Introduction
Fulvestrant is an estrogen-receptor (ER) antagonist with no known agonist effects. It binds, blocks and accelerates degradation of the ER, leading to dose-dependent reductions in cellular ER and proliferation-related antigen Ki67 [
1,
2]. Unlike tamoxifen, which increases progesterone-receptor (PgR) expression, fulvestrant also induces dose-dependent reductions in PgR expression [
1]. Fulvestrant-mediated reductions in tumor biomarkers are indicative of anti-estrogenic and anti-proliferative effects [
1,
3,
4], and may be useful as surrogate endpoints of clinical efficacy. The registration trials of fulvestrant (0020 and 0021) provided the first indication of its dose-related anti-tumor activity. Fulvestrant 250 mg was not significantly different from the aromatase inhibitor (AI) anastrozole, 1 mg daily, for the primary endpoint of time to progression in the treatment of postmenopausal women with advanced breast cancer who had recurred or progressed following prior endocrine therapy [
5‐
7]. Although these trials initially incorporated a fulvestrant 125 mg treatment arm, an interim analysis showed insufficient evidence of clinical activity (no objective tumor responses and numerically lower time to progression (TTP)), and this arm was discontinued [
5‐
8].
As ER down-regulation appears to be dose-dependent, it was hypothesized that ER antagonism may be enhanced by raising fulvestrant steady-state plasma concentrations to a higher level than those achieved by the 250 mg/month regimen [
1,
2]. In addition to this, the biological activity of fulvestrant 500 mg (fulvestrant 500 mg/month plus 500 mg on Day 14 of Month 1) versus 250 mg (fulvestrant 250 mg/month) was investigated in the Neoadjuvant Endocrine therapy for Women with Estrogen-Sensitive Tumors (NEWEST) study. The higher fulvestrant dose was associated with significantly greater down-regulation of Ki67 at Week 4 (primary endpoint), as well as similar reductions in ER and PgR protein expression. The higher fulvestrant dose also produced numerically higher tumor response rates [
9].
The clinical activity of fulvestrant 500 versus 250 mg was further compared in the phase III COmparisoN of Faslodex In Recurrent or Metastatic breast cancer (CONFIRM) study, in which a significantly longer TTP was reported with the 500 mg dose (hazard ratio (HR) = 0.80;
P = 0.006) [
10].
Other questions posed were, first, whether a 500 mg regimen might be better than an AI, given that fulvestrant 250 mg had been equivalent to anastrozole; and second, whether there was any value in combining an AI and fulvestrant, either at the 250 or 500 mg dose. The rationale for the latter approach was that combining fulvestrant with an estrogen-lowering agent may lead to enhanced ER blockade and anti-tumor activity. This possibility was examined in the Fulvestrant loading dose and Anastrozole in Combination Trial (FACT), in which patients at first relapse received either a loading-dose regimen of fulvestrant (500 mg on Day 0; 250 mg on Days 14 and 28; then 250 mg monthly thereafter) plus anastrozole (1 mg daily), or anastrozole alone [
11]. There were no significant differences between the two groups in the primary endpoint of TTP (HR = 0.99;
P = 0.91) or in any of the secondary endpoints [
11]. Critically, there was no underlying biomarker study, and FACT did not compare the combination of fulvestrant plus anastrozole versus fulvestrant alone. In the recently reported SWOG S0226 study, Mehta and colleagues report that the combination of the fulvestrant 250 mg loading-dose regimen with anastrozole was associated with improved progression-free survival versus anastrozole alone in postmenopausal women with untreated advanced breast cancer, indicating that the combination treatment may be effective in some patients [
12].
This pre-surgical study was thus designed to compare the biological activity and safety of fulvestrant 500 mg versus anastrozole versus the combination (fulvestrant 500 mg plus anastrozole) as pre-surgical treatment in women with ER-positive breast cancer. This study was conducted in parallel with the Fulvestrant fIRst-line Study comparing endocrine Treatments (FIRST) phase II trial, which compared the clinical efficacy of fulvestrant 500 mg versus anastrozole for the first-line treatment of advanced hormone receptor-positive breast cancer [
13]. The primary endpoint of FIRST was clinical benefit rate (CBR), which was numerically but not statistically greater for fulvestrant 500 mg compared with anastrozole (72.5% versus 67.0%), with the odds ratio (OR = 1.30;
P = 0.386) in favor of fulvestrant. The secondary endpoint (TTP) was, however, significantly longer for fulvestrant 500 mg (median TTP not reached for fulvestrant
v 12.5 months for anastrozole; HR = 0.63;
P = 0.0496) [
13]. The superior clinical efficacy seen in FIRST makes the present biomarker study even more important, both for its potential to provide a biological rationale for why fulvestrant 500 mg might be better than anastrozole and also to provide an insight into whether adding anastrozole to fulvestrant 500 mg might be a treatment strategy worth pursuing.
