Purpose:

Metastatic hormone receptor (HR)-positive, HER2-negative breast cancer is an important cause of cancer mortality. Endocrine treatment with or without additional targeted therapies has been the mainstay of treatment. This trial was designed to evaluate the combination of fulvestrant plus everolimus versus fulvestrant, everolimus, and anastrozole compared with fulvestrant alone in the first-line treatment of advanced HR-positive, HER2-negative breast cancer.

Patients and Methods:

This randomized placebo-controlled trial included postmenopausal women with HR-positive, HER2-negative advanced breast cancer who had received no prior systemic therapy for metastatic disease. Participants were randomized to one of three treatment arms and the primary outcome was progression-free survival (PFS), comparing combinations of fulvestrant and everolimus with or without anastrozole with fulvestrant alone. Circulating tumor cells (CTC), as measured with two different methods, and circulating tumor DNA (ctDNA) were evaluated serially prior to treatment and the beginning of the second cycle of therapy.

Results:

Due in part to changes in clinical practice, the study was closed after accruing only 37 participants. There was no evidence that everolimus-containing combination treatment improved PFS or overall survival relative to fulvestrant alone. When modeled continuously, an association was observed of baseline CTC and ctDNA with poorer survival.

Conclusions:

Although power of the study was limited, the findings were unable to support the routine use of everolimus combination endocrine therapy in the first-line treatment of advanced hormone-sensitive breast cancer. Prognostic impact of baseline ctDNA and copy-number variations in CTC was demonstrated.

Translational Relevance

SWOG S1222 is a phase III randomized clinical trial of fulvestrant, anastrozole, and everolimus in the front-line treatment of advanced hormone receptor–positive breast cancer. The study evaluated the hypothesis that addition of everolimus with or without anastrozole would improve progression-free survival compared with fulvestrant alone. Translational studies of circulating tumor cells (CTC) were also conducted with measurement by two distinct methods. The expectation was that patients with high CTC and/or high circulating tumor DNA (ctDNA) might benefit from additional therapy while those with low CTC would not and that CTC phenotype, specifically relative expression of estrogen receptor, BCL2, HER2, and Ki67, would predict benefit from endocrine therapy. Because of early study termination, only the prognostic value of the CTC and ctDNA measures could be evaluated. In this limited sample, CTC measures had high concordance and analysis of ctDNA using genomic copy number was shown to indicate poor prognosis.

The mainstay of treatment for endocrine-sensitive metastatic breast cancer has been sequential use of endocrine therapies including selective estrogen receptor (ER) modulators, aromatase inhibitors and a selective ER downregulator. Recent studies have demonstrated favorable results with endocrine agents used in combination (1) or with the addition of targeted therapies including the mTOR inhibitor everolimus (2, 3) or inhibitors of cyclin-dependent kinase 4 and 6 (CDK4/6; refs. 4–7). At initiation of the current study, CDK4/6 inhibitors were not yet FDA-approved therapeutic options. The primary objective of this study was to test the progression-free survival (PFS) benefit of combining fulvestrant with everolimus versus combining fulvestrant with everolimus and anastrozole, each compared with fulvestrant alone in the treatment of postmenopausal women with hormone receptor (HR)-positive metastatic breast cancer. Further objectives included additional comparisons of PFS, overall survival (OS), response rates, toxicities, adherence, and feasibility. Translational studies were also planned to investigate molecular determinants of response to treatment and prognosis in components of liquid biopsies: specifically circulating tumor cells (CTC) and circulating tumor DNA (ctDNA).

Clinical eligibility and trial conduct

Eligible patients were postmenopausal women with histologically confirmed HR-positive and HER2-negative metastatic breast cancer for which no prior systemic treatment had been received in the metastatic or recurrent setting. Prior chemotherapy and endocrine therapy in the adjuvant or neoadjuvant setting were permitted as long as any aromatase inhibitor therapy was completed more than 12 months prior to randomization. Those with prior treatment with fulvestrant or mTOR inhibitors were ineligible. Participants were required to have adequate cardiac, hepatic, renal, and bone marrow function. Those with elevated cholesterol or triglycerides and those with bleeding diathesis or on long-term anticoagulant therapy were excluded.

