Clinical trial NCT02729701
Conditions: Breast Cancer
Sponsor: University of Kansas Medical Center
Start date: 2016-05-01; Completion date: 2019-06-01
Enrollment: 28 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to learn whether daily use of Duavee® is accepted and tolerated by peri- and post-menopausal women at moderate risk for development of breast cancer.

Detailed description:
Duavee® is tissue specific estrogen complex of bazedoxifene plus conjugated estrogen which is FDA approved for relief of menopausal symptoms and prevention of osteoporosis in women with a uterus who have not been diagnosed with estrogen dependent neoplasia. The overall purpose of this research is to demonstrate in a preliminary fashion that despite reduction in menopausal symptoms, (Duavee®) does not increase and may decrease proliferation in benign breast tissue in a cohort of peri- or post-menopausal women at moderately increased risk for breast cancer. If this pilot shows rapid accrual, good retention, and lack of significant increase in the risk biomarker Ki-67 in benign breast tissue, a larger prevention trial is envisioned

Arms: arm 1: Participants will be asked to take Duavee for 6 months while on the study.
Interventions: intervention 1: Duavee
Intervention descriptions: intervention 1: Once daily tablet of Duavee (Bazedoxifene (20 mg) plus conjugated estrogen (0.45 mg))

Protocol documents:
Prot_SAP_000.pdf: 
 
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COVER PAGE 
 
Protocol #: 
 
 
 
 
 
STUDY 00002440 
 
 
 
PROTOCOL TITLE 
 
Pilot Study of the Effect of Duavee® on Benign Breast Tissue Proliferation  
in Peri or Post-Menopausal Women  
at Moderate Risk for Development of Breast Cancer 
 
 
 
Principal Investigator:  
Carol J. Fabian, MD 
Protocol Chair 
 
University of Kansas Medical Center 
 
 
2330 Shawnee Mission Parkway, Mailstop 5015 
 
 
Westwood, KS  66205 
 
 
Phone: 913-588-7791 
 
 
Fax: 913-588-3679 
 
 
cfabian@kumc.edu 
 
Co-Investigators: 
 
Bruce F. Kimler, Ph.D. 
 
 
Carola M. Zalles, M.D. 
 
IND: 
 
FDA IND Exempt (10 April 2015) 
 
 
Agent(s)/Supplier: 
Duavee® (Bazedoxifene/conjugated estrogens) 
 
provided by Pfizer/Wyeth Pharmaceuticals, Inc. 
 
 
Protocol Version:       Revision or Amendment #  
Date   
 
 
Approval 
 
Version 1.0c  
 
 
 
 
24 June 2016 
7 July 2016 
 
Version 1.0b  
 
 
 
 
25 April 2016  
4 May 2016 
 
Version 1.0a  
 
 
 
 
4 March 2016 
10 March 2016 
 
Version 1.0 
 
 
 
 
 
6 July 2015  
10 July 2015 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
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SCHEMA 
Pilot Study of the Effect of Duavee® on Benign Breast Tissue Proliferation  
in Peri or Post-menopausal Women 
at Moderate risk for Development of Breast Cancer 
 
Population 
Peri- or post-menopausal women <65 with a uterus and BMI < 36 kg/m2 who are 
not taking systemic hormone replacement therapy, who exhibit vasomotor symptoms, 
and who are at moderate risk of developing breast cancer based on any of the following: 
1st or 2nd degree relative with breast cancer,  estimated mammographic breast density  
25% or higher , prior biopsy showing proliferative breast disease, multiple prior biopsies, 
or by having either a 5-year risk Gail Risk (as calculated by the NCI Breast Cancer Risk 
Assessment Tool) or 10-year risk by the IBIS Breast Cancer Risk Evaluation Tool of 2X 
or more that of the average for the age group.  
Women at very high risk such as those with a BRCA1/2 mutation or LCIS  
are not eligible. 
 
Screening/Eligibility 
Normal mammogram within 6 months of aspiration  
If performed at KUMC, then eligible for optional volumetric (Volpara) and area (Cumulus) 
assessment of density  
RPFNA yielding >500 cells on the Ki-67 and/or cytology slide; 
Ki-67 ≤4% positivity (500 or more cells) 

Baseline Study Visit 
Fasting blood draw to archive for serum SHBG, FSH, estradiol, testosterone, 
progesterone (progesterone for peri-menopausal only), chemistry profile, cytokine panel, 
and bazedoxifene levels. If peri-menopausal, negative pregnancy test required 
DEXA Scan for bone density and body composition; Medical history and physical; 
Height/weight 
29 item validated Menopause Quality of Life Intervention Questionnaire and hot flash 
assessment 

Total of 40 participants will receive 6-8 months of open-label Duavee® one tablet daily 

End of Study (6-8 months) 
Brief physical, height/weight,  
29 item validated Menopause Quality of Life Intervention Questionnaire and hot flash 
assessment. Study Agent Stopped; Study Agent Returned; Compliance Calculated 
Procedures 
Mammogram for volumetric and area density (if baseline at KUMC); DEXA; RPFNA 
Fasting blood draw to archive for serum SHBG, FSH estradiol, testosterone, 
progesterone (peri-menopausal only), chemistry profile, cytokine panel, and 
bazedoxifene levels  


 
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TABLE OF CONTENTS 
 
 
Page 
COVER PAGE .............................................................................................................................. 1 
SCHEMA ....................................................................................................................................... 2 
TABLE OF CONTENTS ................................................................................................................ 3 
LIST OF ABBREVIATIONS ........................................................................................................... 5 
1.  OBJECTIVES ............................................................................................................. 6 
1.1   Primary Feasibility Objective ................................................................................ 6 
1.2  Secondary Exploratory Objectives ........................................................................ 6 
2.  BACKGROUND .......................................................................................................... 7 
2.1 The Scope of the Problem ..................................................................................... 7 
2.2 Rationale for Present Proposal .............................................................................. 7 
2.3  Proliferation in Hyperplastic Benign Breast Tissue as a Response Endpoint ....... 8 
2.4  Other Risk and Mechanism of Action Biomarkers in Benign Breast Tissue Likely 
to be Modulated by Duavee® ...................................................................................... 9 
2.5  Mammographic Breast Density as Risk and Response Biomarker ..................... 10 
2.6  Serum risk Biomarkers as Response Endpoints ................................................. 10 
3.  SUMMARY OF STUDY PLAN .................................................................................. 12 
4.  PARTICIPANT SELECTION ..................................................................................... 13 
4.1  Inclusion Criteria for Screening RPFNA .............................................................. 13 
4.2   Exclusion Criteria for Screening RPFNA ........................................................... 13 
4.3  Inclusion Criteria for Intervention Phase ............................................................. 14 
4.4  Exclusion Criteria for Study Intervention ............................................................. 14 
4.5  Inclusion of Women and Minorities ..................................................................... 15 
4.6  Recruitment and Retention Plan ......................................................................... 15 
5.  AGENT ADMINISTRATION ...................................................................................... 16 
5.1   Dose Regimen and Dose Groups ...................................................................... 16 
5.2   Duavee® Administration .................................................................................... 16 
5.3  Concomitant Medications .................................................................................... 16 
5.4  Dose Modification ............................................................................................... 16 
5.5  Adherence/Compliance ....................................................................................... 17 
6.  PHARMACEUTICAL INFORMATION ....................................................................... 18 
6.1  Duavee® Provider .............................................................................................. 18 
6.2  Pharmacology ..................................................................................................... 18 
6.3  Mutagenesis, Carcinogenesis and Fertility ......................................................... 19 
6.4  Pfizer Sponsored Clinical Studies ....................................................................... 19 
6.5  Special Considerations ....................................................................................... 19 
6.6  Availability and Agent Distribution ....................................................................... 19 
6.7  Agent Accountability ............................................................................................ 19 
6.8  Packaging and Labels ........................................................................................ 20 
6.9  Storage ............................................................................................................... 20 
6.10  Registration/Randomization .............................................................................. 20 
6.11  Blinding and Unblinding Methods ...................................................................... 20 
6.12  Agent Destruction/Disposal ............................................................................... 20 


 
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7.  CLINICAL EVALUATIONS AND PROCEDURES ..................................................... 21 
7.1  Schedule of Events ............................................................................................. 21 
7.2   Baseline Testing/Pre-study Evaluation ............................................................... 22 
7.3  Evaluations During Study Intervention ................................................................ 23 
7.4  Evaluation at Completion of Intervention ............................................................ 24 
7.5  Post-intervention Follow-up Period ..................................................................... 24 
8.  CRITERIA FOR EVALUATION AND ENDPOINT DEFINITION ................................ 25 
8.1  Primary Endpoint ................................................................................................ 25 
8.2  Secondary Endpoints .......................................................................................... 25 
8.3  Off Agent Criteria ................................................................................................ 25 
8.4 Off Study Criteria ................................................................................................. 25 
8.5  Study Termination ............................................................................................... 25 
9.  CORRELATIVE/SPECIAL STUDIES ........................................................................ 26 
10.  SPECIMEN MANAGEMENT .................................................................................. 28 
11.  REPORTING ADVERSE EVENTS ......................................................................... 29 
12.  STUDY MONITORING ........................................................................................... 31 
12.1  Data Management ............................................................................................ 31 
12.2   Data and Safety Monitoring Committee ........................................................... 31 
12.3 Sponsor Monitoring ............................................................................................ 31 
12.4  Record Retention .............................................................................................. 31 
13.  STATISTICAL CONSIDERATIONS ........................................................................ 33 
13.1 Study Design/Endpoints ..................................................................................... 33 
13.2  Primary Feasibility Endpoints ............................................................................ 33 
13.3  Primary Quantitative Endpoint .......................................................................... 34 
13.4  Secondary Exploratory Endpoints ..................................................................... 34 
13.5  Reporting and Exclusions ................................................................................. 35 
13.6  Interim Analysis ................................................................................................. 36 
14.  ETHICAL AND REGULATORY CONSIDERATIONS .............................................. 37 
14.1  Form FDA 1572 ................................................................................................ 37 
14.2  Other Required Documents .............................................................................. 37 
14.3  Institutional Review Board Approval ................................................................. 37 
14.4  Informed Consent ............................................................................................. 37 
15.  FINANCING, EXPENSES, AND/OR INSURANCE ................................................. 38 
16.  REFERENCES ....................................................................................................... 39 
 
 
 


