Clinical Efficacy and Whole Exome Sequencing of Liquid Biopsies in a Phase IB/II study of Bazedoxifene and Palbociclib in Advanced Hormone Receptor-Positive Breast Cancer

Abstract

Purpose:

Sensitivity to endocrine therapy (ET) is critical for the clinical benefit from the combination of palbociclib plus ET in hormone receptor-positive/HER2-negative (HR+/HER2−) advanced breast cancer. Bazedoxifene is a third-generation selective ER modulator and selective ER degrader with activity in preclinical models of endocrine-resistant breast cancer, including models harboring ESR1 mutations. Clinical trials in healthy women showed that bazedoxifene is well tolerated.

Patients and Methods:

We conducted a phase Ib/II study of bazedoxifene plus palbociclib in patients with HR+/HER2− advanced breast cancer who progressed on prior ET (N=36) (NCT02448771).

Results:

The study met its primary endpoint, with a clinical benefit rate of 33.3%, and the safety profile was consistent with what has previously been seen with palbociclib monotherapy. The median progression free survival (PFS) was 3.6 months (CI_95% 2.0-7.2). An activating PIK3CA mutation at baseline was associated with a shorter PFS (HR = 4.4, CI_95% 1.5-13, P = 0.0026) but activating ESR1 mutations did not impact the PFS. Longitudinal plasma circulating tumor DNA whole exome sequencing (WES) (N=68 plasma samples) provided an overview of the tumor heterogeneity, the sub-clonal genetic evolution and identified actionable mutations acquired during treatment.

Conclusions:

The combination of palbocilib and bazedoxifene has clinical efficacy and an acceptable safety profile in a heavily pre-treated patient population with advanced HR+/HER2− breast cancer. These results merit continued investigation of bazedoxifene in breast cancer.

Introduction

CDK4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) are the standard of care in hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2−) advanced breast cancer(1). The cyclin D1 dependent kinases CDK4 and CDK6 phosphorylate the retinoblastoma (pRb) tumor suppressor, enabling progression through the cell cycle. Cyclin D1 is a key transcriptional target of the estrogen receptor (ER), and the convergence of the ER transcriptional network and cyclin D1-CDK4/6-Rb1 axis contributes to the synergistic activity of ET and CDK4/6i, such as palbociclib(2–4). This synergy is a key component of the clinical efficacy of palbociclib, which has limited clinical benefit as a single agent (5).

The importance of the synergy between ET and palbociclib suggests that improved targeting of ER may enhance the efficacy of regimens combining ET and palbociclib. This notion is supported by recent studies demonstrating that several mechanisms of resistance to ET are also mechanisms of resistance to the combination of ET and palbociclib(6,7). Importantly, in the randomized phase III PADA-1 trial patients with HR+/HER2− metastatic breast cancer with an ESR1 activating ligand binding domain (LBD) mutation, which confers resistance to ET, had double the odds of disease progression while on treatment with an aromatase inhibitor plus palbociclib compared to patients with wild-type (WT) ESR1(8). Furthermore, in the PADA-1 trial, patients with rising plasma circulating tumor DNA (ctDNA) ESR1 mutations had improved progression free survival (PFS) when treatment was switched to fulvestrant and palbociclib versus continued treatment with an aromatase inhibitor and Palbociclib(9). In the PALOMA-3 trial, the Y537S ESR1 LBD mutation was enriched after the acquisition of resistance to fulvestrant alone or to the combination of palbociclib plus fulvestrant in metastatic disease(10). Collectively, these results suggest that better targeting of ER can improve outcomes with the combination of a CDK4/6i and ET, and the Y537S ESR1 mutation has a role in the acquisition of resistance to fulvestrant and palbociclib.

Bazedoxifene is a selective ER modulator (SERM)- selective ER degrader (SERD) hybrid (SSH) (i.e. has ER degrader properties in addition to the SERM activity)(11–13). Bazedoxifene has favorable effects on bone and lipid metabolism without adversely affecting the uterus(14,15). It is the only SERM that is approved in combination with conjugated estrogens for the prevention of post-menopausal symptoms and osteoporosis based on large randomized clinical trials in healthy post-menopausal women(16). Pre-clinical studies showed that bazedoxifene has anti-tumor activity in HR+ breast cancer models, including models of resistance to tamoxifen, resistance to estrogen deprivation, and models harboring a Y537S ER LBD mutation(11,12,17)^,(18). Preclinical studies showed that bazedoxifene had greater inhibitory effects on cell growth and ER-mediated transcription compared to tamoxifen or fulvestrant in the presence of the Y537S ESR1 mutations(13,19). Computational predictions and, more recently, the crystal structure of mutant LBD bound to bazedoxifene showed that the Y537S mutant LBD bound to bazedoxifene leads to the formation of a new S537-E380 hydrogen bond that stabilizes helix 12 (H12) in the activating function-2 (AF-2) cleft of the LBD, and this enables the inhibition of co-activator binding that results in transcriptional inhibition. In contrast, several other SERMS and SERDs, including tamoxifen and fulvestrant, were shown to have limited transcriptional inhibitory activity in the presence of the Y537S mutation. This is likely because H12 is pushed out from the AF-2 cleft of the Y537S mutant LBD after interacting with tamoxifen or fulvestrant, and this allows co-activator binding and consequently a decreased response to these drugs^13,19.

Taken together, based on the pre-clinical data of bazedoxifene in endocrine resistant HR+ breast cancer models, the safety profile of bazedoxifene in randomized clinical trials, and the rationale of combining palbociclib with next-generation ETs, we conducted the first clinical trial with the combination of palbociclib and bazedoxifene. This was a phase Ib/II study designed to investigate the efficacy and safety of palbociclib with bazedoxifene for the treatment of patients with HR+/HER2− advanced breast cancer who had progressed on prior ET. Additionally, we performed serial circulating tumor DNA (ctDNA) whole exome sequencing (WES) to study genetic mechanisms of resistance and the tumor clonal architecture and evolution during treatment with palbociclib and bazedoxifene.