Participants were randomized with equal allocation to three arms: fulvestrant plus placebo for both everolimus and anastrozole (arm 1), fulvestrant plus everolimus with placebo for anastrozole (arm 2), or fulvestrant plus everolimus and anastrozole (arm 3). Fulvestrant dosing was 500 mg intramuscular every 4 weeks with an additional 500 mg loading dose day 15 of cycle 1, everolimus was dosed at 10 mg orally daily and anastrozole dose was 1 mg orally daily. Treatment was continued until disease progression, unacceptable toxicity, treatment delay > 4 weeks, if a need for antiretroviral therapy arose, withdrawal of consent, or study closure. The study was conducted in accordance with U.S. Common rule ethical guidelines with written informed consent obtained from all participants and approval of local Institutional Review Boards.

Translational studies

The identification and enumeration of CTCs has proven to be a clinically useful method of assessing progression in metastatic breast cancer (8, 9). As an integrated translational study, blood was collected separately into CellSave tubes which were sent to the University of Michigan (Ann Arbor, MI) for CellSearch analyses and into Streck tubes which were sent to the USC Michelson CSI-Cancer (Los Angeles, CA) for high-definition single-cell analysis (HD-SCA) and cell-free DNA (cfDNA) analyses at treatment cycle1 day 1 (baseline), cycle 2 day 1and at progression.

CTC enumeration and characterization were performed using the CellSearch CXC Kit and CellSearch system (Menarini Silicon Biosystems, Inc.) at baseline and then follow-up timepoints only if elevated at baseline (10) as described previously (8, 10, 11). CTC levels were enumerated as the average of the CTC levels in the four different aliquots of 7.5 mL whole blood (WB), each of which was used to determine each of the four respective CTC-biomarker expressions to calculate the CTC-endocrine therapy index (CTC-ETI) for that blood draw. The CTC-ETI was calculated as described (10). As per prior studies (8, 9, 11), ≥5 CTC/7.5 mL WB were considered elevated, and 0–4 CTC/7.5 mL WB were designated as low.

The HD-SCA method of CTC enumeration and characterization (12) was to be performed for all samples at all three timepoints (baseline, cycle 2 day 1 and progression). An average volume of 0.55 mL of blood was analyzed per assay and all cells including leukocytes were identified using immunofluorescent stains and enumerated. Cells with high levels of cytokeratin staining were counted as CTCs and scored as a continuous variable ranging from 2.2 to 145.8 CTC/mL blood. Prior to CTC capture, plasma was prepared by centrifugation and archived at −80°C. cfDNA was extracted using the QIAamp Kit (QIAGEN) and cfDNA concentration was measured using Qubit (Thermo Fisher Scientific) as published previously (12). Low-pass DNA sequencing and copy-number profiling were performed as previously described on both cfDNA and isolated single cells (12). ctDNA tumor fractions were estimated using the ichorCNA statistic (13) and scored as a continuous variable. Multiplex proteomic analysis of individual CTC was performed using the Hyperion Imaging Mass Cytometer (Fluidigm) as published previously (14).

Statistical analysis

The primary outcome was PFS defined as time to progression or death due to any cause. The primary aim was to compare the two combination arms to arm 1. Secondary outcomes included OS defined as time from registration to death from any cause, as well as Common Terminology Criteria for Adverse Events toxicity. Survival times were compared using log-rank tests for comparisons of treatment and Cox regression analysis for HR estimation and testing of treatments and biomarkers. Response rates were compared by χ2 testing.

Predictive testing of the role of liquid biopsy results on treatment and subsequent clinical outcomes were planned, using Cox regression, with a hypothesis that participants with high CTC might benefit from combination therapy while those with low CTC would not.

Clinical outcomes according to treatment assignment

The original planned sample size of SWOG1222 (NCT02137837) was 825, assuming PFS medians of 15, 21.5, and 25 months for the three arms, respectively. Accrual of 37 participants occurred between May 2014 and February 2015 (Supplementary Fig. S1). FDA approval of CDK4/6 inhibitors in the first-line treatment of HR-positive metastatic breast cancer in February 2015 made the trial not viable. In October 2015, the study sponsor permanently closed the study, offering participants the option to continue their current active drug therapy after unblinding. All study follow-up concluded December 2019.

