Clinical trial NCT01276704
Conditions: Breast Cancer
Sponsor: Carol Fabian, MD
Start date: 2011-01-01; Completion date: 2017-07-01
Enrollment: 180 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The investigators will study the effect of 12 months of SDG (Brevail) vs placebo on women at increased risk for development of breast cancer.

Detailed description:
The investigators would like to see if women at increased risk for breast cancer are likely to tolerate SDG daily for 12 months without significant side effects or changes in their menstrual cycles. The investigators would also like to determine if Brevail® can reduce breast cell proliferation in pre-menopausal women.

Arms: arm 1: Secoisolariciresinol diglycoside arm 2: Matched Placebo
Interventions: intervention 1: Placebo intervention 2: secoisolariciresinol
Intervention descriptions: intervention 1: The placebo contains same filler materials as commercially available Brevail® but without active SDG. intervention 2: 1 capsule daily of secoisolariciresinol diglycoside (SDG)50mg

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Final Version of Protocol 
for  
NCT01276704 
 
 
Prepared: June 14, 2023 


 
 
PROTOCOL FACE SHEET 
 
Protocol Number:  
KUMC HSC# 12377  
 
Protocol Title: 
Flaxseed Lignan as a Prevention Strategy for Pre-Menopausal Women at 
High Risk for Development of Breast Cancer 
 
Principal Investigator/ 
 Carol J. Fabian, MD 
Protocol Chair: 
 University of Kansas Medical Center 
 
 3901 Rainbow Boulevard MS 3003 
 
 Kansas City, KS 66160 
 
 Phone 913-588-7791 
 
 E-mail: cfabian@kumc.edu 
 
Regulatory/Financial: 
 Bruce F. Kimler, PhD 
 
 University of Kansas Medical Center 
 
 Phone 913-588-3660 
 
 E-mail: bkimler@kumc.edu  
 
KUMC Laboratory Director: Brian K. Petroff, DVM, PhD,  
 
University of Kansas Medical Center 
 
 Phone 913-588-3992 
 
 E-mail: bpetroff@kumc.edu 
 
Minority Accrual/ 
 Jennifer R. Klemp, MPH, PhD 
Quality of Life: 
 University of Kansas Medical Center 
 
 Phone 913-588-3662 
 
 E-mail: jklemp@kumc.edu  
 
 
  
Biostatistician: 
 Henry Yeh, PhD 
 
 University of Kansas Medical Center 
 
 Phone 913-588-0531 
 
 E-mail: hyeh@kumc.edu  
 
 
Collaborators : 
Debra K. Sullivan, PhD (Dietary) 
 
Samuel Kim, MD (Fertility) 
 
Consultants: 
 
Stephen D. Hursting, PhD, MPH (Cytokines/Adipokines) 
 
 
Eldon R. Jupe, PhD (SNPs) 
 
 
Johanna W. Lampe, PhD (Urine Lignans) 
 
 
Gordon B. Mills, MD, PhD (Proteomics) 
 
 
Kenneth D.R. Setchell, PhD (Plasma Lignans) 
 
 
Carola M. Zalles, MD (Cytomorphology) 
 
Agent(s)/Supplier: 
SDG (as Brevail®)/matched placebo) 
 
Lignan Research, Inc.   
 
FDA IND Exempt 
 
 


Flaxseed Lignan as a Prevention Strategy for Breast Cancer 
KUMC HSC# 12377                 Version 2.0 – October  7, 2013                             Page 2 of 53 
 
Protocol Version/Date: 
Version 2.0; October 7, 2013  
Approved November 4, 2013 
 
Version 1.10; August 27, 2013 
Approved September 11, 2013 
 
Version 1.9; August 27, 2012  
Approved August 31, 2012 
 
Version 1.8; May 25, 2012 
 
Approved June 5, 2012 
 
Version 1.7; September 14, 2011 
Approved September 19, 2011 
 
Version 1.6; August 1, 2011  
Approved August 4, 2011 
 
Version 1.5; May 4, 2011 
 
Approved May 10, 2011 
 
Version 1.4; April 5, 2011 
 
Approved April 19, 2011 
 
Version 1.3; February 15, 2011  
Approved February 18, 2011 
 
Version 1.2; December 29, 2010 
Approved January 11, 2011 
 
Version 1.1; December 1, 2010 
Approved December 17, 2010 
 
Minor alterations November 5, 2010 Approved November 12, 2010
 
Version 1.0; September 9, 2010 
Approved October 22, 2010 
 
Collaborating Institutions and Site PIs 
 
Dana Farber Cancer Institute, Boston, MA 
Judy E. Garber, MD, MPH; Site PI 
 
Northwestern University, Chicago, IL 
Seema A. Khan, MD, Site PI 
 
Ohio State University, Columbus, OH 
Lisa D. Yee, MD, Site PI 
 
University of Arkansas, Little Rock, AR 
V. Suzanne Klimberg, MD, Site PI 
 
University of Oklahoma Health Sciences Center, Oklahoma City, OK 
William C. Dooley, MD, Site PI 
 
University of Washington, Seattle, WA 
Larissa A. Korde, MD, MPH, Site PI 
 
Important Contact Information 
 
BCPC Lab  
 
 
Phone: 913-588-3821    
Email:  tmetheny@kumc.edu, tphillip@kumc.edu 
 
 
 
KUMC Study Coordinators 
 
 
 
Jessica Box, BS, PBT (ASCP)CM, CCRP® 
913-588-3622  jbox@kumc.edu 
 
 
Jennifer Nydegger, BS, CCRP® 
 
913-588-3963  jnydegger@kumc.edu 
 
 
 
 
 
CRIS Database Support 
 
 
Phone: 913-588-1210 
 
 
Email:  CRISsupport@kumc.edu 
 
 
 


Flaxseed Lignan as a Prevention Strategy for Breast Cancer 
KUMC HSC# 12377                 Version 2.0 – October  7, 2013                             Page 3 of 53 
 
 
TABLE OF CONTENTS  
 
Page 
COVER PAGE .............................................................................................................................. 1 
COLLABORATING INSTITUTIONS ............................................................................................. 2  
TABLE OF CONTENTS ............................................................................................................... 3 
LIST OF ABBREVIATIONS .......................................................................................................... 5  
SCHEMA ....................................................................................................................................... 6 
 
1. OBJECTIVES .......................................................................................................................... 8 
 
2. BACKGROUND ...................................................................................................................... 9 
2.1  Need for Prevention Alternatives to Tamoxifen in Premenopausal Women ..................... 9 
2.2 Reduction in Breast Epithelial Cell Proliferation and Cancer with Lignans  ....................... 9 
2.3 
Mechanisms by Which Lignans Block Breast Carcinogenesis  .................................... 10 
2.4 
Lignan Pilot Trial ........................................................................................................... 10 
 
3.  SUMMARY OF STUDY PLAN ............................................................................................... 14 
 
3.1    Randomized Trial Schema  .......................................................................................... 14 
 
3.2    Impact and Innovation .................................................................................................. 15 
 
4. PARTICIPANT SELECTION ................................................................................................. 16 
4.1 
Risk Level Required for RPFNA Screening for Eligibility ............................................. 16 
4.2 
Age, Life-Style and Medical Eligibility Criteria for Tissue Screening ............................ 16 
4.3 
Exclusion Criteria for Screening RPFNA and Study Participation ................................ 17 
4.4 
Inclusion Criteria for Study Entry .................................................................................. 18 
4.5 
Screening and Enrollment Process .............................................................................. 19 
4.6 
ID Assignment and Randomization  ............................................................................. 20 
 
5. AGENT ADMINISTRATION .................................................................................................. 21 
5.1 Dose Regimen and Dose Groups ................................................................................ 21 
5.2 Study Agent Accountability ........................................................................................... 21  
5.3 Packaging and Labels .................................................................................................. 21 
5.4 Storage ......................................................................................................................... 21 
5.5 Study Agent Administration .......................................................................................... 21 
5.6 Blinding and Unblinding Methods ................................................................................. 22 
5.7 Agent Destruction/Disposal .......................................................................................... 23 
5.8 Concomitant Medications ............................................................................................. 24 
5.9 Dose Modification ......................................................................................................... 24 
5.10 Interruption or Discontinuation of Treatment ................................................................ 23 
5.11 Adherence/Compliance ................................................................................................ 23  
 
6. CLINICAL EVALUATIONS AND PROCEDURES................................................................ 24 
6.1 Schedule of Events  ........................................................................................................ 24 
6.2 Pre-study Procedures and Evaluations ........................................................................... 25 
6.3 On Study Visit/Baseline Studies ...................................................................................... 26 
6.4 Evaluations During Study Intervention ............................................................................ 28 
6.5 Evaluation at Off-Study 12 Month Visit ............................................................................ 29 
6.6 Post-Intervention Follow-up Period .............................................................................. 31 
 
7. BIOLOGIC SPECIMEN HANDLING, STORAGE AND SHIPPING ...................................... 32 


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8.  SAFETY ASSESSMENTS ..................................................................................................... 33 
8.1 Definition of an Adverse Event ..................................................................................... 33 
8.2 Adverse Event Reporting ............................................................................................. 33 
8.3 Assessment of Relationship of AE to Treatment .......................................................... 34 
8.4 Follow-up of AEs .......................................................................................................... 34 
8.5 Serious Adverse Events ............................................................................................... 34 
8.6 Reporting Serious Adverse Events .............................................................................. 34 
 