Materials and Methods

Patient Population

We enrolled female or male patients aged 18 years or older with histologically confirmed invasive breast cancer that was metastatic or unresectable locally advanced disease. Primary or invasive disease was confirmed to be HR+ (ER >10% and/or progesterone receptor>10%) and HER2− according the American Society of Clinical Oncology/College of Pathology guidelines(20) (defined as 0-1+ by immunohistochemistry or FISH-negative [HER2 copy number < 6 and HER2/CEP17 ratio < 2.0]). Measurable disease was required per RECIST 1.1 criteria(21). Bone-only disease was allowed if there was evidence of lytic lesions(22). Progressive disease on prior ET was required; this was defined as relapse while on adjuvant endocrine therapy or within 1 year of completion of adjuvant endocrine therapy, or progression through at least one line of endocrine therapy in metastatic or locally advanced breast cancer. Post-menopausal and pre-menopausal women who have been on a GnRH agonist were eligible. Prior treatment with up to two lines of chemotherapy in advanced disease was allowed. The protocol was amended to allow up to one line of prior chemotherapy when the eighteenth patient was enrolled. There was no limit to the number of prior endocrine therapies.

Study Design and Treatment

The study was a phase IB/II, single-arm, single institution, and open label study. The objectives were to evaluate the safety and antitumor activity of the combination of palbociclib and bazedoxifene. The primary end point was clinical benefit rate (CBR), defined as the proportion of patients with complete response (CR), partial response (PR), or stable disease (SD) at week 24 (per RECIST version 1.1(21)). A CBR of 20% or less would not be of clinical interest based on a conservative estimate of the percentage of patients who progressed on adjuvant ET or progressed on ET or palbociclib as a single agent in metastatic disease⁵. Secondary end points included objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and safety. Objectives were determined in the intention to treat (ITT) population and the evaluable cohort who reached the first restaging timepoint.

All patients received bazedoxifene 40 mg orally once daily on days 1-28 and palbociclib 125 mg orally once daily on days 1-21 of a 28-day cycle. The bazedoxifene dose of 40 mg was based on published safety data for this dose(23). The first 6 patients enrolled completed a safety run-in, and accrual was paused for safety assessment after these patients completed at least one treatment cycle. Response and progression were evaluated using RECIST 1.1 guidelines(21). For patients with bone-only disease, evaluation was done per MD Anderson bone tumor response criteria(22). After a baseline scan, tumor response was assessed every 8 weeks, with a PR or CR requiring confirmation within 4-6 weeks. After cycle 6, tumor measurements were performed every 12 weeks. Safety was evaluated according to NCI-CTCAE version 4.0. Exploratory translational objectives were to investigate the efficacy of the combination of bazedoxifene and palbociclib in relation to genomic findings in ctDNA.

Statistical Analyses

The study followed a Simon’s “optimal” two-stage design. Based on the published activity of palbociclib as a single agent, a CBR of 20% or less was considered not to be of clinical interest and was the null hypothesis, and a CBR of ≥ 30% was considered worthy of further investigation. The sample size of 37 patients was chosen to have 90% power to declare the combination effective at this rate while controlling for no more than 10% one-sided Type I error under the null hypothesis. The first stage of the study included 17 evaluable patients, and accrual was continued to the second stage after the confirmation that at least 4 patients met the primary end point.

CBR was reported as a proportion and 95% confidence interval (CI) accounting for the two-stage design. Associations between CBR and clinical/correlative factors were assessed using the Wilcoxon test for continuous variables and Fisher’s exact test for categorical variables.

Median PFS in months was estimated using the Kaplan-Meier estimation method and compared between clinical/correlative factors via log-rank test. Hazard ratios were obtained using Cox proportional hazard models. All tests were 2-sided with a type I error of 0.05. All analyses were carried out via SAS, V9.4 (SAS Institute) and R, V4.0.2.

Sample Collection and Processing

Whole venous blood (6–10 mL) was collected from patients into Ethylenediaminetetraacetic acid vacutainer tubes (Becton and Dickinson). Blood was processed within 2 hours of collection. Whole blood was centrifuged for 10 min at 1200 × g and the plasma supernatant was cleared by centrifugation for 10 min at 3000 × g. Cell-free DNA (cfDNA was extracted from 2 to 7 mL of plasma and eluted into 40–80 μL of re-suspension buffer using the Qiagen Circulating DNA kit on the QIAsymphony liquid handling system. Germline DNA (gDNA) was extracted using the Qiasymphony DSP DNA midi kit on the QIAsymphony liquid handling system. Quantification of extracted cfDNA and gDNA was performed using the PicoGreen (Life Technologies) assay on a Hamilton STAR-line liquid handling system. Library preparation was performed using a commercially available kit (KAPA HyperPrep Kit with Library Amplification product KK8504) and IDT’s duplex UMI adapters. Unique 8-base dual index sequences embedded within the p5 and p7 primers (purchased from IDT) were added during PCR. Enzymatic clean-up was performed using Beckman Coultier AMPure XP beads. Samples were sequenced using Illumina sequencing technology.

ctDNA Analysis

Sequenced reads in both ULP-WGS and WES libraries were mapped to the human reference genome (hg19) with BWA-MEM (version 0.17.7; RRID: SCR_010910). PCR duplicates were flagged by MarkDuplicates in Picard tool (version 2.26.10; RRID: SCR_006525). ichorCNA (version 0.1)(24) was run for estimating tumor purity estimation. We applied the hg19 panel of normals provided in the ichorCNA github repo (inst/extdata/HD_ULP_PoN_1Mb_median _normAutosome_mapScoreFiltered_median.rds).

