This is the first prospective, randomized, controlled study evaluating the influence of zoledronic acid on DTC presence and survival in early-stage breast cancer patients. Our results demonstrate that bisphosphonate therapy contributes to eradication of disseminated tumor cells. All patients treated with ZOL became bone marrow negative after 24 months in comparison to 84% in the control group (p = 0.032). This is in accordance with previous studies; Aft et al. reported that ZOL administered with neoadjuvant chemotherapy resulted in a decreased proportion of DTC-positive patients. This effect did not reach statistic significance (p = 0.054); however, in contrast to our study the reevaluation of bone marrow status was performed after a relatively short time (three months) after begin of ZOL treatment [
14]. In the present study no significant correlation between bisphosphonate therapy and clinical outcome could be observed; this is possibly due the relatively small patient number. This is an important limitation of the present trial.
DTC and microenvironment
Despite progress made in the therapy of breast cancer, the prognosis of patients even with small tumor burden is still limited by metastatic relapse often long after removal of the tumor. Current theories on biology of cancer suggest that disseminated tumor cells detected in bone marrow or blood circulation of patients may serve as a surrogate parameter for minimal residual disease and are therefore precursor of metastasis. However, it is currently not predictable which of these cells will evolve into metastases; presumably, the vast majority undergo apoptosis leaving only a minor percentage able to persist in secondary homing sites [
15]. This phenomenon, described as “metastatic inefficiency”, is consistent with the observation that detection of tumor cells in blood or bone marrow does not inevitably cause metastasis; 50% of initially DTC-positive patients do not suffer from a relapse and stay disease-free [
1]. Studies suggest that 0.01% of CTC can ultimately produce a single bone metastasis, and at least 10,000 CTC are required for the development of a metastatic colony [
16,
17]. Nonetheless, approximately one-third of patients fail to clear tumor cells from the blood following removal of primary tumor [
18,
19]. It is not yet clear, which factors determine the fate of DTC and facilitate their persistence. Recently, oncologic research has focused increasingly not only on the cancer cell itself but on its microenvironment; in this context the concept of a premetastatic niche, permissive to development of (micro-)metastases, has gained much interest [
20]. Bone tissue may serve as a suitable microenvironment for the growth of tumor cells derived from the primary tumor in the breast; it represents a dynamic 'habitat’ influenced by a number of parameters, among them angiogenetic mechanisms, osteoclastic bone resorption and hormonal factors [
21]. An imbalance between these parameters leads to decreased bone density and osteoporosis; these changes may affect the role of bone marrow as a homing site for DTC. Consequently, bone-targeted agents, such as BP, have not only a stabilizing effect on the bone density itself, but show antitumorigenic activity as well.
Antitumorigenic effects of bisphosphonates
There is a growing body of evidence of bisphosphonates’ anti-cancer activity
in vivo and
in vitro. BP are assumed to alter bone and BM microenvironment affecting its ability to host tumor cells; their effect in the bone is mainly due to inhibition of osteoclast-mediated bone resorption [
22]. Several preclinical and animal model-based studies have demonstrated BP to exert direct anti-cancer activity, inhibiting tumor cell adhesion [
23], invasion [
24], proliferation and angiogenesis [
25] and inducing apoptosis [
26,
27]. Exposure of cortical bone slices to BP partially inhibited breast cancer cell adhesion
in vitro[
23]; interestingly, cancer cells appeared to be more susceptible to BP treatment than normal cells [
28]. Boissier et al. demonstrated BP to inhibit breast cancer cell invasion in a dose-dependent manner using a matrigel invasion assay. This effect was partly due to observed reduction in proteolytic activity of matrix metalloproteinases (necessary to digest basement membrane). In that study, ZOL had the highest potency with regard to invasion inhibition, followed by ibandronate, risedronate analogue NE-10244 and clodronate [
24]. Wood et al. showed significant antiangiogenic properties of zoledronate
in vitro and
in vivo[
25]. Further, recent data indicate that nitrogen-containing BP can inhibit the proliferation of human cancer cell lines and induce their apoptosis [
26]. Data from animal studies confirm that BP exert potent effects on visceral metastases as well [
27,
29]. These reports suggest that BP affect the invasive behavior of metastatic cells in secondary sites through both direct and indirect effects and have the ability to interact with tumor cells at each step of the metastatic process.
Clinical relevance of bisphosphonate treatment in adjuvant setting
The presence of DTC correlates significantly with increased risks of distant metastasis, locoregional recurrence, and death in breast cancer patients [
1,
30]. Therefore, treatment strategies that target DTC in BM may potentially improve disease-free and overall survival. We hypothesize that bisphosphonates affect the role of bone marrow as a suitable microenvironment for DTCs. Indeed, protective effects of bisphosphonates were reported in clinical trials exploring ZOL as adjuvant therapy in early breast cancer.