9.   BIOMARKER AND LABORATORY ASSESSMENTS ......................................................... 35 
 
10. DATA MANAGEMENT ......................................................................................................... 37 
10.1  Source Documents and CRFs ...................................................................................... 37 
10.2  Data and Safety Monitoring Plan .................................................................................. 37 
10.3 FDA Monitoring ............................................................................................................. 38 
10.4 Record Retention .......................................................................................................... 38 
 
11.  STATISTICAL METHODS .................................................................................................... 39 
11.1 Sample Size and Power   .............................................................................................. 39 
11.2 Statistical Analysis ........................................................................................................ 39 
 
12. REGULATORY ..................................................................................................................... 41 
 
13.  REFERENCE LIST .............................................................................................................. 42 
 
Appendix 1 – Biomarkers and Testing Procedures ............................................................... 51 
 


Flaxseed Lignan as a Prevention Strategy for Breast Cancer 
KUMC HSC# 12377                 Version 2.0 – October  7, 2013                             Page 5 of 53 
 
List of abbreviations 
 
ADH 
Atypical ductal hyperplasia 
AE 
Adverse event 
ALT 
alanine aminotransferase 
AMH 
Antimullerian hormone 
AST 
aspartate aminotransferase 
BCPT 
Breast Cancer Prevention Trial 
b.i.d. 
bis in diem/twice a day 
BMD 
Bone mineral density 
CI 
Confidence interval 
BMI  
Body mass index  
BPQ 
Breast pain questionnaire 
CBC 
Complete blood count 
CRF 
Case Report/Record Form 
DCIS 
Ductal carcinoma in situ 
ECG 
Electrocardiogram 
EDL 
Enterodiol 
ENL 
Enterolactone 
ER 
Estrogen receptor 
FDA 
Food and Drug Administration 
FNA 
Fine needle aspiration 
FSH 
Follicle stimulating hormone 
IGF1 
Insulin-like growth factor 1 
IGFBP3 
Insulin-like growth factor binding protein 3 
IND 
Investigational new drug 
i.v. 
intravenous(ly) 
IRB 
Institutional review board 
IUD 
Intrauterine device 
KUCC 
KUMC 
LCIS 
University of Kansas Cancer Center 
University of Kansas Medical Center 
Lobular carcinoma in situ 
LH 
Lutenizing hormone 
p.o. 
per os/by mouth/orally 
RPFNA 
Random periareolar fine needle aspiration 
SAE 
Serious adverse event 
SDG 
Secoisolariciresinol diglycoside 
SECO 
Secoisolariciresinol 
SHBG 
Sex hormone binding globulin 
SOP 
Standard operating procedure 
 
 
 
 


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SCHEMA 
 
Initial Screening 
Premenopausal women with BMI < 40 kg/m2, age 21-49 who have any of the following risk factors 
suggesting moderate to increased risk of breast cancer: first or second degree relative diagnosed 
with breast cancer under the age of 60; prior breast biopsy showing proliferative breast disease, 
ADH, DCIS, LCIS; multiple biopsies regardless of histology, mammographic density 50% or 
higher; atypia by RPFNA; BRCA1/2 mutation carrier; 5 yr Gail risk of ≥1.67% or 2X risk for age as 
given in model, or 10 yr Tyrer-Cuzick risk 2x population risk as listed in model. 
No pregnancy or lactation for previous 12 months. If using a hormonal type of contraception, 
should have been using the  same type of hormonal contraception for at least 2 months prior to 
RPFNA. . 
RPFNA must be able to be scheduled between day 1 and 10 of the menstrual cycle in women 
having at least 4 menstrual periods in the past year. 
If not menstruating because of prior endometrial ablation or use of progestin only IUD or prior 
hysterectomy with ovaries preserved, must be able to predict timing of follicular phase by 
hormone levels (estradiol, progesterone, FSH) assessed prior to RPFNA. 
If undergoing yearly mammographic screening must have normal mammogram within nine 
months of RPFNA.  If not undergoing routine screening, no mammogram is necessary. 
 
 
 
Screening Visit for Eligibility 
No flaxseed supplements or antibiotics for the 3 weeks prior to RPFNA. 
Random breast FNA performed during follicular phase (Day 1-10) of menstrual cycle or if non-
menstruating additional serum aliquot frozen for confirmation of follicular phase.  
Serum and plasma specimens frozen for later assays in central laboratories. 
CBC and fasting metabolic chemistry profile may be performed if desired and study entry 
anticipated within 3 months of RPFNA  
History and physical exam may be performed if desired and study entry anticipated within 3 
months of RPFNA  
Subject given materials for menstrual diary recording 
 
 
 
Tissue Qualifiers for trial participation 
Epithelial hyperplasia +/- atypia; Masood score ≥13; plus Ki-67 ≥ 2% 
 
 
 
Study Entry Visit (within six months of RPFNA) 
History and physical exam (if not performed within 3 months) 
Complete BCPT symptom checklist, BPQ Breast Pain Questionnaire and Lignan Diet 
Questionnaire.  Collect Pre-study Menstrual Diary Information  
CBC and fasting metabolic chemistry profile (if not performed within previous 3 months) 
Fasting serum and plasma specimens frozen for later assays in central laboratories. 
Spot (+ optional overnight) urine collection and buccal sample for later assays in central 
laboratories. 
Negative urine pregnancy test 
Randomization number assigned (next in sequence) 
 


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Study Agent dispensed – 6 month supply 
 
 
 
 
Phone contacts at 1 and 3 months to monitor adverse events and concomitant meds 
 
 
 
6-month Visit 
Review of adverse events and concomitant meds 
Return study agent for compliance check; New supply of study agent issued 
 
 
 
 
Phone contact at 9 months to monitor adverse events and concomitant meds 
 
 
 
2-4 Weeks Prior to Anticipated Off Study Visit (for non-menstruating women only) 
Assess serum hormone levels (Estradiol, Progesterone, FSH); use for prediction of menstrual 
cycle and scheduling of RPFNA during follicular phase. 
 
 
 
12-month Visit (Off-Study) 
No antibiotics for the 3 weeks prior to RPFNA. 
Fasting blood drawn, preferably within 3-4 hours of last dose of study agent 
 CBC and metabolic chemistry profile 
Serum and plasma specimens frozen for later assays at central laboratories.  
Spot (+ optional overnight) urine collection frozen for assay in central laboratories. 
 Complete BCPT symptom checklist, BPQ Breast Pain Questionnaire, Lignan Diet Questionnaire, 
Collect menstrual Diary Information. 
Return Study Agent. 
RPFNA (Day 1-10 of menstrual cycle); for non-menstruating women, additional serum aliquots 
frozen for confirmation of follicular phase. 
 
 
 
Phone contact at 2 weeks off study agent to monitor adverse events 


Flaxseed Lignan as a Prevention Strategy for Breast Cancer 
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1.0 
OBJECTIVES 
 
The overall objective is to determine whether the natural flaxseed lignan, secoisolariciresinol 
diglycoside (SDG), in amounts providing plasma lignan levels associated with reduced breast 
cancer risk, will favorably modulate risk biomarkers for breast cancer development. This will 
provide biologic evidence of SDG’s potential for breast cancer chemoprevention.  Based on a 
positive pilot study in which both Ki-67 and the proportion of women with atypical cytomorphology 
was reduced with 12 months of SDG 50 mg/day, a randomized, placebo-controlled clinical 
chemoprevention trial will be conducted wherein pre-menopausal women at increased risk for the 
development of breast cancer will receive Brevail® (200 mg, equivalent to 50 mg of SDG) or 
matched, blinded placebo for 12 months.  A total of 242 subjects will be accrued, randomized 2:1 
to SDG vs placebo, with the goal of achieving 220 subjects completing study and evaluable for 
biomarker change. 
 
The primary objective is to determine if women randomized to 12 months of SDG exhibit   greater 
change in the proportion of breast epithelial cells expressing the proliferation marker Ki-67/MIB-1 
in hyperplastic benign breast tissue than do women randomized to placebo. 
 
Secondary objectives are:  
 
1)  to determine if 12 months of SDG is associated with favorable modulation of other risk 
biomarkers including cytologic atypia, and serum levels of bioavailable estradiol and 
testosterone, IGF-I:IGFBP-3 ratio, c-peptide, adiponectin, and leptin.  
2)  to determine if Ki-67 and modulation of other risk biomarkers is correlated with change in 
blood or urine lignans.  
3)   to determine impact or potential impact on quality of life as measured by the  BPQ breast 
pain questionnaire (a modified McGill Pain questionnaire), The Breast Cancer Prevention 
Trial (BCPT) Symptom Checklist a menstrual diary for cyclicity and cycle length for women 
not on cycle modulating contraceptives`; and antimullerian hormone (AMH), a measure of 
follicular reserve.  
 
An exploratory objective is to interrogate SDG’s mechanism of action through measurement of 
changes in hormones, growth factors, gene expression, adipokines and cytokines and proteomic 
signatures. 
 
The overriding hypothesis is that biomarkers assessed both in serum and in hyperplastic breast 
tissue obtained by RPFNA from high risk pre-menopausal women will be favorably modulated by 
12 months of SDG without negatively affecting quality of life. 
 