SNV/INDEL Detection

Somatic mutations on the exome data were called by the Cancer Genome Analysis WES Characterization Pipeline (the CGA pipeline). The CGA pipeline first runs deTiN(25) and ContEst(26) (RRID: SCR_000595) for estimating potential tumor-in-normal contamination and cross-patient contamination, and calls SNVs and indels with MuTect(27) (RRID: SCR_000559) and Strelka(28), (RRID: SCR_005109) respectively. Out of the 68 exome samples, 2 samples were processed as tumor-only due to lack of the matched normals. For the tumor-only runs, CalculateContamination in GATK4 (version 4.0.5.1; RRID: SCR_001876) was used for estimating the cross-patient contamination. For the indel calling step in the tumor-only runs, an unmatched normal sample was used for Strelka, which requires a matched normal input. Potential artifacts were filtered with two panels of normals (created from 298 normal samples and 8334 TCGA normal samples, respectively), the BLAT (RRID: SCR_011919) read-realignment filter, and the read orientation bias filters. The mutations in the tumor-only runs were further filtered if the called mutations overlapped with the germline mutations with the population allele frequencies ≥5% in the gnomAD database(29) (RRID: SCR_014964). Mutations were denoted as pathogenic or likely pathogenic based on OncoKB(30) (RRID: SCR_014782).

Mutational signatures were extracted using two algorithms: (1) hierarchical Dirichlet process (HDP; https://github.com/nicolaroberts/hdp) based on the Bayesian hierarchical Dirichlet process, and (2) SignatureAnalyzer based on Bayesian non-negative matrix factorization(31). The Gibbs sampler was run with 20,000 burn-in iterations (parameter ‘burnin’). With a spacing of 200 iterations (parameter ‘space’), 100 iterations were collected (parameter ‘n’). After each Gibbs sampling iteration, three iterations of concentration parameter sampling were performed (parameter ‘cpiter’). To deconvolute composite signatures and to equate obtained HDP signatures to reference signatures, we used an expectation-maximization algorithm to deconstruct these signatures into reference constituents(31). Because of the low burden of indels, we excluded a mismatch repair deficiency signature (SBS6) and homologous repair deficiency signature (SBS3) from the candidate list using SigFit(32).

Copy number alterations in the WES samples were detected by GATK-CNV (version 4.1.2.0; RRID: SCR_001876) with -number-of-changepoints-penalty-factor=2.0. As the post filtering steps, a panel of normals created from 298 normal samples was used, and potential germline copy number alterations were discarded. The allelic copy number alteration outputs from ModelSegments in GATK-CNV (version 4.1.2.0) were used for estimating tumor purity with ABSOLUTE(33) (RRID: SCR_005198). The estimated tumor purity between the exomes (ABSOLUTE) and the ULP-WGS data (ichorCNA) was highly correlated (Pearson correlation coefficient = 0.98). Tumor mutation burden (TMB) was calculated as the number of coding mutations per Mb of covered exons.

The genomic sites of detected mutations across serial WES in a patient were force-called by investigating the number of reference and alternate bases at the sites. SCNAs in the serial WES were also grouped across the timepoints. PhylogicNDT was run with 500 iterations of Markov chain Monte-Carlo Gibbs sampler on the force-called mutations and SCNAs to assign mutations to clonal clusters for estimating the phylogeny(34).

Compliance with Ethical Standards

The study was conducted in accordance with the International Conference on Harmonization Good Clinical Practice Standards and the Declaration of Helsinki. Institutional review board approval was obtained at Dana-Farber/Harvard Cancer Center (DF/HCC). The study was registered in ClinicalTrials.gov (NCT02448771). The DF/HCC Data and Safety Monitoring Committee, which is composed of clinical specialists with experience in oncology and who had no direct relationship with the study, reviewed and monitored toxicity and accrual data from the study. Participants provided written informed consent prior to the performance of any protocol specific procedures or assessments. The study was an investigator-initiated trial funded by Pfizer. Palbociclib and bazedoxifene were supplied by the manufacturer (Pfizer). The funder had no role in data collection, data analysis or data interpretation.

Data and Materials Availability

TCGA pancancer data are available through a data portal (https://gdc.cancer.gov/node/905/; https://gdc.cancer.gov/about-data/publications/pancanatlas). The whole exome sequencing data from this study was deposited in dbGap accession number is phs002802.v1.p1.

Results

Patients

Between July 2015 and July 2017, we enrolled 36 patients with advanced HR+/HER2− breast cancer (34 females and 2 males) (Supplemental Figure 1). The study met its primary endpoint after enrolling 36 patients. Patient demographics and baseline characteristics are in Table 1 and supplemental Table 1 (Supplemental table 1 includes the study representative information). All patients had ER+, HER2− disease. The median age was 59.5 years (range, 37-79 years). Most of the patients had liver metastases (64%, 23/36) and 33% of patients (12/36) had lung metastases. Only 6% of patients (2/36) had bone-only disease. All patients had disease progression while on ET or disease recurrence within 1 year of completion of adjuvant ET. Most patients were heavily pre-treated, with 50% of patients (18/36) having received 2 or more prior lines of ET and 52% of patients (19/36) having received up to 2 lines of chemotherapy in advanced disease. Nearly all patients (28/36) had disease progression while on treatment with an aromatase inhibitor (AI) or disease recurrence within 1 year of completion of adjuvant treatment with an AI. There were 7 (19%) patients who received prior treatment with fulvestrant and 4 (11%) patients who received prior treatment with everolimus.

Table 1.

Baseline characteristics of study participants in the intention to treat patient population.

Characteristic	Number of Patients (%)
Sex
Male	2 (6%)
Female	34 (94%)
ECOG Performance Status
00	33 (92%)
01	3 (8%)
Estrogen Receptor Status
Low Positive	1 (3%)
Positive (> 10%)	35 (97%)
Progesterone Receptor Status
Unknown	1 (3%)
Low Positive	1 (3%)
Positive (> 10%)	22 (61%)
Negative (≤ 10%)	12 (33%)
HER2 Status
Negative	36 (100%)
Sites of disease
Lymph nodes	13 (36%)
Breast and chest wall	14 (39%)
Lung	12 (33%)
Liver	23 (64%)
Bone	26 (72%)
Other	7 (19%)
Bone Only
Yes	2 (6%)
No	34 (94%)
Visceral Disease
Yes	29 (81%)
No	7 (19%)
Prior Lines of Hormonal Treatment for Metastatic Breast Cancer
0	6 (17%)
1	12 (33%)
2	7 (19%)
3	8 (22%)
4	2 (6%)
5	1 (3%)
Prior Lines of Chemotherapy for Metastatic Breast Cancer
0	17 (47%)
1	16 (44%)
2	3 (8%)
Everolimus for Metastatic Breast Cancer
Yes	4 (11%)
No	32 (89%)

