Therapeutic Targeting of ER Beta in Triple Negative Breast Cancer
Phase: PHASE2
Conditions: Advanced Triple-Negative Breast Carcinoma, Anatomic Stage III Breast Cancer AJCC v8, Anatomic Stage IV Breast Cancer AJCC v8, Metastatic Triple-Negative Breast Carcinoma, Recurrent Breast Carcinoma
Sponsor: Mayo Clinic

 
 
 
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THERAPEUTIC TARGETING OF ER ΒETA IN TRIPLE NEGATIVE BREAST 
CANCER 
Protocol Number 
Mayo Clinic Protocol Number: MC1831 
TBCRC Protocol Number: TBCRC051 
 
 
Protocol Chair 
 
 
 
 
 
 
 
 
Co-Investigators 
 
 
 
 
Statistician 
 
 
 
 
Supporters 
Johns Hopkins University on behalf of the Translational Breast 
Cancer Research Consortium (TBCRC) 
 
Coordinating Center  
 
 
 
 
 
 
Investigational New Drug (IND) Number EXEMPT (142986) 
 

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THERAPEUTIC TARGETING OF ERβ IN TRIPLE NEGATIVE BREAST CANCER 
Study Reference Numbers 
Organization 
Reference number 
Mayo Clinic 
MC1831 (IRB 18-000734) 
TBCRC 
TBCRC051 
ClinicalTrials.gov 
NCT03941730 
FDA 
IND 142986 EXEMPT 
 
 
 
 
 
- Protocol Revision Record - 
Original Protocol: 
03Jul2019 
Activation to Mayo Clinic 
28Aug2019 
MCCC Amendment 1 
31Mar2020 
MCCC Amendment 2 and 
Activation to TBCRC 
24Jun2020 
MCCC Amendment 3 
17Mar2022 
MCCC Amendment 4 
09Sep2022 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
THERAPEUTIC TARGETING OF ER ΒETA IN TRIPLE NEGATIVE BREAST 
CANCER ....................................................................................................................................... 1 
TABLE OF CONTENTS ............................................................................................................ 3 
SCHEMA ....................................................................................................................................... 6 
1. 
STUDY DESIGN/SUMMARY ...................................................................................................................... 7 
2. 
OBJECTIVES ................................................................................................................................................. 8 
2.1 
Primary Objective ......................................................................................................... 8 
2.2 
Secondary Objectives ................................................................................................... 8 
2.3 
Exploratory Objectives ................................................................................................. 8 
3. 
BACKGROUND ............................................................................................................................................. 9 
4. 
PARTICIPANT SELECTION ...................................................................................................................... 16 
4.1 
Pre-Screening Inclusion Criteria (Step 0) ................................................................... 16 
4.2 
Pre-Registration Inclusion Criteria (Step 1) ............................................................... 16 
4.3 
Pre-Registration Exclusion Criteria ............................................................................ 17 
4.4 
Registration Inclusion Criteria (Step 2) ...................................................................... 18 
4.5 
Registration Exclusion Criteria .................................................................................. 18 
4.6 
Inclusion of Underrepresented Populations ................................................................ 18 
5. 
REGISTRATION PROCEDURES ............................................................................................................... 19 
5.1 
Pre-Screening and Pre-Registration (Step 0 and Step 1) ............................................ 19 
5.2 
Registration Procedures (Step 2) ................................................................................ 20 
6. 
STUDY CALENDAR ................................................................................................................................... 23 
7. 
TREATMENT PLAN ................................................................................................................................... 24 
7.1 
Biopsy and enrollment ................................................................................................ 24 
7.2 
Agent Administration ................................................................................................. 24 
7.3 
Concomitant Treatment and Supportive Care Guidelines .......................................... 25 
8. 
EXPECTED ADVERSE EVENTS AND DOSING DELAYS/DOSE MODIFICATIONS .......................... 26 
8.1 
Treatment Administration........................................................................................... 26 
8.2 
Anticipated Adverse Events ....................................................................................... 26 
8.3 
Management of Adverse Events ................................................................................. 26 
8.4 
Dose Modifications/Delays ........................................................................................ 26 
8.5 
Special Considerations ............................................................................................... 29 
8.6 
Adverse Events to be Graded at End of Each Cycle of Treatment............................. 29 
9. 
DRUG FORMULATION/STORAGE/SUPPLY .......................................................................................... 30 
9.1 
Estradiol ...................................................................................................................... 30 
9.2 
Ordering ...................................................................................................................... 31 
9.3 
Drug Accountability ................................................................................................... 31 

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10. 
CORRELATIVE/SPECIAL STUDIES ......................................................................................................... 32 
10.1 
Summary Table of All Biospecimens ..................................................................... 32 
10.2 
Blood Samples ........................................................................................................ 33 
10.3 
Tissue Samples........................................................................................................ 37 
10.4 
Other Samples and/or Devices ................................................................................ 41 
10.5 
Genetic Testing ....................................................................................................... 41 
10.6 
Additional Information ........................................................................................... 42 
10.7 
Specimen Banking .................................................................................................. 42 
11. 
MEASUREMENT OF EFFECT ................................................................................................................... 42 
11.1 
Schedule of Evaluations .......................................................................................... 42 
11.2 
Definitions of Measurable and Non-Measurable Disease ...................................... 42 
11.3 
Guidelines for Evaluation of Measurable Disease .................................................. 43 
11.4 
Measurement of Treatment/Intervention Effect ...................................................... 45 
12. 
TREATMENT/FOLLOW–UP DECISION AT EVALUATION OF PATIENT ........................................... 48 
12.1 
No disease progression or Unacceptable Adverse Events ...................................... 48 
12.2 
No disease Progression, Unacceptable Adverse Events ......................................... 48 
12.3 
Discontinuation of Protocol Therapy ...................................................................... 48 
12.4 
Refuse to begin protocol treatment following registration ..................................... 49 
12.5 
Ineligible prior to the start of protocol treatment after registration ........................ 49 
12.6 
Ineligible after the start of protocol treatment ........................................................ 49 
13. 
ADVERSE EVENT REPORTING REQUIREMENTS ................................................................................ 50 
13.1 
General .................................................................................................................... 50 
13.2 
Definitions............................................................................................................... 51 
13.3 
Reporting Procedures .............................................................................................. 52 
14. 
REGULATORY CONSIDERATIONS ......................................................................................................... 53 
14.1 
Protocol Review and Amendments ......................................................................... 53 
14.2 
Informed Consent.................................................................................................... 53 
14.3 
Ethics and GCP ....................................................................................................... 54 
14.4 
Compliance with Trial Registration and Results Posting Requirements ................ 54 
15. 
MULTI-CENTER GUIDELINES ................................................................................................................. 54 
15.1 
Study Documentation.............................................................................................. 54 
15.2 
Records Retention ................................................................................................... 55 
15.3 
Publication .............................................................................................................. 55 
16. 
STATISTICAL CONSIDERATIONS .......................................................................................................... 55 
16.1 
Study Design ........................................................................................................... 56 
16.2 
Analysis of Secondary Endpoints for each patient cohort independently .............. 57 
16.3 
Monitoring .............................................................................................................. 59 

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16.4 
Other Considerations .............................................................................................. 59 
17. 
REFERENCES .............................................................................................................................................. 61 
APPENDICES ............................................................................................................................. 63 
APPENDIX A: ECOG PERFORMANCE STATUS SCALE ................................................ 64 
APPENDIX B: STUDY DRUG DIARY ................................................................................... 65 
 
 

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SCHEMA 
 
Pre-screening 
 
 
 
 
 
 
 
 
Pre-registration 
 
 
 
 
 
 
 
 
 
 
 
 
 
Confirmation of  
ERα neg, PR neg, and 
HER2 neg 
 
 
Unable to confirm  
ERα neg, PR neg, or 
HER2 neg 
 
 
 
 
 
 
Registration 
 
 
Off study 
 
 
 
 
 
 
Estradiol by mouth  
three times daily 
continuously 
 
 
 
 
 
 
 
 
 
 
Events at any time: 
intolerable adverse effects 
disease progression 
patient refusal 
 
 
 
 
 
 
 
 
 
 
Survival Follow-up/Event 
monitoring 
 
 
 
 
Cycle length = 28 days ±3 days 
 
Generic name: estradiol 
Brand name(s): Cenestin, Enjuvia, Estrace, Femtrace, Ogen, Premarin, etc. 
Mayo Abbreviation: estradiol 
Availability: Commercial  
 

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1. STUDY DESIGN/SUMMARY 
This is a phase II study to determine the anti-tumor activity of estradiol in women with locally 
advanced or metastatic TNBC that expresses ERβ (>25% moderate or strong nuclear staining) 
and who have prior receipt of taxane and anthracycline based chemotherapy. 
The study is divided into pre-screening, pre-registration phase and a registration phase: 
Pre-Screening: Patients with a history of locally advanced or metastatic breast cancer that is 
ERα negative (<1% nuclear staining) and HER2 negative and who have received at most 
3 prior chemotherapy regimens for treatment of metastatic breast cancer may register for the 
pre-screening portion of this trial. Note, pateints with a history of metastatic ERα+ breast 
cancer are not eligible.  An archived FFPE tumor tissue from a locally advanced or metastatic 
site is to be submitted to the Mayo Clinic CAP/CLIA-certified Anatomic Pathology 
Laboratory for ERβ testing. If an archived FFPE tumor tissue specimen from a locally 
advanced or metastatic site is not available, then a specimen from primary tumor must be 
submitted. If the specimen is determined to have >25% moderate or strong nuclear staining 
for ERβ, the patient will be eligible for Pre-registration. 
Pre-Registration: Patients determined to meet the requirements of the pre-screening phase 
and who have not undergone a biopsy of metastatic disease in the year prior to pre-
registration will undergo a standard of care tumor biopsy for assessment of ERα, PR, and 
HER2 as well as three (3) additional research cores (see Section 10.2 and lab manual for 
details for processing). The first core will be FFPE and processed locally for ERα, PR, and 
HER2 as well as sectioning of three (3) unstained slides submitted simultaneously to Mayo 
Anatomic Pathology. Patients determined to be either ERα positive (>1% nuclear staining) or 
HER2 positive (see Section 4.1.2 for definition) are ineligible and should proceed with 
standard of care systemic therapy directed towards these validated targets. Those patients 
with confirmed ERα negative (<1% nuclear staining) and HER2 negative can proceed to 
registration. Patients known to have >25% moderate or strong nuclear staining for ERβ 
demonstrated in the pre-screening phase, and who have had a tissue-based biopsy of 
metastatic disease in the year prior to pre-registration can move directly to registration if all 
of the following are true: 
– tissue from this metastatic biopsy was determined to be ERα negative (<1% 
nuclear staining) and HER2 negative  
sufficient archived tissue is available for translational studies (a minimum of 3 and 
ideally 10 unstained slides cut at 5 microns) 
 
 
 

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2. OBJECTIVES 
2.1 
Primary Objective 
To assess the anti-tumor activity of estradiol in patients with locally advanced or 
metastatic TNBC that expresses ERβ (>25% moderate or strong nuclear staining). 
2.2 
Secondary Objectives 
• To examine the safety profile of estradiol when administered at a dose of 2 mg tid 
to women with locally advanced or metastatic TNBC that expresses ERβ  
• To examine the changes in phospho-ERβ, cystatins 1, 2, 4 and 5, phospho-
Smad2/3 and Ki-67 in tumor biopsies taken before and after the first cycle of 
treatment  
2.3 
Exploratory Objectives 
• To examine changes in plasma estradiol, serum cytokine and cystatin levels 
before/after 1 cycle of estradiol 
• Analyze the global gene expression profiles of paired biopsies prior to and 
following 1 cycle of therapy 
• To develop PDX that are ERα negative, HER2 negative and ERβ positive (Mayo 
only) 
• To examine changes in the relative abundance of circulating immune cell 
populations after the first cycle of treatment and whether these changes differ with 
respect to whether the patient is still on treatment after 6 cycles of treatment or 
not. 
 

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3. BACKGROUND 
Our preliminary data indicate that ERβ expression (defined as >25% cells staining positive for 
moderate or high intensity within the nucleus) is present in up to 30% of TN breast tumors and 
expression is maintained in the majority of matched metastatic tumor lesions. Activation of 
ERβ by estrogen results in potent anti-cancer activity in ERβ expressing TNBC models in part 
through a novel mechanism involving increased cystatin expression which inhibits canonical 
TGFβ signaling, as well as direct suppression of the NFκB pathway by ERβ itself. 
Furthermore, through the establishment of TNBC PDX models, we have shown that ERβ 
positivity is associated with substantially reduced tumor growth rates in estrogen supplemented 
mice compared to ERβ negative TN PDXs. Ellis et al demonstrated the safety and efficacy of 
low dose estradiol (2 mg tid) in the setting of advanced ERα+ breast cancer.(Ellis et al. 2009) 
Therefore, using this dose and schedule, we will perform a prospective phase II study of 
estradiol in women with TNBC that is centrally confirmed (Mayo Clinic CLIA certified 
laboratory) to express ERβ and who have prior progression on taxane and anthracycline based 
chemotherapy. While our preliminary data demonstrate that up to 30% of TNBCs stain positive 
for moderate or high nuclear staining of ERβ, the cut-off for ERβ is unknown as a predictor of 
response. Therefore, we will chose to enroll patients with >25% moderate or strong nuclear 
staining. 
ERβ expression in triple negative human breast tumors: We have developed an ERβ antibody 
that has been validated for clinical use in the Mayo Clinic Immunostains clinical laboratory, 
and have defined the IHC scoring system for ERβ based on extent and intensity of staining. 
Specifically, the extent of staining is scored as: 0: less than 1% positive cells, 1: 1%-25%, 2: 
26%-50%, 3: 51%-75% and 4: 76%-100% while the intensity of staining is scored as none (0), 
weak (1), moderate (2) or strong (3). The resulting scores are summed and grouped into 3 
categories, namely, negative/low (0-2), moderate (3-5) and high (6-7).  
Through the analysis of two separate cohorts of triple negative breast tumors totaling nearly 
500 cases, we have demonstrated that ERβ protein is detectable at high levels in 5% of tumors 
and moderate levels in another 25% of tumors. Representative images of a negative/low, 
moderate and high expressing tumor are shown in Figure 1. Preliminary analysis of androgen 
receptor (AR) expression in these specimens indicates that ERβ+ and AR+ tumors do not 
significantly overlap suggesting that patients with ERβ positive TNBC represent a unique 
cohort. Importantly, we have also analyzed recurrent lesions from 27 patients whose primary 
tumors were ERβ positive and have demonstrated that ERβ protein expression is maintained 
at similar levels in the majority of these cases.  
Figure 1. Representative images of an ERβ negative/low, moderate 
and high triple negative breast tumor. 

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Biological effects of ERβ in triple negative breast cancer cells: The Hawse lab has developed 
ERβ expressing TNBC cell lines and have shown that treatment of these cells with estrogen or 
various ERβ specific agonists results in substantial inhibition of cell proliferation in 
vitro.(Reese et al. 2017; Reese et al. 2014) These inhibitory effects have been confirmed using 
multiple doses of a potent ERβ selective agonist, LY500307 (Figure 2). Inhibition of cell 
proliferation is a result of cell cycle arrest of TNBC cells following ligand mediated activation 
of ERβ (Figure 3). Molecular profiling of these cells has revealed that estrogen and ERβ 
selective agonists result in substantial up-regulation of 
a family of cysteine protease inhibitors known as the 
cystatins, specifically cystatin 1, 2, 4 and 5, at both the 
mRNA and protein level (data not shown).(Reese et al. 
2018) Since cystatins are secreted proteins which elicit 
 
Figure 
2. 
Effects 
of 
increasing 
concentrations of LY500307 on the 
proliferation rates of MDA-MB-231-ERβ 
cells relative to vehicle control treated 
cells. 
 