Discussion
Previous neoadjuvant studies have compared selective ER modulators, such as tamoxifen, with AIs, such as anastrozole [
14] and letrozole [
15]. To our knowledge, this study is the first to compare directly, in a randomized trial, the biological activity of a selective ER antagonist, such as fulvestrant, versus an AI in a pre-surgical setting. Furthermore, the dose of fulvestrant used was 500 mg, which is now the recommended dose in many countries. This study is also the first to compare the activity of fulvestrant 500 mg with and without anastrozole. Following treatment, ER H-scores were significantly reduced from baseline in all groups; however, there were greater reductions with fulvestrant 500 mg and fulvestrant 500 mg plus anastrozole, compared with anastrozole alone. These findings are entirely in accordance with the known mechanism of action (MoA) of these two agents: fulvestrant as a selective ER antagonist reduces tumor ER protein levels, while anastrozole reduces estradiol levels and, therefore, signaling through the ER, but has little or no inhibitory effect on ER levels. No additional reduction in ER expression was observed when fulvestrant was combined with anastrozole, compared with fulvestrant alone.
PgR expression levels were also significantly reduced from baseline in all groups, but there were no significant differences between fulvestrant plus anastrozole and either agent alone. Again, this is in agreement with what is known about the MoA of these drugs. PgR is an estrogen-inducible protein, and removal of ER signaling either by ER down-regulation or by blockade of estradiol synthesis leads to reduced PgR levels. Similarly, either MoA can decrease tumor cell proliferation. Although all treatments impacted substantially on ER function and proliferation, there was still some ER and PgR expression remaining in the post-fulvestrant treatment biopsies.
The fact that combining two endocrine agents did not result in increased reduction of PgR or Ki67 levels confirms that they act with equivalent magnitude on the same signaling pathway, but at different points. The reductions in tumor biomarkers observed after anastrozole treatment are also consistent with a recent randomized phase II study which reports that anastrozole and letrozole led to equally significant reductions in Ki67 expression [
16]. Ellis and colleagues also reported that neoadjuvant treatment with an AI was effective at improving clinical response rates and surgical outcomes in postmenopausal breast cancer.
One of the strengths of this randomized pre-surgical study is that double-blind procedures were extended until all biomarker measurements had been made before the treatment codes were broken.
The initial rationales for this study were that fulvestrant 500 mg might be biologically more potent than an AI and also that a synergistic effect of combining the two could be achieved. As fulvestrant competes with estradiol for ER binding, reducing plasma estrogen levels using anastrozole could feasibly increase fulvestrant-ER binding and increase its efficacy. Using an intratumoral, aromatase-transfected xenograft model, fulvestrant plus anastrozole was found to delay tumor growth more effectively than either agent alone [
17]. In addition, further reduction in ER levels and down-regulation of signaling proteins involved in the development of hormonal resistance (for example, insulin-like growth factor receptor 1 (IGF-1R), MAPK and AKT) were observed with the combination treatment. Despite these preclinical findings, our study found no biomarker evidence that the combined treatment had enhanced biological activity in patients with breast cancer. These results are in line with the FACT study, which reported no benefit in clinical endpoints with the combination of a loading-dose regimen of fulvestrant (250 mg) plus anastrozole versus anastrozole alone [
11]. The recently described SoFEA trial also failed to show any efficacy benefit for the combination of fulvestrant with an AI. Equivalent PFS was demonstrated for fulvestrant (250 mg loading-dose regimen) plus anastrozole compared with fulvestrant alone in postmenopausal patients with advanced breast cancer following progression on non-steroidal AIs [
18]. However, data from the SWOG S0226 study suggest that combination therapy as first-line treatment for advanced breast cancer was associated with efficacy benefits in some patients and may warrant further study [
12]. For the primary endpoint, median PFS was 15.0 months for the combination therapy (fulvestrant 250 mg loading-dose regimen plus anastrozole) compared with 13.5 months for anastrozole alone (HR = 0.80;
P = 0.007). The reason for the different outcomes in FACT and SWOG S0226 has not been fully established. However, this may in part be explained by the proportion of patients who had received prior adjuvant endocrine therapy, which was somewhat higher in the FACT trial. In a retrospective analysis of those patients who had received previous adjuvant tamoxifen treatment in the SWOG S0226 study (280/694; 40.3%), median PFS was 13.5 months in the combination group compared with 14.1 months in the anastrozole-alone group (HR = 0.89;
P = 0.37). In those patients naive to prior tamoxifen therapy (414/694; 59.7%), median PFS was 17.0 months for the combination compared with 12.6 months for anastrozole alone (HR = 0.74;
P = 0.006) [
12]. Importantly, there was no fulvestrant-alone arm in SWOG S0226 and so it is not possible from this study to establish if the difference between the two arms is due to the fulvestrant 250 mg loading-dose regimen being better than anastrozole in the first-line setting or due to the combination of fulvestrant 250 mg and anastrozole.