Specifically, it is hypothesized that SDG administration will be associated with  
 a reduction in proliferation as measured by Ki-67;  
 a reduction in proportion of specimens with cytologic evidence of atypia;  
 a favorable tolerability and quality of life profile;  
 no significant impact on menstrual cycle length;  
 no significant effect on follicular reserves as measured by early follicular phase anti-
mullerian hormone in women entering the study between ages 21-35;  
 a reduction in breast pain in those women entering the study with mastalgia. 
 


Flaxseed Lignan as a Prevention Strategy for Breast Cancer 
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2.0  
BACKGROUND  
 
2.1 Need for Prevention Alternatives to Tamoxifen in Premenopausal Women 
 
It is currently estimated that of the ~194,000 invasive breast cancers  anticipated in the US this 
year, 24% or ~ 40,000 will be in women under 50, and most women <50 will be premenopausal  
[1]. A high proportion of women developing breast cancer have no major risk factors and the 
future in the US of screening mammography for average risk women under 50 is uncertain [2,3].  
Tamoxifen is the only FDA approved drug for risk reduction in premenopausal women considered 
at high risk because of a prior pre-cancerous biopsy or  Gail model estimated 5 year probability of 
cancer 1.67% or greater [4]. This equates to the average risk of a 60 year old but is about 3X that 
of the average 40 year old and 10X that of the average 30 year old [5,6]. Although an overview of 
placebo-controlled trials suggests tamoxifen reduces risk of breast cancer by 38%, no survival 
advantage has yet been demonstrated for primary prevention [7,8]. Further, uptake by even very 
high risk women is poor primarily due to concerns about side effects [9-12]. An economical 
prevention alternative is needed which does not negatively alter quality of life and is so safe that it 
does not require a prescription or medical monitoring. Such an alternative is likely to be accepted 
by a larger proportion of women and thus has the potential for greater impact on cancer 
incidence. Natural products such as lignans with few side effects, which are inexpensive and may 
be purchased without a prescription, are likely to have high uptake if reduced cancer incidence or 
favorable modulation of pathways leading to cancer can be demonstrated. 
 
2.2 Reduction in Breast Epithelial Cell Proliferation and Cancer with Lignans 
 
Lignans are a group of phenolic compounds found in edible plants which after fermentation by gut 
bacteria are converted to derivatives which modulate estrogen dependent and independent 
signaling pathways [13-15]. The typical Western diet is low in lignans, providing 10 mg or less per 
day [16,17]. An important plant lignan is Secoisolariciresinol (SECO) found in highest 
concentration in flaxseed but also in other oil rich seeds, nuts, whole grains, legumes, and certain 
fruits, and vegetables [16, 17].   The primary medicinal use of lignans in young women is to treat 
cyclic breast pain [18] and in Canada, flaxseed is considered first line treatment for this condition 
[19]. Flaxseed or SECO administered as the diglycoside (SDG) is associated with reduced 
mammary gland cell proliferation and incidence of estrogen receptor negative (ER-) and ER+ 
mammary cancer in pre-clinical studies [20-23]. SECO is converted to enterolactone (ENL) and 
enterodiol (EDL) which is readily measured in blood and urine.  There is some inconsistency [24-
38] in human correlative studies which is to be expected given the inherent limitations of dietary 
recall, early use of frequency questionnaires with incomplete validation for lignans, variation in 
lignan metabolism, and single point blood collections [39-43]. However, many studies suggest 
reduced cancer incidence in premenopausal women with higher lignan intakes or plasma ENL 
levels [25-28,30-32,36]. These correlative studies indicate reduction in risk of ER- as well as ER+ 
cancer, including ER- cancer in pre-menopausal women [29,31,33-35]. This is important as ER- 
tumors constitute approximately 40% of cancers in premenopausal women and at present there is 
no clinically available agent which has been demonstrated to reduce ER- breast cancer although 
several are undergoing initial testing [44]. Correlations between lignan exposure and breast 
cancer incidence appear stronger in premenopausal women with 1 or more CYP 17 A2 alleles 
which in turn results in higher endogenous estrogen production [25,26,30,36]. Lignans appear to 
reduce proliferation in established tumors. In a pre-surgical model trial, pre and postmenopausal 
women with newly diagnosed breast cancer randomized to ~30 days of muffins baked with 25 
grams of flaxseed were found to have a significant reduction in their tumor proliferation rate (Ki-
67) at re-excision lumpectomy or mastectomy compared to Ki-67 change in women eating muffins 
without flaxseed [45]. 


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2.3 Mechanisms by Which Lignans Block Breast Carcinogenesis 
 
Pre-clinical investigations suggest lignans have diverse effects which may be important in 
initiation, promotion and progression including  
 
 antioxidant properties [46];  
 enhanced differentiation [47] and increased expression of the tumor suppressor proteins 
BRCA 1 and 2 [48];  
 reduction in tissue estrogen production and estrogen receptor related signaling in part due 
to suppression of inflammatory cytokines and aromatase [49-53];  
 reduction in growth factor receptor related signaling and proliferation [51,54,55]; and  
 favorable modulation of adiponectin [56,57], insulin [58,59], and leptin [56,60].  
 
These latter risk biomarkers for breast cancer [57,61-66] are important modulators of MAP kinase, 
PI3 kinase and mTOR  pathways [67-75] which link extracellular signals to cellular machinery  
controlling proliferation and migration, protein synthesis, energy metabolism, and cell survival [76-
79] and are thought to play an important role in breast cancer promotion and progression.  
 
2.4 Lignan Pilot Trial 
 
2.4.1 Selection of lignan source and dose for pilot trial 
 
An important early step in evaluating a new intervention for preventive activity is to assess its 
effects on risk biomarkers for development of cancer, generally over a few months to a year. 
Tissue risk biomarkers are generally selected as the primary endpoint. If the agent has previously 
been tested in humans and a dose is reasonably well established which is likely to modulate 
markers but unlikely to cause symptoms, a single arm pilot feasibility study is first performed to 
show consistent modulation of markers and feasibility. This is then followed by a Phase II 
placebo-controlled trial [80]. In preclinical studies, either flaxseed or the lignan SDG appeared to 
be equally efficacious in reducing breast cancer [20-22] and in the placebo-controlled clinical trial 
reported by Thompson et al., 25 grams of flaxseed was able to reduce proliferation in cancer cells 
[45].  A pilot study was conducted at KUMC utilizing an SDG dose which would produce plasma 
ENL levels in the range that had been associated with significant reduction in risk for breast 
cancer in clinical correlative studies [24] and would be comparable to the 25 grams of flaxseed 
baked in muffins in the pre-surgical model study showing reduced proliferation in cancer cells [45]. 
The variability of SDG content and bioavailability in raw flaxseed and the propensity for raw 
flaxseed to cause gastrointestinal symptoms at higher doses [81] prompted the selection of a 
commercial preparation (Brevail®) with a consistent SDG content but with reduced likelihood of 
gastrointestinal symptoms. A single 200 mg Brevail® capsule contains 50 mg of highly 
bioavailable SDG providing plasma ENL levels similar to that in 25 grams of whole flaxseed. Daily 
dosing with this formulation produced median ENL levels of 63 nmol/L similar to those in the 
highest quintile of the case control study of Pietinen et al. which was associated with a 2/3 
reduction in cancer incidence when compared to ENL levels in the  lowest quintile [24,82]. 
 
2.4.2  Selection of biomarkers for pilot trial   
 
Given the likelihood that lignans serve as partial estrogen antagonists in a high estrogen 
environment [52,53,55,83], risk biomarkers were selected that were likely to be modulated by 
selective estrogen receptor modulators (SERMs), as well as those likely to be modulated by 
lignans for the pilot trial in premenopausal women [84]. The initial risk biomarker set included 
cytomorphology and Ki-67 in hyperplastic breast epithelium in tissue sampled by RPFNA, 


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mammographic breast density, serum bioavailable estradiol, testosterone, and IGF-I:IGFBP-3 [85-
90]. Subsequently serum adiponectin, leptin and cytokine assays [57,61-66,91,92] were added 
given the emergence of data that several of these markers - particularly adiponectin and possibly 
leptin - may be risk factors for breast cancer. Adiponectin counteracts the promotional effects of 
leptin on the PI3 kinase and mTOR pathways [93], reduces insulin and insulin resistance [94], and 
blocks cell entry into S phase [95], whereas cytokines promote tissue aromatase activity, 
increasing estradiol. Change in Ki-67 in areas of cytologic hyperplasia was chosen as the primary 
endpoint using the following rationale. It had previously been established in a prospective study 
that cytologic atypia in tissue sampled by random periareolar fine needle aspiration (RPFNA) was 
a strong risk biomarker for breast cancer [85]. When utilized in women at increased risk for cancer 
between the ages of 30 and 60, it was associated with a 5-fold increase in the risk of DCIS and 
invasive cancer and could stratify short term risk based on the Gail model despite being used in 
women already at increased risk for the disease [96]. However, hyperplasia with atypia is not a 
practical primary response endpoint for Phase II prevention studies because based on prior 
studies [97] it would require screening aspiration of 4-5 pre-menopausal women to enroll one 
woman with atypia. Further requiring baseline atypia would limit generalizability of results. 
Consequently, the proliferation biomarker Ki-67 assessed in areas of hyperplasia was chosen as 
the primary endpoint.  Ki-67 is currently acceptable as a primary endpoint in Phase II prevention 
trials for the following reasons [80]:  
 
Proliferation plays a fundamental role in carcinogenesis.  
 