Figure 3. Cell Cycle Analysis of MDA-MB-231-ERβ with E2 or LY500307 treatment. Cells were treated 
for 24 hours or 5 days and flow cytometry was performed to determine cell cycle progression. (A) Raw peaks 
determined using ModFit software and (B) quantitation demonstrate an accumulation of cells in G1 and G2 
following E2 or LY500307 treatment. 

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tumor suppressive effects in other types of cancers (Alvarez-Diaz et al. 2009; Shridhar et al. 
2004; Briggs et al. 2010; Wegiel 
et al. 2009; Zhang et al. 2004; Jin 
et al. 2012)3-8, we determined if 
conditioned media isolated from 
estrogen and ERβ agonist treated 
MDA-MB-231-ERβ expressing 
cells 
could 
inhibit 
the 
proliferation of parental MDA-
MB-231 cells that do not express 
any 
form 
of 
the 
ERs. 
Furthermore, we determined if 
depletion 
of 
cystatins 
from 
conditioned media reversed this 
effect. Indeed, IgG depleted 
conditioned media isolated from 
1nM estrogen and 100nM ERβ 
agonist treated MDA-MB-231-
ERβ cells significantly inhibited 
the proliferation rates of parental 
MDA-MB-231 cells (Figure 4). 
These effects were completely 
reversed following depletion of cystatins from the media (Figure 4). We have also 
demonstrated that conditioned media isolated from estrogen and ERβ agonist treated cells 
inhibits 
TGFβ 
signaling 
as 
measured 
by 
the 
activity 
of 
a 
Smad 
Figure 4. Effects of IgG or cystatin 1, 2, 4 and 5 depleted 
conditioned media isolated from 1nM E2 or 100nM WAY 
treated MDA-MB-231-ERβ cells on the proliferation rates of 
ERβ negative MDA-231 cells. Data are depicted relative to 
conditioned media isolated from vehicle treated cells following 
depletion with IgG. 

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Binding Element (SBE) luciferase reporter construct (Figure 5A). Interestingly, depletion of 
cystatins from this conditioned media also reversed this effect (Figure 5A) suggesting that 
modulation of TGFβ signaling by ERβ may in part explain its tumor-suppressive effects.(Reese 
et al. 2018) Furthermore, pre-treatment of MDA-MB-231 cells with 0.125 μg/mL of 
recombinant cystatins 1, 2, 4 and 5 following transfection of the SBE significantly inhibited 
the ability of TGFβ to induce this reporter construct (Figure 5B). These observations are of 
particular significance since TGFβ signaling is known to promote tumor progression, 
metastasis and chemotherapy resistance in TNBC (Bhola et al. 2013; Bierie and Moses 2009; 
Giampieri et al. 2009; Matise, Pickup, and Moses 2009). 
 
Figure 5. A). Conditioned media isolated from 1nM E2 and 100nM WAY treated MDA-MB-231-ERβ 
expressing cells significantly inhibits TGFβ mediated activation of a Smad Binding Element luciferase 
reporter construct.  This inhibitory effect on the TGFβ pathway is lost when cystatins 1, 2, 4 and 5 
(CSTs) are depleted from the media.  B). A combination of recombinant CSTs 1, 2, 4 and 5 (0.125 
mg/mL each) significantly inhibits TGFβ (1ng/mL) mediated activation of the Smad Binding Element 
reporter construct. 

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Identification of the canonical NFκB pathway as a target of ERβ: Our gene expression 
studies and Ingenuity Pathway Analyses have also identified the canonical NFκB signaling 
pathway as being highly suppressed in response to E2 (Figure 6A). Indeed, the majority of 
down-regulated genes following E2 
treatment consisted of well-known 
NFκB 
targets 
including 
multiple 
chemokines and cytokines (Figure 6B). 
Decreased expression of NFκB target 
genes following E2 treatment was 
confirmed by RT-PCR in both MDA-
MB-231-ERβ and Hs578T-ERβ cell 
lines (Figure 6C). Gene set enrichment 
analysis (GSEA) showed that ERβ-
regulated 
genes 
were 
positively 
correlated with signatures that are 
decreased in basal-like breast tumors or 
decreased 
in 
metastatic 
lesions 
suggesting that ERβ induces a more 
differentiated 
and 
less 
aggressive 
phenotype (Figure 6D). In parallel to these studies, we also performed chromatin 
immunoprecipitation assays for ERβ followed by sequencing (ChIPseq). We identified 
26,896 ERβ binding sites across the genome in E2 treated MDA-MB-231-ERβ cells (Figure 
7A and B), of which the large majority of binding sites resided at enhancer elements located 
within intergenic and intronic regions of chromatin (Figure 7C). Not surprisingly, the most 
Figure 6. ERβ suppresses NFκB target genes in TNBC cells. (A) IPA analysis of the E2 regulated 
ERβ transcriptome in MDA-MB-231-ERβ cells indicated alterations in canonical NFκB signaling. (B) 
Heatmap depicting the top 20 E2 repressed (blue) and induced (red) genes. Gene names in red are 
known to be up-regulated by NFκB. (C) RT-PCR confirmation of E2-mediated suppression of NFκB 
target genes in 2 different ERβ expressing TNBC cell lines. (D) GSEA of the ERβ regulated 
transcriptome in MDA-MB-231-ERβ cells show positive correlation with signatures that are anti-
metastatic and anti-basal-like in breast cancer.  
Figure 7. E2-induced ERβ cistrome in MDA-MB-231-ERβ cells. 
(A) Heatmap indicating E2-induced ERβ chromatin binding sites. (B) 
Number of ERβ binding sites identified and (C) their location in 
relation to specific gene regions. (D) Top motifs that are significantly 
enriched within ERβ binding locations. (E) Relative proportion of all 
ERβ bound chromatin sites that encode NFκB response elements. 

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common motif that ERβ associated with was a classic estrogen response element, but 
interestingly, ERβ was also enriched at NFκB binding motifs (Figure 7D). In fact, over 12% 
of all ERβ binding sites contained a consensus NFκB response element (Figure 7E), further 
implicating this pathway as a target of ERβ signaling. 
Tumor suppressive effects of ERβ in vivo: We have also demonstrated that treatment of 
ERβ expressing MDA-MB-231 xenograft tumors with estrogen inhibits tumor growth 
relative to vehicle treated control animals (Figure 8).(Reese et al. 2018)  
 
 
 
Figure 8. Estrogen suppresses MDA-MB-231-ERβ-Luc tumor growth in vivo. (A) Tumor progression 
was monitored at 8 weeks via IVIS2000 xenogen imaging and (B) average total flux was quantified. 
(C) Time to tumor doubling was determined between E2 treated animals and the placebo controls. The 
level of ERβ expression was determined in residual tumors at the time of sacrifice by (D) RT-PCR 
normalized to No Dox/Veh control cells, (E) western blotting and (F) immunohistochemistry.

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Similar effects have been observed in a second TNBC cell model system (Hs578T) (data 
not shown). Estrogen treatment of mice 
was also shown to completely block the 
development of metastatic lesions in 
nude mice following tail vein injection of 
ERβ expressing MDA-MB-231 cells 
(Figure 9). Using PDX models 
established from women with newly 
diagnosed high-risk breast cancer who 
were enrolled in a prospective 
neoadjuvant study at Mayo Clinic, we 
have now identified both ERβ+ and ERβ- 
triple negative breast tumors. 
Interestingly, during establishment of 
these PDXs in estrogen treated NOD 
Scid mice, ERβ+ tumors exhibit reduced 
growth rates compared to ERβ- tumors. 
Representative growth curves for three 
ERβ- and three ERβ+ PDX are shown in 
Figure 10. These data further support the 
hypothesis that ERβ is a potent tumor 
suppressor and that therapeutic targeting 
of this receptor may elicit anti-cancer 
effects in patients with ERβ+ TNBC.  
 
 
Figure 9. Estrogen treatment of ERβ-positive TNBC prevents lung tumor metastasis in vivo. OVX mice 
challenged with MDA-MB-231-ERβ-Luciferase cells via tail vein injection and randomized to treatment. 
(A) Presence of lung metastasis at week 8 via IVIS2000 xenogen imaging. At the termination of the 
study, lung dissections revealed the (B) presence of nodules in the no dox placebo and dox placebo 
groups (10x magnification). (C) Percent incidence, (D) number of lung nodules and (E) average tumor 
volume were also measured for each treatment group.
Figure 10. Representative growth curves of 
ERβ- and ERβ+ TNBC PDXs in NOD SCID 
mice supplemented with estrogen. 
 

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4. PARTICIPANT SELECTION 
4.1 
Pre-Screening Inclusion Criteria (Step 0) 
4.1.1 
Women of age ≥18 years. 
4.1.2 
History of locally advanced or metastatic breast cancer that is ERα negative 
(<1% nuclear staining) and HER2 negative (see note below)  
NOTE: HER2 Negative Disease per 2018 ASCO/CAP guidelines, one of the 
following must apply: 
1) 0 or 1+ by IHC and not amplified by ISH 
2) 0 or 1+ by IHC and ISH not done 
3) 2+ by IHC and ISH results are: <6.0 HER2 signals/cell with HER2/CEP17 
ratio <2.0   
4) IHC not done and not amplified by ISH 
4.1.3 
≤3 prior chemotherapy regimens for treatment of metastatic breast cancer 
NOTE: Prior use of monoclonal antibodies targeting PD1, PDL1 is allowed (if 
administered as monotherapy it is not counted as a chemotherapy regimen). 
4.1.4 
ECOG Performance status 0 or 1 (see Appendix A) 
4.1.5 
Willing to submit a biopsy specimen from locally recurrent or metastatic site 
(or primary if metastatic site not available) of breast cancer for ERβ staining 
to Mayo Clinic Anatomic Pathology. 
4.1.6 
No prior history of metastatic ERα positive breast cancer (≥1%) 
4.2 
Pre-Registration Inclusion Criteria (Step 1) 
4.2.1 
Presence of moderate or strong nuclear ERβ staining in >25% of cells in 
specimen submitted during Pre-Screening Step. 
4.2.2 
For patients who did not have a biopsy or lacking ERα, PR, and HER2 
results from a locally advanced or metastatic site performed ≤12 months prior 
to Pre-Registration: Willing to undergo a standard of care biopsy of locally 
recurrent or metastatic breast cancer for ERα, PR, and HER2 as well as 
additional research cores. 
4.2.3 
Measurable or non-measurable disease as defined by RECIST criteria 
(Section 11.2) that will be assessed using imaging-based evaluations 
(Section 11.3).  
NOTE: The tumor lesion biopsied during the pre-registration period is not 
considered measurable disease nor a target lesion. 

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4.2.4 
If history of brain metastases must meet the following criteria: 
- 
Patients with a history of brain metastases are eligible only if they are 
asymptomatic and have stable disease for ≥3 months, including <28 days 
prior to Pre-Registration. 
- 
Not receiving steroids for brain metastases 
4.2.5 
ECOG Performance status 0 or 1 (see Appendix A) 
4.2.6 
≤3 prior chemotherapy regimens for treatment of metastatic breast cancer. 
NOTE: Prior use of monoclonal antibodies targeting PD1, PDL1 is allowed 
4.2.7 
Women must be postmenopausal. 
NOTE: Postmenopausal status is verified by: 
1) Prior bilateral surgical oophorectomy, or 
2) Age ≥60 years, or 
3) Age <60 years with no menses for >1 year with estradiol levels within 
postmenopausal range, according to institutional standard 
4.2.8 
Able to swallow oral medications. 
4.2.9 
Willingness to stop use of strong inducers or inhibitors of CYP3A4 prior to 
registration. 
NOTE: Use of strong inducers or inhibitors is allowed during Pre-Registration as 
long as patient will complete course prior to Registration. 
4.3 
Pre-Registration Exclusion Criteria 
4.3.1 
Uncontrolled intercurrent illness including, but not limited to: 
− Ongoing or active infection 
− Symptomatic congestive heart failure 
− Unstable angina pectoris 
− Uncontrolled symptomatic cardiac arrhythmia 
− Uncontrolled hypertension (defined as blood pressure >160/90) 
4.3.2 
Any of the following co-morbid conditions: 
- DVT/PE ≤12 months prior to pre-registration 
Note: Patients who are on anticoagulant therapy for maintenance are 
eligible as long as the DVT and /or PE occurred >6 months prior to Pre-
Registration, and there is no evidence for active thrombosis (either 
DVT or PE). 
- stroke ≤6 months prior to pre-registration 
- two or more episodes of DVT and/or PE ≤5 years prior to pre-registration 
- abnormal uterine bleeding ≤6 months prior to pre-registration 
- history of coagulopathy 

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4.3.3 
Other active second malignancy other than non-melanoma skin cancers 
within 3 years prior to pre-registration.  
NOTE: A second malignancy is not considered active if all treatment for that 
malignancy is completed and the patient has been disease-free for ≥3 years 
prior to pre-registration. 
4.4 
Registration Inclusion Criteria (Step 2) 
4.4.1 
For patents who had a biopsy taken from a metastatic site ≤12 months prior 
to Pre-Registration: Confirmation from the local lab that the tumor from this 
biopsy was ERα negative (<1% nuclear staining) and HER2 negative. 
4.4.2 
For patients who underwent a pre-registration biopsy: Histologic 
confirmation from local lab that tumor is ERα negative (<1% nuclear 
staining), and HER2 negative (see prior note in Section 4.1.2). 
4.4.3 
Laboratory values ≤14 days prior to registration: 
• Hemoglobin  
≥8 g/dL 
• Platelet Count  
≥75,000/mm3 
• Creatinine 
 
≤1.5 x upper limit of normal ULN 
• Total Bilirubin 
≤1.5 x upper limit of normal (ULN) 
• AST/SGOT 
 
≤2.5 x upper limit of normal (ULN)* 
*For patients with liver metastasis: ≤5 x upper limit of normal (ULN) 
4.5 
Registration Exclusion Criteria 
4.5.1 
None of the following therapies are allowed ≤14 days prior to registration*: 
• 
Chemotherapy  
• 
Immunotherapy  
• 
Biologic therapy  
• 
Hormonal therapy  
• 
Monoclonal antibodies  
• 
Anti-HER2 or other “targeted” (e.g. mTOR) therapy  
*NOTE: Any adverse events derived from these therapies must be ≤Grade 2 prior 
to starting study therapy (exception: alopecia). 
4.6 
Inclusion of Underrepresented Populations 
Individuals of all races and ethnic groups are eligible for this trial. There is no bias 
towards age or race in the clinical trial outlined. This trial is open to the accrual of 
women. 