Three large prospective studies evaluated the impact of addition of ZOL to systemic treatment on survival (Table
4) [
6,
8,
31]. The randomized open-label Austrian Breast and Colorectal Cancer Study Group (ABCSG)-12 trial (NCT00295646) evaluated the influence of adding ZOL to adjuvant endocrine therapy [
31]. In this four-arm trial, 1,803 premenopausal women with hormone receptor positive early stage (stage I-II) breast cancer were randomized to receive goserelin 3.6 mg every 28 days plus either tamoxifen 20 mg daily or anastrozole 1 mg daily, with or without ZOL 4 mg every 6 months for 3 years. At a median follow-up of 62 months, the addition of ZOL reduced risk of disease-free survival events compared with endocrine therapy alone (p = 0.009). The reduction in recurrence was observed locally and distantly both in and outside the bone. The risk of death was also reduced but did not reach statistical significance, although it approached significance in women older than 40 years (p = 0.057).
Table 4
Studies evaluating antitumor activity of bisphosphonates in breast cancer
N
| 1065 | 1803 | 3023 | 3323 | 3360 | 299 | 302 | 953 | 1069 |
Bisphosphonate
| Zoledronate i.v. | Zoledronate i.v. | Ibandronate p.o. | Clodronate p.o. | Zoledronate i.v. | Clodronate p.o. | Clodronate p.o. | Pamidronate p.o. | Clodronate p.o. |
Duration of therapy
| 5 years | 3 years | 2 years | 3 years | 5 years | 3 years | 2 years | 4 years | 2 years |
Adjuvant therapy
| Endocrine (AI) | Endocrine (Tam vs. AI +GnRH-Anal.) | Dose-dense doseintense CTX | CTX +/- Endocrine | CTX +/- Endocrine | CTX +/- Endocrine | CTX +/- Endocrine | CTX | CTX +/- Endocrine |
Follow up
| 60 months | 84 months | 39 months | 91 months | 60 months | 120 months | 103 months | n.d. | 66 months |
Premenopausal
| - | Yes (DFS, OS) | No | No | No | No | Yes (OS) | No | Yes (OS, bonemetastasis free survival) |
Postmenopausal
| Yes (DFS) | - | No (Trend > 60 yrs.) | Yes (> 50 yrs. DFS, but not OS) | Yes (DFS, OS) | | | | |
In the Zometa-Femara Adjuvant Synergy Trials (Z-FAST/ZO-FAST/E-ZO-FAST), designed to investigate the bone-protective effects of zoledronic acid, an exploratory analysis was conducted to assess the anticancer potential of zoledronic acid. A total of 2,194 postmenopausal women with hormoneresponsive early breast cancer received letrozole 2.5 mg daily [
6]. Patients were randomized to receive zoledronic acid 4 mg administered every 6 months for 5 years starting either upon randomization (up-front) or upon a predetermined measure of bone loss (delayed start). After 36 months of follow-up, a 34% reduced incidence of disease-free survival (DFS) events with up-front zoledronic acid treatment compared with delayed treatment was observed in the ZO-FAST trial (
P = 0.0375). These studies show a DFS benefit or overall survival (OS) benefit in postmenopausal women or women who have chemical ovarian suppression.
In contrast to ABCSG-12 and ZO-FAST trials, the Adjuvant Zoledronic Acid to Reduce Recurrence (AZURE; BIG 01/04) trial (NCT00072020) did not show a benefit from adding bisphosphonates to adjuvant therapy in the overall patient population. The AZURE trial evaluated the antitumor activity of ZOL combined with (neo)adjuvant chemotherapy as well as endocrine therapy in 3,360 pre- and postmenopausal patients with stage II/III breast cancer [
4]. Patients were randomly assigned to receive standard adjuvant systemic therapy either with or without ZOL (every 3–4 weeks for 6 doses and then every 3 to 6 months to complete 5 years of treatment). In a second interim analysis, ZOL was not associated with a significant improvement in DFS in the overall population. However, in a subgroup analysis, ZOL improved OS by 29% (p = 0.017) and reduced DFS events in and outside the bone in women who were more than 5 years postmenopausal [
8]. Interestingly, in the subset of 195 patients who received neoadjuvant chemotherapy addition of ZOL to neoadjuvant chemotherapy reduced mean residual invasive tumor size by approximately 43% compared with chemotherapy alone. The adjusted mean residuum was 12 mm lower in the ZOL group (15.5 mm) than in the group not receiving ZOL (27.4 mm; p = 0.006), suggesting potential antitumor benefit from combining bisphosphonates with cytotoxic treatment [
8].
Further, Aft et al. [
14] demonstrated that DTC-free BC patients treated with ZOL (4 mg every 3 weeks) were more likely to remain DTC free at 3 months (
P = 0.03), and that the subset of patients with estrogen receptor-negative and epidermal growth factor receptor-2–negative disease were more likely to have pathologic complete response with ZOL versus no ZOL. In small studies, ZOL (4 mg/month) increased the proportion of DTC-free patients who remained DTC-free at 6 months versus no ZOL [
36], and significantly decreased DTC levels versus baseline at 12 (
P < 0.0006) and 24 months (
P = 0.0026) [
37] in DTC-positive BC patients. Therefore, it is reasonable to hypothesize that ZOL may delay disease recurrence.
Extensive data on the adjuvant effects of another bisphosphonate, clodronate, were provided by Diel et al. [
33]. In that prospective clinical trial 302 patients were randomized to receive either oral clodronate 1600 mg/day for 2 years or standard follow-up. An updated survival analysis with a median follow-up of 103 months showed patients in clodronate group to perform better with regard to OS (p = 0.049).