Patient characteristics are shown in Table 1. One participant received no protocol treatment and is not evaluable for clinical benefit or adverse events. Among 36 evaluable patients, no grade 3 or higher toxicity was observed in the fulvestrant arm; 1 patient receiving fulvestrant plus everolimus experienced grade 4 toxicity (hypophosphatemia) and an additional 10 participants receiving fulvestrant and everolimus with or without anastrozole experienced grade 3 toxicities. PFS appeared similar for all arms (Fig. 1A; log-rank P = 0.88) with an overall median of 11.2 months. At the end of study follow-up at 5 years, 3 patients (one in each arm) were still receiving protocol assigned treatment and had not progressed. There was also no evidence of a difference in OS (Fig. 1B; log-rank P = 0.81) with an overall median of 42 months. Median follow-up time for those still alive was 56 months. Among those with measurable disease, there were two responses in 9 patients on arm 1 (22.2%), six in 10 patients on arm 2 (60.0%), and four in 9 patients on arm 3 (44.4%). Though suggestive of better response on combination therapy, these differences were not statistically different (P = 0.25).

Table 1.

Patient characteristics by study arm.

FulvestrantFulvestrant + EverolimusFulvestrant + Everolimus + Anastrozole
(n = 13)(n = 12)(n = 12)
AGE 
 Median 63.4  62.6  60.5  
 Minimum 54  45  48  
 Maximum 74  87  69  
HISPANIC 
 Yes 8% 17% 0% 
 No 12 92% 10 83% 12 100% 
RACE 
 White 69% 10 83% 75% 
 Black 15% 0% 25% 
 Asian 0% 8% 0% 
 Multi-Racial 8% 0% 0% 
 Unknown 8% 8% 0% 
DISEASE 
 Measurable 69% 10 83% 75% 
 Evaluable non-measurable disease 31% 17% 25% 
PRIOR HORMONE 
 Prior adjuvant hormonal therapy completed more than 5 years ago 23% 8% 17% 
 Prior adjuvant hormonal therapy completed 1–5 years ago 31% 42% 50% 
De novo presentation of metastatic disease or no prior adjuvant hormonal therapy 46% 50% 33% 
FulvestrantFulvestrant + EverolimusFulvestrant + Everolimus + Anastrozole
(n = 13)(n = 12)(n = 12)
AGE 
 Median 63.4  62.6  60.5  
 Minimum 54  45  48  
 Maximum 74  87  69  
HISPANIC 
 Yes 8% 17% 0% 
 No 12 92% 10 83% 12 100% 
RACE 
 White 69% 10 83% 75% 
 Black 15% 0% 25% 
 Asian 0% 8% 0% 
 Multi-Racial 8% 0% 0% 
 Unknown 8% 8% 0% 
DISEASE 
 Measurable 69% 10 83% 75% 
 Evaluable non-measurable disease 31% 17% 25% 
PRIOR HORMONE 
 Prior adjuvant hormonal therapy completed more than 5 years ago 23% 8% 17% 
 Prior adjuvant hormonal therapy completed 1–5 years ago 31% 42% 50% 
De novo presentation of metastatic disease or no prior adjuvant hormonal therapy 46% 50% 33% 
Figure 1.

PFS and OS by randomized treatment groups. Data cutoff was December 31, 2019. Log-rank test compared all three treatment groups. A, PFS. B, OS.

Figure 1.

PFS and OS by randomized treatment groups. Data cutoff was December 31, 2019. Log-rank test compared all three treatment groups. A, PFS. B, OS.

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Liquid biopsy analyses

Because of a regulatory issue which delayed the ability of the University of Michigan laboratory to accept specimens, only 13 patients had CTC evaluation by CellSearch at baseline. Two (15.4%) patients had elevated CTC prior to treatment (Fig. 2) and were tested again after one cycle. CTC levels declined dramatically for both patients. For one, assigned to fulvestrant and everolimus, CTC declined from a baseline level of 18 to first follow-up level of 4 CTC/7.5 mL WB. The second patient, assigned to fulvestrant only, had an average of 46 CTC at baseline which declined at first follow-up to 8 CTC/7.5mL WB. PFS did not differ between these 2 patients with elevated CTC at baseline compared with those without elevated CTC at baseline (log-rank P = 0.47). Because only 2 patients evaluated by the CellSearch assay had elevated CTC levels, CTC-ETI analysis was determined, but association with outcomes was not performed.