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5. REGISTRATION PROCEDURES 
5.1 
Pre-Screening and Pre-Registration (Step 0 and Step 1) 
5.1.1 
TBCRC Institutions other than Mayo Clinic 
To pre-screen or pre-register a patient, fax (
) a completed eligibility 
checklist to the Mayo Clinic Cancer Center (MCCC) Registration Office between 8 
a.m. and 4:30 p.m. Central Time Monday through Friday excluding holidays. 
5.1.2 
Mayo Clinic Institutions 
To pre-screen or pre-register a patient, access the Mayo Clinic Cancer Center (MCCC) 
web page and enter the registration/randomization application. The registration/ 
randomization application is available 24 hours a day, 7 days a week. Back up and/or 
system support contact information is available on the website. If unable to access the 
website, call the MCCC Registration Office at 
between the hours of 8 
a.m. and 5:00 p.m. Central Time (Monday through Friday excluding holidays). 
The instructions for the registration/randomization application are available on the 
MCCC web page (
) and detail the process for 
completing and confirming patient registration. Prior to initiation of protocol treatment, 
this process must be completed in its entirety and an MCCC subject ID number must be 
available as noted in the instructions. It is the responsibility of the individual and 
institution registering the patient to confirm the process has been successfully 
completed prior to release of the study agent. Patient registration via the 
registration/randomization application can be confirmed in any of the following ways: 
• Contact the MCCC Registration Office 
 If the patient was 
fully registered, the MCCC Registration Office staff can access the 
information from the centralized database and confirm the registration. 
• Refer to “Instructions for Remote Registration” in section 
“Finding/Displaying Information about A Registered Subject.” 
5.1.3 
All Institutions:  
5.1.3.1 
Verification 
Prior to accepting the pre-screening/pre-registration, the registration application will 
verify the following: 
• IRB approval at the registering institution 
• Patient pre-registration eligibility  
• Existence of a signed consent form  
• Existence of a signed authorization for use and disclosure of protected 
health information. 
5.1.3.2 
Pre-screening tests/procedures 
Pre-screening/pre-registration tests/procedures (see Section 6) must be completed 
within the guidelines specified on the test schedule. 

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5.1.3.3 
Biospecimen Banking - Patient Permissions 
At the time of registration, the following will be recorded: 
• Patient has/has not given permission to store and use his/her information and 
sample(s) for future research to learn about, prevent, or treat cancer. 
• Patient has/has not given permission to store and use his/her sample(s) for 
future research to learn, prevent, or treat other health problems. 
• Patient has/has/not given permission to have coded genetic information and 
coded medical information placed in password protected, secured database for 
research analyses. 
• Patient has/has not given permission for study doctor or someone from the 
research team at the study doctor’s site to contact them about future 
participation in more research 
5.2 
Registration Procedures (Step 2) 
5.2.1 
TBCRC Institutions other than Mayo Clinic 
5.2.1.1 
Registration 
To register a patient, fax (
) a completed eligibility checklist to the 
Mayo Clinic Cancer Center (MCCC) Registration Office between 8 a.m. and 4:30 
p.m. Central Time Monday through Friday. 
5.2.1.2 
Correlative Research 
A mandatory correlative research component requiring blood and tissue (see 
Section 10) is part of this study. The patient will be registered onto this component 
by the MCCC Registration Office. 
5.2.2 
Mayo Clinic Institutions 
5.2.2.1 
Registration 
To register a patient, access the Mayo Clinic Cancer Center (MCCC) web page and 
enter the registration/randomization application. The registration/ randomization 
application is available 24 hours a day, 7 days a week. Back-up and/or system support 
contact information is available on the Web site. If unable to access the Web site, call 
the MCCC Registration Office at 
 between the hours of 8 a.m. and 5:00 
p.m. Central Time (Monday through Friday). 
The instructions for the registration/randomization application are available on the 
MCCC web page (
) and detail the process for 
completing and confirming patient registration. Prior to initiation of protocol 
treatment, this process must be completed in its entirety and a MCCC subject ID 
number must be available as noted in the instructions. It is the responsibility of the 
individual and institution registering the patient to confirm the process has been 
successfully completed prior to release of the study agent. Patient registration via the 
registration/randomization application can be confirmed in any of the following 
ways: 

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• Contact the MCCC Registration Office 
 If the patient was 
fully registered, the MCCC Registration Office staff can access the 
information from the centralized database and confirm the registration. 
• Refer to “Instructions for Remote Registration” in section 
“Finding/Displaying Information about A Registered Subject.” 
5.2.2.2 
Correlative Research 
A mandatory tissue and blood correlative research component is part of this study. 
The patient will be automatically registered onto this component (see Section 10). 
5.2.3 
All Institutions 
5.2.3.1 
Documentation 
Documentation of IRB approval must be on file in the MCCC Registration Office 
before an investigator may register any patients. 
In addition to submitting initial IRB approval documents, ongoing IRB approval 
documentation must be on file (no less than annually) at the Registration Office (fax: 
). If the necessary documentation is not submitted in advance of 
attempting patient registration, the registration will not be accepted and the patient 
may not be enrolled in the protocol until the situation is resolved. 
When the study has been permanently closed to patient enrollment, submission of 
annual IRB approvals to the Registration Office is no longer necessary. 
5.2.3.2 
Verification 
Prior to accepting the registration, registration/randomization application will verify 
the following: 
• IRB approval at the registering institution 
• Patient eligibility 
• Existence of a signed consent form 
• Existence of a signed authorization for use and disclosure of protected health 
information  
5.2.3.3 
Biospecimen Banking - Patient Permissions 
At the time of registration, the following will be recorded: 
• Patient has/has not given permission to store and use his/her information and 
sample(s) for future research to learn about, prevent, or treat cancer. 
• Patient has/has not given permission to store and use his/her sample(s) for 
future research to learn, prevent, or treat other health problems. 
• Patient has/has/not given permission to have coded genetic information and 
coded medical information placed in password protected, secured database for 
research analyses. 
• Patient has/has not given permission for study doctor or someone from the 
research team at the study doctor’s site to contact them about future 
participation in more research 

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5.2.3.4 
Treatment on this protocol must commence at a TBCRC institution under 
the supervision of a medical oncologist. 
5.2.3.5 
Treatment cannot begin prior to registration and must begin ≤7 days after 
registration. 
5.2.3.6 
Pretreatment tests/procedures (see Section 6) must be completed within 
the guidelines specified on the test schedule. Prior to registration (during 
pre-registration period) a research biopsy of metastatic breast tissue must 
be taken for central pathologic review. 
5.2.3.7 
All required baseline symptoms must be documented and graded. 
5.2.3.8 
Blood draw kit is available on site for this patient. 
5.2.3.9 
Descriptive Factor (to be recorded on On Study Form) 
Tissue submission type: New biopsy at time of pre-registration vs. existing 
tissue from a metastatic site ≤12 months old  
NOTE: Participants MUST be registered with the Lead Institution prior to the 
start of protocol treatment. For sites outside of Mayo Clinic, registration can only 
be conducted during the business hours of 8 a.m. and 4:30 p.m. Central Time, 
Monday through Friday (excluding holidays). 
Note: Same day treatment registrations will only be accepted with prior notice and 
discussion with the Lead Institution. 
 

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6. STUDY CALENDAR 
Parameter 
ERβ 
screening 
(Pre-
Screening)1 
Prior to 
Pre-
registration 
Pre-
registration 
period  
(prior to 
registration) 
Prior to 
Day 1 of 
each 
treatment 
cycle2 
End of 
treatment 
(EOT) 
 for any 
reason3 
Window 
N/A 
≤21 days 
≤28 days 
±3 days 
N/A 
CLINICAL EVALUATIONS: 
 
 
 
 
 
History and Physical 
 
X 
 
X 
X 
Height 
 
X 
 
 
 
Vital signs and Weight 
 
 
 
X 
X 
Performance status 
 
X 
 
X 
X 
LAB/RADIOLOGIC EVALUATIONS: 
 
 
 
 
 
Hematology (CBC/diff incl ANC, PLT) 
 
 
X 
X 
X 
Comprehensive Metabolic Panel4 
 
 
X 
X 
X 
Imaging/tumor measurements/ evaluation 
of indicator lesions5,6 
 
 
X 
X 
X 
CORRELATIVE STUDIES: (See Section 10) 
 
 
 
 
 
Tissue submission 
X1 
 
X 
X7 
X8 
Tumor/Tissue Biopsy 
 
 
X9 
X10 
 
Blood samples 
 
 
 
X11 
X 
ADDITIONAL INFORMATION: 
 
 
 
 
 
Adverse Events/Toxicity Assessment 
 
 
X 
X 
X 
Patient Study Drug Diary (Appendix B) 
 
 
 
X 
X 
 
 
1  Pre-screening for ERβ may occur at any time prior to Pre-Registration using archival tissue from a local recurrence or 
metastatic site (or primary if local recurrence/metastatic site not available) of breast cancer. 
2  Cycle length is 28 (±3) days. Labs and AEs completed prior to registration may be used for Cycle 1 Day 1 tests if obtained 
<14 days prior to registration. For subsequent cycles, labs, scans, tests and observations may be obtained ≤3 days prior to 
Day 1 of treatment. 
3  Visit should be completed as soon as possible after last treatment and prior to starting any other treatment. Treatment may end 
due to progressive disease, intolerable adverse events, patient refusal, etc. See Section 12 
4  Panel includes: albumin, AST, ALT, alk phos, t bili, BUN, fasting calcium, creatinine, fasting glucose, sodium 
5  Acceptable imaging modalities for measurable disease include: CT or MRI. PET scanning is allowed to complement CT 
scanning in assessment of progressive disease. 
6  Tumor measurements are required during the Pre-Registration period (Days -28 to -1), at completion of Cycles 2, 4, 6, and 
about every 8 weeks until disease progression. The same imaging method used at baseline must be used at all disease 
assessments and must include all target and non-target lesions recorded at baseline. 
7  Research biopsy prior to Cycle 2 (end of Cycle 1) only 
8  If clinical biopsy performed at progression, please submit tissue sample (10 unstained slides) 
9  Patients must have had a tissue-based biopsy of a local recurrence or metastatic site ≤12 months prior to Pre-Registration and 
residual tissue must be available for testing. If a tissue-based biopsy was not performed ≤12 months prior to Pre-Registration, a 
new biopsy must be performed. Tissue  must be submitted for central review at Mayo Clinic per Section 10 and Lab Manual. 
10 Research biopsy required prior to Cycle 2 (end of Cycle 1) only 
11 Blood samples collected prior to treatment on C1D1; prior to Cycle 2 (end of Cycle 1); and EOT – See Section 10 

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7. TREATMENT PLAN 
7.1 
Biopsy and enrollment 
7.1.1 
Prescreening for ERβ  
Patients with archived tumor tissue available from a prior biopsy of locally recurrent 
or metastatic breast cancer site (or primary if locally recurrent/metastatic site not 
available) must submit a minimum of three (3) unstained 5 micron slides and ideally 
ten (10) unstained slides to the study central laboratory (Mayo Clinic) for staining, 
review and assessment of ERβ prior to full pre-registration. (See Section 10 and Lab 
Manual for submission instructions.)  
Patients with ERβ moderate or strong nuclear staining >25% will be considered 
eligible to proceed to Pre-Registration. 
7.1.2 
Pre-Registration 
All patients will need to consent to the full study prior to the biopsy. 
Patients for whom tissue from a biopsy of locally recurrent or metastatic disease is 
not available for assessment of ERα, PR, and HER2 obtained in the 12 months prior 
to Pre-Registration will undergo a standard of care tumor biopsy for assessment of 
ERα, PR, and HER2 as well as three (3) additional research cores (see Section 10 and 
Lab Manual for details for processing). The first core will be FFPE and processed 
locally for ERα, PR, and HER2 and sectioning of three (3) unstained slides 
(submitted simultaneously to Mayo Anatomic Pathology).  
Patients determined to be either ERα positive (>1% nuclear staining) or HER2 
positive (see Section 4.1.2 for definition) are ineligible and should proceed with 
standard of care systemic therapy directed towards these validated targets.  
Patients with confirmed ERα alpha negative (<1% nuclear staining), HER2 negative 
disease may proceed to Registration. 
7.1.3 
Registration 
Verify labs and remaining items for eligibility. 
7.2 
Agent Administration 
Agent 
Dose 
Route 
Day 
Continuation Cycle 
Estradiol 
2 mg 
1 tablet three times 
daily by mouth 
Days 1-28 
(continuous) 
Every 28 days (±3 days) 
Instruct patient to take medication as consistently as possible 
Instruct patient not to consume grapefruit or grapefruit juice while on study drug. 
If a dose is missed, skip that dose and take the next scheduled dose. 
If the patient vomits after taking a dose, skip that dose and resume with next 
scheduled dose. 

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7.3 
Concomitant Treatment and Supportive Care Guidelines 
7.3.1 
Full Supportive Care 
Patients should receive full supportive care while on this study, including blood 
product support, antibiotic treatment, and treatment of other newly diagnosed or 
concurrent medical conditions. All blood products and concomitant medications such 
as antidiarrheals, analgesics, and/or antiemetics received from the first day of study 
treatment administration until 30 days after the final dose should be recorded in the 
patient’s institutional medical records. 
7.3.2 
Vaginal hemorrhage (bleeding/spotting) 
7.3.2.1 
Management of Grade 1 vaginal hemorrhage 
Patients should continue taking the current dose of estradiol, and clinicians should 
manage according to institutional standards of care. 
Suggestions include: 
Use of a short course of medroxyprogesterone -- Clinicians may consider the use 
of medroxyprogesterone, 5-10 mg per day for 5-10 days until vaginal bleeding 
stops. Consideration should be for use of the lowest dose and duration of 
medroxy-progesterone as the effects of medroxyprogesterone on the antitumor 
effects of estradiol in TNBC are uknown. 
7.3.2.2 
Management for Grade ≥2 vaginal hemorrhage 
Temporarily discontinue estradiol and start short course (5-10 days) of medroxy-
progesterone as described in Section 7.3.2.1 above. 
If AE returns to ≤Grade 1, restart estradiol at next lower dose. 
If AE does not return to ≤Grade 1 within 14 days, discontinue estradiol. 
7.3.3 
Participation in Other Clinical Trials 
Patients participating in this clinical trial are not to be considered for enrollment in 
any other study involving pharmacologic agents whether for symptom control or 
therapeutic intent. 
7.3.4 
Radiation Therapy 
Patients must terminate study treatment if they are to receive radiation therapy for 
palliative reasons to the indicator lesion (s), as it impacts upon assessing the primary 
endpoint. 
 