Ki-67 expression is positively associated with pre-cancerous changes of histologic and 
cytologic atypia [86].  
 
Higher Ki-67 in hyperplastic and atypical hyperplastic specimens predicts cancer 
development [87,88].  
 
Ki-67 can be modulated quickly in both cancer treatment and prevention trials [98-100].  
 
2.4.3  Eligibility criteria for SDG pilot study 
 
Eligibility criteria included regular menstrual cycles, no oral contraceptives, a 3-fold or greater 
increase in 5-year estimated risk for breast cancer relative to SEER statistics for age group, and 
normal mammogram performed in the follicular phase of the menstrual cycle within 3 months of 
baseline RPFNA. Subjects on oral contraceptives were excluded in order to reduce subject 
hormonal heterogeneity and because it might be more difficult to demonstrate biomarker 
modulation from SDG in the face of exogenous hormones. Baseline RPFNA was also to be 
performed in the follicular phase and had to show unequivocal hyperplasia +/- atypia as well as a 
minimum Ki-67 expression of 2% in hyperplastic ducts. The Ki-67 value of 2% was chosen since 
this is the median Ki-67 value in hyperplastic tissue obtained by RPFNA from high risk pre-
menopausal women in the follicular phase of the cycle [86]. Further, a Mayo Clinic series 
suggests that women having a biopsy showing atypical hyperplasia and a Ki-67 of 2% or higher 
have a significantly higher 10-year risk of breast cancer (14% versus 3%) [88]. The early-mid 
follicular phase is utilized due to greater subject comfort and less bleeding during RPFNA. 
 
2.4.4  SDG pilot study results 
 
Forty-nine premenopausal women were accrued over 2.3 years to the single institution; NCI R21-
funded pilot study of 12 months of SDG. Forty-five subjects completed the study and were 
evaluable for biomarker change.  Median age was 43 years, BMI 22.8 kg/m2, and 5 year Gail risk 
1.6% in biomarker evaluable subjects. Seventy-five percent had one or more first degree relatives 
with breast cancer, and 22% had a prior biopsy with atypical ductal hyperplasia or lobular 
carcinoma in situ.  
 


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2.4.4.1  Change in lignan levels 
 
Compliance was excellent (median = 96%) with few serious adverse events (4% grade 3). Median 
plasma ENL increased ~ 9-fold (11 nmol/L to 99 nmol/L). Even greater increases were observed 
in SECO and EDL, often not detectable at baseline with median levels of 5.7 and 23.3 nmol/L, 
respectively, at 12 months.  
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
2.4.4.2.  Change in Ki-67 and cytomorphology 
 
Thirty-six (80%) of the 45 evaluable 
subjects demonstrated a decrease 
in Ki-67, from a median of 4% 
(range 2-16.8%) to 2% (range 0-
15.2%) (p<0.001 by Wilcoxon signed 
rank test). A decrease from baseline 
in the proportion of women with 
atypical cytology from 62% to 42% 
(p=0.035) was also observed. 
 
 
 
 
 
 
 
 
2.4.4.3  Change in blood adipokines and cytokines, growth factors and hormones  
 
Significant increases in serum adiponectin and reductions in leptin were observed along with 
borderline changes in IGFBP3 and bioavailable testosterone by ELISA techniques [101]. 
Additional preliminary data on selected serum adipokines and cytokines performed on reserved 
serum from 37 women using Luminex Multiplex Cytokine Assays, and Multiplex Endocrine Assay 
kits by Dr. Hursting at the University of Texas at Austin are promising and mirror ELISA data with 
increases in adiponectin, a significant reduction in leptin, a significant increase in the adiponectin 
to leptin ratio, and a numerical decrease in the inflammatory cytokine TNF alpha.  
 
Plasma Lignan 
N 
Pre-
Study, 
nmol/L 
Mid-Study 
(6 mo), 
nmol/L 
Post-Study 
nM (12 mo), 
nmol/L 
Difference 
Baseline to 12 
mo, nmol/L 
Secoisolariciresinol 
42 
0.0 
0.61 
 1.16 
4.4 
32.8 
 73.7 
5.7 
33.7 
 66.5 
5.5 
32.8 
 66.5 
Enterodiol 
42 
0.0 
0.69 
 1.29 
34.1 
55.2 
62.5 
23.3 
84.3 
 133.0 
23.3 
83.7 
 133.0 
Enterolactone 
42 
11.1 
15.8 
 17.4 
95.2 
108.9 
 67.0 
99.2 
132.7 
 120.3 
74.8 
117.0 
 117.7 


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2.4.4.4  Change in mammographic density  
 
There were no significant changes in follicular phase percent mammographic density.   
 
2.4.4.5  Adverse events  
 
The main expected AEs from SDG were diarrhea, flatulence, and irregular menses. GI symptoms, 
usually grade 1, were encountered in 42%, irregular menses in 26%, and hot flashes in 6% of 
subjects. Only two grade 3 AEs were reported. One subject was diagnosed with DCIS on the 
basis of the mammogram performed prior to the 12 month RPFNA.  This was not considered 
related to the study agent.  One subject discontinued study after 3 months because of grade 3 
pelvic pain that was thought to be possibly related to study agent 
 
Frequency of adverse events by worst reported per subject. 
 
 
CTC Grade 
Number 
0 
1 
2 
3 
4 or 5 
Total 
Total 
7 
(14%) 
17 
(35%) 
23 
(47%) 
2 
(4%) 
0 
(0%) 
49 
 
Change in plasma lignans did not correlate with change in Ki-67, adverse events (AEs), or 
compliance [101]. 
 
Analyte 
Pre- 
Treatment 
Post-
Treatment 
Pre-Post 
Treatment 
P value 
Leptin (ng/ml) 
15.7 ± 5.3 
12.9 ± 5.2 
1.22 
0.03 
Adiponectin (g/ml) 
23.6 ± 10.8 
29.3 ± 11.4 
0.82 
0.03 
Leptin:adiponectin Ratio 
0.67 ± 0.2 
0.49 ± 0.2 
1.37 
0.02 
TNF- (pg/ml) 
5.2 ± 2.3 
4.5 ± 1.9 
1.15 
0.16 


Flaxseed Lignan as a Prevention Strategy for Breast Cancer 
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3.0  
SUMMARY OF STUDY PLAN  
 
The favorable outcome of the pilot study suggests progression to a Phase II multi-institutional 
placebo-controlled trial of 12 months of Brevail® (50 mg SDG) in moderate and high risk 
premenopausal women. Modulation of Ki-67 in women with baseline Ki-67 of 2% or greater in 
hyperplastic tissue is the primary endpoint rather than normalization of atypia, so as to limit the 
number of women who would need to be screened by RPFNA to 2-3 for every woman going on 
trial (vs. 4-5 if all had to have atypia). Other risk biomarkers will be measured, as well as impact 
on quality of life, change in breast pain in those with mastalgia at baseline, and surrogate markers 
of fertility. These considerations will be important for eventual uptake of SDG as a prevention 
agent. Mechanisms of action will also be explored. Similar eligibility criteria are used as in the pilot 
study, where the average percentage of cells expressing Ki-67 at baseline was 6.4% (median 4%) 
with an average change of -3.7% with a common standard deviation of 5.5%.  
 
The study eligibility is being altered approximately 2.5 years after study start to allow inclusion of 
women on oral contraceptives. This will both widen application of results should they be positive 
and is expected to at least double the rate of accrual.    Assessment of difference in change in Ki-
67 between women randomized to SDG versus placebo can be accomplished with a 2:1 
randomization of 242 women. Assuming a dropout rate of 10%, with 220 evaluable subjects there 
will be 83% power to detect a 2.5% reduction in Ki-67 for the treatment group compared to no 
reduction in the control group; this will be our primary endpoint. This is based on an assumption of 
a common standard deviation of 6% for change in Ki-67 in both the no oral contraceptive and oral 
contraceptive groups and a Type I error rate of 5%   
 
3.1  Randomized Trial Schema 
 
 
 


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3.2  Impact and Innovation 
 
The potential impact of this trial is high in that it addresses unmet needs in the area of breast 
cancer prevention for premenopausal women. It has the potential to provide an intervention 
which:  
1) has excellent tolerability with low likelihood of inducing menopause symptoms, 
gynecologic problems, or reducing quality of life.   
2) is low cost and available without a prescription or need for medical monitoring.   
3) is likely to reduce ER- as well as ER+ breast cancers and  
4) has potential to reduce ovarian cancer [102].   
 
The combination of lack of objective or subjective side effects, lack of need for prescription or 
medical monitoring and low cost (Brevail® is less than $20 per month) makes it an intervention 
that can and is likely to have high proportional uptake and compliance in  average as well as high 
risk women. This contrasts with tamoxifen, the only approved agent for risk reduction in 
premenopausal women, which still requires a prescription, medical monitoring, and because of 
side effects is approved  only for high risk individuals [4].  Even if the magnitude of SDG related 
relative risk reduction is only 25% (compared to 38% for tamoxifen), with high uptake 10,000 new 
cases of breast cancer per year could be prevented in women under age 50. There is no 
prescription drug likely to  provide the uptake of an over-the-counter natural product, and thus 
even if the relative risk reduction with SDG is less than tamoxifen the impact on cancer incidence 
with a positive trial is likely to be higher.  Given the 2/3 reduction in breast cancer in case control 
studies for women with high ENL levels (similar to that achieved with 50 mg SDG/day in the pilot 
study) relative to women with low levels (similar to baseline ENL levels in the pilot), a 25% 
reduction in risk is a reasonable expectation [24,101].  
 