Figure 2.

CTC expression of ER in 2 patients with elevated CTC levels. CTC enumeration and ER expression determined using CellSearch. See text for details.

Figure 2.

CTC expression of ER in 2 patients with elevated CTC levels. CTC enumeration and ER expression determined using CellSearch. See text for details.

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Using the HD-SCA assay, 25 of 34 cases (74%) had measurable non-leukocyte cell counts and seven of the 34 (21%) had CTCs with high cytokeratin expression. The presence of CTCs was not associated with poorer PFS [HR = 1.40; 95% confidence interval (CI), 0.59–3.32; P = 0.45]. However, if the count of high cytokeratin CTC at baseline is modeled as a continuous variable in the Cox regression, there is a significant decrease in PFS with each unit of CTC by HD-SCA (HR = 1.02; 95% CI, 1.00–1.04; P = 0.043). For draw 2 after one cycle of treatment, 3 of 32 (9.4%) patients measured by the HD-SCA assay were positive for high cytokeratin CTC, including 2 of the patients elevated at baseline. For draw 3 at the time of progression 5 of 18 (27.8%) patients had positive CTC. For the 13 patients evaluated for CTC by both methods, there was perfect concordance of the two assays: the same 2 patients had elevated CTC by both assays and the remaining 11 did not.

In all detectable cases, copy-number profiles of ctDNA represented an aggregate of all clones detected on a single-cell level. Baseline ctDNA (n = 25), measured as tumor DNA fraction, was also modeled continuously, and was statistically associated with poorer PFS (HR = 1.08; 95% CI, 1.02–1.15; P = 0.005), while total cf DNA as purified from the plasma was not (HR = 1.04; 95% CI, 0.99–1.09; P = 0.11).

Because of the evolving landscape of first-line therapy for metastatic hormone-sensitive breast cancer, the current study was unable to complete accrual or determine the impact of the addition of everolimus to fulvestrant alone or to combination endocrine therapy in this setting. The observed median PFS of 11.2 months in this study (S1222) compares favorably with the 5.6-month PFS with fulvestrant 500 mg in the CONFIRM study which enrolled patients who progressed within 12 months of adjuvant therapy or while on first-line endocrine therapy (15), and unfavorably with the median time to progression of 23.4 months observed with first-line fulvestrant alone in the FIRST study (16), highlighting difficulties in cross-study comparisons. The addition of everolimus to endocrine therapy in the current study, S1222, was associated with increased toxicity. In spite of an impressive impact on PFS in previous studies, an OS benefit with everolimus in the treatment of metastatic breast cancer has yet to be established. The recent demonstration of improved OS with CDK4/6 inhibitors (17, 18) firmly establishes their inclusion early on in the treatment of metastatic HR-positive breast cancer. Everolimus remains an option in subsequent lines of therapy as suggested by the PreE0102 study, in which the PFS was 10.3 versus 5.1 months with the addition of everolimus to fulvestrant following progression aromatase inhibitor therapy (3). Furthermore, everolimus toxicity in the form of stomatitis may be reduced with the use of oral dexamethasone mouthwash (19), which was not mandated in S1222.

The planned translational liquid biopsy studies were likewise severely limited by the low accrual and by a regulatory issue that prevented analysis of the entire population of participants. Nonetheless, enumeration of CTC by two different methods (CellSearch and HD-SCA) was completely concordant (2 elevated, 11 not). No statistically significant difference in PFS was observed between the 2 patients with elevated CTC by both methods compared to the 11 with non-elevated CTC at baseline. However, both of these patients experienced a “CTC response,” which has been associated with a better prognosis when compared to patients whose CTC remain elevated at the end of the first cycle of therapy (9, 11). When modeled as a continuous variable, elevated levels of CTC by HD-SCA were associated with a worse prognosis, consistent with several prior studies that have demonstrated that the presence of CTC enumerated with CellSearch prior to start of therapy is associated with a worse outcome in metastatic breast cancer (8, 11).

