. As others reported [18, 31], Bcl-2 expression was positively correlated with ER and PR expression, but negatively correlated with HER2 expression, grade, and tumor size, confirming Bcl-2’s association with favorable prognostic factors or differentiated markers in both groups with and without tamoxifen (Table 1). In patients who did not receive adjuvant therapy, Bcl-2 expression significantly correlated with premenopausal status (Table 1). Correlation of Bcl-2 expression with premenopausal status has been previously reported by Zhang et al. [36], whereas Hellmans et al. found no relation [23]. Higher premenopausal serum estrogen may promote Bcl-2 expression through ER [37], although this may be offset by the commonly observed correlation of ER expression with postmenopausal status [38], leading to inconsistent results.

In the tamoxifen-treated group, Bcl-2 positivity correlated with better OS in overall patients and in the subgroup with ER-positive and/or PR-positive tumors, but with poor OS in the subgroups with ER-negative and PR-negative tumors or with triple-negative tumors (Fig. 3); however, Bcl-2 was not an independent predictor of clinical outcome in overall patients or subgroups when ER/PR or HER2 status is taken into consideration (Table 3). In the group without adjuvant therapy, there was no evidence that Bcl-2 positivity was a favorable prognostic factor in the entire group or in the subgroup with ER-positive and/or PR-positive tumors (Fig. 2). The finding that a favorable prognosis with Bcl-2 positivity was more evident in the tamoxifen-treated group than in the no adjuvant group is consistent with other reports [18, 21–26, 31]. The favorable prognosis reported for Bcl-2-positive tumors therefore seems to at least partly reflect the indirect effect of coexpressed hormone receptors.

In the group of patients that did not receive adjuvant therapy, Bcl-2 positivity significantly correlated with poor clinical outcome in patients with hormone receptor-negative (ER-negative and PR-negative) or triple-negative tumors (Fig. 2). In multivariate analysis, Bcl-2 positivity independently predicted recurrence/mortality in the entire group of patients and in hormone receptor-negative or triple-negative cases, but not in ER-positive and/or PR-positive cases (Table 2). It seems that the anti-apoptotic effect of Bcl-2, which usually correlates with poor clinical outcome or resistance to therapy in tumors other than breast cancer [5, 7, 8], is evident only in cases without hormone receptors and without adjuvant therapy. In the tamoxifen-treated group, Bcl-2 positivity was also significantly correlated with poor OS among patients with hormone receptor-negative or triple-negative tumors, but that did not reach statistical significance in DFS, probably due to the small number of cases in that group (Fig. 3). Only a few studies have examined the clinical importance of Bcl-2 in subgroups considering the status of hormone receptors or HER2. Tawfik et al. reported that Bcl-2 expression was an independent poor prognostic factor in 124 triple-negative breast cancers; however, multivariate analysis in that study did not include tumor size or nodal status, which are the most powerful prognostic factors. Further, information about adjuvant therapy was not described in that study [39]. Ryu et al. did not find Bcl-2 useful in predicting clinical outcome in 94 triple-negative cancers [40]. In a study by Dawson et al., the hazard ratio for Bcl-2 positive vs. negative expression was reportedly larger in ER-negative, PR-negative, and triple-negative than ER-positive, PR-positive, and non-triple-negative cancers, respectively, which is in line with our present study; however, adjuvant therapy was not considered in each comparison [28].

Interestingly, the prognostic value of Bcl-2 in hormone receptor-negative or triple-negative cases without any adjuvant therapy was more evident in postmenopausal, but diminished in premenopausal, women (Fig. 4). In other words, postmenopausal patients with Bcl-2-negative and hormone receptor-negative (or triple-negative) cancers exhibited quite favorable clinical outcome even without adjuvant chemotherapy (Fig. 4a-d). The reason is not known; however, the present results suggest the need for a reevaluation of adjuvant chemotherapy for these patients.

In the present study, a 30 % cut-off was used to determine Bcl-2 status, as proposed by Silvestrini et al., who examined the outcome predictive power of Bcl-2 status using several cut-offs, and showed that a 30–40 % cut-off yielded the best result; however, a 10 % cut-off has been more frequently used by others [20]. We obtained similar results using a 10 % cut-off for Bcl-2, but the outcome predictive power decreased compared with the Bcl-2 status determined by a 30 % cut-off (data not shown). The reason for this is not known, but the usefulness of a 30 % cut-off for Bcl-2 status as reported in our study seems to validate Silvestrini’s results. Since the overall results were essentially the same irrespective of whether the cut-off value was 10 % or 30 %, it is unlikely that the different cut-off value we used in this study is the reason why our results are different from studies showing that bcl-2 positivity is a predictor of favorable outcome.

Bcl-2 antisense therapy has been suggested for various tumors in an in vitro setting [11–13]. The development of Bcl-2 inhibitors has been explored [41, 42], and small-molecule inhibitors of Bcl-2 such as ABT–737 and ABT-199 have recently been introduced [10, 15]. These Bcl-2 inhibitors were shown to be effective in prolonging survival in animal models bearing lymphoid malignancies [10, 14]. Emerging evidence also suggests the usefulness of this type of therapy in breast cancer [9, 43, 44]. ABT-199 has been reported to improve the response of ER-positive tumors to tamoxifen [43, 44]. Oakes et al. reported that ABT–737 sensitized primary basal-like breast cancers with elevated Bcl-2 levels to docetaxel and improved response and OS in an in vivo setting, suggesting that elevated Bcl-2 expression constitutes a predictive response marker in breast cancer [9]. This recently demonstrated usefulness of Bcl-2 inhibitors in basal-like breast cancers expressing Bcl-2, together with our present finding that Bcl-2 positivity is correlated with poor clinical outcome in patients with hormone receptor-negative or triple-negative tumors, suggest that Bcl-2-targeted therapy may improve the poor clinical outcome of patients with such tumors expressing Bcl-2. This evidence warrants a clinical study of Bcl-2-targeted therapy in breast cancer. Bcl-2 examination is expected to improve prediction of the clinical outcome or to predict response to Bcl-2-targeted therapy in breast cancer.

Bcl-2 expression is an independent poor prognostic factor in patients with hormone receptor-negative or triple-negative breast cancers, especially in the absence of adjuvant therapy, suggesting that the anti-apoptotic nature of Bcl-2 is clearly exhibited under such conditions. The prognostic value of Bcl-2 is more evident in postmenopausal women. The favorable prognosis previously observed in Bcl-2-positive cancer seems to reflect the indirect effect of frequently coexpressed hormone receptors and adjuvant endocrine therapy.