Abbreviations: ECOG - Eastern Cooperative Oncology Group

Treatment Administration and Safety

At the data cutoff in August 2020, with a median follow-up of 29.6 months, all patients had discontinued palbociclib and bazedoxifene treatment. A median of 4 cycles were given per patient (range 1-34). The first 6 patients who were included in the safety run-in did not experience any dose limiting toxicities (DLTs) during their first treatment cycle. Thus, all patients were given 125 mg palbociclib for the remainder of the study. Neutropenia (61%) and fatigue (22%) were the most common adverse events associated with study treatment. A total of 16 patients (44%) experienced grade 3 neutropenia, and 1 patient (3%) experienced grade 4 neutropenia (Table 2 and Supplemental Table 2). There were no deaths attributed to the study treatment. Most patients discontinued treatment due to disease progression (83%). Only 1 patient (3%) discontinued treatment due to protocol-specified unacceptable toxicity (neutropenia). Palbociclib dose reductions were required in 14 (38.9%) patients due to toxicity. Doses were held in 23 (63.9%) patients for a median of 10 days (range 1-45 days). There were no bazedoxifene dose reductions, and bazedoxifene doses were held in 13 (36.1%) patients for a median of 12 days (range 5-22 days) (Supplemental Tables 3 and 4).

Table 2.

Adverse events related to study treatment in at least 5% of patients

Adverse Event	All grades (%)	Grade ≥ 3 (%)
Neutrophil count decreased	22 (61.1%)	17 (47.2%)
Fatigue	8 (22.2%)	0 (0%)
Anemia	3 (8.3%)	1 (2.8%)
Mucositis oral	3 (8.3%)	0 (0%%)
White blood cell decreased	3 (8.3%)	1 (2.8%)
Gastroesophageal reflux disease	2 (5.6%)	0 (0%)
Thrombocytopenia	2 (5.6%)	1 (2.8%)

Efficacy

Of the 36 patients, 1 patient was unevaluable (this patient developed grade 4 neutropenia during the first cycle) and was included in the intention to treat (ITT) analysis (Table 3). In the first stage of the study, 17 evaluable patients were enrolled; 5 of these patients achieved a clinical benefit (SD≥24 weeks). The study continued to the second stage and accrued 19 additional patients. Accounting for the two-stage design, the best overall response in the ITT analysis was SD in 20 patients (20/36, 55.6%), progressive disease in 14 patients (14/36, 38.9%) and a confirmed PR in 1 patient. Among the patients with SD, three patients had unconfirmed PR (Figure 1A). The CBR at ≥ 24 weeks was 33.3% (12/36; CI_95% 9.8-56.4%) accounting for two-stage design. This exceeded the predetermined boundary of at least 11 patients experiencing clinical benefit and therefore met the primary endpoint. These included 1 patient with a confirmed PR, and 11 patients with SD ≥ 24 weeks. When including only evaluable patients, the CBR at ≥ 24 weeks was 34.3% (12/35, CI_95% 20.7-58.9%) accounting for two-stage design. Of the patients who achieved a clinical benefit at ≥ 24 weeks, 4 patients (4/36, 11.1%) had an exceptional clinical benefit with disease stability for ≥ 12 months (Figure 1A–B, Table 3). The median PFS was 3.6 months (CI_95% 2.0-7.2) (Figure 1C), and the median OS was 26.5 months (CI_95% 20.7-not reached) (Figure 1D). There was no association between prior treatment regimens in the metastatic setting and the clinical benefit, including; prior tamoxifen treatment (prior tamoxifen, 30.8% [8/26] vs. no prior tamoxifen, 40% [4/10]; P=0.7, Fisher’s exact test for all comparisons in this section), prior fulvestrant (prior fulvestrant, 28.6% [2/7] vs. no prior fulvestrant, 34.5% [10/29]; P = 0.8), or prior chemotherapy treatment (prior chemotherapy, 36.8% [7/19] vs. no prior chemotherapy, 29.4% [5/17]; P = 0.7). There was also no association between the number of prior treatment regimens in the metastatic setting and clinical benefit (0-1 lines of treatment, 33.3% [3/9] vs ≥ 2 lines of treatment, 33.3% [9/27]; P = 1.0). However, liver metastases versus no liver metastases (17.4% [4/23] vs 61.5% [8/13] respectively), or 3-4 organs with metastatic involvement versus 2 organs versus 1 organ with metastatic involvement (14.2% [3/21] vs 60% [6/10] vs 60% [3/5], respectively) were associated with decreased clinical benefit (P = 0.01).

Table 3.

Summary of efficacy in Intention to Treat and Evaluable patients

Response	Intention to Treat Cohort (n=36)	Evaluable Cohort (n=35)
Best Overall response n (%)
Complete response (CR)	0 (0%)	0 (0%)
Partial response (PR)
Total	4 (11.1%)	4 (11.4%)
Confirmed	1 (2.8%)	1 (2.9%)
Unconfirmed	3 (8.3%)	3 (8.6%)
Stable Disease (SD)	20* (55.6%)	20 (57.1%)
SD ≥ 24 weeks	11 (30.6%)	11 (31.4%)
Progressive Disease	14 (38.9%)	14 (40%)
Clinical Benefit Rate (CR+PR+SD≥24weeks)	12 (33.3%)	12 (34.3%)
Response Duration ≥ 12 months	4 (11.1%)	4 (11.5%)
Median PFS, months (95%CI)	3.6 (2-7.2)
Median OS, months (95% CI)	26.5 (20.7-NA)

Abbreviations: PFS - progression free survival; CI - confidence interval; OS - overall survival

^*includes the 3 unconfirmed PR patients. They all had SD≥24weeks.

Figure 1.