Findings of CTC-ETI analyses of four participants with elevated CTC at baseline are of interest, suggesting that further genomic and proteomic analysis might be valuable in treatment selection and outcomes (Figs. 2 and 3). For patient 1, who was assigned to fulvestrant and everolimus, all of the CTC detected by CellSearch and by HD-SCA at baseline were negative for ER expression, which we hypothesized would predict for lack of benefit from endocrine therapy (Fig. 2). However, she experienced a “CTC response,” raising the speculation that blocking the mTOR pathway may be successful even if the cancer has reverted to a HR-negative phenotype. In contrast, in patient 2, approximately 60% of the CTC were ER positive at baseline. She was assigned to fulvestrant alone and also had a CTC response (Fig. 2).

Figure 3.

CTC genomic and phenotypic analysis in 2 patients with elevated CTC levels. CTC enumeration and genomic and phenotypic characterization determined using HD-SCA. A, Galleys of CTC of blood collected from patients 3 and 4, showing cytokeratin, ER, DNA, and CD45 expression. B, ER expression in CTC in patients 3 and 4. C, Genomic analysis of CTC in patients 3 and 4. D, Galleys of CTC of blood collected from patient 4 showing expression of HER2, e-cadherin, EpCam, and TWIST in three epithelial cells. See text for details.

Figure 3.

CTC genomic and phenotypic analysis in 2 patients with elevated CTC levels. CTC enumeration and genomic and phenotypic characterization determined using HD-SCA. A, Galleys of CTC of blood collected from patients 3 and 4, showing cytokeratin, ER, DNA, and CD45 expression. B, ER expression in CTC in patients 3 and 4. C, Genomic analysis of CTC in patients 3 and 4. D, Galleys of CTC of blood collected from patient 4 showing expression of HER2, e-cadherin, EpCam, and TWIST in three epithelial cells. See text for details.

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Similarly, 2 other patients with with the highest level of CTC elevation as identified by the HD-SCA assay provided provocative findings (Fig. 3). In a manner similar to patient 1, patient 3 had a high percentage of ER-positive CTC as well as a copy-number profile suggestive of a luminal subtype (Fig. 3AC). This patient was treated with fulvestrant, anastrozole, and everolimus, and had a remarkable PFS of over 2 years. In contrast, patient 4's CTC were were almost entirely ER negative with a basal-like genomic subtype (Fig. 3BD). Furthermore, proteomic analysis demonstrated that many of her CTC expressed HER2 and TWIST (Fig. 3D). This patient, treated with fulvestrant and everolimus, progressed within 18 days from the time of entry onto the trial.

Taken together, these data suggest an intriguing hypothesis that CTC-ER phenotype might help select patients who could be treated with endocrine therapy alone or who are better treated with combination endocrine and other pathway (such as mTOR or CDK4/6) inhibition. Of course, these speculations require substantial validation. Further assessment of ctDNA showed promise as a prognostic marker for PFS, similar to previously published reports (20).

While the genomic and proteomic analyses are only exploratory given the small number of CTC-positive patients, our findings indicate that while CTC enumeration alone can be of prognostic value, deeper characterization of CTC combined with ctDNA analysis may provide further insight into the mechanisms underlying treatment response. Future studies should lead to improved understanding of molecular determinants of response and progression which may help to select which patients are most likely to benefit from the various therapeutic options.