 

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8. EXPECTED ADVERSE EVENTS AND DOSING DELAYS/DOSE 
MODIFICATIONS 
8.1 
Treatment Administration 
Estradiol is an oral formulation designed to be taken three times per day with water 
on a continuous basis. 
8.2 
Anticipated Adverse Events 
Consult the package insert for the most current and complete information. 
Less common but serious side effects include endometrial cancer, ovarian cancer, 
breast cancer, stroke, myocardial infarction, venous thrombosis and pulmonary 
embolism, dementia and gallbladder disease. Other side effects include 
hypercalcemia, visual abnormalities, elevated blood pressure, hypertriglyceridemia, 
hypothyroidism, edema, exacerbation of other conditions (i.e. endometriosis, asthma, 
diabetes mellitus, epilepsy, migraine or porphyria, systemic lupus erythematosus, 
hepatic hemangiomas), breast pain, irregular vaginal bleeding or spotting, 
stomach/abdominal cramps, bloating, nausea and vomiting. 
8.3 
Management of Adverse Events  
Strictly follow the modifications in this table for the first two cycles, until individual 
treatment tolerance can be ascertained. Thereafter, these modifications should be 
regarded as guidelines to produce mild-to-moderate, but not debilitating, side effects. 
If multiple adverse events are seen, administer dose based on greatest reduction 
required for any single adverse event observed. Reductions or increases apply to 
treatment given in the preceding cycle and are based on adverse events observed 
since the prior dose. 
8.4 
Dose Modifications/Delays 
8.4.1 
Dose Levels 
Dose Level 
Estradiol 
0* 
2 mg three times per day 
-1 
1 mg three times per day 
-2 
0.5 mg three times per day 
-3 
Discontinue 
*Level 0 is the starting dose 
Dose Modificatins for Adverse Events Associated with Estradiol. Note, In the 
case of adverse events persisting ≥14 days but in patients experiencing clinical 
benefit, contact the overall PI. 
A maximum of two dose reductions are allowed. 

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8.4.2 
Table of Actions for Adverse Events Associated with Estradiol 
CTCAE 
System/Organ/Class 
Adverse Event 
Grade 
Action for Estradiol 
Blood and lymphatic 
system disorders 
Anemia 
≥3 
Omit until resolved or return to ≤Grade 2 
Then resume at next lower dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 2, then 
discontinue 
Gastrointestinal 
disorders 
Nausea 
≥3 
Omit until resolved or return to ≤Grade 2 
Then resume at current dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 1, then 
discontinue 
If AE recurs despite appropriate 
antiemetic therapy then resume at next 
lower dose 
Gastrointestinal 
disorders 
Vomiting 
≥3 
Omit until resolved or return to ≤Grade 2 
then resume at current dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 1, then 
discontinue 
If AE recurs despite appropriate 
antiemetic therapy then resume at next 
lower dose 
Investigations 
Lymphocyte 
count decreased 
≥3 
Omit until resolved or return to ≤Grade 2 
Then resume at next lower dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 2, then 
discontinue 
 
Neutrophil count 
decreased 
≥4 
Omit until resolved or return to ≤Grade 2 
Then resume at next lower dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 2, then 
discontinue 
 
Platelet count 
decreased 
≥3 
Omit until resolved or return to ≤Grade 2 
Then resume at next lower dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 2, then 
discontinue 

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CTCAE 
System/Organ/Class 
Adverse Event 
Grade 
Action for Estradiol 
Investigations 
White blood cell 
decreased 
≥3 
Omit until resolved or return to ≤Grade 2 
Then resume at next lower dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 2, then 
discontinue 
Metabolism and 
nutrition disorders 
Hypercalcemia 
≥2 
Omit until resolved or return to ≤Grade 1 
or baseline 
Then resume at next lower dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 1, then 
discontinue 
 
Hyponatremia 
≥2 
Omit until resolved or return to ≤Grade 1 
Then resume at next lower dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 1, then 
discontinue 
Nervous system 
disorders 
Ischemia, 
cerebrovascular 
2 
Omit and initiate antiplatelet or 
anticoagulant therapy per local guidelines 
If return to ≤Grade 1, then resume at next 
lower dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 1, then 
discontinue 
 
Stroke 
≥2 
Discontinue 
 
Transient 
ischemic attacks 
≥2 
Omit and initiate antiplatelet or 
anticoagulant therapy per local guidelines 
If return to ≤Grade 1, then resume at next 
lower dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 1, then 
discontinue 
Reproductive system 
and breast disorders 
Vaginal 
hemorrhage 
≥2 
Omit estradiol and initiate 
medroxyprogesterone per Section 7.3.2 
If return to ≤Grade 1, then restart 
estradiol at next lower dose 
If estradiol is omitted for >14 days and 
the AE does not return to ≤Grade 1, then 
permanently discontinue estradiol 

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CTCAE 
System/Organ/Class 
Adverse Event 
Grade 
Action for Estradiol 
Vascular disorders 
Thromboembolic 
event 
≥2 
Omit until appropriate anticoagulation 
therapy can be initiated and patient is 
medically stable 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 1, then 
discontinue  
Discontinue if event recurs (second 
episode) while on anticoagulation therapy 
All other 
Any event not 
specified above 
≥3 
Omit until resolved or return to ≤Grade 1 
Then resume at next lower dose 
If drug is omitted for ≥14 days and the 
AE does not return to ≤Grade 1, then 
discontinue 
Discontinue if event recurs 
 
8.5 
Special Considerations 
• 
The treating investigator may reduce a subject’s dose for an AE of any 
grade/duration where s/he believes it to be in the best interests of the subject. 
• 
Any consideration to modification of the above dose modification guidelines 
should be discussed with the Principal Investigator for approval or disapproval in 
advance. 
8.6 
Adverse Events to be Graded at End of Each Cycle of Treatment 
Use NCI Common Terminology for Adverse Events (CTCAE) v.5.0  
CTCAE 
System/Organ/Class 
(SOC) 
Adverse Event/Symptoms 
Baseline 
Each 
Evaluation 
Gastrointestinal  
# of stools at baseline 
X 
 
disorders 
Diarrhea 
 
X 
 
Nausea 
X 
X 
 
Vomiting 
X 
X 
Investigations 
Platelet count decreased 
X 
X 
 
Neutrophil count decreased 
X 
X 
 
White blood cell decreased 
X 
X 
Metabolism and  
Hypercalcemia 
X 
X 
nutrition disorders 
Hyponatremia 
X 
X 
Reproductive system 
and breast disorders 
Vaginal hemorrhage 
X 
X 
 

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9. DRUG FORMULATION/STORAGE/SUPPLY 
9.1 
Estradiol 
Estradiol is an approved agent for the treatment of metastatic breast cancer. Estradiol is 
commercially available. 
9.1.1 
Background 
Estradiol is the principal intracellular human estrogen and is substantially more potent 
than its metabolites, estrone and estriol at the receptor level. Estrogens act through 
binding to nuclear receptors in estrogen-responsive tissues. Circulating estrogens 
modulate the pituitary secretion of the gonadotropins, luteinizing hormone (LH) and 
follicle stimulating hormone (FSH), through a negative feedback mechanism. 
9.1.2 
Formulation 
NOTE: Will depend on generic brand dispensed at local pharmacy. 
Each tablet, for oral administration, must contain 0.5, 1 or 2 mg of estradiol.  
9.1.3 
Storage 
Instruct patient to store at room temperature.  
9.1.4 
Administration 
Refer to the treatment section (Section 7) for specific administration instructions.  
9.1.5 
Pharmacokinetic information 
Distribution: Estrogens are widely distributed in the body and are generally found in 
higher concentrations in the sex hormone target organs. Estrogens circulate in the blood 
largely bound to sex hormone binding globulin (SHBG) and albumin. 
Metabolism: Estrogens also undergo enterohepatic recirculation via sulfate and 
glucuronide conjugation in the liver, biliary secretion of conjugates into the intestine and 
hydrolysis in the gut followed by reabsorption. 
Excretion: Estradiol, estrone, and estriol are excreted in the urine along with glucuronide 
and sulfate conjugates. 
9.1.6 
Potential Drug Interactions 
In vitro and in vivo studies have shown that estrogens are metabolized partially by 
cytochrome P450 3A4 (CYP3A4). Therefore, inducers or inhibitors of CYP3A4 may 
affect estrogen drug metabolism. Inducers of CYP3A4 such as St. John’s Wort 
preparations (Hypericum perforatum), phenobarbital, carbamazepine, and rifampin may 
reduce plasma concentrations of estrogens, possibly resulting in a decrease in therapeutic 
effects and/or changes in the uterine bleeding profile. Inhibitors of CYP3A4 such as 
erythromycin, clarithromycin, ketoconazole, itraconazole, ritonavir and grapefruit juice 
may increase plasma concentrations of estrogens and may result in side effects.  

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9.1.7 
Known potential adverse events 
Consult the package insert for the most current and complete information. 
Less common but serious side effects include endometrial cancer, ovarian cancer, breast 
cancer, stroke, myocardial infarction, venous thrombosis and pulmonary embolism, 
dementia and gallbladder disease. Other side effects include hypercalcemia, visual 
abnormalities, elevated blood pressure, hypertriglyceridemia, hypothyroidism, edema, 
exacerbation of other conditions (i.e. endometriosis, asthma, diabetes mellitus, epilepsy, 
migraine or porphyria, systemic lupus erythematosus, hepatic hemangiomas), breast pain, 
irregular vaginal bleeding or spotting, stomach/abdominal cramps, bloating, nausea and 
vomiting. 
9.2 
Ordering 
Patients will be given a prescription for estradiol to fill at the pharmacy of their choice. 
9.3 
Drug Accountability 
Drug accountability will be performed via patient pill diaries and patient appointments. 

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10. CORRELATIVE/SPECIAL STUDIES  
10.1 
Summary Table of All Biospecimens 
 
 
When to submit 
Specimen 
Pre-
Screening 
During Pre-
Reg Period 
Baseline (C1D1 
pre-treatment) 
End of Cycle 1 
(prior to 
Cycle 2) 
End of 
Treatment  
for any 
reason 
Archived FFPE 
slides 
(Section 10.3.1 
and Section 
10.3.4) 
Mandatory 
 
 
 
Optional 
Frozen and FFPE 
(and PDX)* tissue 
(Section 10.3.1 
and Section 
10.3.2) 
 
Mandatory 
 
Mandatory 
 
Blood for DNA 
(Section 10.2.1) 
 
 
Mandatory 
 
 
Blood for other 
correlatives 
(Section 10.2.1) 
 
 
Mandatory 
Mandatory 
Mandatory 
*PDX tissue is only collected at Mayo Clinic in Rochester, Minnesota. 
NOTE: Optional items are strongly recommended. 
 
 

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10.2 
Blood Samples 
10.2.1 Summary of the blood biospecimen collection schedule 
NOTE: Blood collection is mandatory for this study at all timepoints specified. 
 
Cycle 1, Day 1 prior to 
start of treatment 
(Baseline) 
At the end of Cycle 1 
(prior to start of 
Cycle 2) 
At end of treatment for 
any reason  
Submit to: 
Estradiol, 
cytokine, 
cystatin 
X 
X 
X 
Mayo Clinic 
(per lab 
manual) 
CTC 
X 
X 
X 
Mayo Clinic 
cfDNA 
X 
X 
X 
Mayo Clinic 
SNP 
X 
 
 
Mayo Clinic 
PBMC 
X 
X 
X 
Mayo Clinic 
Thymidin
e Kinase 
x 
x 
x 
Mayo Clinic 
Kits will be provided for blood collection. 
Instructions for collection and shipping are in each kit. 
See Lab Manual for instructions.  
Samples should be collected and shipped Monday – Friday.  
However, if the subject can only be seen on Fridays, please ensure that Saturday 
delivery is marked clearly on the shipping boxes and Mayo Clinic staff are notified.  
10.2.2 Methods 
10.2.2.1 Circulating Tumor Cell Capture and Phenotyping 
The relative abundance of CTCs in the peripheral blood has strong correlation with tumor 
recurrence and distant metastases in both early stage and advanced breast tumors. We 
will prospectively isolate CTCs from blood specimens collected from study participants 
at three time points for phenotypic and possibly genotypic analyses.  
One blood sample will be collected in the CellSave® Preservation Tube for CTC 
isolation using the Veridex CellSearch™ system (Janssen; Raritan, NJ). CellSave® 
contains an optimized preservative that stabilizes cells for up to 96 hours at room 
temperature. The CellSearch™ system (
) allows for the 

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immunomagnetic selection, fluorescence staining, concentration, and enrichment of 
CTCs for enumeration and analysis. The FDA cleared process uses the CellSearch™ 
Epithelial Cell Kit, which contains anti-EpCAM (epithelial cell adhesion molecule) 
Ferrofluid, a fluorescent nucleic acid dye (DAPI), and fluorescently labeled monoclonal 
antibodies specific for leukocytes (CD45) and epithelial cells (cytokeratins 8, 18, and 19) 
to identify CTCs as those that are cytokeratin positive, DAPI positive, and CD45 
negative with the appropriate morphology. At least two-thirds of patients with metastatic 
breast cancer have >1 CTC per 7.5 mL blood assayed with this technology, and the 
enumeration of CTCs relative to a threshold of 5 CTCs per 7.5 mL blood is associated 
with clinical outcomes in this patient population27-29. The standard process has been 
modified to allow for CTC retrieval and subsequent assessment for additional protein 
markers of interest (e.g., Aurora A kinase, ER, p-SMAD5, and p-SOX2). 
CTCs vary in the degree of EpCAM expression, so malignant cells in the circulation may 
be missed by the CellSearch technology. Toward that end, other methodologies that do 
not rely on EpCAM for capture are now available. Therefore, a second blood sample will 
be collected in the AccuCyte® Blood Collection Tube for CTC isolation using the 
AccuCyte® Platform (RareCyte; Seattle, WA). The blood collection tube contains an 
optimized preservative that stabilizes cells for up to 96 hours at ambient temperature. The 
AccuCyte® Platform (
) is a comprehensive, reproducible, and highly 
sensitive platform for enriching, identifying, and isolating CTCs for downstream 
molecular analyses (ref: Campton DE et al. BMC Cancer 2015. 15:360.). The system 
relies on density gradient centrifugation and allows for virtually complete harvesting of 
the buffy coat into a small volume for application to a microscopic slide without cell lysis 
or washing. Immunofluorescent staining for selected markers (e.g., cytokeratins, 
EpCAM, DAPI, CD45, HER2, ER, Aurora A kinase, p-SMAD5, and p-SOX2) is used for 
the efficient positive and negative identification of CTCs via automated scanning digital 
microscopy and image analysis. An integrated device provides for mechanically precise 
CTC retrieval for advanced genomic analyses, including next generation sequencing, on 
single or pooled CTCs. 
10.2.2.2 Peripheral blood mononuclear cell (PBMC) phenotyping using CyTOF 
Analyses of tumor-host immune interactions have largely focused on exploring 
interactions between cancer cells and immune cells at the tumor tissue level. Efforts to 
understand perturbations in the systemic immune response occurring in the peripheral 
blood, and their influence on prognosis and chemotherapy response have been limited. It 
has been observed that decreased levels of circulating effector immune cells (e.g., CD8+ 
CTLs) or increased levels of circulating immune suppressive cells (e.g., monocytes, 
regulatory T cells, myeloid-derived suppressor cells) are associated with disease 
progression, and that anticancer therapy has an impact on the relative abundance of 
specific immune cell populations detectable in peripheral blood. The availability of 
multiplexed technologies such as Cytometry by time-of-flight (CyTOFTM) has greatly 
improved the ability to evaluate a large number of surface markers at the single cell level, 
allowing for deep characterization of circulating immune cell populations in peripheral 
blood specimens. 
Cytometry by time-of-flight (CyTOFTM, Fluidigm) or mass cytometry is a platform that 
uses mass spectrometry to allow the evaluation of over 35 simultaneous parameters at a 

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single-cell resolution. This technology uses nonradioactive non-biological metal isotopes 
as reporters tagged to monoclonal antibodies. Measurements based on mass spectrometry 
largely avoids the hurdles of interference and spectral overlap experienced with 
fluorochrome-based flow cytometry. This constitutes an ideal platform for peripheral blood 
immune monitoring given its ability to assess a large number of parameters and resolve 
small differences in a heterogeneous population of cells. 
Mononuclear cells will be isolated from peripheral blood via gradient separation using a 
Ficoll sodium diatrizoate solution (Ficoll-PaqueTM PLUS, GE Healthcare). The resulting 
mononuclear single-cell suspension will be suspended in freezing media containing 10% 
dimethyl sulfoxide and stored in liquid nitrogen or -150°C freezer for analysis. Specimens 
will be stained in batches of 4-5 patients. Unstimulated live single-cell suspensions will be 
stained with a cocktail of metal-tagged antibodies designed to recognize 29 cell surface 
proteins optimized for immune monitoring of human peripheral blood. Nucleated cellular 
events will be identified using a DNA intercalator conjugated to natural abundance iridium 
(191Ir and 193Ir). Cisplatin (195Pt) will be used for dead-live cell discrimination and 
calibration beads containing natural abundance cerium (140/142Ce), europium 
(151/153Eu), holmium (165Ho), and lutetium (175/176Lu) will be used for normalization 
of acquired data. Our panel will be constructed using commercially available metal-
conjugated antibodies (Fluidigm Corporation) and stained cells will acquired on the 
CyTOF2 (Fluidigm Corporation). 
We will stain a minimum of 3 x 106 cells per specimen and acquire a minimum of 1 x 106 
events on the CyTOF. We will initially perform high-level manual gating to exclude dead 
or apoptotic cells, debris, beads and doublets. Additional multiparametric analysis will be 
done using platforms such as Astrolabe Diagnostics, Spanning-tree Progression Analysis 
of Density-normalized Events (SPADE), and visualization of unbiased clustering of events 
using the t-SNE (t-Distributed Stochastic Neighbor Embedding) algorithm or viSNE. 
 