 
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LIST OF ABBREVIATIONS 
 
ADH 
Atypical Ductal Hyperplasia 
AE 
Adverse Event 
AH 
Atypical Hyperplasia 
AI 
Aromatase Inhibitor 
AUC 
Area Under the Curve 
BMD 
Bone Mineral Density 
BMI 
Body Mass Index 
CBC 
Complete Blood Count 
CBE 
Clinical Breast Exam 
CRF 
Case Report Form 
CSA 
Clinical Supply Agreement 
CTA 
Clinical Trials Agreement 
CTCAE 
Common Terminology Criteria for Adverse Events 
DARF 
Drug Accountability Record Form 
DCF 
Data Collection Form 
DCIS 
Ductal Carcinoma In Situ 
DEXA 
Dual-Energy X-ray Absorptiometry 
DSMC 
Data Safety and Monitoring Committee 
EGF 
Epithelial Growth Factor 
EGFR 
Epithelial Growth Factor Receptor 
EH 
Epithelial Hyperplasia 
ELISA 
Enzyme-Linked Immunosorbent Assays 
ER 
Estrogen Receptor 
FDA 
Food and Drug Administration 
FSH 
Follicle Stimulating Hormone 
GCP 
Good Clinical Practice 
HRT 
Hormone Replacement Therapy 
HSC 
Human Subjects Committee 
IGF-1 
Insulin-like Growth Factor-1 
IGFBP-3 
Insulin-like Growth Factor Binding Protein - 3 
IGFR  
Insulin-like Growth Factor Receptor 
IRB 
Institutional Review Board 
KUMC 
University of Kansas Medical Center 
LCIS 
Lobular Carcinoma In Situ 
LDL 
Low Density Lipid 
LH 
Luteinizing Hormone 
NCI 
National Cancer Institute 
NSAID 
Non-Steroidal Anti-Inflammatory Drug 
PgR 
PI 
Progesterone Receptor 
Principal Investigator 
PID 
Participant Identification number 
QNS 
Quantity Not Sufficient 
qRT-PCR 
Quantitative Real Time – Polymerase Chain Reaction 
RIA 
Radioimmunoassay 
RPFNA 
Random Periareolar Fine Needle Aspiration 
SAE 
Serious Adverse Event 
SERM 
Selective Estrogen Receptor Modulator 
SHBG 
Sex Hormone Binding Globulin 


 
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1.  OBJECTIVES 
 
Duavee® is tissue specific estrogen complex of bazedoxifene plus conjugated estrogen which is 
FDA approved for relief of menopausal symptoms and prevention of osteoporosis in women with 
a uterus who have not been diagnosed with estrogen dependent neoplasia.  The overall 
purpose of this research is to demonstrate in a preliminary fashion that despite reduction in 
menopausal symptoms, (Duavee®) does not increase and may decrease proliferation in benign 
breast tissue in a cohort of peri- or post-menopausal women at moderately increased risk for 
breast cancer.  If this pilot shows rapid accrual, good retention, and lack of significant increase 
in the risk biomarker Ki-67 in benign breast tissue, a larger prevention trial is envisioned in the 
Southwest Oncology Group 
 
1.1   Primary Feasibility Objective 
To determine feasibility for a larger clinical trial in women at moderately increased risk we will: 
1.1.1 Determine if accrual is brisk as demonstrated by accrual of at least 20 moderate risk 
women in the initial 6 months (3.5 entrants per month) in a single institution; 
1.1.2 Determine if there is a 15% or lower dropout rate over the 6 month intervention; and  
1.1.3 Determine if 20% or fewer women with paired RPFNA specimens exhibit a 
meaningful increase in benign breast cell proliferation (Ki-67) where meaningful increase is 
defined as an increase in Ki-67 to 2% or higher in individuals with initial Ki-67< 1% or a doubling 
of Ki-67 in individuals with initial Ki-67 of 1% or higher.   
                                                                                                                                  
1.2  Secondary Exploratory Objectives 
A number of analyses will be conducted in an effort to document and descript the effects of 
Duavee® on a variety of variables that are actual or potential risk biomarkers for development of 
breast cancer.   
 
 
Assess benign breast tissue for decrease in Ki-67 in women with Ki-67 of 1% or higher 
 
Assess changes in benign breast tissue estrogen responsive genes by RTqPCR 
 
Assess changes in benign breast tissue peptides and phosphopeptides.  
 
Assess changes in serum bioavailable hormones and cytokines 
 
Assess change in bazedoxifene serum levels as a function of BMI, percent body fat,  
and compliance (by tablet count) 
 
Document change if any in body composition (DEXA), and waist circumference 
 
Document change if any in volumetric breast density (Volpara) and compare to % dense 
area (Cumulus)    
 


 
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2.  BACKGROUND 
 
2.1 The Scope of the Problem 
 
Vasomotor and other menopausal symptoms are a significant problem impacting quality of life 
for up to 75% of women undergoing natural menopause transition usually between age 45 and 
55 and generally last about 5 years [Freeman, 2011].  Approximately 1/3 of women have severe 
enough symptoms to prompt them to seek medical attention.  These women and those who 
develop vasomotor symptoms early in transition are likely to be those who are bothered with 
these symptoms for 10 or more years [Freeman, 2011; Freeman, 2014].  Hormone replacement 
therapy, either estrogen alone for women without a uterus or estrogen and progesterone for 
women with a uterus, is the most effective means of relief but concerns about increasing the risk 
for breast cancer make providers reluctant to provide prescriptions for hormone replacement 
therapy for even average risk women much less those at increased risk.  Reluctance is largely 
based on results of the Women’s Health Initiative  study in which an average of 5 years of 
combined conjugated estrogen 0.625 mg plus a progestin resulted in a 24% increase in breast 
cancer risk overall and a 34% increase in risk for women beginning it in their 50’s (HR 1.34; 
0.82-2.19) [Manson 2013; Anderson 2012]. No increase in risk of breast cancer was observed 
for conjugated estrogen 0.625 mg/day alone [Manson 2013; Anderson 2012].   
 
At least 25% of women 45 and older are at increased risk for breast cancer because of family, 
reproductive, or personal health history. In addition to counseling for lifestyle modifications 
(normal weight, exercise, reduce alcohol intake), peri- and post-menopausal women 35 and 
older at moderately or higher increased risk may be counseled to take an anti-estrogen to 
reduce their risk [Visvanathan 2013].  Overall only 4% of women eligible for antihormonal 
primary prevention therapy agree to take it although it can reduce the risk of breast cancer by 
40% or more  [Fisher 1998; Vogel 2006; Ropka 2010; Goss 2011; Cuzick 2013; Cuzick 2014].   
 
Given that medications such as tamoxifen and aromatase inhibitors often increase vasomotor 
symptoms and are not associated with a survival advantage in the primary prevention setting 
[Visvanathan 2013; Goss 2011; Cuzick 2014], it is likely that an even lower percentage of 
women currently having difficulty with hot-flashes will agree to take anti-estrogens as primary 
prevention therapy. The development of tissue selective estrogen complexes combining 
estrogen (for hot flashes and bone health) plus a selective estrogen receptor modulator (SERM) 
with breast and uterine antagonist activity presents a potential solution to this conundrum for 
women with both vasomotor symptoms and an increased risk for development of breast cancer. 
 
2.2 Rationale for Present Proposal 
 
In clinical trials of bazedoxifene plus conjugated estrogen (now marketed as Duavee®) versus 
placebo, relief of hot flashes and improvement of both hip and lumbar spine bone density was 
accomplished without development of uterine hyperplasia, increase in breast tenderness, or 
increase in mammographic breast density [Harvey 2013; Pinkerton 2013; Pinkerton 2014a; 
Pinkerton 2014b; Pinkerton 2014c; Abraham 2014; Komm 2014]. Contra-indications to use of 
Duavee® include estrogen dependent neoplasia (such as breast, ovarian, and endometrial 
cancer), predisposition to or prior thromboembolism, hepatic impairment, and abnormal uterine 
bleeding. There is no explicit label warning about women who may be at increased risk for 
breast cancer [Duavee® Prescribing Information, 2014]. 
 


 
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Preclinical studies in MCF-7 breast cancer cells suggests that bazedoxifene down-regulates ER 
alpha and decreases cyclin D1 as well as cell growth [Lewis-Wambi 2011]. 
 
In non-human primates, 6 months of bazedoxifene or bazedoxifene combined with conjugated 
estrogen was associated with reduced Ki-67 compared to conjugated estrogens alone in normal 
mammary tissue [Ethun 2012]. Bazedoxifene combined with conjugated estrogen still functions 
as an estrogen antagonist in the macaque monkey uterus with reduction of Estrogen Receptor 
alpha (ER alpha) related genes GREB1 and pS2 (Trefoil Factor 1) [Ethun 2013]. There was little 
effect on lipids or coronary or peripheral atherosclerosis for bazedoxifene alone but 
bazedoxifene partially antagonized the protective effects of conjugated estrogens on coronary 
and peripheral but not cerebral/carotid atherosclerosis [Clarkson 2014].  
 
There is no FDA approved chemopreventive agent for breast cancer risk reduction which 
reduces menopause symptoms, in fact the SERMs tamoxifen (for pre and postmenopausal 
women) and raloxifene (for postmenopausal women), as well as aromatase inhibitors (for 
postmenopausal women), all increase menopause symptoms [Visvanathan 2013]. If 
bazedoxifene and conjugated estrogens were actually found to reduce proliferation in benign 
breast tissue similar to what has been observed in animal models, then it should be explored in 
placebo-controlled Phase IIB trials for change in tissue proliferation and in Phase III trials for 
change in cancer incidence.  The combination of menopause symptom relief and breast cancer 
risk reduction would be a major advance for women in early menopause. Even if breast cell 
proliferation did not decrease but was at least stable (not increased) after Duavee®, this would 
be a major advance as women at increased risk for breast cancer (and their physicians) would 
likely be more comfortable with its use. 
 
2.3  Proliferation in Hyperplastic Benign Breast Tissue as a Response Endpoint 
 
Women with diagnostic biopsy evidence of proliferative breast disease following an abnormal 
finding on physical exam or mammogram are at increased risk for breast cancer ranging from ~ 
2 fold for hyperplasia without atypia to a 4-5 fold increase in relative risk for atypical ductal or 
lobular hyperplasia [Dupont 1985; Tavessoli 1990; Drystad 2015; Hartmann 2014]. However, 
women age 50-69 who simply have a diagnostic breast biopsy without evidence of proliferative 
breast disease or family history may also have a 20% up to a 2 fold increase in the risk for 
breast cancer [Drystad 2015; Castells 2015].  Proliferation as measured by detection of mitotic 
index and/or detection of nuclear associated proliferation antigens such as Ki-67 (MIB-l) 
progressively increases from normal to hyperplasia to atypical hyperplasia and DCIS [Shrestha 
1992; Mommers 1999; Allred 1998].  
 
In normal pre-menopausal breast epithelium, proliferation (Ki-67) correlates with systemic levels 
of progesterone, and is lowest in the follicular phase (0.2-0.7%) and highest in the luteal phase 
(~2%) [Soderqvist 1997; Shoker 1999]. Ki-67 labeling averages 0-1% in normal 
postmenopausal epithelium [Shoker 1999]. In diagnostic biopsies from proliferative breast 
disease Ki-67 has been reported to vary between 1 and 6% [Shaaban 2002; Allred 1998]. 
 