This will be the only placebo-controlled clinical trial of SDG or flaxseed with sufficient subjects 
such that a significant difference in the primary endpoint risk biomarker could potentially be 
observed. Further, it is the only trial taking a comprehensive approach to assessing the molecular 
mechanisms of action for lignans and their effects on risk biomarkers, quality of life, breast pain, 
menstrual cycle length and surrogate measures of fertility. The application of state of the art 
molecular techniques to the exploration of mechanisms of action and critical pathways affected 
will contribute to acceptance of the trial results by regulatory agencies, peer reviewers, and the lay 
public. 


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4.0    PARTICIPANT SELECTION 
 
4.1  Risk Level Required for RPFNA Screening for Eligibility  
 
Given the low probability of side effects and the desire to be able to generalize results to a 
moderate as well as high risk population, the target cohort is pre-menopausal women who have a 
relative risk for breast cancer which is 2-fold or greater than that of the average woman in their 
age group by virtue of any one of the following conditions:  
 
4.1.1  A 1st or 2nd degree relative with breast cancer diagnosed under the age of 60;  
 
4.1.2  A prior biopsy indicating proliferative breast disease, atypical hyperplasia, DCIS or LCIS; 
 
4.1.3  Multiple prior breast biopsies regardless of histology; 
 
4.1.4   50% or higher estimated mammographic density on visual inspection; 
 
4.1.5  Prior or current RPFNA evidence of atypia; or  
 
4.1.6  Known carrier of a BRCA1 or 2 mutation. 
 
4.1.7 Projected probability of developing breast cancer by either the Gail model (5-year risk) or 
the Tyrer–Cuzick model (10-year risk) that is at least 2-fold greater than that for the 
population.  For the Gail model any value of 5-year risk ≥1.67% satisfies the requirement.  
 
4.2  Age, Life-Style and Medical Eligibility Criteria for Tissue Screening 
 
Candidates for tissue screening for this study are pre-menopausal women who meet the risk 
criteria above and all of the following demographic and medical criteria: 
 
4.2.1  Age 21 to 49 (limiting the maximum age to 49 in women who are not already 
perimenopausal will reduce the possibility of reduction in Ki-67 due to entry into 
menopause transition during the study). 
 
4.2.2  BMI < 40 kg/m2.  
 
4.2.3   Stable hormone status for 8 weeks prior to aspiration and willing to maintain same status 
while on study. This means no change in an oral or non-oral hormonal contraceptive within 
the past 8 weeks prior to RPFNA. . 
 
4.2.4  Timing of follicular phase of menstrual cycle: 
 
a) For women who are actively menstruating, at least 4 menstrual periods in past 12 
months such that there is a reasonable expectation of being able to perform the 
aspiration in the follicular phase of the cycle (sometime between day 1 and day 10 
inclusive, with day one being the first day of bleeding). Very light periods and spotting 
count.  
OR 
b) Women who have not had 4 menstrual periods in the past year due to Mirena type 
IUDs or endometrial ablation may be screened, but hormone levels must be assayed 
2-4 weeks prior to RPFNA in order to predict when to perform RPFNA so as to be in 


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KUMC HSC# 12377                 Version 2.0 – October  7, 2013                             Page 17 of 53 
 
the follicular phase. Lab results and institutional normal ranges must be reviewed by 
the Protocol Chair, who will provide an acceptable time window within which to 
conduct the RPFNA. Women who have had their uterus removed, but still have at least 
one functioning ovary may be screened using the same hormone level check. Must be 
willing to have same assessment of hormones and prediction of follicular phase 
repeated for the off-study RPFNA. 
 
4.2.5  If undergoing annual screening mammography, must have been performed within 9 
months prior to baseline RPFNA and interpreted either as not suspicious for breast cancer 
or with any supplementary imaging performed and interpreted as not suspicious for breast 
cancer. 
 
4.2.6   Breast exam interpreted as normal (not suspicious for cancer). 
 
4.2.7  There has been a sufficient interval between a breast procedure and the RPFNA: at least 
2 months for a prior RPFNA, at least 2 months for a core needle biopsy, at least 3 months 
for an excisional biopsy. 
 
4.3  Exclusion Criteria for Screening RPFNA and Study Participation 
 
Candidates are ineligible for tissue screening if they meet any of the following conditions: 
 
4.3.1 Have changed type of hormonal contraception  during the previous 8 weeks.  
 
4.3.2 Have experienced lactation or pregnancy during the previous 12 months,   
 
4.3.3 Considered to be perimenopausal and/or entering the menopause transition 
 
4.3.4 Chronic use of systemic antibiotics. Systemic antibiotics reduce intestinal bacteria and 
thus the ability to convert SECO to ENL. Topical lotions which include antibiotics are 
permitted. Occasional use of antibiotics is allowed, but must be stopped for 3 weeks prior 
to RPFNA and for 3 weeks prior to collecting blood or urine specimens for the pre-study 
(i.e., prior to dispensing/starting study agent) assessment of lignan levels.   
 
4.3.5 Consumption of systemic antibiotics or commercial supplements containing SDG (e.g. 
flaxseed or sesame seed supplements) during the 3 weeks prior to baseline RPFNA. 
Consumption of foods containing flaxseed or sesame seed are permitted 
 
4.3.6 Use of any selective estrogen receptor modulator or aromatase inhibitor (tamoxifen, 
raloxifene, arzoxifene, acolbifine, anastrozole, exemestane, letrozole) within the previous 6 
months. 
 
4.3.7 Currently enrolled on an interventional investigational study.  
 
4.3.8 Bilateral breast implants.  
 
4.3.9 Invasive cancer diagnosis within five years, excluding squamous or basal cell skin cancer. 
Subjects with DCIS or stage I invasive cancer are eligible if they are at least 2 months 
from radiation or surgery and at least 1 yr from chemotherapy or hormone therapy. 
RPFNA will be performed on the contralateral breast only in these instances. 
 


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4.3.10 Any malignancy metastatic to other organ, excluding Hodgkin’s or non-Hodgkin's 
lymphoma. 
 
4.3.11   Regular consumption of non-prescription anticoagulants, such as aspirin, NSAIDS or fish 
oil during the 3 weeks prior to baseline RPFNA is strongly discouraged, but occasional use 
will not exclude subject from participation.  
 
4.3.12  Any consumption of prescription anticoagulants, including Coumadin and Lovenox, during 
the 3 weeks prior to baseline RPFNA.   
 
4.3.13 Any other condition or intercurrent illness that in the opinion of the investigator makes the 
subject a poor candidate for RPFNA or the trial. 
 
4.4  Inclusion Criteria for Study Entry 
 
4.4.1 RPFNA performed within 6 months of the Study Entry Visit and in the follicular portion (day 
1-10) of the menstrual cycle. Note that day 1 is defined as the first day of bleeding.  For 
non-menstruating women, RPFNA during the follicular phase must be confirmed by 
hormone levels drawn on the day of RPFNA.  Either clinical laboratory results are sent to 
Protocol Chair for assessment of menstrual cycle phase; or an additional frozen serum 
aliquot is sent to KUMC for assay of hormone levels and phase confirmation.  
Confirmation of follicular phase will be included in the eligibility report for the potential 
subject. 
 
4.4.2 
RPFNA specimen exhibits hyperplasia +/- atypia; Masood score of >13 with >500 cells on 
the cytology slide. 
 
4.4.3 Ki-67 ≥2% positivity (>500 cells). 
 
4.4.4  Willing to continue on the same or similar type of contraception during the 12 month 
course of study. If using non-hormonal contraception, continue that and if hormonal 
contraception continue that. If heterosexually active, must be agreeable to use some form 
of contraception during the trial unless husband or partner  has had a vasectomy or 
subject has had uterus removed. Safety of SDG during pregnancy has not been 
documented.   
 
4.4.5 Have reasonable organ function as documented by CBC and metabolic chemistry profile 
(within 3 months prior to Study Entry Visit). 
 
4.4.6 Willing to undergo a history and physical at baseline and 12 months and be contacted 
periodically by the trial coordinator during the 12 month study period.  
 
4.4.7 Willing to repeat RPFNA at 12 months following initiation of study agent.  
 
4.4.8 Willing to have blood drawn at baseline and 12 months. If non-menstruating, an additional 
draw will be performed at approximately 11 months so as to predict when to perform the 
RPFNA 
 
4.4.9  Able to understand and willing to provide informed consent for the RPFNA’s and study 
participation. 
 


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4.5    Screening, Consent and Enrollment Process  
 
4.5.1   Cohort and Accrual: Potential subjects will be screened in the Breast Cancer Prevention  
  
     Center at the University of Kansas Medical Center (KUMC), Kansas City, Kansas; and at          
   
     similar high risk clinics at collaborating institutions. Recruitment will be conducted over  
  
     approximately 4.5 years for a total accrual of n=242. 
 
4.5.2  Consent at KUMC: At KUMC, potential subjects will sign KUMC HSC#4601 consent for  
          screening RPFNA procedure; subjects qualifying for randomization will sign the trial  
          specific consent document.  
 