10.2.2.3 
Thymidine Kinase 
Serum thymidine kinase activity measurements have shown changes in levels during 
progression and treatment of metastatic breast cancer and other cancers. Thymidine 
kinase 1 (TK1) is a key enzyme in DNA synthesis. TK1 catalyzes the conversion of 
thymidine to deoxythymidine monophosphate (dTMP), which is further phosphorylated 
to di- and triphosphates preceding its incorporation into DNA. Expression of TK1 is E2F-
dependent and peaks in the S-phase of the cell cycle, and CDK4/6 inhibition prevents 
E2F-dependent transcription of TK1 (Thomas, 2016). Due to uncontrolled cellular 
proliferation and higher replication rates compared to normal tissues, solid tumors can 
secrete pathological levels of TK1 that can be detected in serum. Studies have shown that 
serum TK1 levels were predictive of response, progression free survival (PFS) and breast 
cancer-free survival in both the neoadjuvant and metastatic settings (all disease subtypes).  
This was recently demonstrated in a secondary analysis of the SWOG S0226 trial, where patients 
with baseline elevated TKI using the DiviTum® TK assay had significantly worse PFS [median 
11.2 vs. 17.3 months, HR = 1.76; 95% confidence interval (CI; 1.43-2.16); P < 0.0001] and OS 
[median 30 vs. 58 months, HR = 2.38; 95% CI (1.91-2.98); P < 0.0001] compared to those with 
low TK1 levels. Furthermore, at serial timepoints, high versus low sTK1 had significantly worse 

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subsequent PFS and OS [at cycle 2: PFS HR = 1.70, 95% CI (1.34-2.17); P < 0.0001, OS HR = 
2.51, 95% CI (1.93-3.26); P < 0.0001].  (Paoletti et al.  Clin Canc Res 2021).   
 
We will measure thymidine kinase activity (TKa) in serum samples obtained at baseline, 
end of cycle 1 and end of treatment using the DiviTum® TKa  (DiviTum® TKa ELISA 
kit, Biovica Cat #950). TK activity is determined from a calibration curve established 
using TK1 standards.  The units are expressed as DiviTum units (DuA). The DuA value 
is established so that 1 DuA is the TK activity of 1 pg/mL of standard recombinant TK 
under the standard DiviTum® TKa assay conditions. 
 
 
 
 

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10.3 
Tissue Samples 
10.3.1 Pre-screening: ERβ pretesting (mandatory) 
Correlative Study 
(Section for more 
information) 
Type of Tissue 
to Collect/ 
Send 
Process at 
site?  
(Yes or No) 
Temperature 
conditions for 
Storage /Shipping 
Submission of archived 
tumor specimen from 
locally recurrent or 
metastatic breast cancer for 
ERβ staining 
10 unstained 
slides 
(5 micron) 
Cut sections at 
local site 
Send to Mayo 
Coordinator 
Room Temperature 
(Unstained slides) 
For patients who have registered for the prescreening phase and have archived 
FFPE tumor tissue from a locally advanced or metastatic site (or primary if 
locally recurrent/metastatic site not available) available for ERβ testing in the 
Mayo CAP/CLIA certified Anatomic Pathology Laboratory, the local site must 
submit 10 unstained sections (cut at 5 microns) per instructions in the Lab 
Manual. 
Requirements for using archived tissue: 
• Biopsy must have been tissue-based. Fine needle aspirations (FNA) are 
NOT allowed for this submission. 
• Ideally 10 unstained sections or a minimum of 3 unstained sections (cut at 
5 microns) must be available for submission. 
If all the above conditions cannot be met, then the patient cannot proceed to pre-
registration. 
Results of testing (either positive or negative for ERβ), will be reported to the 
local study coordinator. 
If the result is negative for ERβ, patient may be directed to another study or may 
choose to have a new biopsy to confirm the result. 
If the result is positive for ERβ (>25% moderate or strong nuclear staining) 
patients should be encouraged to proceed to Pre-Registration phase. 
 
 

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10.3.2 Pre-Registration Tumor Tissue Collection  
10.3.2.1  Tissue Collection for Patients undergoing Pre-Registration Biopsy ONLY 
Correlative Study 
Mandatory 
or Optional 
Type of 
Tissue to 
Collect 
Process 
at site? 
(Yes or 
No) 
Temperature 
Conditions for 
Storage 
/Shipping 
Assay to be 
performed 
Core 1 (FFPE) 
Mandatory 
Fresh tumor 
biopsy for 
FFPE 
Yes 
Room 
Temperature 
(Unstained 
slides) 
ER, PR, HER2 (tested 
locally) and 3 
additional unstained 
sections (sent to Mayo 
Clinic) 
Core 2 (FFPE) 
Mandatory  
Fresh tumor 
biopsy for 
FFPE 
Yes- 
FFPE 
Room 
Temperature 
For example: Ki-67, 
phospho-ERβ, 
phospho-smad 2/3, 
Cystatins 1, 2, 4, and 5 
Core 3 (Frozen) 
Mandatory  
Fresh tissue 
core (core) 
for flash 
freezing 
Yes 
Frozen/ dry ice 
DNA and RNA 
sequencing 
Core 4 (PDX for 
Rochester 
patients only) 
Mandatory 
Fresh tissue 
biopsy 
placed in 
media 
Yes 
Fresh 
PDX 
A total of 3 or 4 cores will be obtained as outlined in Table 10.3.2 (above). The 
first core will be FFPE and processed as a standard of care test for ERα, PR, and 
HER2 as well as sectioning of three (3) unstained slides submitted simultaneously 
to Mayo Anatomic Pathology.  
Patients determined to be either ERα positive (>1% nuclear staining) or HER2 
positive (see Section 4.1.2 for definition) are ineligible and should proceed with 
standard of care systemic therapy directed towards these validated targets.  
Patients confirmed to have ERα negative (<1% nuclear staining), and HER2 
negative disease. Mayo Anatomic Pathology will proceed with ERβ testing using 
the same tumor block; however, these results will not be used for eligilblity. Core 
number 2 (Research FFPE) will be stored in Mayo Clinic BAP and processed 
(along with biopsy samples from Cycle 1) at a later time for antibodies such as: 
phospho-ERβ, cystatins 1, 2, 4 and 5, phospho-Smad2/3 and Ki-67. Core number 
3 will be frozen and processed later for DNA and RNA sequencing. Core number 
4 (Mayo Rochester only) will be processed for PDX generation. 
10.3.2.2 For Patients with Tumor Tissue Available from a Biopsy/Excision 
Performed ≤12 months Prior to Pre-Registration 
Submit archived FFPE tissue from biopsy/excision performed ≤12 months prior to 
Pre-Registration. 
• Biopsy must have been tissue-based. Fine needle aspirations (FNA) are 
NOT allowed for this submission. 

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• Ideally 10 unstained sections or a minimum of 3 unstained sections (cut at 
5 microns) must be available for submission. 
Note: If tissue-based biopsy of metastatic disease was performed ≤12 months 
prior to Pre-Registration, and there is sufficient archived tissue for a minimum of 
3 and ideally 10 unstained slides cut at 5 microns, then a new biopsy is not 
required. 
10.3.3 Guidelines for tissue acquisition and shipping  
See Lab Manual for specific information 
Image guided biopsies are recommended where feasible. The amount of tissue 
collected will follow the guidelines listed below. If a patient has more than one 
site of disease, only one site needs to be biopsied. 
Skin/breast/chest wall: An incisional/excisional biopsy is preferred. If punch 
biopsy is performed, a minimum of 4 punch biopsies (≥5mm diameter). 
Lymph node: A goal of 4-6 core biopsy specimens obtained using an 18-gauge 
needle or larger. Smaller or less samples will be obtained at the discretion of the 
physician performing the biopsy. 
Liver: A goal of 4-6 core biopsy specimens obtained using an 18-gauge needle. 
Lung: Because of the risk of pneumothorax associated with core needle biopsies 
of lung nodules, biopsies are not recommended. 
Bone: Because the yield of malignant tissue from bone biopsies tends to be 
relatively low, if a patient has another accessible site of disease (i.e., skin, lymph 
node, liver, other metastatic lesions outside of the bone), that site should be 
biopsied preferentially. If bone is the only biopsy-accessible site, then a goal of 3-
6 core biopsy specimens will be obtained using an 18-gauge needle. 
A series of high throughput assays including exome sequencing, and RNA seq 
will be applied using tumor samples baseline (DNA and RNA seq) and after 
Cycle 1 (RNA). 
10.3.3.1 Xenograft mice model (Patient derived xenograft [PDX]) – Pre-Reg only 
In Mayo Clinic (Rochester) Subjects Only: 
For Mayo Clinic patients in Rochester, MN, registered to 
TBCRC051/MC1831, tumor biopsy material from core number 4 will be 
immediately injected into NGS mice for the creation of human tissue 
xenografts (see IACUC A49111 and IACUC A00003279-17). The xenograft 
tumor samples will be used to determine the functional implications of tumor 
alterations identified from tumor sequencing and for future drug screening and 
cytotoxicity assay. These lines will be also useful for future research purposes, 
such as testing novel compounds and different regimens. All the tumor tissues 
from xenografts will be stored on Gonda 19-466E.  

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10.3.4 Research Biopsy for ALL Patients Obtained after Cycle 1: 
NOTE: Tissue collection is mandatory for this study 
Correlative Study 
Mandatory 
or Optional 
Type of 
Tissue to 
Collect 
Process at 
site?  
(Yes or No) 
Temperature 
Conditions for 
Storage/ 
Shipping 
Planned Assays  
Core 1 (FFPE) 
Mandatory 
Fresh tumor 
biopsy for 
FFPE 
Yes to 
create FFPE 
Room 
Temperature 
(Unstained 
slides) 
Ki-67, phosphor 
ERβ, phospho-
smad 2/3, 
Cystatins 1, 2, 4, 
and 5 
Core 2 (FFPE) 
Mandatory 
Fresh tumor 
biopsy for 
FFPE 
Yes to 
create FFPE 
Room 
Temperature 
DNA and RNA 
sequencing 
Core 3 (Frozen) 
Mandatory 
Fresh tissue 
core for flash 
freezing 
Yes 
Frozen/ dry ice 
Storage for 
sequencing later 
See Lab Manual for specific instructions for collection/shipping 
10.3.4.1 Evaluation of changes in phospho-ERβ, cystatins 1, 2, 4 and 5, phospho-
Smad2/3 and Ki-67 
As shown in the preliminary data, we have demonstrated that ligand mediated 
activation of ERβ leads to increased expression of cystatins 1, 2, 4 and 5 as well 
as suppression of phospho-Smad2/3 and downstream markers of canonical TGFβ 
signaling. Additionally, based on recent data that phosphorylation of tyrosine 36 
of ERβ (but not total ERβ) in breast tumors is associated with activation of ERβ,  
we will use core biopsies obtained after Cycle 1 (along with the baseline biopsies) 
to assess for changes in the phosphorylation of ERβ Y36 and Smad2/3, as well as 
the expression levels of cystatins 1, 2, 4 and 5 and Ki67. 
Our hypothesis is that estradiol will activate ERβ resulting in 1) down-regulation 
of Ki-67; 2) reduction in phospho-Smad2/3; 3) increase in phosphorylation 
(activation) of ERβ; and finally 4) increase in expression levels of cystatins 1, 2, 
4, and 5. 
Additionally, these core biopsies will be used for RNA sequencing, to assess and 
compare changes in gene expression comparing tumors sensitive and resistant to 
estradiol.  
10.3.4.2 Frozen Tissue for Sequencing/Exome (Core 3) 
Tumor DNA will be isolated from specimens obtained and baseline. DNA will be 
processed and plated at Mayo BAP lab. After the first 10 patients have been 
enrolled, DNA and RNA and RNA yields will be reviewed. If inadequate, the 
protocol will be changed to use one core for DNA and another core for RNA 
sequencing. 
 