A Ki-67 labeling index of 2% or higher in usual duct hyperplasia (cross sectional study from 
Shaaban) or atypical hyperplasia (Mayo prospective cohort study Santisteban) is associated 
with increased risk of breast cancer compared with women with hyperplasia or atypical 
hyperplasia with lower levels of proliferation [Shaaban 2002; Santisteban 2010]. We have used 
random periareolar fine needle aspiration to obtain benign breast tissue from women at 
increased risk for breast cancer for risk stratification and to obtain tissue for Phase II prevention 


 
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trials for over 2 decades [Fabian 2000, 2002, 2005, 2007, 2010, 2013].  Similar to diagnostic 
biopsy series, [Dupont 1985; Hartmann 2014] women with cytologic evidence of hyperplasia 
with atypia have an increased risk of DCIS and invasive cancer as well as a higher proliferative 
rate [Fabian 2000; Khan 2005]. We observed a median Ki-67 labeling index of 1.4% in RPFNA 
samples exhibiting hyperplasia with or without atypia from high-risk post-menopausal women in 
whom there were sufficient cells for Ki-67 to be attempted [Khan 2005].  Inter- and intra-
observer variation in interpretation of staining results was low with a Cronbach’s alpha of 0.99 
[Khan 2005].     
 
A number of antihormonal treatments effective in breast cancer treatment and prevention have 
been shown to reduce proliferation indices in benign and cancerous breast epithelial cells by 17-
50% relative to baseline. [Clarke 1993; Chang 2000; Dowsett 2001; Dowsett 2005; Decensi 
2003]. Reduction in proliferation (Ki-67) at two weeks and 12 weeks in the neoadjuvant IMPACT 
Trial (tamoxifen, anastrozole, combination) was positively associated with disease free survival 
[Dowsett 2005]. Tamoxifen has been shown to reduce proliferation (Ki-67) in benign breast 
tissue of postmenopausal women [de Lima 2003].  In a single arm pilot trial six months of the 
aromatase inhibitor letrozole reduced proliferation by 66% in benign tissue from high-risk 
women despite continuing hormone replacement [Fabian 2007].  
 
Ki-67 is quantitative, exhibits low intra- and inter-observer variance, and is favorably modulated 
in benign breast tissue with agents known to be effective in primary and secondary breast 
cancer prevention. Consequently, change in proliferation as measured by Ki-67 
immunohistochemistry is often used as a primary response endpoint in Phase II and pilot 
prevention studies of premenopausal women and postmenopausal women on hormone 
replacement therapy [Fabian 2005; Fabian 2007].  We and others have shown that random 
periareolar fine needle aspiration is accepted by high risk women and is a reproducible means 
to sample breast tissue [Fabian 2000; Ibarra-Drendall 2009]. However, given the extent of 
lobular involution in definitively postmenopausal women not on hormone replacement therapy, a 
sizable number of women may need to be screened by RPFNA to detect those with a Ki-67 of 1-
2% or higher, making reduction in Ki-67 an impractical endpoint for primary prevention trials in 
definitively postmenopausal women not on hormone replacement.  Ki-67 has not been 
adequately studied in moderate to high risk peri-menopausal women < 60 having menopausal 
symptoms in order to determine the proportion of women with a Ki-67 of >1% in their RPFNA 
specimen.  
 
2.4  Other Risk and Mechanism of Action Biomarkers in Benign Breast Tissue Likely to 
be Modulated by Duavee®  
 
Sampling of benign breast tissue also provides the opportunity to assess fresh frozen tissue for 
assessment of change in gene or protein expression to better define mechanism of action.  We 
will assess a number of estrogen response genes including ER alpha, progesterone receptor, 
pS2 (Trefoil factor 1) cMYC, GREB-1, amphiregulin, known to be increased by estrogen and 
antagonized by bazedoxifene in mice benign and malignant mammary tissue [Song 2012]. 
Several additional genes known to be increased at the mRNA level by estrogen such as 
amphiregulin, FOS, and JAK2 will also be assessed by RTqPCR [Chang 2010; Fabian 2010; 
Phillips 2013; Ibarra-Drendall 2012; Fabian 2013]. Reverse phase protein arrays will also be 
used to identify changes in proteins and phosphoproteins in pathways important in breast 
carcinogenesis [Akbani 2014; Meric-Bernstam 2014; Fabian2013]. 
 
 


 
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2.5  Mammographic Breast Density as Risk and Response Biomarker  
 
Mammographic density which is composed histologically of ductal and stromal tissue, and fluid 
is recognized as a risk factor for breast cancer particularly when corrected for age and Body 
Mass Index (BMI) [Boyd 1993; Boyd 1995; Boyd 1998; Byrne 1995; Warwick 2014].  Although 
absolute dense area along with % dense area are both risk biomarkers, % density appears to 
be the stronger risk biomarker [Pettersson 2014]. In a recent analysis, Boyd et al. suggest that 
women with a high density in the highest tertile of mammographic density corrected for BMI 
have a risk of 2.63x that of women in the lowest tertile (0-20% density), and women in the mid 
tertile (20-41%) a risk of 2.15X that of women in the lowest tertile [Boyd 2014]. Given these 
updated results it is reasonable to include women with an estimated breast density of 25% or 
higher in the risk criteria for consideration of participation in this study.  
 
Change in mammographic density is validated as a surrogate response biomarkers for trials 
involving the selective estrogen receptor tamoxifen which is known to reduce risk for breast 
cancer by 40% or more [Cuzick 2011; Cuzick 2013].  However, change in breast density is not 
modulated by raloxifene, a selective estrogen receptor modulator known to reduce risk for 
breast cancer in postmenopausal women almost to the same extent as tamoxifen [Martino 2004; 
Freedman 2001; Pearman 2010]. Nor is density modulated with exemestane, an aromatase 
inhibitor that is shown to reduce breast cancer risk by 65%. [Cigler 2011; Goss 2011].  Weight 
reduction may actually increase percent dense area [Fabian 2013; Boyd 1997]. Breast density, 
even with computer assisted calculations, is subjective and pre and post intervention 
assessments should be performed by the same individual at the same setting with the rater 
blinded as to which image was baseline [Stone 2003]. In addition there are a number of 
technical factors that can introduce error when change is being evaluated including variation in 
type of imaging, positioning and degree of compression.  
 
Breast density (% dense area)  was not significantly altered compared to controls over 24 
months in a centrally read retrospective analysis of digital mammograms from the SMART 1 
study which compared bazedoxifene + 0.45 mg CE or 0.625 mg CE vs placebo to placebo or 60 
mg of raloxifene. (Mean reductions of 0.05-.41%). In a prospective cohort of over 900 women in 
the SMART 5 study ages 40-65 with a mean baseline density of ~25% there was no change in 
centrally read digital mammographic density over a 12 month period comparing 0.45 or 0.625 
mg CE + 20 mg bazedoxifene vs placebo (both declined by ~ 0.3 %). A comparator of CE + 
medoxyprogesterone acetate increased by 1.66% [Pinkerton 2013]. Automated volumetric 
mammographic breast density assessments appear to have less variation than computer 
assisted operator density assessments [Eng 2014; Alonzo-Proulx 2015; Brand 2014; Ekpo 
2014].  Automatic Volpara volumetric density measurements utilized in this study will be 
compared to the Cumulus % dense area to explore whether small changes in density with 6 
months of Duavee® may be detected with the volumetric method.  
 
2.6  Serum risk Biomarkers as Response Endpoints 
 
Higher endogenous bioavailable estradiol and testosterone levels have long been associated 
with breast cancer risk in postmenopausal women [Toniolo 1994; Pearce 1989; Tworoger 2014; 
Verkasalo 2001; Missmer 2006; Farhat 2011].   Several serum biomarkers associated with 
metabolism are known to be risk biomarkers for proliferative breast disease and breast cancer 
in postmenopausal women including serum insulin, markers of insulin resistance such as 
HOMA-IR, adiponectin, leptin: adiponectin ratio [Fabian 2012; Catsburg 2014; Gunter 2009; 
Hernandez 2014; Vona-Davis 2007]. Lipocalin 2 (Lcn2) and retinol binding protein 4 (RBP4) are 


 
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also increased in insulin resistance. Lipocalin 2 has been shown to promote breast cancer 
development and progression primarily through activation of the estrogen receptor in preclinical 
models [Guo 2012; Yang 2009; Shi 2008]. An increase in the serum ratio of retinol binding 
protein to STA6 is oncogenic and is mediated through the actions of JAK2 and Stat 3 and 5 at 
the cellular level [Berry 2014]. Treatment of ovariectomized mice with conjugated estrogen (CE), 
bazedoxifene (BZA) or the combination vs control suggests that both CEE and BZA alone or in 
combination have favorable effects on leptin, adiponectin, Lcn2, and RBP4. The most dramatic 
effects appear to be for the leptin: adiponectin ratio (reduced by over 10 fold in the 
ovariectomized mouse) [Kim 2014]. Ovariectomized mice subjected to 10-12 weeks of diets 
producing obesity showed attenuation of weight gain, fat mass gain, and measures of insulin 
resistance when treated with BZA and CE [Barrera 2014].    


 
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3.  SUMMARY OF STUDY PLAN 
 
Peri- or post-menopausal women meeting the risk and medical eligibility criteria (see below) will 
undergo random periareolar fine needle aspiration (RPFNA) under the umbrella screening 
protocol HSC 4601. Women with at least 500 cells on their cytomorphology and/or Ki-67 slide, 
but with Ki-67 expression less than 4% will be eligible to proceed to the intervention phase of 
this study. At a baseline on-study visit, women will have a physical exam, DEXA for body 
composition, complete a 29 item Menopause Quality of Life Intervention Questionnaire [Lewis 
2005] and hot flash assessment, have fasting blood obtained for biomarkers as well as to 
assure reasonable normal organ function. Key symptoms assessed at baseline will be hot 
flashes. Forty women will be entered on the treatment portion of the trial and will take Duavee® 
one table daily. Drug will be supplied by Pfizer. After 6-8 months of study agent, biomarkers will 
be reassessed. Mammogram, DEXA for body composition, RPFNA, and blood draw will be 
repeated. A complete side effects assessment will be performed. Entry of 40 women should 
allow 36 women to complete the study and be evaluable for the primary endpoint. Trial accrual 
is anticipated to take 6 months with completion of clinical aspects of the study by 12 months. 
Since this is not a blinded study, analysis of feasibility and changes in Ki-67 can take place 
immediately.  
 