4.5.3 Consent at collaborating sites: Collaborating sites may use the trial specific consent prior to  
         screening RPFNA for obtaining biospecimens for baseline assays, or may use a separate             
         approved research/clinical consent for that purpose. 
 
4.5.4 All prospective subjects undergoing RPFNA for screening at collaborating sites will be  
         registered in a site screening log by the site study coordinator or principal investigator. The  
         study coordinator will assign a unique identifier from a pre-designated series specific to that  
         institution.  This identifier, which will include subject initials, serves as the subject “name” for  
         screening purposes and along with the date will be attached to all subject related  
         biospecimens sent to KUMC. A screening checklist will also accompany the screening  
         RPFNA specimen. This will also be used for pre-enrollment blood, urine questionnaires, and  
         data collection. No protected health information should be attached to these items. 
 
4.5.5 Subjects may not be stratified and randomized until they have completed the entire eligibility 
process including RPFNA and collection of baseline biospecimens, and the site study 
coordinator has received confirmation from KUMC that the subject is tissue eligible, and for 
non-menstruating women that the RPFNA was performed in the follicular phase. 
 
4.5.6 The RPFNA results provided to the site will include the cytomorphology descriptor (non- 
         proliferative, hyperplasia, or hyperplasia with atypia), cytology index score and Ki-67 percent  
         positive cells; as well as a statement that the subject is or is not eligible on the basis of  
         RPFNA to be randomized and enrolled. 
 
4.5.7 At that time subjects will have an on study visit scheduled at which the remaining clinical  
         eligibility criteria will be confirmed; at least a spot urine (and preferably also a 10-12 hr  
         overnight) urine, fasting blood, and a buccal swab for polymorphism testing will be collected  
         (collection materials provided by KUMC), and baseline questionnaires will be completed.  
 
4.5.8 Once all biospecimens have been collected, all questionnaires completed, and all eligibility  
         criteria have been met, the subject may be randomized and a completed eligibility checklist  
         communicated to the KUMC Project Manager. Subjects will not be considered enrolled on  
         the intervention phase of this study until they have been randomized.  
 
4.5.9 Subjects who have undergone RPFNA tissue screening and are not eligible or who have  
         decided not to participate in the intervention phase prior to randomization will have the  
         primary reason for non-participation recorded on the post-aspiration screening case report  
         form.  
 
 


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4.6  ID Assignment and Randomization   
 
4.6.1 Each site will have a series of unique study IDs that provide the study agent assignment.   
         The site study coordinator will assign the subject the next available sequential study ID.  All  
         forms, questionnaires and biospecimens subsequent to randomization are to be labeled with  
         this study ID as well as the date. 
 
4.6.2 Each site investigational pharmacy will have a randomization schedule which has been  
         prepared and provided by the Study Statistician. Study agent randomization to placebo or  
         active SDG will be provided for each sequential post-randomization study ID number.  Other  
         than the investigational pharmacist(s) and the central biostatisticians, the enrolling site staff,  
         the coordinating site staff, and subjects will be blinded as to the assignment to active agent  
         or matched placebo.   
 
4.6.3 For subjects not on oral contraceptives, sequential study ID numbers are assigned starting 
at X001 and then increasing  (e.g., X002, X003, etc.).  Subjects using oral contraceptives will be 
assigned a study ID number starting with the last number provided to the institution and then in 
decreasing order (e.g., X099, X098, X097, etc.).


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5.0 
 
AGENT ADMINISTRATION 
 
5.1  Dose Regimen and Dose Groups 
 
Both Brevail® and matched placebo are to be provided by Lignan Research, Inc. to the 
investigational pharmacy at each participating institution. 
 
 
 
Brevail® (containing 50 mg of the active ingredient SDG) or placebo (containing the same filler 
material but without SDG) will be given to all trial participants for the 12 months of participation. All 
subjects should have off-study procedures no later than within 36 hours of the last dose of SDG or 
placebo. However, if a participant stops study agent prematurely in an unscheduled situation, she 
should come in for biomarker re-assessment as early as possible. 
 
 5.2  Study Agent Accountability 
 
It is necessary to maintain a careful record of the inventory and disposition of all study agent using 
a drug accountability record form. The Investigational Pharmacy at each participating site is 
required to maintain adequate records of receipt, dispensing and final disposition of study agent. 
Include on receipt record from whom the agent was received 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.  
 
5.3  Packaging and Labels 
 
Brevail® and matched placebo will be supplied by Lignan Research, Inc. in a form ready for 
dispensing. Each bottle of 100 capsules will be labeled with a label identifying study specific 
information, such as protocol number, dosing instructions, recommended storage conditions, 
randomization number, 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. 
             
5.4  Storage   
 
Storage in the Investigational Pharmacy should be at room temperature. 
 
5.5  Study Agent Administration 
 
The Institutional Investigational Pharmacy will be responsible for receiving, inventory and 
dispensing of study agent after documentation of local IRB approval of the KUMC-approved 
protocol and consent.  
 
Study agent will be dispensed with the study number of each subject. All subjects will be given 
200 capsules (two bottles) of study agent at the baseline visit. This provides sufficient study agent 
to accommodate a 2-3 week delay in scheduling of procedures or travel. If additional study agent 
is required because of extended delays in scheduling, it may be shipped to the subject. At the 6-
month visit, the unused study agent will be counted for compliance, but then returned to the 
subject.  An additional 200 capsules (two bottles) will be dispensed for the remainder of the study.  
Again, if additional study agent is required because of extended delays in scheduling, it may be 
shipped to the subject. 
 


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The study coordinator will give the study agent to the subject with instructions to take one capsule 
at approximately the same time of day in conjunction with intake of food and at least eight ounces 
of water and to keep track of missed doses or lost capsules. 
 
5.6  Blinding and Unblinding Methods 
 
In the event of a serious adverse event or other circumstance requiring unblinding, in the opinion 
of the responsible physician, the site investigator will notify KUMC. The KUMC statistician will 
unblind the subject to the site investigator if necessary. If the treatment blind is broken, the reason 
and the date should be recorded and signed by the investigator.  
 
5.7  Agent Destruction/Disposal 
 
At the completion of investigation, all unused study agent will be destroyed in accordance with 
each site’s investigational pharmacy regulations 
 
5.8  Concomitant Medications 
 
Subjects are not allowed to start flaxseed, or lignan supplements, tamoxifen, raloxifene, 
anastrozole, letrozole or any other SERM or aromatase inhibitor or investigational drug while 
participating on this trial. Subjects may not alter their use of oral or non-oral hormonal 
contraceptives. 
 
Subjects 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.   
 
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. 
 
5.9 Dose Modification 
 
Dose modifications will not be utilized for this study. Because study agent is only available in a 
single dose capsule, no dose modification option is available.  If a subject is experiencing adverse 
events or is having a medical procedure that requires a brief suspension of SDG, this must be 
noted in the CRF on the dose suspension page. At the discretion of the PI, subjects may be 
removed from participation for significant dose suspension time periods. 
 
Subjects with grade 2 adverse events considered to be related to study agent will have the agent 
suspended until the toxicity has resolved.  The agent will then be resumed.  If toxicity again 
occurs, and is thought to be agent-related, the agent will be permanently stopped.  Grade 2 hot 
flashes or menstrual irregularities will not be cause for suspending study agent.  
 
Subjects who experience grade 3-4 toxicity will have the study agent suspended. The agent may 
be re-instituted after resolution of the adverse event, providing the investigator feels relatively 
certain that the toxicity is not due to the study agent and that resuming agent will not affect 
recovery from the condition 
 
 


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5.10  Interruption or Discontinuation of Treatment 
 
A genuine effort must be made to determine the reason(s) why a subject fails to return for the 
necessary visits or is discontinued from the trial. Information regarding the reason for not 
completing the trial will be recorded on the appropriate case report forms. 
 
It will be documented whether or not each subject completed the clinical study. If for any subject 
study treatment or observations were discontinued the reason will be recorded on the appropriate 
case report form. Reasons that a subject may discontinue participation in a clinical study are 
considered to constitute one of the following: 
 
adverse event(s) 
 
abnormal laboratory value(s) 
 
abnormal test procedure result(s) 
 
subject develops breast cancer 
 
protocol violation 
 
subject withdrew consent 
 
lost to follow-up 
 
administrative problems 
 
physician decision 
 
death 
 
All subjects that discontinue trial prematurely require notification of the Protocol Chair or her 
designee at KUMC within 72 hours of known discontinuation. 
 
Any subject who receives at least one dose of study agent will be included in the safety analysis. 
 
5.11  Adherence/Compliance 
 
 
At the end of treatment the actual quantity of unused study agent will be compared to the 
anticipated amount of unused study agent. At the six month visit, after being counted by the study 
coordinator, the unused agent will be returned to the subject. 
 
Subjects that maintain 70% of study agent intake as measured through patient reports and tablet 
counts conducted by the trial coordinator will be considered “compliant”. Percent intake will be 
computed as the number of tablets taken divided by the number of days subject should have 
taken capsules. Pill counts and compliance will be considered separately for the first six and the 
last six months in the analysis. 
 