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10.3.5 Tissue Submission at End of Treatment for Any Reason 
This submission is optional if archived tissue is available. 
Correlative Study  
(Section for more 
information) 
Type of 
Tissue to 
Collect/ 
Send 
Process at site? 
(Yes or No) 
Temperature 
Conditions for 
Storage/ 
Shipping 
Submission of archived 
tumor specimen from 
locally recurrent or 
metastatic breast cancer 
for ERβ staining 
5 unstained 
slides 
(5 micron) 
Cut sections at 
local site 
Send to Mayo 
Pathology 
Coordinator 
Room 
Temperature 
(Unstained 
slides) 
For patients who have enrolled in this study, and for whom a clinical biopsy is 
performed at time of suspected disease progression, and for whom archived FFPE 
tumor tissue from a metastatic site is available, the local site should submit 
5 unstained sections (cut at 5 microns) per instructions in the Lab Manual 
10.4 
Other Samples and/or Devices 
10.4.1 Patient Derived Xenograft (Mayo Clinic Rochester patients only) 
Xenografts will be generated in 
 Her lab has already 
successfully generated a series of breast cancer models for all subtypes from both 
primary breast cancer as well as metastatic breast cancer using core biopsy or 
surgical samples. In this study, to generate individual tumor xenograft line for drug 
screening, drug cytotoxicity assay, specimen obtained during the biopsy will be 
immediately injected into NGS mice for the creation of human tissue xenografts. 
The xenograft tumor samples will be used to determine the functional implications 
of tumor alterations identified from tumor sequencing and for future drug screening 
and cytotoxicity assay. These lines will be also useful for future research purposes, 
such as testing novel compounds and different regimens. All the tumor tissues from 
xenografts will be stored on Gonda 19 466E. 
10.5 
Genetic Testing 
Participants will be given information as part of the informed consent process that 
samples will be used for research tests that will include genetic studies and testing. The 
intent is not to give participants (or his/her medical providers) the results of any testing 
done for research purposes; however, incidental germline (heritable) mutations may be 
identified of which a participant may or may not already be aware. In the case that an 
incidental genetic finding is identified, the Protocol Chair of this project will be 
notified. The possible decisions for handling incidental findings may include 
notification of the participant (and provider); recommendation for genetic counseling, 
which may or may not include genetic testing (e.g., if the finding was not done in a 
CLIA certified laboratory); or, neither. In general, a member of the participant’s 
treating team will be given the information to help with notification. In all cases, the 
current policy of the Mayo Clinic and local/participating site IRB, as applicable, will 

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be followed and any additional approvals that may be required prior to participant 
notification will be secured in advance. 
10.6 
Additional Information 
Submission of data for Genome Wide Association Studies (GWAS) is not currently 
planned; however, subjects will be asked for permission in the informed consent 
process. A revision to the protocol and/or any regulatory approvals will be secured prior 
to any GWAS submission or inclusions in the future, if applicable. 
10.7 
Specimen Banking 
The study Protocol Chair and collaborators have approval by the TBCRC to use all 
research bio-specimens collected during the conduct of this trial to address the research 
questions described in the protocol document. All future use of residual or repository 
specimens collected in this trial for purposes not prospectively defined will require 
review and approval by the TBCRC according to its established policies, whether the 
specimens are stored in a central site or at a local institution in a virtual repository. 
Secondary use of biospecimens for new endpoints must be submitted to the TBCRC 
Central Office for possible review by the TBCRC Correlative Science Review 
Committee. 
11. MEASUREMENT OF EFFECT 
Response and progression will be evaluated in this study using the international criteria 
proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guidelines 
(version 1.1) Changes in the largest diameter of the tumor lesions and the short axis 
measurements in the case of lymph nodes are used in the RECIST guideline. 
11.1 
Schedule of Evaluations 
For the purposes of this study, patients should be re-evaluated every 8 weeks (at the end 
of every 2 cycles) until progression.  
11.2 
Definitions of Measurable and Non-Measurable Disease 
11.2.1 Measurable Disease 
A non-nodal lesion is considered measurable if its longest diameter can be 
accurately measured as 2.0 cm with chest x-ray, or as ≥1.0 cm with CT scan, CT 
component of a PET/CT, or MRI.  
A superficial non-nodal lesion is measurable if its longest diameter is ≥1.0 cm in 
diameter as assessed using calipers (e.g. skin nodules) or imaging. In the case of 
skin lesions, documentation by color photography, including a ruler to estimate the 
size of the lesion, is recommended. 
A malignant lymph node is considered measurable if its short axis is >1.5 cm when 
assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 
mm).  

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Tumor lesions in a previously irradiated area are not considered measurable 
disease. 
The tumor lesion biopsied during the pre-registration period is not considered 
measurable disease. 
Lytic bone lesions, with an identifiable soft tissue component, evaluated by X-ray, 
CT (with bone windows) or MRI, can be considered as measurable lesions if the 
soft tissue component otherwise meets the definition of measurability described 
above 
11.2.2 Non-Measurable Disease 
All other lesions (or sites of disease) are considered non-measurable disease, 
including pathological nodes (those with a short axis ≥1.0 to <1.5 cm). Bone 
lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis 
cutis/pulmonis, inflammatory breast disease, and abdominal masses (not followed 
by CT or MRI), are considered as non-measurable as well. 
Note: ‘Cystic lesions’ thought to represent cystic metastases can be considered as 
measurable lesions, if they meet the definition of measurability described above. 
However, if non-cystic lesions are present in the same patient, these are preferred 
for selection as target lesions. In addition, lymph nodes that have a short axis 
<1.0 cm are considered non-pathological (i.e., normal) and should not be recorded 
or followed. 
11.3 
Guidelines for Evaluation of Measurable Disease 
11.3.1 Measurement Methods:  
• 
All measurements should be recorded in metric notation (i.e., decimal 
fractions of centimeters) using a ruler or calipers. 
• 
The same method of assessment and the same technique must be used to 
characterize each identified and reported lesion at baseline and during follow-
up. For patients having only lesions measuring at least 1 cm to less than 2 cm 
must use CT imaging for both pre- and post-treatment tumor assessments. 
11.3.2 Acceptable Modalities for Measurable Disease: 
- Conventional CT and MRI: This guideline has defined measurability of 
lesions on CT scan based on the assumption that CT slice thickness is 5 mm 
or less. If CT scans have slice thickness greater than 5 mm, the minimum size 
for a measurable lesion should be twice the slice thickness. 
As with CT, if an MRI is performed, the technical specifications of the 
scanning sequences used should be optimized for the evaluation of the type 
and site of disease. The lesions should be measured on the same pulse 
sequence. Ideally, the same type of scanner should be used and the image 
acquisition protocol should be followed as closely as possible to prior scans. 
Body scans should be performed with breath-hold scanning techniques, if 
possible. 

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- PET-CT: If the site can document that the CT performed as part of a PET-CT 
is of identical diagnostic quality to a diagnostic CT (with IV and oral 
contrast), then the CT portion of the PET-CT can be used for RECIST 
measurements and can be used interchangeably with conventional CT in 
accurately measuring cancer lesions over time.  
- Chest X-ray: Lesions on chest x-ray are acceptable as measurable lesions 
when they are clearly defined and surrounded by aerated lung. However, CT 
scans are preferable. 
- FDG-PET: FDG-PET scanning is allowed to complement CT scanning in 
assessment of progressive disease [PD] and particularly possible 'new' disease. 
A ‘positive’ FDG-PET scanned lesion is defined as one which is FDG avid 
with an update greater than twice that of the surrounding tissue on the 
attenuation corrected image; otherwise, an FDG-PET scanned lesion is 
considered ‘negative.’ New lesions on the basis of FDG-PET imaging can be 
identified according to the following algorithm: 
a. Negative FDG-PET at baseline with a positive FDG-PET at 
follow-up is a sign of PD based on a new lesion. 
b. No FDG-PET at baseline and a positive FDG-PET at follow-up:  
- If the positive FDG-PET at follow-up corresponds to a 
new site of disease confirmed by CT, this is PD.  
- If the positive FDG-PET at follow-up is not confirmed 
as a new site of disease on CT at the same evaluation, 
additional follow-up CT scans (i.e., additional follow-
up scans at least 4 weeks later) are needed to determine 
if there is truly progression occurring at that site. In this 
situation, the date of PD will be the date of the initial 
abnormal PDG-PET scan.  
- If the positive FDG-PET at follow-up corresponds to a 
pre-existing site of disease on CT that is not 
progressing on the basis of the anatomic images, it is 
not classified as PD. 
11.3.3 Measurement at Follow-up Evaluation: 
• A subsequent scan must be obtained not less than 4 weeks following initial 
documentation of an objective status of either complete response (CR) or partial 
response (PR).  
• In the case of stable disease (SD), follow-up measurements must have met the 
SD criteria at least once after study entry at a minimum interval of not less 
than 6-8 weeks (see Section 11.44). 
• The cytological confirmation of the neoplastic origin of any effusion that 
appears or worsens during treatment when the measurable tumor has met 
criteria for response or stable disease is mandatory to differentiate between 

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response or stable disease (an effusion may be a side effect of the treatment) 
and progressive disease. 
• Cytologic and histologic techniques can be used to differentiate between PR 
and CR in rare cases (e.g., residual lesions in tumor types such as germ cell 
tumors, where known residual benign tumors can remain.) 
11.4 
Measurement of Treatment/Intervention Effect 
11.4.1 Target Lesions & Target Lymph Nodes 
• 
Measurable lesions (as defined in Section 11.2.1) up to a maximum of 5 lesions 
representative of all involved organs, should be identified as “Target Lesions” 
and recorded and measured at baseline. These lesions can be non-nodal or nodal 
(as defined in 11.2.1), where no more than 2 lesions are from the same organ 
and no more than 2 malignant nodal lesions are selected.  
Note: If fewer than 5 target lesions and target lymph nodes are identified (as there 
often will be), there is no reason to perform additional studies beyond those 
specified in the protocol to discover new lesions.  
• 
Target lesions and target lymph nodes should be selected on the basis of their 
size, be representative of all involved sites of disease, but in addition should be 
those that lend themselves to reproducible repeated measurements. It may be 
the case that, on occasion, the largest lesion (or malignant lymph node) does 
not lend itself to reproducible measurements in which circumstance the next 
largest lesion (or malignant lymph node) which can be measured reproducibly 
should be selected.  
• 
Baseline Sum of Dimensions (BSD): A sum of the longest diameter for all 
target lesions plus the sum of the short axis of all the target lymph nodes will 
be calculated and reported as the baseline sum of dimensions (BSD). The BSD 
will be used as reference to further characterize any objective tumor response 
in the measurable dimension of the disease.  
• 
Post-Baseline Sum of the Dimensions (PBSD): A sum of the longest diameter 
for all target lesions plus the sum of the short axis of all the target lymph nodes 
will be calculated and reported as the post-baseline sum of dimensions (PBSD). 
If the radiologist is able to provide an actual measure for the target lesion (or 
target lymph node), that should be recorded, even if it is below 0.5 cm. If the 
target lesion (or target lymph node) is believed to be present and is faintly seen 
but too small to measure, a default value of 0.5 cm should be assigned. If it is 
the opinion of the radiologist that the target lesion or target lymph node has 
likely disappeared, the measurement should be recorded as 0 cm. 
• 
The minimum sum of the dimensions (MSD) is the minimum of the BSD and 
the PBSD. 

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11.4.2 Non-Target Lesions & Non-Target Lymph Nodes 
Non-measurable sites of disease (Section 11.2.2) are classified as non- target 
lesions or non-target lymph nodes and should also be recorded at baseline. These 
lesions and lymph nodes should be followed in accord with Section 11.4.3.3. 
11.4.3 Response Criteria 
11.4.3.1 Measurement 
All target lesions and target lymph nodes followed by CT/MRI/PET-
CT/Chest X-ray must be measured on re-evaluation. Specifically, a change in 
objective status to either a PR or CR cannot be done without re-measuring 
target lesions and target lymph nodes. 
Note: Non-target lesions and non-target lymph nodes should be evaluated at 
each assessment, especially in the case of first response or confirmation of 
response. In selected circumstances, certain non-target organs may be 
evaluated less frequently. For example, bone scans may need to be repeated 
only when complete response is identified in target disease or when 
progression in bone is suspected. 
11.4.3.2 Evaluation of Target Lesions 
• Complete Response (CR): All of the following must be true: 
a. Disappearance of all target lesions. 
b. Each target lymph node must have reduction in short axis to 
<1.0 cm. 
• Partial Response (PR): At least a 30% decrease in PBSD (sum of the 
longest diameter for all target lesions plus the sum of the short axis of all 
the target lymph nodes at current evaluation) taking as reference the BSD 
(see Section 11.4.1). 
• Progression (PD): At least one of the following must be true: 
a. At least one new malignant lesion, which also includes any lymph 
node that was normal at baseline (<1.0 cm short axis) and increased 
to ≥1.0 cm short axis during follow-up.  
b. At least a 20% increase in PBSD (sum of the longest diameter for 
all target lesions plus the sum of the short axis of all the target lymph 
nodes at current evaluation) taking as reference the MSD (Section 
11.4.1). In addition, the PBSD must also demonstrate an absolute 
increase of at least 0.5 cm from the MSD.  
c. See Section 11.3.2 for details in regards to the requirements for PD 
via FDG-PET imaging. 
• Stable Disease (SD): Neither sufficient shrinkage to qualify for PR, nor 
sufficient increase to qualify for PD taking as reference the MSD. 

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11.4.3.3 Evaluation of Non-Target Lesions & Non-target Lymph Nodes 
• Complete Response (CR): 
All of the following must be true: 
a. Disappearance of all non-target lesions.  
b. Each non-target lymph node must have a reduction in short axis to <1.0 cm. 
• Non-CR/Non-PD: Persistence of one or more non-target lesions or non-
target lymph nodes. 
• Progression (PD): At least one of the following must be true: 
a. At least one new malignant lesion, which also includes any lymph node that 
was normal at baseline (<1.0 cm short axis) and increased to ≥1.0 cm short 
axis during follow-up.  
b. Unequivocal progression of existing non-target lesions and non-target lymph 
nodes. (NOTE: Unequivocal progression should not normally trump target 
lesion and target lymph node status. It must be representative of overall 
disease status change.)  
c. See Section 11.3.2 for details in regards to the requirements for PD via FDG-
PET imaging. 
11.4.4 Overall Objective Status 
The overall objective status for an evaluation is determined by combining the 
patient’s status on target lesions, target lymph nodes, non-target lesions, non-target 
lymph nodes, and new disease as defined in the following tables: 
For Patients with Measurable Disease 
Target Lesions & 
Target Lymph Nodes 
Non-Target Lesions & 
Non-Target Lymph 
Nodes 
New Sites of 
Disease 
Overall 
Objective Status 
CR 
CR 
No 
CR 
CR 
Non-CR/Non-PD 
No 
PR 
PR 
CR 
Non-CR/Non-PD 
No 
PR 
CR/PR 
Not All Evaluated* 
No 
PR** 
SD 
CR 
Non-CR/Non-PD 
Not All Evaluated* 
No 
SD 
Not all Evaluated 
CR 
Non-CR/Non-PD 
Not All Evaluated* 
No 
Not Evaluated 
(NE) 
PD 
Unequivocal PD 
CR 
Non-CR/Non-PD 
Not All Evaluated* 
Yes or No 
PD 

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Target Lesions & 
Target Lymph Nodes 
Non-Target Lesions & 
Non-Target Lymph 
Nodes 
New Sites of 
Disease 
Overall 
Objective Status 
CR/PR/SD/PD/Not all 
Evaluated 
Unequivocal PD 
Yes or No 
PD 
CR/PR/SD/PD/Not all 
Evaluated 
CR 
Non-CR/Non-PD 
Not All Evaluated* 
Yes 
PD 
* See Section 11.4.3.1 
11.4.5 Symptomatic Deterioration:  
Patients with global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time, and not 
either related to study treatment or other medical conditions, should be reported as 
PD due to “symptomatic deterioration.” Every effort should be made to document 
the objective progression even after discontinuation of treatment due to 
symptomatic deterioration. 
12. TREATMENT/FOLLOW–UP DECISION AT EVALUATION OF PATIENT 
12.1 
No disease progression or Unacceptable Adverse Events 
Patients who have not had disease progression and have not developed unacceptable 
adverse events will be eligible to continue treatment at their current dose level until disease 
progression, unacceptable adverse events, or refusal.  
12.2 
No disease Progression, Unacceptable Adverse Events  
Those patients who have not had disease progression, but have experienced unacceptable 
adverse events may be eligible to continue treatment at a lower dose (see Section 8.0). 
12.3 
Discontinuation of Protocol Therapy  
Reasons why the protocol therapy may be discontinued: 
• Tumor progression 
• Request by the patient to withdraw 
• Unacceptable adverse events 
• Inter-current illness  
• Administration of alternative treatment  
Patients who discontinue treatment for any of the above reasons will proceed to event 
monitoring. Patients will be followed yearly for a maximum of 5 years from study 
registration. Once a patient has discontinued study treatment, future therapy is at the 
discretion of the treating physician.  