 
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4.  PARTICIPANT SELECTION 
 
4.1  Inclusion Criteria for Screening RPFNA 
 
Note that the screening RPFNA may be conducted under a separate IRB-approved research 
protocol with its own consent form.  Potential participants are not required to make a decision 
regarding participation in the Duavee® trial until after receipt of the results of their RPFNA. 
However, if a woman is undergoing an RPFNA expressly for participation for this protocol, the 
following should be kept in mind for eligibility 
 
4.1.1  Women with vasomotor symptoms with a uterus who are postmenopausal (no menstrual 
period for 12 months) or in late menopause transition (no period for 3 months and 
elevated FSH) [Sherman 2005].  
 
4.1.2  Age ≤65 
 
4.1.3  BMI: <36 kg/m2  
 
4.1.4 Breast Imaging: Class I-III mammogram within 6 months of RPFNA. If Class 0 or 4, must 
be resolved with additional procedures. If breast imaging pre and post study is  
performed at KUMC, then volumetric assessment by  Volpara software will be performed.  
 
4.1.5 If previously on oral contraceptives or hormone replacement, off for 8 weeks or more 
prior to baseline RPFNA. The exception is low dose vaginal hormones (estring, vagifem, 
or 0.5 gram or less of conjugated estrogen vaginal cream twice weekly or less often).  
These vaginal preparations may be continued at the same dose.  
 
4.1.6 Risk Factors/Level. Moderate risk of developing breast cancer based on either by having 
at least one of following: 
 
  
 
First or second degree relative with breast cancer age 60 or younger; 
 
Prior breast biopsy; 
 
Prior RPFNA atypia; 
 
Estimated Mammographic Density of 25% or higher; 
 
Gail 5-year risk of >1.7% (as calculated by the NCI Breast Cancer Risk 
Assessment Tool) or a 5 year Gail Risk of 2X that for age group; and/or   
 
IBIS Breast Cancer Risk Evaluation (http://www.ems-trials.org/riskevaluator/) 
[Tyrer–Cuzick 2004]. 10-year relative risk of >2X that for the population for age 
group.  
 
4.2   Exclusion Criteria for Screening RPFNA 
 
4.2.1  Risk: A prior biopsy showing LCIS, DCIS, or invasive breast cancer, or individual with 
BRCA1/2 deleterious mutation. 
  
4.2.2 Medical Conditions:  
 
Have a predisposition to or prior history of thromboembolism, deep venous 
thrombosis, pulmonary embolism, or stroke 
 
History of renal or liver disease 


 
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 
Prior ovarian or endometrial cancer 
 
Stopped or started hormone replacement within 8 weeks 
 
Any other condition or intercurrent illness that in the opinion of the investigator 
makes the woman a poor candidate for RPFNA.  
 
4.2.3  Medications   
 
Current anticoagulant use (must discontinue for 3 weeks prior to FNA) 
 
Taking systemic hormones within two months (eight weeks) prior to screening 
RPFNA. 
 
Taken tamoxifen, raloxifene, or an aromatase inhibitor within the past 6 months 
 
Participation on any chemoprevention trial within 6 months 
 
4.3  Inclusion Criteria for Intervention Phase 
 
4.3.1  RPFNA Results: An RPFNA performed less than six months prior to enrollment on study  
that exhibits at least 500 cells on the cytology or Ki-67 slide, and a Ki-67 positivity 
measured on at least 500 cells that is less than 4%. 
 
4.3.2  Baseline chemistry profile showing reasonably normal renal and hepatic function. 
Creatinine <2.0, Bilirubin < 2.5, and albumin > 3.4 
 
4.3.3  Willingness to comply with study procedures. 
 
Willing to have a blood draw for serum to archive bioavailable estradiol, SHBG, 
FSH, and bazedoxifene levels as well as a chemistry profile to ensure 
reasonable normal organ function at baseline and 6 month visits (approximately 
four tubes of blood collected) 
 
Willing to have a DEXA scan for body composition and waist measurement at 
baseline and 6-8 months. 
 
Willing to have a repeat mammogram and RPFNA at 6-8 months following 
initiation of study drug 
 
Willing to undergo a history, physical, vitals and breast exam at baseline and 6 
month visits  
 
Willing to be contacted by the trial coordinator at months 1 and 3 during the 6 
month study period. 
 
Willing to complete a 29 item validated Menopause Quality of Life Intervention 
Questionnaire and hot flash assessment at baseline and 6 month visits 
 
Willing to sign and able to understand separate consents for the RPFNAs and for 
study participation 
  
4.4  Exclusion Criteria for Study Intervention 
 
4.4.1  RPFNA Criteria:  
 
 
Cells suspicious for malignancy 
 
4.4.2  Medical: Intercurrent illness which makes potential participant unsuitable for study or 
started hormone replacement therapy between RPFNA and enrollment on study.    
  
 


 
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4.5  Inclusion of Women and Minorities 
 
All women who meet eligibility criteria and members of all races and ethnic groups are eligible 
for this trial.   
 
4.6  Recruitment and Retention Plan 
  
Potential participants will be screened in the Breast Cancer Prevention Center at the University 
of Kansas Medical Center (KUMC), Westwood, Kansas. All potential participants will be involved 
in the High Risk Breast Program at KUMC. The duration of accrual is expected to take 6 months, 
accruing 6.7 participants per month. 
 
Study participants will be contacted by phone at month 1 and month 3 during the months 
between clinic visits in order to insure active participation and to collect adverse event, dosing 
and other pertinent information. It is expected that the dropout rate will be less than 10%. 
 


 
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5.  AGENT ADMINISTRATION 
 
Intervention will be administered on an outpatient basis.  
 
5.1   Dose Regimen and Dose Groups 
 
 
 
Open label Bazedoxifene (20 mg) plus conjugated estrogen (0.45 mg) marketed as Duavee® 
for six months will be given to all trial participants. There will be no randomization as this is a 
one arm trial. All participants will be given eight months (two months of overage) of Duavee® to 
allow for scheduling study procedures.   
 
5.2   Duavee® Administration 
 
The KUMC Investigational Pharmacy will be responsible for receiving, inventory and dispensing 
of study agent. 
 
A study number will be assigned to each participant and provided to the investigational 
pharmacy. Study agent will be dispensed with the study number and initials of each participant. 
Eight months of Duavee® will be dispensed. The study coordinator will then give the study 
agent to the participant with instructions to take one tablet at approximately the same time of 
day in conjunction with intake of food and to keep track of missed doses or lost tablets. 
 
5.3  Concomitant Medications 
 
Study participants are strongly encouraged to continue any routine medications throughout the 
duration of the study to avoid confounding adverse events due to starting and stopping other 
medications while on study. Participants are not allowed to start tamoxifen, raloxifene, 
anastrozole, letrozole or any other SERM or aromatase inhibitor (or other chemopreventive) 
while participating on this trial. 
 
All medications (prescription and over-the-counter), vitamin and mineral supplements, and/or 
herbs taken by the participant will be documented on the concomitant medication CRF and will 
include: 1) start and stop date, dose and route of administration, and indication. Medications 
taken for a procedure (e.g., biopsy) should also be included. 
 
5.4  Dose Modification 
 
Dose modifications will not be allowed on this study. Because study agent is only available in a 
single dose tablet, no dose modification option is available.  If a participant is experiencing 
adverse events or is having a medical procedure that requires a brief suspension of Duavee®, 
this must be noted in the CRF on the dose suspension page. At the discretion of the PI, 
participants may be removed from participation for significant dose suspension time periods. 
 
Participants with grade 2 adverse events considered to be related to study agent will have the 
agent suspended until the toxicity has resolved.  If the adverse event resolves within one month, 
the agent will then be resumed.  If toxicity again occurs, and is thought to be agent-related, the 
agent will be permanently stopped.   
 


 
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Participants who experience grade 3 toxicity will have the study agent suspended until the 
adverse event resolves.  If the investigator is reasonably certain that the toxicity is not due to the 
agent, the agent may be re-instituted after resolution of the adverse event.   
 
Development of a grade 4 adverse event will require suspending the study agent.  If the 
investigator feels the event is not study agent related, then it may be re-instituted at full dose 
once the adverse event is resolved.   
 
Examples of grade 3 or 4 adverse events which would require permanent study agent 
discontinuation and withdrawal from the trial include development of breast or any other type of 
cancer (other than skin) that requires definitive treatment; deep venous thrombosis; 
symptomatic ovarian cysts requiring hospitalization or narcotic pain management.  Examples of 
grade 3 or 4 adverse events requiring temporary discontinuation and possible re-institution 
include bronchitis/pneumonia, broken bones, nausea-vomiting due to influenza, and rash of 
unknown cause. 
 
5.5  Adherence/Compliance 
 
 
Participants that maintain 70% compliance throughout the duration of the trial will be considered 
“compliant”. Compliance will be measured through tablet counts conducted by the trial 
coordinator and recorded in the CRF. 
 


 
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6.  PHARMACEUTICAL INFORMATION 
 
Duavee® is the combination of 0.45 mg of conjugated estrogen and 20 mg of bazedoxifene and 
was approved for relief of menopausal symptoms and prevention of postmenopausal 
osteoporosis in women with a uterus in October 2013. Contraindications listed in prescribing 
information include undiagnosed abnormal uterine bleeding,  past history of breast cancer, 
current estrogen-dependent neoplasia, active or history of venous or arterial thromboembolism,  
hypersensitivity to estrogen or bazedoxifene,  known hepatic impairment or disease,  known 
protein C, protein S, or anti-thrombin deficiency or other thromboembolic disorders,  or 
pregnancy and nursing.  
 
6.1  Duavee® Provider 
 
Duavee® will be provided for this study by Pfizer, Inc., the manufacturer.  
 
6.2  Pharmacology 
 
6.2.1  Absorption: Both bazedoxifene and conjugated estrogen are well absorbed from the 
gastrointestinal tract.  Bio-availability of bazedoxifene is 6%. Steady state concentrations 
are obtained by day 10. At day 10 steady state Cmax for the estrogen component is 2.6 
ng/ml estrone and 6.9 ng/ml bazedoxifene with Tmax after a single dose of 6.5 and 2.5 
hours respectively in postmenopausal women.   
 
6.2.2  Food Effect: Duavee® may be taken without regard to meals but a high fat meal 
increased the AUC by 25%. 
 
6.2.3  Distribution: The estrogen component is bound to SHBG and albumin and is widely 
distributed. Bazedoxifene is also highly distributed and circulates highly bound (97-98%) 
to plasma proteins but not SHBG. A radio-ligand assay has been developed for 
bazedoxifene [Komm 2005 Endocrinology, Harris 2002] 
 
6.2.4  Metabolism: The estrogen component is metabolized similarly to endogenous estrogen 
with conversion reversibly to estrone which can be converted to 17 B estradiol or estriol, 
or estrone sulfate. Bazedoxifene is metabolized via glucuronidation with little cytochrome 
P450 mediated metabolism.  The concentration of the 5-glucuronidated metabolite in the 
circulation is 10x that of the parent compound.  
 