Recently, a growing body of evidence has suggested that fulvestrant 500 mg would offer efficacy benefits over the existing 250 mg regimen. NEWEST was the first study to demonstrate a higher biological activity (depletion of ER, PgR and Ki67) for fulvestrant 500 mg versus 250 mg [
9]. In addition, results from the CONFIRM study indicate greater clinical efficacy with the 500 mg regimen, without increased toxicity [
10]. It, therefore, appears that fulvestrant 500 mg results in both increased biological activity and clinical efficacy.
The median day of biopsy across all patients in the present study was Day 18 following a single fulvestrant 500 mg dose. However, previously published data suggest that steady-state plasma fulvestrant levels are achieved after approximately 28 days with the fulvestrant 500 mg dose regimen (which also includes a 500 mg dose on Day 14) [
8], and hence exposures following a single 500 mg dose would be lower than those achieved at steady state. This would suggest that the biological effect seen with a single fulvestrant 500 mg dose may be an underestimate compared with the fulvestrant 500 mg dose regimen approved for clinical practice.
The biological results from the present study may shed light on the results from FIRST, in which the secondary endpoint of TTP was significantly longer for fulvestrant 500 mg over anastrozole (
P <0.05) [
13], an effect that was maintained in prolonged follow-up data (HR = 0.66;
P = 0.01) [
19]. The primary endpoint showed a numerical, but not statistically significant, difference in CBR. In the current study, there was a greater decrease in ER (but not in Ki67) for fulvestrant 500 mg compared with anastrozole. The similar substantial initial decrease in proliferation (that is, Ki67) would be in keeping with the initial CBRs (
de novo response) seen in FIRST. The improvement in TTP in FIRST occurred after six months (due to prolongation of acquired resistance in tumors in which initial clinical benefit was shown). In model systems, a mechanism implicated in acquired resistance to endocrine therapy (including estrogen deprivation) is cross-talk between ER and other growth factor pathways (for example, HER2, IGF-1R and downstream signaling kinases) [
20‐
23]. The activity of residual ER (or growth factor signaling elements) following fulvestrant treatment remain largely unexplored, but the greater reduction in ER seen here with fulvestrant might feasibly hinder instigation of such cross-talk mechanisms and thus delay emergence of acquired resistance, compared with tumors treated with anastrozole alone.
When tolerability was assessed, the AE profile was similar in all treatment groups, with no emerging safety concerns for fulvestrant 500 mg. However, only very limited safety data were available due to the short treatment duration in the study. The safety profile of fulvestrant 500 mg has previously been described in FIRST and CONFIRM, where the safety profile of the higher dose was similar to that of the 250 mg dose. The comparable AE profile between fulvestrant 500 mg plus anastrozole and anastrozole alone was in keeping with the side-effect profiles of fulvestrant 250 mg plus anastrozole versus anastrozole alone reported in FACT [
11]. There were, therefore, no new safety concerns for the higher-dose fulvestrant regimen, when used alone or in combination with anastrozole [
10,
13,
24].
Competing interests
JFRR has acted as a consultant on advisory boards for AstraZeneca and Bayer HealthCare, and received research funding from Amgen, AstraZeneca, Bayer HealthCare and Novartis. He has received honoraria for speaking at symposia organized by AstraZeneca and GlaxoSmithKline. He has provided expert testimony on fulvestrant before the European Medicines Agency. IOE is the Medical Director of Source Bioscience plc and has acted as a consultant on advisory boards for Roche. EC has received payment from Nottingham University using AstraZeneca research funding. RIN and JMWG currently hold research grants from AstraZeneca. JMD, DMS, AJ and PH-T have no conflicts of interest to declare. This was an investigator-initiated study, sponsored by the University of Nottingham and supported by a research grant from AstraZeneca Pharmaceuticals. The article-processing charge is being financed by AstraZeneca.
Authors' contributions
JFRR and IOE designed the concept of this study. JFRR, JMD, DMS, AJ, IOE, EC, PH-T and JMWG collected and assembled the trial data. All biomarker immunohistochemistry was performed under the laboratory direction of JMWG. JFRR, EC, RIN and JMWG performed the data analysis and interpretation. All authors were involved in the writing of the manuscript and approved the final version of the manuscript.