H.C.F. Moore reports grants from SWOG Cancer Research Network during the conduct of the study and other support from AstraZeneca, Roche/Genentech, Daiichi-Sankyo, and Sermonix outside the submitted work. W.E. Barlow reports grants from NCI and other support from AstraZeneca during the conduct of the study. J.R. Gralow reports other support from Roche/Genentech, AstraZeneca, Sandoz/Hexal AG, Puma, Novartis, SeaGen, Genomic Health/Exact Sciences, and Radius outside the submitted work. D.F. Hayes reports grants and non-financial support from Janssen Diagnostics and personal fees from Janssen Diagnostics during the conduct of the study. D.F. Hayes also reports non-financial support from inbiomotion; grants and personal fees from cepheid; personal fees from Cellworks, BioVeca, EPIC Sciences, L-Nutra, OncoCyte, Turnstone Biologics, predictus BioSciences, and Tempus; and grants from Merrimack pharma, Eli Lilly, and AstraZeneca outside the submitted work; in addition, D.F. Hayes has a patent regarding circulating tumor cells, for which the rights were licensed to the manufacturer of CellSearch: first Janssen and then Menarini Silicon Biosystems, and D.F. Hayes received annual royalties through January 2021. P. Kuhn reports grants from Hope Foundation for Cancer Research and Breast Cancer Research Foundation during the conduct of the study and grants and personal fees from Epic Sciences outside the submitted work; in addition, P. Kuhn has a patent for Systems, methods and assays for outlier clustering unsupervised learning automated report (ocular) pending, licensed, and with royalties paid from Epic Sciences and a patent for Methods for detection of circulating tumor cells and methods of diagnosis of cancer in a mammalian subject pending, licensed, and with royalties paid from Epic Sciences. J.B. Hicks reports grants from Breast Cancer Research Foundation during the conduct of the study and personal fees from Epic Sciences outside the submitted work; in addition, J.B. Hicks has a patent for OCULAR Technologies asn Software for Rare Cell Identification and Classifiation licensed and with royalties paid from Epic Sciences, Inc. L. Welter reports grants from Breast Cancer Research Foundation, Hope Foundation for Cancer Research, and Alan Joseph Endowed Fellowship during the conduct of the study. A.K. Conlin reports personal fees from AstraZeneca and SeaGen outside the submitted work. D.L. Lew reports grants from AstraZeneca during the conduct of the study. D. Tripathy reports grants and personal fees from Novartis during the conduct of the study; personal fees from AstraZeneca, Gilead, GlaxoSmithKline, Exact Sciences, and OncoPep; and grants and personal fees from Pfizer outside the submitted work. L. Pusztai reports personal fees from AstraZeneca, Merck, Novartis, Bristol-Myers Squibb Genentech, Eisai, Pieris, Immunomedics, Seattle Genetics, Clovis, Syndax, H3Bio, and Daiichi outside the submitted work. G.N. Hortobagyi reports grants and personal fees from Novartis during the conduct of the study and outside the submitted work. No disclosures were reported by the other authors.

H.C.F. Moore: Conceptualization, data curation, writing–original draft, writing–review and editing. W.E. Barlow: Conceptualization, data curation, formal analysis, methodology, writing–original draft, writing–review and editing. G. Somlo: Conceptualization, resources, data curation, investigation, methodology, writing–review and editing. J.R. Gralow: Conceptualization, writing–review and editing. A.F. Schott: Conceptualization, resources, writing–review and editing. D.F. Hayes: Conceptualization, resources, investigation, methodology, writing–review and editing. P. Kuhn: Resources, data curation, investigation, methodology, writing–review and editing. J.B. Hicks: Resources, data curation, investigation, methodology, writing–review and editing. L. Welter: Resources, data curation, investigation, methodology, writing–review and editing. P.A. Dy: Resources, writing–review and editing. C.H. Yeon: Resources, writing–review and editing. A.K. Conlin: Resources, writing–review and editing. E. Balcueva: Resources, writing–review and editing. D.L. Lew: Conceptualization, data curation, formal analysis, writing–review and editing. D. Tripathy: Conceptualization, resources, writing–review and editing. L. Pusztai: Conceptualization, resources, writing–review and editing. G.N. Hortobagyi: Conceptualization, resources, supervision, writing–review and editing.

SWOG Clinical Trials Partnerships (CTP) manages the non-federally funded components of SWOG Cancer Research Network under The Hope Foundation for Cancer Research. This study received support from AstraZeneca plc and from Novartis Pharmaceuticals. D.F. Hayes received research funding support from Janssen Diagnostics during the conduct of this trial. P. Kuhn and J.B. Hicks received research funding from the Breast Cancer Research Foundation. L. Welter was supported by the Alan Joseph Endowed Fellowship.

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Supplementary data