6.2.5  Excretion: Total estrone is eliminated primarily in the urine with a half-life of 17 hours. 
Bazedoxifene is eliminated via biliary excretion and feces with <1% in urine.  The 
elimination half-life of bazedoxifene is 30 hours. It is likely that it undergoes an extensive 
entero-hepatic circulation and that some drugs might interfere with elimination. However, 
commonly used antibiotics, NSAIDs, and antacids had minimal tested effect on 
Bazedoxifene concentrations with increases in AUC of Bazedoxifene of 6-7% with co-
administration of ibuprofen, atorvastatin, and aluminum and magnesium hydroxide and 
decease of bazedoxifene by 15% with azithromycin.  
 
 
 
 
 


 
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6.3  Mutagenesis, Carcinogenesis and Fertility  
 
There is no evidence that bazedoxifene is mutagenic or genotoxic.  In female rats and mice 
there is evidence of increase in benign ovarian, and ovarian granulosa cell tumors. Estrous 
cycles and fertility is impaired in female rats.  
 
6.4  Pfizer Sponsored Clinical Studies 
 
6.4.1  Relief of Menopausal Symptoms in Women: a 12 week double blind placebo controlled 
trial in women age 42-62 (mean 53) who had moderate to severe hot-flashes showed a 
reduction in mean number of hot flashes of 5.9 per day compared to a reduction of -2.8 
for the placebo by 4 weeks as well as a reduction in severity.  
 
6.4.2  Uterine Side Effects in Women: The incidence of endometrial hyperplasia was < 1 % and 
spotting showed little difference from placebo.  
 
6.4.3  Bone Density: Mean % change in BMD with Duavee® at 24 months was + 1.72 vs -1.90 
for placebo in women menopausal for 1-5 years.  
 
6.5  Special Considerations 
 
6.5.1  Hepatic or Renal Impairment: Duavee® is contraindicated in women with hepatic 
impairment (AUC of bazedoxifene increases by 100 % in moderate and over 300 % in 
severe hepatic impairment) and has not been evaluated in women with renal impairment. 
Women with significant hepatic or renal abnormalities will not be eligible for this study. 
 
6.5.2  Women with high BMI: a 17% reduction in bazedoxifene is predicted in women with a 
BMI < 27 kg/m2 compared those with < 27 kg/m2. Reduction in bazedoxifene levels may 
be associated with increased risk of uterine hyperplasia in women using the Duavee® 
long term but it is unlikely to be an issue in our short term trial.  Nonetheless we will 
restrict eligibility to women with a BMI of < 36 kg/m2. 
 
6.5.3  Advanced Age: No difference in safety or effectiveness were noted in the small number 
of women tested between 65-74 in clinical trials with Duavee® and Duavee® has not 
been tested in women over 75. Due to concerns about obtaining adequate amounts of 
breast tissue by RPFNA we will limit study entry to those 65 and younger.  
 
6.6  Availability and Agent Distribution  
 
Duavee® comes from Pfizer as tablets containing 20 mg of bazedoxifene and 0.45 mg of CE. 
240 tablets containing 20 mg of bazedoxifene and 0.45 mg of CE will be dispensed at entry of 
the participant onto the study so as to provide an adequate number of tablets for 8 months.  The 
8 months is based on the planned 6 months of administration, plus two additional months if 
required to accommodate scheduling of procedures. 
  
6.7  Agent Accountability 
 
The Protocol Lead Investigator (Fabian) is required to maintain adequate records of receipt, 
dispensing and final disposition of study agent. This responsibility has been delegated to the 
KUMC Investigational Pharmacy. Include on receipt record from whom the agent was received 


 
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and to whom study agent was shipped, date, quantity and batch or lot number. On dispensing 
record, note quantities and dates study agent was dispensed to and returned by each 
participant. 
 
6.8  Packaging and Labels 
 
Duavee® will be supplied by Pfizer in a form ready for dispensing. For dispensing, each 
container will be labeled with a one-part label identifying study specific information, such as 
Study title, HSC protocol number, subject ID number, dosing instructions, recommended storage 
conditions, and a caution statement indicating that the agent is limited by United States law to 
investigational use only and the agent should be kept out of reach of children. 
 
6.9  Storage 
 
Study agent will be stored in the controlled access Investigational Pharmacy, at room 
temperature. 
 
6.10  Registration/Randomization 
 
There is no randomization for this open-label, single arm study. Registration will be in an 
enrollment log managed by the study coordinator, with matching participant study numbers and 
initials in the Investigational Pharmacy at KUMC. 
 
All participants signing consent forms will be assigned a Study ID number, have the Registration 
CRF completed, and be entered into the CRIS database.   
 
6.11  Blinding and Unblinding Methods 
 
Not applicable. 
 
6.12  Agent Destruction/Disposal 
 
At the completion of investigation, all unused study agent will be returned to the PI for return to 
the KUMC Investigational Pharmacy and destroyed in accordance with KUMC investigational 
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7.  CLINICAL EVALUATIONS AND PROCEDURES 
 
7.1  Schedule of Events 
 
SCHEDULE OF EVENTS 
 
Evaluation/ Procedure 
Pre-Study/ 
Screening 
RPFNA 
Baseline
Contact 
End of 
Month 1 & 3 
Month 
6 
Follow Up 
Phone 
Contact 
Mammogram  
X 
 
 
X 
 
RPFNA for cytomorphology, 
Ki-67, RTqPCR, proteomics 
X 
 
 
X 
 
Fasting blood archived for 
SHBG, estradiol, 
testosterone, progesterone, 
FSH, adipocytokines, and 
Bazedoxifene levels  
 
X 
 
X 
 
Assess Eligibility 
X 
X 
 
 
 
Informed Consent 
 
X 
 
 
 
Medical History 
 
X 
 
 
 
Physical Exam/CBE 
 
X 
 
 
 
Vital Signs/ Height and 
Weight, BMI, waist 
measurement 
 
X 
 
X 
 
29 item validated 
Menopause Quality of Life 
Intervention Questionnaire 
and hot flash assessment 
 
X 
 
X 
 
Blood Chemistry 
 
X 
 
X 
 
DEXA body composition 
and waist measurement 
 
X 
 
X 
 
Symptom Assessments 
 
X 
X 
X 
X 
Concomitant Medications 
 
X 
X 
X 
X 
Pregnancy test if peri-
menopausal 
 
X 
 
 
 
Dispense Study Agent 
 
X 
 
 
 
Collect Study Agent 
 
 
 
X 
 
Adverse Events 
 
 
X 
X 
X 
Telephone or Email Contact 
 
 
X 
 
X 
 
 
 
 
 
 


 
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7.2   Baseline Testing/Pre-study Evaluation 
 
7.2.1 Pre-study Eligibility RPFNA 
 
Women who potentially meet medical eligibility criteria for trial and are interested will un-
dergo a pre-study screening visit. Women may take vitamin K 10 mg/day for 3 days prior 
to the aspiration to help prevent hematoma formation but this is not required. 
 
 
 
RPFNA will be performed under local anesthesia from both breasts (two sites on each 
breast) and all cells pooled from both breasts (one breast in the case of prior DCIS or 
invasive cancer) into a single 15 cc tube containing modified Cytolyt™  (9 cc Cytolyt™ 
and 1 cc 10% neutral buffered formalin). After cleansing the breast with betadine and 
alcohol swabs, the breast is anesthetized with epinephrine and lidocaine using a 
tuberculin syringe. After the surface has been anesthetized, epinephrine and lidocaine 
are injected deeper into the breast at two sites, approximately at the 10 and 2 o’clock 
locations. Then, 5-6 needle passes are made into the anesthetic puncture sites to 
withdraw epithelial cells lining the ductal tree. 
 
After the procedure, participants will have ice packs placed on the breast for 10-15 
minutes to control swelling and bleeding. Participants are then wrapped with gauze cling 
wrap to create pressure on the puncture sites. A snug bra is also strongly recommended. 
Participants are advised to limit strenuous physical activity over the next 24 hours and to 
report any pain or signs of infection. 
 
Participants are offered Ativan 1 mg prior to the procedure for anxiety, and analgesics 
after the procedure in order to control any pain that may occur. These medications are 
optional. Participants should also be instructed to eat a full breakfast the morning of the 
RPFNA, to abstain from vitamin E, NSAIDS and fish oil products for three weeks prior to 
RPFNA, and if planning on taking Ativan, to have someone with them to drive them to 
and from the clinic. 
 
7.2.2 Pre-study risk assessment  
 
 
Gail Risk assessment: A 5-year projected probability of breast cancer development 
(“Gail risk”) will be calculated using the NCI website algorithm 
(http://www.cancer.gov/bcrisktoolmobile). The risk is expressed as relative to the 
average risk for women in the same age-group, based on values calculated from the 
Surveillance, Epidemiology, and End Results (SEER) Program provided by the NCI 
(http://srab.cancer.gov/devcan/) 
 
IBIS Risk assessment: a 10 year The IBIS Risk Assessment will be performed. The 
IBIS Breast Cancer Risk Evaluation Tool can be found (http://www.ems-
trials.org/riskevaluator/).   
 
 
Mammographic Density Estimation: Mammographic Density will be estimated from 
the pre-study digital mammogram left cranial caudal view. Note that a digital 
mammogram must have been performed within 6 months of RPFNA. 
 
   
 
 


 
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7.2.3 Baseline Studies 
 
7.2.3.1 Brief Physical and Medical History 
 
 
All women will have a brief physical (including clinical breast exam) performed by a 
study-associated clinician. This may be performed at the time of the RPFNA. All 
abnormal findings will be recorded on the physical source document, including duration 
and severity.  Height and weight, BMI, and waist measurement will also be recorded. 
Women will have a medical history collected by study personnel, including event dates, 
duration, and severity. 
 
7.2.3.2 DEXA Scan 
 
 
Dual energy x-ray absorptiometry (DEXA; Lunar Corp.) will be performed within 90 days 
prior to study initiation and at the completion of study participation to analyze change in 
body fat mass, fat free mass, and percent body fat. DEXA uses very low X-ray doses 
(0.02 mrem), that is, less than several hours of background exposure, and is able to 
detect changes in body composition on the order of 1.6-3.8%. DEXA will also be used to 
estimate visceral adipose composition. All calculations use GE Prodigy 9.1 software. 
 
7.2.3.3  Bloodwork  
 
 
 
Fasting (10-12 hours) blood will be drawn for  
 
Archived serum for FSH, testosterone, estradiol, SHBG, and for perimenopausal 
women progesterone, as well as adipocytokines and bazedoxifene levels. 
 