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12.4 
Refuse to begin protocol treatment following registration 
If an eligible patient refuses to begin treatment following registration (and is classified as 
a cancel by the Research Base), all on-study materials and the End of Active 
Treatment/Cancel Notification Form must be submitted. For those patients who also 
consent to the correlative studies, research biospecimens are not to be submitted. No further 
data submission is necessary. Future therapy is at the discretion of the treating physician. 
12.5 
Ineligible prior to the start of protocol treatment after registration 
If a patient is determined not to have satisfied each and every eligibility criteria for study 
entry after registration but prior to the start of protocol treatment, on-study materials must 
be submitted. For those patients who also consent to the correlative studies, research 
biospecimens are not to be submitted. Bio-specimens should not be submitted. No further 
data submission is necessary. Future therapy is at the discretion of the treating physician. 
12.6 
Ineligible after the start of protocol treatment 
If a patient is determined not to have satisfied each and every eligibility criteria but has 
received some study treatment and is having some clinical benefit, the patient may be able 
to continue study treatment per protocol after consultation with the study chair.  
If the patient does continue on study treatment, study forms and bio-specimens should be 
submitted using the same schedule as eligible patients.  
If the patient does not continue study treatment, complete the End of Active Treatment 
Form. No further data submission is necessary. Future therapy is at the discretion of the 
treating physician. 
 
 

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13. ADVERSE EVENT REPORTING REQUIREMENTS 
Summary of SAE Reporting for this study  
(please read entire section for specific instructions): 
WHO: 
WHAT form: 
WHERE to send: 
All sites 
Pregnancy Reporting 
 
Mayo Sites – complete and 
attach to MCCC Electronic 
SAE Reporting Form 
Will automatically be sent to 
 
Non Mayo sites – complete and 
forward to 
 
Mayo 
Clinic Sites 
MedWatch 3500A: 
AND attach to 
Mayo Clinic Cancer Center SAE Reporting 
Form:
  
Will automatically be sent to 
 
 
 
 
Non-Mayo 
Clinic Sites 
  
 
Send to: 
 
 
 
13.1 
General 
Adverse event collection and reporting is a routine part of every clinical trial. This study 
will use the descriptions and grading scales found in the NCI Common Terminology 
Criteria for Adverse Events current version (CTCAE v5.0) that is available at 
 
Information on all adverse events, whether reported by the participant, directly observed, 
or detected by physical examination, laboratory test or other means, will be collected, 
recorded, followed and reported as described in the following sections.  

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Adverse events experienced by participants will be collected and reported from initiation 
of study medication, throughout the study, and within 30 days of the last dose of study 
medication. Participants who experience an ongoing adverse event related to a study 
procedure and/or study medication beyond 30 days will continue to be contacted by a 
member of the study team until the event is resolved, stabilized, or determined to be 
irreversible by the participating investigator.  
Participants should be instructed to report any serious post-study event(s) that might 
reasonably be related to participation in this study. The investigator should notify the IRB 
and any other applicable regulatory agency of any unanticipated death or adverse event 
occurring after a participant has discontinued or terminated study participation that may 
reasonably be related to the study.  
13.2 
Definitions 
13.2.1 Adverse Event (AE) 
An adverse event is any undesirable sign, symptom or medical condition or 
experience that develops or worsens in severity after starting the first dose of study 
treatment or any procedure specified in the protocol, even if the event is not 
considered to be related to the study.  
Abnormal laboratory values or diagnostic test results constitute adverse events only 
if they induce clinical signs or symptoms or require treatment or further diagnostic 
tests.  
13.2.2 Serious adverse event (SAE) 
A serious adverse event is an undesirable sign, symptom, or medical condition 
which: 
• 
is fatal;  
• 
is life-threatening; 
• 
requires or prolongs inpatient hospitalization for ≥24 hours; 
• 
results in persistent or significant disability/incapacity to conduct normal life 
functions; 
• 
constitutes a congenital anomaly or birth defect; or 
• 
jeopardizes the participant and requires medical or surgical intervention to 
prevent one of the outcomes listed above; 
• 
Important Medical Events (IME) that may not result in death, be life 
threatening, or require hospitalization may be considered serious when, based 
upon medical judgment, they may jeopardize the patient or subject and may 
require medical or surgical intervention to prevent one of the outcomes listed 
in this definition. 
Events not considered to be serious adverse events are hospitalizations for: 
• 
routine treatment or monitoring of the studied indication, not associated with 
any deterioration in condition, or for elective procedures 
• 
elective or pre-planned treatment for a pre-existing condition that did not 
worsen, or that is required per protocol 

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• 
emergency outpatient treatment for an event not fulfilling the serious criteria 
outlined above and not resulting in inpatient admission 
• 
respite care 
• 
Death due to disease progression unless attributable to the study drug(s) 
• 
A hospitalization due to an expected adverse event (e.g., hospitalization due to 
expected febrile neutropenia). 
13.2.3 Expectedness 
• 
Expected: Expected adverse events are those that have been previously 
identified as resulting from administration of the agent. For the purposes of this 
study, an adverse event is considered expected when it appears in the current 
adverse event list, the Investigator’s Brochure, the package insert or is included 
in the informed consent document as a potential risk. 
• 
Unexpected: An adverse event is considered unexpected when it varies in 
nature, intensity or frequency from information provided in the current adverse 
event list, the Investigator’s Brochure, the package insert or when it is not 
included in the informed consent document as a potential risk 
13.2.4 Attribution  
Attribution is the relationship between an adverse event or serious adverse event 
and the study treatment. Attribution will be assigned as follows: 
• 
Definite – The AE is clearly related to the study treatment. 
• 
Probable – The AE is likely related to the study treatment. 
• 
Possible – The AE may be related to the study treatment.  
• 
Unlikely - The AE is doubtfully related to the study treatment. 
• 
Unrelated - The AE is clearly NOT related to the study treatment. 
13.3 
Reporting Procedures 
13.3.1 General 
All adverse events will be captured on the appropriate study-specific case report 
forms (CRFs).  
13.3.2 Serious Adverse Events 
All serious adverse events, regardless of causality to study drug, will be reported to 
the Principal Investigator and/or the Study Coordinator at each institution, and also 
to the Coordinating Center. 
All serious adverse events must be reported to the Coordinating Center within 
1 business day after the investigator becomes aware of the event. Events should be 
reported using a MedWatch form (3500A) as available on the FDA website (see 
link below).  
Follow-up information must also be reported within 1 business day of receipt of the 
information by the investigator. 

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The Coordinating Center will disseminate information regarding serious adverse 
events to the participating sites within 5 days of review of the information by the 
Protocol Chair (or her designee in the event of extended absence) only in the case 
that the event(s) is believed to be related (i.e., possibly, probably, or definitely) to 
the study medication. The Coordinating Center will be responsible for reporting of 
events to the FDA and supporters, as appropriate (outlined below). 
13.3.3 Institutional Review Board 
All adverse events and serious adverse events will be reported to the IRB per 
current institutional standards. If an adverse event requires modification of the 
informed consent, these modifications will be provided to the IRB with the report 
of the adverse event. If an adverse event requires modification to the study protocol, 
these modifications will be provided to the IRB as soon as is possible. 
13.3.4 Food and Drug Administration (FDA) 
In this trial, unexpected adverse events believed to be definitely, probably, or 
possibly related to the medications will be reported to the Food and Drug 
Administration via MedWatch. The Coordinating Center will be responsible for 
correspondence regarding adverse events with the FDA for all participating sites. 
Sites will be instructed the method by which to report events to the Coordinating 
Center and per what forms (e.g., mandatory MedWatch 3500/3500A forms 
available at: 
 
14. REGULATORY CONSIDERATIONS 
14.1 
Protocol Review and Amendments 
Unless otherwise specified, each participating institution must obtain its own IRB approval. 
It is expected that the IRB will have the proper representation and function in accordance 
with federally mandated regulations. The IRB should approve the consent form and 
protocol. 
Information regarding study conduct and progress will be reported to the Institutional 
Review Board (IRB) per the current institutional standards of each participating center. 
Any changes to the protocol will be made in the form of an amendment and must be 
approved by the IRB of each institution prior to implementation.  
The Protocol Chair (or his designee) is responsible for the coordination and development 
of all protocol amendments, and will disseminate this information to the participating 
centers. 
14.2 
Informed Consent 
The investigator (or his/her designee) will explain to each subject the nature of the study, 
its purpose, the procedures involved, the expected duration, the potential risks and benefits 
involved and any discomfort it may entail. Each subject will be informed that participation 
in the study is voluntary, that s/he may withdraw from the study at any time, and that 

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withdrawal of consent will not affect her subsequent medical treatment or relationship with 
the treating physician(s) or institution. The informed consent will be given by means of a 
standard written statement, written in non-technical language, which will be IRB approved. 
The subject should read and consider the statement before signing and dating it, and will 
be given a copy of the document. No subject will enter the study or have study-specific 
procedures done before his/her informed consent has been obtained. 
In accordance with the Health Information Portability and Accountability Act (HIPAA), 
the written informed consent document (or a separate document to be given in conjunction 
with the consent document) will include a subject authorization to release medical 
information to the study sponsor and supporting agencies and/or allow these bodies, a 
regulatory authority, or Institutional Review Board access to subjects’ medical information 
that includes all hospital records relevant to the study, including subjects’ medical history. 
14.3 
Ethics and GCP 
This study will be carried out in compliance with the protocol and Good Clinical Practice, 
as described in: 
1. ICH Harmonized Tripartite Guidelines for Good Clinical Practice 1996. 
2. US 21 Code of Federal Regulations dealing with clinical studies (including parts 50 
and 56 concerning informed consent and IRB regulations). 
3. Declaration of Helsinki, concerning medical research in humans (Recommendations 
Guiding Physicians in Biomedical Research Involving Human Subjects, Helsinki 1964, 
amended Tokyo 1975, Venice 1983, Hong Kong 1989, Somerset West 1996). 
The investigator agrees to adhere to the instructions and procedures described in it and 
thereby to adhere to the principles of Good Clinical Practice that it conforms to. 
14.4 
Compliance with Trial Registration and Results Posting Requirements 
Under the terms of the Food and Drug Administration Modernization Act (FDAMA) and 
the Food and Drug Administration Amendments Act (FDAAA), the sponsor-investigator 
of the trial is solely responsible for determining whether the trial and its results are subject 
to 
the 
requirements 
for 
submission 
to 
the 
Clinical 
Trials 
Data 
Bank, 
 Information posted will allow subjects to identify potentially 
appropriate trials for their disease conditions and pursue participation by calling a central 
contact number for further information on appropriate trial locations and study site contact 
information. 
15. MULTI-CENTER GUIDELINES 
15.1 
Study Documentation 
Each participating site is responsible for submitting copies of all relevant regulatory 
documentation to the Coordinating Center. The required documents include, but are not 
limited to the following: local IRB approvals (i.e., protocol, consent form, amendments, 
patient brochures and recruitment material, etc.), IRB membership rosters, summary of 
unanticipated problems or protocol deviations, and documentation of expertise of the 
investigators. The Coordinating Center will provide each participating site with a 

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comprehensive list of the necessary documents. It is the responsibility of the participating 
sites to maintain copies of all documentation submitted to the Coordinating Center. 
The requirements for data management, submissions, and monitoring are outlined in the 
Data Safety Monitoring Plan [provided separately]. 
15.2 
Records Retention 
Following closure of the study, each participating center will maintain a copy of all site 
study records in a safe and secure location. The Coordinating Center will inform the 
investigator at each site at such time that the records may be destroyed. 
15.3 
Publication 
It is understood that any manuscript or releases resulting from the collaborative research 
must be approved by the Protocol Chair and will be circulated to applicable participating 
sites/investigators prior to submission for publication or presentation.  
Additionally, any publication of study data and results must conform to the publications 
policy as stated the Translational Breast Cancer Research Consortium’s (TBCRC) 
“Policies and Procedures”. 
16. STATISTICAL CONSIDERATIONS 
An archived FFPE tumor tissue from a locally advanced or metastatic site or from the 
primary tumor specimen if a locally advanced or metastatic site is not available will be 
submitted to the Mayo Clinic CAP/CLIA-certified Anatomic Pathology Laboratory for 
ERβ testing. If the specimen is found to be ERβ+,  the patient may registered to the study.   
 
If the specimen submitted was from a tissue-based biopsy of a local recurrence or 
metastatic site taken at most 12 months prior to Pre-Registration, a new biospy is not 
required prior to the start of treatment.  These patients will be included in the ERβ+ 
cohort for analysis purposes. 
 
If the specimen submitted is not from a tissue-based biopsy of a local recurrence or 
metastatic site taken at most 12 months prior to Pre-Registration, a new biopsy of a local 
recurrence or metastatic site must be performed prior to the start of treatment and the 
specimen submitted for central laboratory testing and correlative research aims.   The 
results of this ERβ testing does not impact whether the patient remains on protocol 
treatment.  It does however impact how the primary and secondary endpoints are 
accessed.  If this new tissue specimen is found to be ERβ+, the patient will be included in 
the ERβ+ cohort for analysis purposes.  If this new tissue specimen is found to be ERβ-, 
the patient will be included in the ERβ- cohort for analysis purposes.   
 
All eligible patients who begin treatment will be included in the analyses of the primary 
and secondary clinical endpoints. 
 
 
 
 

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16.1 
Study Design 
Primary endpoint of this trial:  6 month clinical benefit rate 
A patient is said to have derived clinical benefit rate at the 6 month time point if the 
patient’s disease meets the RECIST criteria for complete response (CR), partial response 
(PR), or stable disease (SD) for >6 months following initiation of treatment. The 6 month 
clinical benefit rate is the percentage of patients who are found to meet the criteria for 
clinical benefit at least 6 months among all the patients who have started estradiol 
treatment. 
 
ERβ+ cohort 
A single-stage, phase II trial will be conducted to assess independently the 6 month 
clinical benefit rate (CBR6m) among patients with moderate to strong ERβ expressing 
metastatic TNBC treated with estradiol.  
With a sample size of 38 patients with moderate to strong ERβ expressing TNBC, a one-
sided α=0.10 test of binomial proportions would have a 90% chance of rejecting the null 
hypothesis that the CBR6m is ≤5% when the true CBR6m is ≥20% in this patient 
population. 
 
ERβ- cohort 
As the number of patients with discordant ERβ findings are expected to be small,  a 90% 
exact binomial confidence interval will be constructed for the proportion of patients who 
were found to have no to weak ERβ expressing metastatic TNBC and who derived 
clinical benefit rate at the 6 month time point. 
 
For patients with a history of moderate to strong ERβ expressing TNBC who are 
registered onto this study, it is unknown what percentage of these patients will not have 
moderate to strong ERβ expression found in their pre-registration biopsy specimen. After 
every 5th patient is registered to the study (up to 30 patients), we will examine the number 
of the patients who do not have moderate to strong ERβ expression found in their pre-
registration biopsy specimen as follows: 
If ≥2 of the first 5 patients registered,  
≥2 of the first 10 patients registered,  
≥2 of the first 15 patients registered,  
≥3of the first 20 patients registered,  
≥3 of the first 25 patients registered,  
or ≥4 of the first 30 patients registered  
are found to have no or weak ERβ expression, the trial will temporarily close to assess 
whether the screening/pre-registration process should be modified to ensure accrual only 
for those patients with moderate to strong ERβ expressing metastatic TNBC. 