 


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 6.0  CLINICAL EVALUATIONS AND PROCEEDURES  
 
6.1  
Schedule of Events 
Evaluations 
Procedures 
Eligibility/ 
Pre-study 
visit 
On 
Study 
Visit 
Phone 
Contacts 
Months 
1,3,9 
6 
Month 
Visit 
11 
Month 
Visit 
12 
Month 
Visit 
2 Week 
Follow up 
Phone 
Contact 
Informed Consent 
X 
X 
 
 
 
 
 
Height/Weight, Vital Signs 
X 
X 
 
 
 
X 
 
Breast Exam 
Xc 
Xc 
 
 
 
X 
 
Serum hormone assay only 
if non-menstruating 
X d 
 
 
 
X d 
 
 
RPFNA (day 1-10 of 
menstrual cycle) 
Xa  
 
 
 
 
Xa  
 
Serum/Plasma collection for 
hormone + lignan assays 
Xb 
 
 
 
 
Xb 
 
Screening eligibility 
checklist 
X 
 
 
 
 
 
 
CBC + Fasting blood for 
chem. Profile, lignan and 
cytokine c-peptide 
Xbc 
 
 
 
Xb 
 
Urine collection (spot 
required, additional 
overnight preferred, 
materials to subject for 
menstrual diary) 
 
Xb 
 
 
 
Xb 
 
Medical History 
Xc 
 
 
 
 
 
Physical Exam 
Xc 
 
 
 
X 
 
Urine Pregnancy Test 
 
X 
 
 
 
 
 
Buccal sample 
 
Xb 
 
 
 
 
 
BCPT Symptom Checklist, 
BPQ Breast pain and lignan 
intake questionnaires 
 
X 
 
 
 
X 
 
Concomitant Meds. 
 
X 
X 
X 
 
X 
X 
Dispense Study Agent 
 
X 
 
X 
 
 
 
Adverse Events 
 
 
X 
X 
 
X 
X 
Return Study 
Agent/Evaluate Compliance 
 
 
 
X 
 
X 
 
Off Study Evaluation 
 
 
 
 
 
 
X 
a  See specimen management section 7.0 for details on shipping of RPFNA materials to KUMC 
b To be shipped to KUMC for central analysis. See specimen management section 7.0. Notify 
KUMC by fax and email prior to shipment. 
c Physical, Breast exam, Baseline CBC, Medical history and fasting chem profile may be 
performed at RPFNA or Study Entry Visit as long as within 3 months prior to study entry. 
d If non-menstruating, blood assayed for hormone levels 2-4 weeks prior to anticipated RPFNA so 
as to predict timing of follicular phase. 


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6.2  Pre-Study Procedures and Evaluations 
 
6.2.1 Mammography and Clinical Breast Exam.  
 
Pre-study mammography is not a requirement for this study unless the potential subject is of 
sufficient risk for development of breast cancer that they are undergoing annual screening.  In that 
case potential subjects must have had a normal mammogram within 9 months of baseline 
RPFNA. Subjects must have a clinical breast exam at baseline that is judged as being non-
suspicious for breast cancer. 
 
6.2.2  Pre-RPFNA instructions 
 
Use of systemic antibiotics must be stopped for 3 weeks prior to RPFNA.  Subjects will be told to 
discontinue aspirin products, fish oil, vitamin E, and NSAIDs at least 3 weeks prior to the RPFNA 
to reduce risk of excessive bleeding.  Subjects should also be told to take 10 mg of vitamin K for 3 
days prior to the RPFNA and to eat a good breakfast the day of the procedure. Vitamin K is 
strongly advised to reduce bleeding, but failure to take Vitamin K will not render a subject 
ineligible.  Likewise, failure to discontinue fish oil or Vitamin E, or ingestion of an occasional 
aspirin or NSAIDS, will not render a subject ineligible. If subjects take an anti-anxiety medication 
such as ativan the day of the procedure they must have someone drive them home from the 
procedure. Otherwise they should be able to drive themselves. 
 
For non-menstruating women (see section 4.2.4), hormone levels (estradiol, progesterone, and 
FSH) must be assayed 2-4 weeks prior to RPFNA in order to predict when to perform RPFNA so 
as to be in the follicular phase. Lab results and institutional normal ranges must be reviewed by 
the Protocol Chair, who will provide an acceptable time window within which to conduct the 
RPFNA.    
 
6.2.3 Pre-study Eligibility Visit - RPFNA Procedure  
 
 
 
Breast tissue acquisition will be performed between day 1 and 10 of the menstrual cycle.  RPFNA 
will be performed under local anesthesia from both breasts (two sites on each breast or one 
unaffected breast in the case of prior DCIS or invasive cancer). The breast skin will first be 
cleansed with betadine and alcohol swabs. Two weals per breast with local anesthesia (typically 
1% lidocaine with added epinephrine 1:100,000 buffered with bicarbonate) are made using a 
tuberculin (1 cc) syringe in the skin at ~ 2 and 10 o’clock within 4 cm of the areola, avoiding 
obvious blood vessels. After skin weals are placed, 4-5 cc of the same mixture is injected deeper 
into the breast tissue at the 2 sites. For women who have a hypersensitivity to epinephrine, 
lidocaine without added epinephrine should be used.   
 
Six needle passes per site for the outer quadrants and four for the inner quadrants are performed 
with pre-wetted 10-12 cc syringes (sterile RPMI or PBS) with 21 gauge 1½ inch needle, within the 
anesthetic weal sites to withdraw epithelial cells lining the ductal tree. The clinician will 
preferentially sample areas in which resistance (density) is appreciated with the tip of the needle. 
 
At KUMC and institutions with liquid nitrogen available in the clinic, the first two passes from each 
site in the breast will be expelled into labeled cryovials containing PBS (provided by KUMC) and 
immediately flash frozen by placing in liquid nitrogen. It is critical that bloody material not be 
placed into these cryovials as it ruins the assays.  
 


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Material from the remaining 14 passes (20 passes for institutions not participating in the flash 
frozen RPFNA substudy) in both breasts will be expelled and  pooled  into a single labeled 15 cc 
tube containing modified Cytolyt™ (9 cc Cytolyt™ and 1 cc 10% neutral buffered formalin). Fluid 
from the Cytolyt™ tube should be drawn into the needle/syringe hub approximately four times 
when expelling material into tube solution.  Cell material should not be expressed against the side 
of the tube. Tubes are then placed on a tube rocker for approximately 12-24 hours prior to 
shipment to KUMC. 
 
After the procedure, subjects will have ice packs placed on the breast for 10-15 minutes to control 
swelling and bleeding. Subjects are then wrapped with gauze cling wrap to create pressure on the 
puncture sites. A snug bra is also strongly recommended. Subjects are advised to limit strenuous 
physical activity over the next 24 hours and to report any pain or signs of infection.  
 
Subjects may be offered analgesics after the procedure in order to control any pain that may 
occur.  
 
6.2.4 Pre-study Blood work performed at that time of the RPFNA visit 
 
Note: blood drawing kits will be provided to sites with tubes and vials, plus processing and 
storage information. These kits may be updated as needed and new directions for processing will 
take precedence over instructions listed below. 
 
6.2.4.1 Pre-study serum + plasma collection for assays at central laboratories (non-fasting) 
 
Two 4 ml gold top gel clot tubes will be collected for estradiol, SHBG, testosterone and 
progesterone. Blood is allowed to clot for 30 minutes at room temperature and is then centrifuged 
at ~2200 x g for 15 minutes. A minimum of 0.5 to 1 ml of serum is placed in each of at least four 
cryovials pre-labeled with subject screening identifier, date, and type of specimen (e.g., serum). 
They are to be frozen at -20C until can be transported to a -80C freezer within 2 hours for 
storage until they can be shipped to KUMC.  For non-menstruating women, an additional cryovial 
containing at least 0.5 ml of serum is sent frozen immediately to KUMC for confirmation of 
follicular phase. Alternatively, immediate assessment of estradiol, progesterone, and FSH may be 
conducted at the institutional clinical laboratory and the results sent to the Protocol Chair for 
interpretation and confirmation of follicular phase. 
 
One lavender top 4 ml EDTA plasma tube for lignans should be drawn and centrifuged at ~400 
x g for 15 minutes. Two cryovials with a minimum of 0.5 to 1 ml of plasma should be labeled with 
Study ID, date, “plasma”. These may be temporarily stored at 4C but should be moved to -20C 
within 1 hour and to -80C within 2 hours of collection.     
 
The total tubes for this visit are two 4-ml gold top and one 4-ml lavender top; except for 
non-menstruating women for whom an additional 1-2 two 4-ml gold top tubes are required.. 
 
6.2.5 Completion of Risk Information 
 
Subjects should be given a copy of the family history form to complete. This information is used to 
calculate and IBIS risk profile (central office). This worksheet should be completed and sent back 
to the central office prior to, or the day of the Study Entry Visit. The completed calculation will then 
be sent back to the investigator. The risk information CRF should be completed after the site 
receives the completed calculation. 
 


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6.2.6 Menstrual Diary 
 
Menstrual diary materials will be given to women to begin completing prior to on study visit, 
indicating days of blood flows or spotting. 
 
6.3  On Study Visit/Baseline Studies 
 
 
6.3.1  Physical and Medical History  
 
Subjects will have a brief medical history and physical (including clinical breast exam) performed 
by a study-associated clinician. Forms which may be used as source documents for medical 
history and physical will be provided by KUMC. 
 
6.3.2 Clinical Laboratory Assessments 
 
Blood for CBC and a fasting metabolic chemistry profile may be obtained and assayed at the 
institution or any CLIA approved lab at the RPFNA visit or at any time within 3 months prior to 
randomization. 
 