Chemistry profile will also be drawn and processed real time at the KUMC 
Westwood lab to ensure reasonable normal organ function.  The chemistry profile 
will be approved by the PI or study associated Nurse Practitioner prior to dispensing 
of study agent. 
 
7.2.3.4  Baseline symptom assessment 
 
A baseline symptom assessment will be made, including such relevant conditions as hot 
flashes and other vasomotor symptoms. A 29 Item validated Menopause Quality of Life 
Intervention Questionnaire will be utilized at baseline and end of study.  We will also 
utilize the hot flash scoring system developed by Dr. Charles Loprinzi at Mayo Clinic 
[Loprinzi 2002]. Adverse events will be recorded using NCI common terminology criteria 
for adverse events version CTCAE 4.0. 
 
7.2.3.5 Negative Pregnancy Test 
 
For peri-menopausal women with most recent menstrual period less than 12 months, a 
negative pregnancy test is required prior to dispensing study agent.  This may be either 
blood or urine based. 
 
7.3  Evaluations During Study Intervention 
 
Participants will be contacted after 1 and 3 months by the study coordinator by telephone or e-
mail as the subject prefers to review dosing and adverse events. These will be recorded in the 
source documents and subsequently entered onto CRFs. 


 
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Women who report grade 3-4 adverse events (other than menopausal symptoms) will be 
evaluated in person to fully assess adverse events; and will be followed at intervals deemed 
necessary by the investigator until the adverse event has resolved.   
 
7.4  Evaluation at Completion of Intervention 
 
The off-study fine needle aspiration is scheduled to occur after 6 months of study agent. 
However, aspiration may be delayed for scheduling purposes and study agent may be 
continued for up to two additional months, until the aspiration is performed.  If this occurs, then 
studies associated with the repeat aspiration and completion of study will be conducted at that 
time, not at a calendar six months. 
 
Participants will have a mammogram, DEXA, RPFNA, and a brief physical (including clinical 
breast exam) performed by a study-associated clinician.  An off study mammogram will be 
obtained at KUMC paid for by the study if it is not time for the regular screening mammogram.   
All abnormal findings will be recorded on the physical source document, including duration and 
severity. Vital signs, height, weight, BMI, and waist measurement will also be recorded. 
 
The collection of fasting blood for biomarker assay should be done same day if at all possible 
and will be performed prior to the RPFNA. Women will eat prior to having the RPFNA performed 
 
When the participant has completed taking study agent, any remaining supply will be returned to 
the Investigational Pharmacy and the number of tablets returned will be counted for assessment 
of compliance. 
 
7.5  Post-intervention Follow-up Period 
 
Approximately four weeks after last consumption of study agent, the participant will be contacted 
by the trial coordinator to assess adverse events and answer any remaining questions. 
 


 
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8.  CRITERIA FOR EVALUATION AND ENDPOINT DEFINITION 
 
8.1  Primary Endpoint 
 
Prior to formal analysis, there are two aspects of a feasibility trial that are based on simple 
numbers.  If we cannot accrue 40 eligible women within a reasonable time (no more than 12 
months) and/or if more than 15% of enrolled participants do not complete the study, then it 
would not be practical to propose future studies. 
For the Specific Aim of documenting that Duavee® does not cause a meaningful increase in 
proliferation, only participants for whom paired specimens evaluable for Ki-67 will be included.  
An increase in Ki-67 will be defined by one of two scenarios: a) from a baseline Ki-67 <1% to a 
Ki-67 >2%; or b) for a baseline Ki-67 >1%, at least a doubling of the baseline value.  
8.2  Secondary Endpoints 
 
For the second Specific Aim (Does Duavee® cause a reduction in Ki-67?), we will consider only 
participants having baseline Ki-67 >1%; and thus the potential to experience a decrease. 
Any remaining assessments of modulation due to Duavee® would be considered exploratory for 
purposes of informing future applications and trial designs. This would include change in breast 
cytomorphology index score; changes in gene expression (specifically, pS2, PGR, ER alpha, 
GREB-1, Amphiregulin, SDF-1, cyclin D1) by RTqPCR; changes in levels of ER alpha and cyclin 
D1 and possibly other proteins and phosphoproteins by proteomics; change in serum 
bioavailable hormones, adipocytokines, and change in menopause specific quality of life. For 
these analyses, all participants with paired pre-study and post-study data will be included. 
8.3  Off Agent Criteria 
 
Participants may stop taking study agent for the following reasons: completed the protocol-
prescribed intervention, adverse event or serious adverse event, inadequate agent supply, 
noncompliance, concomitant medications, medical contraindication, or physician determination. 
Participants will continue to be followed, if possible, for safety reasons and in order to collect 
endpoint data according to the schedule of events.   
8.4 Off Study Criteria 
 
Participants may go ‘off-study’ for the following reasons: the protocol intervention and any 
protocol-required follow-up period is completed, adverse event/serious adverse event, lost to 
follow-up, non-compliance, concomitant medication, medical contraindication, withdraw consent, 
death, or participant wishes.  If a participant goes off-study for any other reason, details will be 
recorded. 
8.5  Study Termination 
 
A frequency of greater than grade 2 adverse events (exclusive of hot flashes and menstrual 
irregularities) that exceeds 20% will be cause for consideration of stopping the trial; unless there 
is adequate information to suggest that the adverse events are not related to the study agent. 


 
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9.  CORRELATIVE/SPECIAL STUDIES 
 
9.1  Ki-67 and Cytomorphology 
 
Tissue for Ki-67, cytomorphology (4/5 of aspirate), are placed in a modified Cytolyt solution as 
indicated below and from ~ 1/5th of the aspiration placed immediately in PBS and is immediately 
frozen in liquid nitrogen for gene and protein expression studies.  
 
9.1.1  Ki-67 Primary Endpoint 
 
Cells are acquired by RPFNA following informed consent. Cells are pooled in a 15 cc 
conical tube containing 10 cc of formalin modified Cytolyt™ and processed via a liquid 
based thin layer cell preparation system (ThinPrep™ 2000, Cytyc Corporation). A 
minimum of two slides will be made: at least one each for cytology and one for Ki-67. 
Total epithelial cells will be delineated with Mayer’s hematoxylin counterstain and 
proliferating cells with a MIB-1 (DAKO M7240) antibody for Ki-67. We have adapted a 
staining technique using high temperature antigen retrieval [Khan 2005]. 
 
A DAKO automatic stainer will be used for all stains except hematoxylin to insure 
staining consistency. Ki-67 is visualized as punctate brown dots in the nucleus. Manual 
counting will be performed by two laboratory technicians employed and scoring 
assessed by two laboratory technicians. The scores will consist of number of cells 
assessed, and number of positive cells for Ki-67. Hyperplastic ducts with the highest 
concentration of MIB-1 positively staining cells will be selected for analysis and 500 total 
ductal cells will be scored. Numbers of nuclei expressing Ki-67 will be expressed as a 
percentage of total numbers of cells counted. If hyperplasia is not present in the follow-
up specimen at 6 months, non-proliferative areas will be assessed. Cell clusters 
assessed are viewed with a digital imaging system and representative images archived. 
A separate score for each technician and a consensus score will be recorded. The 
consensus score will be utilized for analysis of the primary endpoint. 
 
9.1.2  Cytomorphology 
 
Cytologic assessment of all screened and on-study participants will be performed by Dr. 
Carola Zalles, the designated cytopathologist for this study. She will provide a traditional 
characterization (QNS, normal, apocrine metaplasia, epithelial hyperplasia, or 
hyperplasia with atypia) and a semi-quantitative cytology index score (modified Masood 
score [Masood 1990])  
 
9.2  Serum Assays  
 
Blood will be drawn fasting for adipocytokines and hormones. All baseline and end of study 
serum assays will be run together along with pooled serum to control for batch variation. 
Adipocytokines are run using customized Luminex kits in the breast cancer prevention 
laboratory. Estradiol and testosterone will either be performed in the Breast Cancer Prevention 
laboratory or, alternatively, at the Ligand Assay and Core Analysis Laboratory at the University 
of Virginia (UVA). The lower limit of detection for estradiol at the Ligand Lab is 1.5 pg/ml and the 
intraassay variation is 4.3%. SHBG is assayed by an immunometric assay using the Immulite 
analyzer (Diagnostic Products Corp), with a lower limit of detection 0.2 nmol/L and intraassay 
variation of 2.4%. 


 
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9.3  Exploratory Molecular Markers 
 
Depending upon funds availability, exploratory studies may be conducted of molecular markers 
from archived frozen tissue stored at -80C.  This includes RTqPCR for estrogen inducible gene 
transcripts and a proteomics panel.  
 
If residual cellular material is available, cells may be processed to slides and processed for 
evaluation of ER expression by immunocytochemistry. ER expression will be assessed with ID5 
antibody from Dako diluted at 1:100 and applied for 30 minutes.  The ER protocol has been 
optimized for fine needle aspirate specimens processed as ThinPrep slides [Petroff 2006].  This 
protocol involves use of low temperature (90C) retrieval for two minutes with a 0.2X nuclear 
decloaker (Biocare) and use of a 0.01% glucose oxidase blocking reagent for 30 minutes at 
37C.  ER expression will be hand scored by two readers utilizing 100 cells from a hyperplastic 
duct exhibiting ER staining.  The proportion of cells staining at each intensity (0-4+) will be 
assessed.  The proportion of cells staining at each intensity is then multiplied by the intensity to 
achieve proportion/intensity sub-scores. The weighted intensity score (IS) is computed by 
adding together the proportion/ intensity sub-scores.  Under this system, scores can 
theoretically range from 0-4.  However, for benign hyperplastic cells which average 30% ER 
expression with 2+ staining, our median intensity score is approximately 0.6. 


 
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10.  SPECIMEN MANAGEMENT 
 
10.1  Specimen Processing and Laboratories 
 
10.1.1 RPFNA 
 
RPFNA is performed in the Breast Cancer Prevention Center as outlined above. 
Cytolyt/formalin fixed specimen is placed on a rocker and frozen specimen is kept in 
liquid nitrogen prior to transport (few hours) and processing in the Breast Cancer 
Prevention Laboratory. The KUMC Breast Cancer Prevention Center Laboratory, under 
the direction of Carol Fabian MD. Is located on the first floor of the Cancer Research 
Building with a total of 1597 square feet plus 400 square feet of office space. The 
laboratories are fully equipped for cell culture, the processing and assessment of 
cytology and immunocytochemistry slides, multiple serum and tissue assays (ELISA’s 
RIAs), laser microdissection, RT-qPCR, and various other molecular biology techniques. 
Staff located in the administrative/laboratory unit include an experienced cytology 
technician, a masters prepared biologist, and an additional technician experienced in 
multiple molecular techniques.    
 