Efficacy of bazedoxifene and palbociclib in evaluable patients. A. Swimmer plot of 34 patients evaluable for time to event end points. Vertical dotted line indicates 6 months. The left panel denotes patients who have liver metastases (liver mets) in red, the number of organs involved with metastases (Number of mets), number of lines of treatment in the metastatic setting (patients who had not received treatment in metastatic disease developed disease recurrence during or within 1 year of adjuvant ET), prior fulvestrant (Fulv) treatment indicated in green and prior chemotherapy treatment indicated in blue (Chemo). † denotes patient 28 who stayed on treatment for 18 cycles despite of the PD assessment after cycle 2. ‡ denotes patient 29 who stayed on treatment for additional 14 days after the last disease evaluation. Neither patient 28 nor patient 29 is considered as having clinical benefit. B. Waterfall plot with best percentage of tumor changes from baseline sum of the longest diameter in target lesions of the 34 evaluable patients. Response is based on RECIST 1.1 criteria. Horizontal dotted line indicates −30% change of targeted lesion. C. Kaplan-Meier curve representing progression-free survival (PFS) for intention to treat patient population (n=36). Median PFS 3.6 months, 95% CI: 2-7.2 months. D. Kaplan-Meier curve representing overall survival (OS) for the intention to treat patient population (n=36). Median OS 26.5 months (95% lower confidence limit 20.7 months).

Abbreviations: PD, progressive disease; PR – partial response; SD – stable disease

Cell free DNA Tumor Fraction Dynamics

Plasma samples were collected at baseline (n=36), day 1 of cycle 2 (C2D1) (n=33) and at the time of disease progression at the end of treatment (EOT) (n=33) to identify genetic alterations associated with primary or acquired resistance to bazedoxifene and palbociclib (Supplemental Figure 2A–B). All samples (n=102) were subjected to ultra-low passage whole genome sequencing (ULP-WGS, mean coverage 0.19-0.57X) to determine cell free DNA (cfDNA) tumor fraction (TF) (see Methods) for the selection of samples with detectable ctDNA for subsequent whole exome sequencing (WES), and to determine the relationship between the TF and clinical outcomes (Supplemental Figure 3A). The median tumor fraction in all samples was 0.04 (range [0, 0.85]). Baseline cfDNA TF was higher in patients with liver metastases (median=0.07 [0, 0.85]) versus patients without liver metastases (median=0.04 [0, 0.3]) (Wilcoxon P=0.01) (Supplemental Figure 3B) and patients who had 3-4 organs involved with metastases (median=0.06 [0, 0.85]) versus 1 organ involved with metastases (median=0.03 [0, 0.04]) (Wilcoxon P=0.03). Moreover, baseline cfDNA TF correlated with PFS; Patients with a high baseline cfDNA TF (≥0.1) had a lower PFS (n = 12, median PFS = 1.94 months) compared to patients with a low cfDNA TF ( <0.1) (n = 24, median PFS=5.56 months) (HR=2.29, CI_95% 1.08-4.89, P = 0.027, log-rank test) (Supplemental Figure 3C). There was no significant association between the changes in cfDNA TF after 1 month of treatment and PFS (ratio between C2D1/baseline TF > 1 [n=12], median PFS = 2.04 months; versus C2D1/baseline TF ≤ 1 [n=19], median PFS = 3.67 months, HR=0.9, CI_95% 0.4-1.9, P = 0.71) (Supplemental Figure 3D). Comparison of the TF at C2D1 and the EOT showed an increase in TF in most cases (58%, 18/31). The TF was unchanged in 7 patients (23%) and in 6 patients (19%) there was a decrease at the EOT. In most of the cases in which the TF decreased at the EOT, the absolute change was marginal and the TF either remained low (in the range of 0.03) or very high (above 0.45) except for one patient (patient 25) who had a decrease in the TF from 0.6 at C2D1 to 0.045 at the EOT. This patient had an increase in the size of the liver metastases but a mixed response of bone metastases with evidence of decreased fluorodeoxyglucose (FDG) uptake in several bone metastases based on a positron emission tomography (PET)-CT scan. Thus, although the early cfDNA TF dynamics were not indicative of PFS, the cfDNA TF was overall reflective of metastatic burden and in most cases of disease progression.

Genomic Alterations and Clinical Benefit

Samples with detectable ctDNA (cfDNA TF > 0.03) were subjected to WES (mean target coverage=90-461X, median=193X). WES was performed in 68 samples (baseline 28/36 [78%], C2D1 19/34 [56%] and EOT 23/32 [78%])(Mutation Annotation Format in Supplemental table 5). These included 20 matched pairs of baseline and EOT samples. Sample-to-sample correlation of the somatic single nucleotide variants (SSNVs) in the 68 samples showed that the matched samples from an individual patient had the strongest correlation in nearly all cases except for two samples that had a low number of mutations (Supplemental Figure 4A). As expected, most of the SSNVs in exonic regions were unique to a single patient (4205 out of 4227 non-silent SSNVs, 99.5%; 3190 out of 3211 silent SSNVs, 99.3%). The most frequent SSNVs were oncogenic hotspot mutations in ESR1 and PIK3CA (Supplemental Figure 4B).