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Based on the experience of TBCRC 011 enrolling patients with histologically confirmed 
ERα negative, PgR-negative MBC who consented for AR testing, we expect 20% of 
patients to have moderate to strong ERβ expression found in the archived locally 
recurrent or metastatic TNBC specimen they submit in the prescreening phase. Also, we 
anticipate that 5-10% of these patients may be found to have no or weak ERβ expression 
found in their pre-registration biopsy specimen. Thus, we anticipate pre-screening as 
many as 210 patients over 24-30 months to enroll 38 patients whose pre-registration 
biopsy specimen is found to have moderate to strong ERβ expression. 
16.1.1 Interim analysis for futility and toxicity 
An interim analysis for futility among the patients whose pre-registration biopsy 
specimen is found to have moderate to strong ERβ expression will be carried out 
based on Simon Two-stage Optimum Phase II trial design for testing the null 
hypothesis that the true CBR6m is at most 5% against the alternative hypothesis 
that the true CBR6m is at least 20% with the significance level set at 0.10 and the 
power at 0.90. Specifically, after the first 19 eligible patients have enrolled and 
either completed 6 months of estradiol or discontinued treatment (for any reason) 
prior to completing 6 months of treatment, an interim analysis will be performed. 
If at most 1 patient has remained on treatment more than 6 months, then the trial 
will be closed to enrollment due to futility. The probability of early termination is 
0.755.  
Safety Stopping Rule for both patient cohorts combined (as the ERβ results for those 
whose submit a second biopsy sample will be delayed) 
16.1.2  
If treatment intolerability is encountered, the trial will temporarily suspend 
enrollment so that the study team can review the toxicity data and develop a 
recommendation to present to MCCC DSMB and IRB for approval. 
We will temporarily halt enrollment: 
- If 2 or more of the first 6 patients enrolled, develop a Grade 3+ 
hypercalcemia, thrombosis, or pleural effusions, or any Grade 4+ event 
considered to be at least possibly related to treatment during the first cycle 
of treatment. 
- If at any point in the trial after the first 6 patients have been enrolled, the 
percentage of patients who develop a Grade 3+ hypercalcemia, 
thrombosis, or pleural effusion, or any Grade 4+ event considered to be at 
least possibly related to treatment at any point in their treatment exceeds 
30%. 
16.2 
Analysis of Secondary Endpoints for each patient cohort independently 
16.2.1 Adverse Events 
An adverse event (AE) is any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease temporally associated with the 

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use of a medical treatment or procedure that may or may not be considered related 
to the medical treatment or procedure. All Grade 2, 3, 4 or 5 adverse events will 
be documented and assigned an attribute by treating clinician as to its relationship 
to treatment. 
For a given AE, the proportion of patients who report developing a Grade 2-5 of 
this AE will be determined. 
The number of dose reductions per patient and the reasons for the dose reduction 
will be summarized. 
16.2.2 Tumor Response Rate among those patients with measurable disease 
The tumor response rate is defined as the 100% time the number of patients with a 
CR or PR (as defined by the RECIST criteria) on 2 consecutive evaluations at 
least 8 weeks apart divided by the total number of eligible patients who began 
study treatment. A 90% binomial confidence interval will be constructed for the 
true response rate. 
16.2.3 Progression-free survival distribution  
Progression-free survival time is defined as the time from registration to the first 
of the following events: local, regional, or distant recurrence, second primary 
disease or death due to any cause. The distribution of PFS times will be estimated 
using the method of Kaplan-Meier. 
16.2.4 Overall survival distribution  
Overall survival time is defined as the time from registration to death due to any 
cause. The distribution of survival times will be estimated using the method of 
Kaplan-Meier. 
16.2.5 Evaluation of changes in phospho-ERβ, cystatins 1, 2, 4 and 5, phospho-
Smad2/3 and Ki-67.  
Patients will undergo tumor biopsies prior to the start of treatment and at 
completion of cycle 1 treatment. These specimens will be undergo IHC staining 
with the following antibodies: phospho-ERβ, cystatins 1, 2, 4 and 5, phospho-
Smad2/3 and Ki-67. These data will be used to examine the hypothesis that 
estradiol will activate ERβ resulting in (1) downregulation of Ki-67, (2) reduction 
in phospho-Smad2/3 (3) increase in phosphorylation (activation) of ERβ and 
finally (4) increase in expression levels of cystatins 1, 2, 4, and 5. 
For each of these biomarkers, a times series plot will be constructed so that an 
individual patient’s data will be represented using the same color for each of the 
five graph. These graphs will be visually inspected for trends within each of the 
graphs (variation between individuals) as well as across the five graphs (profile of 
biomarker changes within an individual). 

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16.2.6 Changes in serum cystatin levels in response to treatment 
Blood specimens will be acquired prior to the start of treatment and at completion 
of cycle 1 treatment. Serum will undergo ELISA testing to obtain cystatins 1, 2, 4 
and 5 levels. These data will be used to examine the hypothesis that the clinical 
benefit of estradiol will be associated with an increased in cystatins 1, 2, 4 and 5. 
For each of the cystatins, the percent change in its level following one cycle of 
treatment will examined using signed rank tests and the difference in the percent 
change in its level following one cycle of treatment between those patients who 
derived clinical benefit and those who did not will be examined using a two sample 
Wilcoxon rank sum test.  
16.3 
Monitoring  
The study chair and the faculty statistician will review the trial data every 3 months 
to identify accrual, toxicity, and endpoint problems that might be developing. The 
faculty and secondary statistician will prepare a report containing accrual, adverse 
events, and efficacy data which will be submitted to the Data and Safety Monitoring 
Board on a semi-annual basis. 
16.4 
Other Considerations 
Adverse events, patterns of treatment failure observed in this study and scientific 
discoveries or changes in standard care will be taken into account in any decision 
to terminate this trial earlier than anticipated. 
16.5 
Inclusion of Women and Minorities 
This study will be available to all eligible patients regardless of race or ethnic 
group. The expected number of patients per racial/ethnicity categories are 
presented in the following table. The sample size for this trial was not increased in 
order to provide additional power for analyses by race or ethnicity. 
NOTE: We have excluded men from enrollment in this trial for two reasons:  
1) Breast cancer is not common in males so only one or two patients are likely to 
be enrolled; and  
2) Because this trial is small, the addition of one or two male patients may 
confound the results without providing meaningful data. 
 
 

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Accrual Targets 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
21 
 
21 
Not Hispanic or Latino 
189 
 
189 
Ethnic Category: Total of all subjects 
210 
0 
210 
Racial Category 
American Indian or Alaskan Native 
 
 
 
Asian 
8 
 
8 
Black or African American 
8 
 
8 
Native Hawaiian or other Pacific Islander 
 
 
 
White 
194 
 
194 
Racial Category: Total of all subjects 
210 
0 
210 
 
Ethnic 
Categories: 
Hispanic or Latino – a person of Cuban, Mexican, Puerto Rican, South or 
Central American, or other Spanish culture or origin, regardless of race. The 
term “Spanish origin” can also be used in addition to “Hispanic or Latino.” 
Not Hispanic or Latino 
Racial 
Categories: 
American Indian or Alaskan Native – a person having origins in any of the 
original peoples of North, Central, or South America, and who maintains tribal 
affiliations or community attachment. 
Asian – a person having origins in any of the original peoples of the Far East, 
Southeast Asia, or the Indian subcontinent including, for example, Cambodia, 
China, India, Japan, Korea, Malaysia, Pakistan, the Philippine Islands, 
Thailand, and Vietnam. (Note: Individuals from the Philippine Islands have 
been recorded as Pacific Islanders in previous data collection strategies.)  
Black or African American – a person having origins in any of the black 
racial groups of Africa. 
Native Hawaiian or other Pacific Islander – a person having origins in any of 
the original peoples of Hawaii, Guam, Samoa, or other Pacific Islands. 
White – a person having origins in any of the original peoples of Europe, the 
Middle East, or North Africa. 
 
 
 

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17. REFERENCES 
Alvarez-Diaz, S., N. Valle, J. M. Garcia, C. Pena, J. M. Freije, V. Quesada, A. Astudillo, F. Bonilla, C. 
Lopez-Otin, and A. Munoz. 2009. 'Cystatin D is a candidate tumor suppressor gene induced by 
vitamin D in human colon cancer cells', J Clin Invest, 119: 2343-58. 
Bhola, N. E., J. M. Balko, T. C. Dugger, M. G. Kuba, V. Sanchez, M. Sanders, J. Stanford, R. S. Cook, and 
C. L. Arteaga. 2013. 'TGF-beta inhibition enhances chemotherapy action against triple-negative 
breast cancer', J Clin Invest, 123: 1348-58. 
Bierie, B., and H. L. Moses. 2009. 'Gain or loss of TGFbeta signaling in mammary carcinoma cells can 
promote metastasis', Cell Cycle, 8: 3319-27. 
Bjohle J, Bergqvist J, Gronowitz JS, Johansson H, Carlsson L, Einbeigi Z, et al. Serum thymidine 
kinase activity compared with CA 15-3 in locally advanced and metastatic breast cancer 
within a randomized trial. Breast cancer research and treatment. 2013;139(3):751-8. 
Briggs, J. J., M. H. Haugen, H. T. Johansen, A. I. Riker, M. Abrahamson, O. Fodstad, G. M. Maelandsmo, 
and R. Solberg. 2010. 'Cystatin E/M suppresses legumain activity and invasion of human 
melanoma', BMC Cancer, 10: 17. 
Chen F, Tang L, Xia T, He E, Hu G, Li Y, et al. Serum thymidine kinase 1 levels predict cancer-
free survival following neoadjuvant, surgical and adjuvant treatment of patients with 
locally advanced breast cancer. Molecular and clinical oncology. 2013;1(5):894-902. 
Ellis, M. J., F. Gao, F. Dehdashti, D. B. Jeffe, P. K. Marcom, L. A. Carey, M. N. Dickler, P. Silverman, G. 
F. Fleming, A. Kommareddy, S. Jamalabadi-Majidi, R. Crowder, and B. A. Siegel. 2009. 'Lower-
dose vs high-dose oral estradiol therapy of hormone receptor-positive, aromatase inhibitor-resistant 
advanced breast cancer: a phase 2 randomized study', JAMA, 302: 774-80. 
Giampieri, S., C. Manning, S. Hooper, L. Jones, C. S. Hill, and E. Sahai. 2009. 'Localized and reversible 
TGFbeta signalling switches breast cancer cells from cohesive to single cell motility', Nat Cell Biol, 
11: 1287-96. 
Jin, L., Y. Zhang, H. Li, L. Yao, D. Fu, X. Yao, L. X. Xu, X. Hu, and G. Hu. 2012. 'Differential secretome 
analysis reveals CST6 as a suppressor of breast cancer bone metastasis', Cell Res, 22: 1356-73. 
Matise, L. A., M. W. Pickup, and H. L. Moses. 2009. 'TGF-beta helps cells fly solo', Nat Cell Biol, 11: 
1281-4. 
Reese, J. M., E. S. Bruinsma, D. G. Monroe, V. Negron, V. J. Suman, J. N. Ingle, M. P. Goetz, and J. R. 
Hawse. 2017. 'ERbeta inhibits cyclin dependent kinases 1 and 7 in triple negative breast cancer', 
Oncotarget, 8: 96506-21. 
Reese, J. M., E.S. Bruisnma, A.W. Nelson, I. Chernukhin, J.S. Carroll, Y. Li, M. Subramaniam, V. J. 
Suman, V. Negron, D. G. Monroe, J. N. Ingle, M. P. Goetz, and J. R. Hawse. 2018. 'ERβ-mediated 
induction of cystatins results in suppression of TGFβ signaling and inhibition of triple negative 
breast cancer metastasis', PNAS. 
Reese, J. M., V. J. Suman, M. Subramaniam, X. Wu, V. Negron, A. Gingery, K. S. Pitel, S. S. Shah, H. E. 
Cunliffe, A. E. McCullough, B. A. Pockaj, F. J. Couch, J. E. Olson, C. Reynolds, W. L. Lingle, T. 
C. Spelsberg, M. P. Goetz, J. N. Ingle, and J. R. Hawse. 2014. 'ERbeta1: characterization, 
prognosis, and evaluation of treatment strategies in ERalpha-positive and -negative breast cancer', 
BMC Cancer, 14: 749. 
Shridhar, R., J. Zhang, J. Song, B. A. Booth, C. G. Kevil, G. Sotiropoulou, B. F. Sloane, and D. Keppler. 
2004. 'Cystatin M suppresses the malignant phenotype of human MDA-MB-435S cells', Oncogene, 
23: 2206-15. 
Thomas S, Liu N, Luo RJ, Hoog J, Bergqvist M, Neumüller M, Suh EM, Guo Z, Vij K, Sanati S, 
Ellis M, Ma CX. Serum Thymidine Kinase 1 Activity as a Pharmacodynamics Marker of 

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Cyclin-Dependent Kinase 4/6 Inhibition in Patients with Early Stage Breast Cancer 
Receiving Neoadjuvant Palbociclib. Presented at the annual San Antonio Breast Cancer 
Symposium (SABCS), December 6-10 2016, San Antonio TX. Abstract P5-04-02. 
Wegiel, B., T. Jiborn, M. Abrahamson, L. Helczynski, L. Otterbein, J. L. Persson, and A. Bjartell. 2009. 
'Cystatin C is downregulated in prostate cancer and modulates invasion of prostate cancer cells via 
MAPK/Erk and androgen receptor pathways', PLoS One, 4: e7953. 
Zhang, J., R. Shridhar, Q. Dai, J. Song, S. C. Barlow, L. Yin, B. F. Sloane, F. R. Miller, C. Meschonat, B. 
D. Li, F. Abreo, and D. Keppler. 2004. 'Cystatin m: a novel candidate tumor suppressor gene for 
breast cancer', Cancer Res, 64: 6957-64. 
 
 

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APPENDICES 
Appendix A: ECOG Performance Status Scale 
Appendix B: Study Drug Diary 
 
 

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APPENDIX A: ECOG Performance Status Scale 
 
ECOG PERFORMANCE STATUS* 
Grade ECOG 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out 
work of a light or sedentary nature, e.g., light house work, office work 
2 
Ambulatory and capable of all selfcare but unable to carry out any work 
activities. Up and about more than 50% of waking hours 
3 
Capable of only limited selfcare, confined to bed or chair more than 50% of 
waking hours. 
4 
Completely disabled. Cannot carry on any selfcare. Totally confined to bed or 
chair. 
5 
Dead 
*As published in Am. J. Clin. Oncol.: 
Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., McFadden, E.T., Carbone, P.P.: 
Toxicity And Response Criteria Of The Eastern Cooperative Oncology Group. Am J Clin Oncol 5:649-
655, 1982. 
The ECOG Performance Status is in the public domain therefore available for public use. To duplicate the 
scale, please cite the reference above and credit the Eastern Cooperative Oncology Group, Robert Comis 
M.D., Group Chair. 
 
 
 
 
 

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APPENDIX B: Study Drug Diary 
PLEASE NOTE: Study Drug Diary for patient care use is posted as a standalone document. 
Remember to submit to your local IRB before using with a patient.