10.1.2  Blood Based Assays  
 
Initial processing for blood tests occurs in the Breast Cancer Prevention Center where 
we have all necessary equipment for phlebotomy, and initial processing including a 
refrigerated centrifuge and immediate access to a -80C freezer. 
 
Blood for the chemistry profile will be drawn in a mint gel clot tube performed by a 
current CLIA certified clinical laboratory. Generally the clinical laboratory at the Cancer 
Center Clinical Laboratory at the Westwood campus. 
 
Hormones: Two full gel clot tubes are allowed to clot at room temperature for thirty 
minutes, then spun at 3500 rpm for 15 minutes.  1-1.5 ml aliquots are placed into four 
polypropylene cryovials. Tubes are then stored in the -80C freezer until all specimens 
from a participant can be analyzed in the same batch.   
 
Adipocytokine panel: 1 red serum tube (no gel stopper) is allowed to clot at room 
temperature for thirty minutes, then centrifuged at 4,100 RPM for 5 minutes at 4C.  
Place 1-1.5 ml aliquots into three 2 ml cryovials. Tubes are then stored in the -80C 
freezer until all specimens from a participant can be analyzed in the same batch.   
 
Blood for Bazedoxifene Levels: 1 EDTA plasma tube is obtained.  Centrifuge at 3000 
RPM for 10 minutes.  Place 1ml aliquots into two 2 ml cryovials.  Tubes are then stored 
in the -80C freezer until all specimens from a participant can be analyzed in the same 
batch.   
 
10.3  Shipping instructions 
 
Samples to be assayed at the University of Virginia will be shipped on dry ice. 
 
 


 
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11.  REPORTING ADVERSE EVENTS 
 
DEFINITION: An adverse event (AE) is any untoward medical occurrence in a study participant.  
An AE does not necessarily have a causal relationship with the treatment or study participant.  
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with participation in a study, whether or not 
related to that participation. This includes any death that may occur while a participant is on a 
study. 
 
11.1 Adverse Events 
 
11.1.1 Reportable Adverse Events 
 
 All adverse events that occur after the informed consent is signed must be recorded on 
the adverse event CRF whether or not related to study agent. 
 
11.1.2  AE Data Elements 
 
 
AE reported date 
 
AE Verbatim Term 
 
CTCAE Term (v 4.0) 
 
Event onset date and event ended date 
 
Severity grade 
 
Attribution to study agent (relatedness) 
 
Whether or not the event was reported as a Serious Adverse Event (SAE) 
 
Action taken with the study agent 
 
Outcome of the event 
 
If participant discontinues trial because of AE 
 
Comments 
 
11.1.3  Severity of AEs 
 
 
 
Identify the adverse event using the NCI Common Terminology Criteria for Adverse 
Events (CTCAE) version 4.0.  The CTCAE provides descriptive terminology and a 
grading scale for each adverse event listed.  A copy of the CTCAE can be found at 
http://ctep.cancer.gov. 
 
AEs will be assessed according to the CTCAE grade associated with the AE term.  AEs 
that do not have a corresponding CTCAE term will be assessed according to their 
impact on the participant’s ability to perform daily activities as follows: 
 
 
 
Grade 
Severity 
Description 
1 
Mild 
Barely noticeable, does not influence functioning 
Causing no limitations of usual activities 
2 
Moderate 
Makes participant uncomfortable, influences functioning 
Causing some limitations of usual activities 
3 
Severe 
Severe discomfort, treatment needed 
Severe and undesirable, causing inability to carry out 
usual activities 


 
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4 
Life 
threatening 
Immediate risk of death 
Life threatening or disabling 
5 
Fatal 
Causes death of the participant 
 
11.1.4  Assessment of relationship of AE to treatment 
 
 
The possibility that the adverse event is related to study drug will be classified as one of 
the following:  not related, unlikely, possible, probable, definite. 
  
11.1.5   Follow-up of AEs 
 
All AEs, including lab abnormalities that in the opinion of the investigator are clinically 
significant, will be followed according to good medical practices and documented as 
such. 
 
11.2  Serious Adverse Events 
 
11.2.1  Definition 
 
ICH Guideline E2A and Fed. Reg. 62, Oct. 7, 1997 define serious adverse events as 
those events, occurring at any dose, which meet any of the following criteria: 
 
Results in death 
 
Is life threatening (Note: the term life-threatening refers to an event in which the 
patient was at risk of death at the time of the event; it does not refer to an event 
which hypothetically might have caused death if it were more severe). 
 
Requires inpatient hospitalization or prolongation of existing hospitalization 
 
Results in persistent or significant disability/incapacity 
 
Is a congenital abnormality/birth defect 
 
Events that may not meet these criteria, but which the investigator finds very 
unusual and/or potentially serious, will also be reported in the same manner. 
 
11.2.2  Reporting Serious Adverse Events to  
 
As per institutional procedures, SAEs will be reported to the University of Kansas Cancer 
Center Data Safety and Monitoring Committee and to the Human Subjects Committee 
(KUMC’s IRB) 
 
Elements to be reported may include 
 
Date and time of the SAE 
 
Date and time of the SAE report 
 
Name of reporter 
 
Call back phone number 
 
Protocol number 
 
Title of protocol 
 
Description of the SAE, including attribution to drug and expectedness 
 
 
All SAE’s will be followed until there is resolution or stability based on the PI’s judgment. 
 
 
 
 
 


 
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12.  STUDY MONITORING 
 
12.1  Data Management 
 
Investigator/staff will also keep a log of potentially eligible individuals screened for study and if 
not entered the primary reason they declined participation.  
 
Eligibility checklist and all clinical data capture form (including current medications) will be 
completed by the PI or clinical staff. Laboratory reports and data will be kept in the participant 
specific study binders as well. Co-existent disease and adverse events will be coded using the 
NCI CTCAE, version 4.0.  
 
Source documents will consist of reports (mammogram reports, laboratory reports, etc.), and 
forms created specifically for this study that will capture information such as physical exam, 
phone contact information, adverse events, concomitant medications, demographics, etc. 
Source document files/chart or electronic medical records must be kept available for monitoring 
and throughout data analysis.  
 
All participants will be registered in the Comprehensive Research Information System (CRIS) 
managed by the Department of Biostatistics and the Cancer Center’s Biostatistics and 
Informatics Shared Resource.  Questionnaires will be completed online by participants for direct 
entry into REDCAP.  Where this is not possible, paper forms will be used and will be directly 
photocopied (assuring that no personal identifiers such as name or medical record number are 
retained) and these will be sent for data entry. All data forms will be stored in subject specific 
binders in the data office. All CRFs and questionnaires will be identified by participant initials 
and study ID number only. Data from laboratory and other research assessments will be entered 
into appropriate databases for eventual relay to the biostatistician for merging together with 
clinical information collected in REDCAP for statistical analyses, including quality control checks. 
 
All data will be audited and verified prior to being considered final.  When the database has 
been declared to be complete and accurate, the database will be locked and sent for statistical 
analysis.   
 
12.2   Data and Safety Monitoring Committee 
 
This study will be monitored by the University of Kansas Cancer Center Data Safety and 
Monitoring Committee, as per institutional guidelines.  There is a quarterly review of subject 
recruitment, subject retention, adverse events, and serious adverse events. 
 
12.3 Sponsor Monitoring 
 
Any required clinical monitoring by the provider of the study agent will be accommodated and 
specific procedures incorporated into the protocol. 
 
12.4  Record Retention 
 
Clinical records for all participants, including CRFs, all source documentation (containing 
evidence to study eligibility, history and physical findings, laboratory data, results of 
consultations, etc.), as well as IRB records and other regulatory documentation will be retained 


 
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by the Protocol Lead Investigator (Dr. Carol Fabian) in a secure storage facility in compliance 
with HIPAA, OHRP, FDA regulations and guidelines.  
 
 


 
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13.  STATISTICAL CONSIDERATIONS 
 
13.1 Study Design/Endpoints 
 
The experimental design for the study is an open label, single arm pilot study of Duavee® as a 
prevention drug for breast cancer in high risk peri- or post-menopausal women at high risk for 
development of breast cancer, with Ki-67 positivity of <4%.  Participants will receive Duavee® 
one tablet daily for six months.  No randomization or stratification is involved. The primary 
endpoint is demonstration of feasibility for subsequent trials of Duavee® using a series of pre-
set criteria having to do with accrual rate, completion, and lack of evidence for an increase in 
breast epithelial cell proliferation.  A secondary endpoint will examine the specific potential for 
Duavee® to actually reduce cell proliferation and thus function as a breast cancer 
chemopreventive agent.  A series of exploratory endpoints will document clinical efficacy (relief 
of vasomotor symptoms) and will examine modulation of other quantitative biomarkers 
potentially related to breast cancer development. 
 
13.2  Primary Feasibility Endpoints 
 
The primary endpoint overall is an assessment of feasibility that will inform the decision about 
and the design of subsequent trials.  This will be based on considerations of 1) ease of accrual 
of the target population; 2) acceptance and tolerability of the intervention. Basically. If either fail, 
then it would indicate that future trials with this approach are not feasible to consider 
 
For the first aspect of accrual, a criterion has been set at 20 participants accrued in 6 months 
(starting from when the trial is actually open to enrollment). If the accrual rate is slower than this, 
it would be impractical to conduct a similar trial at a single institution. While total accrual will be 
used for this initial criterion, the relative ease of accrual into the two cohorts (Cohort A, Ki-
67<1%; Cohort B, 1% ≤ Ki-67 < 4%) will be taken into consideration. Certainly it is anticipated 
that accrual to Cohort A will be easier and faster, but if accrual to Cohort B is not at least half the 
overall rate (i.e., 20 participants per 12 months) then it would indicate that a trial requiring 
measureable Ki-67 >1% would not be a feasible option.  For this assessment, accrual is 
considered as meeting all eligibility criteria, signing an informed consent, and receiving the first 
dose of study agent.  This number will then be used for consideration of the second feasibility 
aspect. 
 
The second aspect of feasibility is retention of participants on trial, completion of the planned 6 
months of intervention, and acquisition of repeat RPFNA specimen (plus blood, mammogram, 
etc.) for assessment of modulation in quantitative biomarkers.  A criterion has been set at 85% 
completion overall.  While the ultimate goal is paired samples for comparison of biomarkers (for 
sample size and power calculation purposes), a high incidence of early problems with 
participant compliance in general (adverse events, complaints about the study agent or the 
schedule, etc.) would serve as an indication that the intervention would not be suitable for future 
trials.  
 