We next focused on known cancer-related genes (list of genes and the detected amino acid changes in Supplemental Table 6). At baseline, the most frequent mutations in the cancer-related genes included pathogenic or likely-pathogenic mutations in ESR1 (Figure 2B), pathogenic PIK3CA mutations (Figure 2D), EPAS1 (none of these are known to be pathogenic) and TP53 mutations (Supplemental Table 6). Other baseline pathogenic or likely pathogenic mutations identified in at least two patients included AKT1 and KMT2C mutations (Figures 2A–C; Supplemental Table 6). Pathogenic ESR1 mutations were found in 32% (9/28) of baseline samples, with the missense mutations Y537 and D538 accounting for the majority of the ESR1 mutations (Figure 2B). ESR1 mutations were detected in patients who had or did not have a clinical benefit at ≥ 24 weeks (Figure 2A), and there was no association between the ESR1 mutations and PFS (wild-type median PFS=3.6 months [n=19], mutant median PFS=2.0 months [n=9]; HR=1.0, CI_95% 0.4-2.4, P=0.94, log-rank test for all comparisons in this section) (Figure 2D). In contrast, baseline hotspot PIK3CA mutations were not detected in patients who had a clinical benefit at ≥ 24 weeks (Figure 2A), and patients with a baseline PIK3CA mutation had a lower PFS compared to patients with wild-type PIK3CA at baseline (wild-type median PFS=3.9 months [n=22], mutant median PFS = 1.8 months [n=6]; HR=0.2, CI_95% 0.06-0.6, P=0.0019) (Figure 2E). This difference remained significant in a multivariate analysis correcting for significant clinical variables including the presence of liver metastases versus no liver metastases and 1 versus 3-4 organs with metastases (mutant versus wild-type HR = 3.82, CI_95% 1.18-12.33, P = 0.025) (Supplemental Table 7). In addition, patients who had a baseline truncating mutation, including mostly pathogenic or likely pathogenic mutations in tumor suppressor genes (TP53, MEN1, RB1, CDKN1B, NF1, TP53, BP1, TP63, SMAD2, SMAD4, ARID1A, and KMT2C), had a worse PFS in a univariate analysis (no truncating mutation PFS = 3.8 months [n=21], truncating mutation median PFS=1.7 months [n=7], HR=0.3, CI_95% 0.1-0.7, P = 0.006) and in a multivariate analysis (mutant versus wild-type HR=3.01, CI_95% 1.09-8.3, P=0.034) (Figure 2F, Supplemental Table 7).

Figure 2:

Whole exome sequencing. A. Baseline somatic mutations in cancer-related genes detected by ctDNA whole exome sequencing (WES) (n=28) in at least two patients. B. Lollipop diagram denoting the ESR1 mutations detected in the baseline ctDNA samples. C. Lollipop diagram denoting the PIK3CA mutations detected in the baseline ctDNA samples. D. Kaplan-Meier curve representing progression-free survival (PFS) during treatment with palbociclib and bazedoxifene in patients with baseline ctDNA WES stratified by no/yes baseline ESR1 ctDNA mutation. The median PFS for patients with WT (wild-type) ESR1 versus mutant ESR1 is 3.6 months and 2 months, respectively. HR = 1.0 (95% CI: 0.4 – 2.4); P = 0.94; log-rank test. E. Kaplan-Meier curve representing PFS on treatment for patients with baseline WES ctDNA analysis stratified by no/yes baseline ctDNA PIK3CA mutation. The median PFS for patients with WT PIK3CA versus mutant PIK3CA was 3.9 months versus 1.8 months, respectively. HR= 0.2 (95% CI: 0.06-0.6); P=0.0019, log-rank test. F. Kaplan-Meier curve representing PFS on treatment for patients with baseline WES ctDNA analysis stratified by no/yes baseline ctDNA truncating mutation. The median PFS for patients without a truncating mutation versus with a truncating mutation was 3.8 months versus 1.7 months, respectively. HR = 0.3 (95% CI 0.1-0.7); P=0.0059, log-rank test. G. End of treatment (EOT) somatic mutations in cancer-related genes detected by ctDNA whole exome sequencing (WES) (n=22) in at least two patients. H. Correlation of the frequency of mutations in cancer-related genes that were detected in at least two patients and including patients with matched baseline and EOT treatment ctDNA WES (n= 20), r=0.7, P=0.01, Pearson correlation. Abbreviations: ctDNA – circulating tumor DNA

Consistent with the baseline ctDNA, the most common SSNVs in cancer-related genes at the EOT were PIK3CA (32%, 7/22) and ESR1 (27%,6/22), followed by PTEN, TP53 and GATA3 (Figure 2G). Comparison of the matched baseline and EOT ctDNA pairs showed a high correlation between the frequency of the mutations found in at least two different patients (r = 0.7, P=0.01, Pearson correlation) (Figure 2H). However, when comparing EOT ctDNA versus baseline ctDNA in individual patients, several patients gained actionable driver mutations at the EOT that are suggestive of putative mechanisms of acquired resistance and offer potential therapeutic targets. As examples, one patient (patient 30) who was on treatment for 7 months gained 2 BRAF missense mutations (G469A and D287H) and 2 hotspot PIK3CA mutations (E365K and N1044K). A second patient (patient 10) was on treatment for 3 months and gained pathogenic mutations in ERBB2 (S310Y) and PIK3CA (H1047L), and a missense mutation in FGFR2 (L309V) (Supplemental Figure 5).

Somatic copy number alteration (SCNA) analysis was performed in 30 plasma ctDNA samples from 16 patients with a TF ≥ 0.15 (baseline n = 12, C2D1 n = 9, EOT n = 9) (Figure 3A). Two patients (patient 1 and patient 14) were found to have genome doubling, which is associated with poor outcomes(35). In all samples, there were SCNAs in at least two cancer-related driver genes. In addition, in all samples we identified SCNAs in at least one gene related to a potential mechanism of resistance to ET and/or CDK4/6i. These included SCNAs in CCND1, CCNE2, MYC, ESR1, CDKN2B, CDKN2A, FOXA1, PIK3CA, AURKA, AURKB, CDKN1A, FGFR1, CDK6, CDK4, IGF1R or PTEN(7,36–38). Amplifications or gains in CCND1 and other genes that are within the same genomic region on 11q13.3 (FGF19, FGF3 and FGF4) were among the most common SCNAs. In addition, ELF3 amplifications or gains were identified in most of the patients (69%, 11/16). In line with the high frequency of ELF3 SCNAs that we identified, amplifications of ELF3 are more frequent in ER+/HER2− metastatic breast cancers in the Metastatic Breast Cancer Project(39,40) versus the ER+/HER2− primary breast cancers in the TCGA cohort(41) (31%, 22/70 in metastatic tumors versus 6%, 29/471 in primary tumors, P < 0.0001, Fisher’s exact test). Overall, amplifications and losses at baseline or C2D1 compared to EOT within the matched pairs were comparable. Only a small number SCNA were acquired at the EOT. As an example, one patient (Patient 4) acquired losses of CDKN2A and CDKN2B (genes that encode the cell cycle inhibitors P16 and P15, respectively, and are both in the same region in 9p).

Figure 3:

Copy number variations, tumor mutational burden (TMB), and mutational signatures. A. Somatic copy number variations (amplifications (AMP), gains, loss, and neutral loss of heterozygosity) in cancer-related genes detected in at least two patients. Samples are from 16 patients and include matched samples from at least two time points (baseline, C2D1 (cycle 2 day1) and end of treatment (EOT)) in 9 patients. B. Serial TMB at baseline (n=28), cycle 2 day1 (C2D1) (n=18), and end of treatment (EOT) (n=22). C. Mutational signatures detected in cfDNA samples.

Abbreviations: cfDNA – cell-free DNA

We next assessed additional genomic features, including TMB(42,43) and mutational signatures(44,45) in the 30 ctDNA WES with sufficient TF. These features are associated with breast cancer outcomes and could potentially predict sensitivity or resistance to bazedoxifene plus palbociclib. The median TMBs at baseline (n=12) and EOT (n=9) were comparable and low (baseline median of 1.3 mutations/Mb, range [0.74,19.5]; EOT median of 0.87 mutations/Mb, range [0.73,12.0]; Wilcoxon P=0.84)(Figure 3B) and there was no significant difference in the TMB in patients who had a clinical benefit compared to patients who did not across all time points (Wilcoxon P=0.7).

As for the mutational signatures in the cohort (Figure 3C), the most common mutational signatures (SBS1, SBS5 and SBS40) can be attributed to clock-like and aging processes. The patients with a high TMB (Patient 2, 10, and 30) had enriched APOBEC signatures (SBS2 and SBS13). The same set of APOBEC signatures was detected in patients 21 and 31. Overall, most of the patients with APOBEC signatures (4 of 5 patients) did not have a clinical benefit from bazedoxifene and palbociclib. Albeit a small number of patients, these results are consistent with previous studies showing that the APOBEC signature is associated with endocrine resistance(44) and a high TMB(46). We also detected the mutational signature (SBS17b) that is associated with exposure to chemotherapeutic agents, 5-Fluorouracil (5-FU) and capecitabine in a patient who received treatment with capecitabine followed by eribulin prior to enrollment on the study.

Clonal architecture and sub-clonal evolution during treatment

To better understand how tumor heterogeneity and the sub-clonal evolution contributes to treatment resistance, we performed an evolutionary analysis (see Methods). Seven patients had sufficient ctDNA (cfDNA TF≥0.15) in three time points or at baseline and EOT (Figure 4A–D and Supplemental Figure 6A–C). In at least 3 of these patients the clonal evolution helped to shed light on potential mechanisms of treatment resistance. Patients 17 (Figure 4A) and 2 (Figure 4B) had clonal PIK3CA and ESR1 pathogenic mutations. Patient 17 had a subclone with losses of the tumor suppressor genes, RB1 and PBRM1, and the abundance of this subclone increased at the time of disease progression (cancer cell fraction [CCF] of 0.10 at baseline increased to 0.52 at EOT). Patient 2 had two major subclone branches (light blue and red in Figure 4B). Interestingly, within less than 2 months at the time of disease progression, the subclone with an ESR1 D538G and an NF1 truncal mutation increased from a CCF of 0.37 at baseline to 1. Loss of neurofibromin was shown to be a mechanism of treatment resistance in HR+ breast cancer(47). On the other hand, the second subclone with a SMAD2 truncating mutation and VHL missense mutation decreased from a CCF of 0.45 at baseline to 0.02 at EOT and is likely not a key driver of the disease progression.

Figure 4:

Evolutionary analysis of longitudinal plasma ctDNA samples. A-D. Left panel showing the phylogenetic trees representing the clonal architecture in plasma ctDNA samples. The number of somatic alterations assigned to each clone and detected alterations in known cancer genes are denoted in each branch. A-D. right panel showing the estimated cancer cell fraction (CCF) of each clone at baseline, after 1 cycle of treatment (C2D1) and end of treatment (EOT). The color of the clone matches the respective clone in the phylogenetic trees. * denotes truncating mutations

Abbreviations: ctDNA – circulating tumor DNA

Patient 1 had rapid disease progression within less than a month and was found to have a clonal pathogenic RB1 mutation (E54*) at baseline and at EOT (Figure 4C). Although this RB1 mutation is monoallelic, it has been shown that the loss of even one copy of Rb1 can lead to genomic instability(48). Patient 1 and patient 5 had clonal ZFHX3 (zinc finger homeobox 3) mutations (Figure 4C and 4D). In addition, three other patients in our study had non-silent ZFHX3 mutations (clonal status could not be evaluated in these other patients), making ZFHX3 a common mutation in our dataset (16% [5/31]). In contrast, ZFHX3 mutations, mainly deletions and truncating mutations, were found in only 4% of the ER+ breast cancers in the TCGA cohort(39–41).

Discussion

We provide the first study report of the clinical efficacy and safety of bazedoxifene in combination with palbociclib for the treatment of endocrine-resistant HR+/HER2− advanced breast cancer. We observed SD as the best response in 56% of the ITT patient population and 57% in the evaluable patient population. The CBR ≥ 24 weeks was 33% and 34% in the ITT and the evaluable patients, respectively, in a heavily pre-treated patient population. The CBR at 24 weeks exceeded the predefined threshold, meeting the primary end point of the study.

In this study, >50% of the patients had prior chemotherapy in the metastatic setting, and >40% of the patients had at least two prior lines of ET. Prior chemotherapy is associated with decreased benefit from subsequent treatments(49,50). In addition, most of the patients had liver metastases, which is also associated with worse outcomes in HR+ advanced breast cancer(51). Importantly, the results of this study compare favorably to a phase II study of palbociclib as monotherapy in patients with HR+ advanced breast cancer, in which the CBR ≥ 24 weeks was 21% (7/33)(5) , providing evidence for the clinical efficacy of bazedoxifene in this setting.