The above criteria are intended to inform consideration of future biomarker trials with Duavee®; 
not to provide conclusive proof of principle.  Thus, pre-set values for “success” or “failure” have 
been established; rather than a formal approach of statistical analysis and sample size power 
calculations.  The total number of 40 participants represents a reasonable number for a pilot trial.  
Given our experience with similar biomarker trials using a variety of SERMs or an AI, this size 


 
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trial will readily document efficiency in accrual and identify problems with participant acceptance 
(compliance, retention, completion).  
 
13.3  Primary Quantitative Endpoint 
 
 
 
The primary quantitative (but secondary overall) endpoint addresses the specific question that 
Duavee® might produce the deleterious effect of increasing breast cell proliferation. While there 
is no evidence for this (and the FDA does not consider this a risk to be included in labelling) it is 
still a perceptual concern by women and providers.  Thus, an assessment will be made of the 
frequency of participants that exhibit a likely clinically meaningful increase in Ki-67.  This will be 
defined differently for the two cohorts, but both will contribute to the analysis of potential 
increase. For Cohort A (initial Ki-67 < 1%), a meaningful increase is defined as any value of 2% 
or greater on the off-study aspiration.  For Cohort B (initial Ki-67 1% but <4%), a meaningful 
increase is defined as a doubling or greater of the initial rate (i.e., a repeat aspiration value of at 
least 2%).  Historically, for women in our High Risk Breast Clinic similar to those being enrolled 
in this study, approximately an 11% incidence of Ki-67 increasing is observed in the absence of 
any intervention.  Therefore, with a minimum (per feasibility endpoint #2) of 34 paired 
specimens in which to assess change, an observed frequency of 10% or less would provide 
assurance that the true rate does not exceed 20% (95% confidence interval). Conversely, an 
observed frequency as high as 20% could still accommodate a true rate of 10%.  Thus, as long 
as the observed frequency is ≤20%, subsequent trials could be designed with a placebo-control 
arm. Only for an observed frequency >20% would it be impractical to design future biomarker 
trials. 
 
13.4  Secondary Exploratory Endpoints 
This study is not specifically powered to address the objectives of estimating favorable change 
in quantitative biomarker endpoints that may be related to risk for development of breast cancer.   
The most critical biomarker from a breast cancer prevention standpoint is that of cell 
proliferation, specifically a reduction in value for participants whose baseline specimen exhibited 
sufficient Ki-67 staining (1%) that there is the potential for a decrease).  Thus, this analysis will 
be limited to those participants in Cohort B.  Also this parameter will be analyzed first and 
separately from all other biomarkers.  Descriptive statistics will be used to summarize the values 
of Ki-67 (percent of cells staining positive) in the baseline specimen, the 6-month (off-study) 
specimen, and the change (both absolute and relative) from baseline to off-study.  For statistical 
analysis, the Wilcoxon signed-rank test will be used to accommodate the expected small 
sample size and to avoid any assumptions regarding normal distributions.  Only participants 
who provide paired specimens evaluable for Ki-67 will be included in this analysis.  However, for 
off-study specimens where the cell yield is so low (<100 cells per slide) that assessment of Ki-
67 is not even possible, an anti-proliferative effect can be inferred.  Thus, a value of 0.5% will be 
imputed as a reasonable compromise between imputing a value of 0% (no evidence of any 
proliferation) and a value of 1% (e.g., only 1 positive cell out of 100 counted).  Since a non-
parametric approach is used, the exact imputed value has minimal impact given that the 
criterion for inclusion is a baseline value of 1%.  For this primary evaluation of modulation of a 
quantitative measure, p<0.05 will be considered the criterion for statistical significance.   
Should there be adequate, paired specimens, further exploration of this primary endpoint 
variable, relative change in Ki-67, will be accomplished by developing an appropriate model and 
testing covariates.  If necessary, an appropriate transformation will be used in this regression.  


 
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For these possible analyses, as for the exploratory analyses below, no adjustment will be made 
for multiple comparisons, but the results will be interpreted with caution. 
Secondary to analysis of Ki-67, other variables will be assessed that represent a wide variety of 
cellular, molecular, and physical factors that have been proposed to have some level of 
connection with the process of breast cancer development.  Categorized by the “specimen” 
being assessed, the variables are: 
Participant 
 
Mammographic breast density 
 
Weight and BMI, 
 
 DEXA body composition and waist measurement 
Blood 
 
Hormones  
 
Adipocytokines 
 
Bazedoxifene levels 
RPFNA 
 
Cytomorphology (categorical) 
 
Peptides and phosphopeptides (proteomics) 
 
Gene expression (RT-qPCR) 
Descriptive statistics will be used to summarize the values at baseline (prior to start of 
intervention), at 6 months (after completion of intervention), and the change (both absolute and 
relative) from baseline to off-study.  Medians, ranges, and interquartile ranges will be employed 
for all variables, along with mean and standard deviation for variables shown to be normally 
distributed.  As appropriate, 95% confidence intervals will also be computed. 
It should be noted that these variables are anticipated to be assessed for all participants that 
provide paired specimens, and thus the total should be close to 40.  This provides additional 
opportunity to achieve normal distributes and to conduct exploratory correlational analyses.  
Thus paired t-test will be used for normally distributed values (initial or after appropriate 
transform), but nonparametric tests such as the Wilcoxon signed rank test will be used when 
necessary 
Since these analyses are considered as exploratory, no adjustments will be made for multiple 
comparisons and p<0.05 will be considered the criterion for statistical significance.  However, 
caution will be employed in the interpretation and presentation of the results. 
Lastly, an assessment of menopause symptom relief in this cohort will be conducted using a 29 
item validated Menopause Quality of Life Intervention Questionnaire and hot flash assessment.  
The same statistical methods as above will be employed. The intent is not statistically significant 
“proof” of efficacy as Duavee® is already FDA-approved for this indication.   
 
13.5  Reporting and Exclusions 
All participants who receive any Duavee® will be analyzed for adverse events.  For biomarker 
modulation, all participants that have evaluable pre-study and post-study specimens and 
assessments will be included in the analysis,  
Compliance will be determined by tablet counts, which will be done at the end of treatment.  
Participants that maintain 70% compliance throughout the duration of the trial will be considered 
“compliant”.  Compliance, in terms of the number of tablets missed and percent of participants 
compliant, will be summarized with descriptive statistics.  Supporting tests for the analyses will 


 
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be performed including only these participants who are compliant and have taken Duavee® for 
at least 3 months and have taken Duavee® within 2 weeks prior to the post-study aspiration. 
 
 13.6  Interim Analysis 
 
There will be no formal interim analysis of the endpoints of the study.  Particularly, the 
quantitative endpoints (other than cytomorphology and Ki-67) are routinely batch-processed at 
the completion of study with pre- and post-intervention samples assayed together.  However, it 
is possible that the feasibility endpoints could be evaluated early.  For example, if six 
participants drop out of the study early, one would not require enrolment of the full planned 40 
participants to conclude that the drop-out rate of 15% was unacceptably high. If it becomes 
obvious that the trial will not demonstrate feasibility, then accrual to the trial can be terminated 
early. 
Safety data will be reviewed quarterly by the KUMC KUCC Data Safety and Monitoring 
Committee. 
 


 
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14.  ETHICAL AND REGULATORY CONSIDERATIONS 
 
14.1  Form FDA 1572 
 
 
Prior to initiating this study, the Protocol Investigator will provide a signed Form FDA 1572 
stating that the study will be conducted in compliance with regulations for clinical investigations. 
 
14.2  Other Required Documents 
 
 Signed and dated current (within two years) Curriculum Vitae or biosketch for all 
investigators listed on the Form FDA 1572. 
 Current medical licenses for all investigators listed on Form FDA 1572. 
 Lab certification (e.g., CLIA, CAP) and lab normal ranges for all labs listed on Form FDA. 
 IRB Membership list/letter from IRB. 
 Documentation of training in “Protection of Human Research Subjects” for all investigators 
listed on the FDA Form 1572. 
 
14.3  Institutional Review Board Approval 
 
Prior to initiating the study and receiving agent, the Principal Investigator must obtain written 
approval to conduct the study from the University of Kansas Medical Center’s Human Subjects 
Committee (IRB). Should changes to the study become necessary, amended protocol must be 
approved by the KUMC HSC prior to implementation. 
 
14.4  Informed Consent 
 
An informed consent will be signed by every participant that undergoes an RPFNA. This 
informed consent is used in conjunction with the KUMC HSC# 4601 study, which is the High 
Risk Protocol. If a participant is interested and eligible for study participation, she will then be 
given a copy of the study-specific informed consent document. 
All potential study participants will be given a copy of the KUMC HSC-approved study-specific 
Informed Consent to review. The investigator will explain all aspects of the study in lay language 
and answer all questions regarding the study. If the participant decides to participate in the study, 
she will be asked to sign the study-specific Informed Consent document. The study agent will 
not be released to a participant who has not signed the study-specific Informed Consent 
document. Participants who refuse to participate or who withdraw from the study will be treated 
without prejudice. 
As part of the informed consent, participants will allow the use of blood samples and tissues 
collected for this study to be used for further research purposes. 
The study-specific informed consent document must be reviewed and approved by the KUMC 
HSC prior to study initiation. Any subsequent changes to the study-specific informed consent 
must be approved by the KUMC HSC prior to implementation. 
 
. 
 


 
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15.  FINANCING, EXPENSES, AND/OR INSURANCE 
 
All laboratory tests and clinic visits required specifically for study participation will be done at no 
cost to the participant or her insurance company. 


 
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16.  REFERENCES 
 
Abraham L, Pinkerton JV, Messig M, Ryan KA, Komm BS, Mirkin S. Menopause-specific quality 
of life across varying menopausal populations with conjugated estrogens/bazedoxifene. 
Maturitas. 2014; 78:212-218. 
Akbani R, Becker KF, Carragher N, Goldstein T, de Koning L, Korf U, Liotta L, Mills GB, 
Nishizuka SS, Pawlak M, Petricoin EF 3rd, Pollard HB, Serrels B, Zhu J. Realizing the 
promise of reverse phase protein arrays for clinical, translational, and basic research: a 
workshop report: the RPPA (Reverse Phase Protein Array) society.   Mol Cell Proteomics. 
2014; 13:1625-43.  
Alonzo-Proulx O, Mawdsley GE, Patrie JT, Yaffe MJ, Harvey JA. Reliability of Automated Breast 
Density Measurements.Radiology. 2015; 275:366-76. 
Allred DC, Hilsenbeck SG. Biomarkers in benign breast disease: risk factors for breast cancer.  J 
Natl Cancer Inst. 1998;90:1247-8.  
Anderson GL, Chlebowski RT, Aragaki AK, Kuller LH, Manson JE, Gass M, Bluhm E, Connelly S, 
Hubbell FA, Lane D, Martin L, Ockene J, Rohan T, Schenken R, Wactawski-Wende J. 
Conjugated equine oestrogen and breast cancer incidence and mortality in postmenopausal 
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