The need for new ETs when combined with a CDK4/6i that better target the ER mutations in advanced breast cancer is supported by the recent results from the PADA-1 clinical trial. In PADA-1, switching from an aromatase inhibitor plus palbociclib to fulvestrant plus palbociclib in patients with HR+ advanced breast cancer and rising levels of ctDNA ESR1 mutations, resulted in improved PFS(9). In retrospective analyses, the presence of an ESR1 mutation did not lead to inferior outcomes with the combination of fulvestrant and a CDK4/6i(52). However, pre-clinical studies(53) and clinical studies(54,55) showed decreased activity of single agent fulvestrant in the presence of the ESR1 mutations, particularly the Y537S ER mutation. On the other hand, pre-clinical studies showed that bazedoxifene has superior activity in inhibiting the Y537S ER mutation compared to fulvestrant and biophysical and structural studies provided mechanistic insights explaining these differences^13,18. In line with these pre-clinical studies, our clinical trial showed that the presence of an ESR1 mutation was not associated with decreased benefit from the combination of bazedoxifene and palbociclib. Taken together, these published clinical and pre-clinical studies, along with the results from our clinical trial support a future randomized clinical trial comparing the combination bazedoxifene plus a CDK4/6i versus fulvestrant plus a CDK4/6i in patients with advanced breast cancer who have rising levels of ctDNA ESR1 mutations.

The EMERALD trial was a randomized phase III study investigating the new a SSH, elacetsrant, versus standard of care ET (AI or fulvestrant)(56). In this trial, patients with HR+ advanced breast cancer who progressed on prior treatment with ET and a CDK4/6i and were randomized to receive elacestrant had improved PFS compared to patients that received standard of care ET. The magnitude of this improvement was higher in patients that had an ESR1 mutation. These results provided evidence for improved clinical outcomes with a single agent next generation ET, particularly in patients that have disease that remains ER dependent and support further development of this class of drugs. However, the median PFS was limited in the EMERALD trial and future studies will need to identify the patients who continue to benefit from ET after treatment with CDK4/6i and investigate novel treatment combination that incorporate the next generation ETs. Increasing the dose of ET is another potential strategy to improve efficacy from ET, especially in the presence of the Y537S ER mutation, which is characterized by decreased affinity to multiple SERMs and SERDS(19,57). Since bazedoxifene was well tolerated in large clinical trials with healthy women and in our study in advanced breast cancer there were no significant gastrointestinal or other dose limiting toxicities, a trial to investigate the pharmacokinetics, safety and efficacy of higher doses of bazedoxifene in patients with HR+ advanced breast cancer and an ESR1 mutation is warranted.

We performed a prospective analysis of serial ctDNA WES to search for genetic determinants of sensitivity and resistance to the combination of bazedoxifene and palbociclib in advanced breast cancer after multiple lines of treatment. We showed that pathogenic PIK3CA mutations were associated with decreased PFS. Pre-clinical studies support the role of the PI3-kinase pathway in endocrine resistance(58,59), and the α-specific PI3-kinase inhibitor alpelisib in combination with fulvestrant is approved for the treatment of PIK3CA mutant HR+ metastatic breast cancer(60). A pre-clinical study showed that inhibition of PI3-kinase can prevent adaptive resistance to palbociclib, suggesting that the activation of the PI3-kinase pathway is involved in resistance to palbociclib(61). In the phase 3 PALOMA-III trial in which patients were randomized to fulvestrant plus palbociclib or fulvestrant plus placebo, there was an enrichment of plasma ctDNA PIK3CA mutations at the EOT. However, baseline plasma ctDNA PIK3CA mutations were not predictive of decreased PFS(10,62). Our results suggest that in HR+ advanced breast cancer, the pathogenicity of the PIK3CA mutations increases in more advanced metastatic disease after multiple lines of treatment. This is in keeping with previous studies that showed that in metastatic disease, the presence of a PIK3CA mutation is associated with treatment resistance and tumor progression(63), whereas in early-stage, treatment-naïve HR+ breast cancer, PIK3CA mutations are associated with improved outcomes(64,65).

The ctDNA WES analyses also identified genetic alterations that are potential novel drivers of disease progression and treatment resistance. As an example, we identified a high rate of ELF3 SCNAs. ELF3 is a member of the ETS transcription factors and is located within 1q32, a region commonly gained in breast cancer. In preclinical studies, ELF3 was shown to be a transcriptional repressor of ER, suggesting a possible role in endocrine resistance(66). We also identified a relatively high rate of ZFHX3 mutations. ZFHX3 encodes the transcription factor AT-motif binding factor-1 (ATBF1), which has key roles in protein-protein interactions and is a tumor suppressor in prostate cancer(67). In HR+ breast cancer, ATBF1 inhibits ER mediated transcription(68), implying that aberrations in this gene may have a role in resistance to ET.

We acknowledge several limitations in our study, including the small number of patients that limits our ability to make strong conclusions about the efficacy of the combination of bazedoxfene and palbociclib, and the determinants of sensitivity or resistance. Additionally, not all the plasma samples had sufficient ctDNA TF for TMB, SCNV, mutational signature, and phylogenetic analyses. Nonetheless, herein we demonstrate the utility and breadth of data that can be gleaned from WES of ctDNA. In summary, our results showed: (i) The safety of bazedoxifene in combination with palbociclib; (ii) a signal of the efficacy of bazedoxifene in HR+ breast cancer; (iii) PIK3CA mutations are a candidate biomarker of resistance to this combination that can be applicable to other next generation ETs; and (iv) the advantages of WES of ctDNA that enabled us to capture the tumor heterogeneity and sub-clonal evolution during disease progression. WES also allowed us to identify potential novel genomic mechanisms of tumor progression that warrant future investigation.

Translational relevance:

In this clinical trial we show that bazedoxifene plus palbociclib has clinical efficacy in patients with advanced hormone receptor-positive breast cancer and this combination is well tolerated. Pathogenic PIK3CA mutations are a candidate biomarker of resistance to bazedoxifene plus palbociclib. In addition, we demonstrate the utility of ctDNA whole-exome sequencing and the significance of determining the sub-clonal evolution in individual patients.