6.3.3  Specimens collected for central laboratory assays 
Systemic use of antibiotics must be stopped for 3 weeks prior to collecting blood or urine 
specimens for the pre-study (i.e., prior to dispensing/starting study agent) assessment of lignan 
levels.   
 
6.3.3.1  Urine collection 
 
A spot urine sample should be collected during the clinic visit.  Approximately three drops should 
be used to perform a urine pregnancy test and the results should be recorded. Urine lignans are 
stable at room temperature for up to a week, but site personnel should decant two 12-ml aliquots 
in labeled 15 ml tubes as soon as possible and store at -80C until shipment to KUMC. Site 
personnel should record urine volume as well as the time of collection.   
 
Preferably, a 10-12 hour overnight urine specimen will also be collected and brought to the clinic. 
Site personnel should decant two 12-ml aliquots in labeled (Study ID, date, “urine”) 15 ml tubes as 
soon as possible and store at -80C until shipment to KUMC. Site personnel should record urine 
volume as well as the beginning and ending times of collection.   
 
6.3.3.2  Fasting blood collection (for on study visit) 
 
Subjects should be fasting for at least 10 hours.  The time of last food consumption and the time 
of collection should be recorded.   
 
6.3.3.2.1  Fasting serum for Adipokines, Cytokines and C-peptide 
 
Two red top 4 ml serum clot tubes should be drawn, allowed to clot for 30 min at room 
temperature, and then centrifuged at ~3000 x g for 5 minutes (at 4º C, a refrigerated centrifuge is 
needed). Three to four 0.5 to 1-ml aliquots of serum should be placed in cryovials labeled with 
Study ID number, date, and ”serum”.  As soon as possible these should be stored at -20C until 
moved within 2 hours to -80C for storage until shipment to KUMC. 
 
 


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6.3.3.2.2  CBC + Fasting plasma Comprehensive Metabolic Panel + plasma lignan levels  
 
Two lavender top 4 ml EDTA plasma tubes should be drawn and one should be sent to a 
commercial lab for a CBC, the other should be centrifuged at ~400 x g for 15 minutes and two 0.5 
to 1-ml plasma aliquots should be placed into cryovials labeled with Study ID, date, “plasma”. 
These may be temporarily stored at 4C but should be moved to -20C within 1 hour and to -80C 
within 2 hours of collection. A 4 ml mint plasma separator tube is also needed for  the metabolic 
panel and should be sent to a commercial lab with the lavender top tube for CBC. 
 
6.3.3.2.3  Fasting serum for Antimullerian hormone, IGF-1 and IGFBP3 
 
One 4 ml gold top clot tube should be drawn, allowed to clot for 30 min at room temperature, 
and then centrifuged at ~2200 x g for 15 minutes. Place 0.5 to 1-ml aliquots into two cryovials and 
freeze at -20C until transferred to -80C within 2 hours. 
 
The total tubes for this visit are two 4-ml red top, one 4-ml gold top, two 4-ml lavender top 
and one 4-ml mint top. 
 
6.3.4 Buccal cell collection 
 
It is possible that single nucleotide  polymorphisms (SNPs) may affect biologic response or 
modulate lignan concentration (e.g., CYP17). Subjects will not be stratified by specific SNPs at 
randomization as this would interfere with efficient study conduct, but results will be utilized in a 
multivariate analysis assessing change in Ki-67and lignan levels. A DNA sample for assessment 
of SNPs will be obtained by rinsing with Scope® mouthwash and placing the effluent in containers 
provided by KUMC. Samples are stable in the mouthwash for years at room temperature with no 
special handling. Specimens will be shipped to the KUMC Central Collection Lab for logging and 
shipment to Intergenetics, Inc. for assessment by PCR-based methodology of a panel of SNPs 
thought to affect breast cancer risk and/or metabolic processing [114,115].  
 
6.3.5  Lignan intake questionnaire 
 
A lignan dietary/supplement questionnaire which we have tested in a premenopausal high risk 
population is based on a validated instrument by Thompson et al. [16,42], and queries intakes and 
amounts of ~125 foods containing significant amounts of lignans. This questionnaire, completed 
with a web based tool or on paper at the same time plasma and urine are obtained for lignans, will 
help us interpret unexplained increases in these specimens in women not randomized to SDG.  
 
6.3.6  Quality of life questionnaires 
 
A baseline symptom assessment will be made via the BCPT symptom checklist for overall 
measures of quality of life, the BPQ for breast pain and discomfort. The two questionnaires will be 
completed by subjects, with an estimated competition time of 30 minutes: 
 
 
BPQ breast pain questionnaire, a modified McGill Pain questionnaire [116],  
 
 
Breast Cancer Prevention Trial (BCPT) Symptom Checklist [117,118]  
 
6.3.7   Menstrual diary 
 
Women will continue to record days of menstrual bleeding or spotting throughout the trial. 


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6.3.8  Baseline symptom assessment 
 
Symptoms (e.g. diarrhea, hot flashes, headaches) existing prior to initiation of study agent will be 
recorded using NCI common terminology criteria for adverse events version CTCAE 4.0. This will 
assist the on site coordinator in determining whether symptoms arising during the course of the 
study qualify as adverse events (new or worsening symptoms). 
 
6.4  Evaluations During Study Intervention 
 
6.4.1  Telephone contact 
 
 
Subjects will be contacted by the study coordinator at months 1, 3, and 9, to review dosing, 
concomitant meds and adverse events. Subjects who are difficult to reach or do not respond to 
messages and other means of contact may be considered non-compliant. These will be recorded 
in the source documents and subsequently entered onto CRFs.  
 
6.4.2  6-Month Intermediate Visit 
 
After 6 months of study agent, a clinic visit will be scheduled to conduct an in-person review of  
 Symptoms  
 Compliance 
 Concomitant Meds 
 Adverse Events 
 
The subject should bring all remaining study agent to the visit so that the number of capsules can 
be counted and then returned to the subject. An additional supply of study agent will be dispensed 
to the subject. 
 
6.5  Evaluation at Off-Study 12 Month Visit (see section 6.1 for Study Calendar) 
 
If necessary for synchronizing the scheduling of RPFNA with the menstrual cycle, study agent 
may be continued for up to two additional months, until the RPFNA is performed.  If this occurs, 
then studies associated with the repeat RPFNA and completion of study will be conducted at that 
time, not at a calendar 12 months. Systemic use of antibiotics must be stopped 3 weeks prior to 
the off-study RPFNA procedure and for 3 weeks prior to collecting blood or urine specimens for 
the off-study assessment of lignan levels.  The procedures may be delayed by up to two months if 
necessary to facilitate this. 
 
For non-menstruating women (see section 4.2.4), hormone levels (estradiol, progesterone, and 
FSH) must be assayed 2-4 weeks prior to RPFNA in order to predict when to perform RPFNA so 
as to be in the follicular phase. Lab results and institutional normal ranges must be reviewed by 
the Protocol Chair, who will provide an acceptable time window within which to conduct the 
RPFNA. 
 
6.5.1 Off-study procedures and evaluations 
 
Subjects should take study agent the day of the off-study visit approximately 3-4 hours prior to the 
time of anticipated blood draw. Specimen collection, processing, and storage; procedures; and 
assessments will be performed as at baseline (see sections 6.2 and 6.3) and include: 
 
Physical exam (with clinical breast exam)  


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 
RPFNA – note: subject may (should) eat breakfast after blood draws but prior to RPFNA 
 
Concomitant medications 
 
Adverse events 
 
Any remaining supply of study agent will be returned by the subject, counted by the study 
coordinator and recorded, and returned to the Investigational Pharmacy. The assessment of 
compliance will be performed on the data entered onto the CRF from the pill counts. 
 
 
6.5.2 Specimens collected for central laboratory assays 
 
6.5.2.1 Urine collection for central lab assays 
 
A spot urine sample should be collected during the clinic visit.  Urine lignans are stable at room 
temperature for up to a week, but site personnel should decant two 12-ml aliquots in labeled 15 ml 
tubes as soon as possible and store at -80C until shipment to KUMC. Site personnel should 
record urine volume as well as the time of collection.   
 
Preferably, a 10-12 hour overnight urine specimen will also be collected and brought to the clinic. 
Site personnel should decant two 12-ml aliquots in labeled (Study ID, date, “urine”) 15 ml tubes as 
soon as possible and store at -80C until shipment to KUMC. Site personnel should record urine 
volume as well as the beginning and ending times of collection.   
 
6.5.2.2 Fasting blood collection for central lab assays 
 
Subjects should be fasting for at least 10 hours (but should take morning dose of Brevail for off 
study blood draw).  The time of last food consumption and the time of collection should be 
recorded.   
 
Two red top 4 ml serum clot tubes should be drawn, allowed to clot for 30 min at room 
temperature, and then centrifuged at ~3000 x g for 5 minutes (at 4º C, a refrigerated centrifuge is 
needed). Three 0.5 to 1-ml aliquots of serum should be placed in cryovials labeled with Study ID 
number, date, and ”serum”.  As soon as possible these should be stored at -20C until moved 
within 2 hours to -80C for storage until shipment to KUMC. 
  
One 4 ml lavender top EDTA plasma tube should be centrifuged at ~400 x g for 15 minutes and 
two 0.5 ml to 1-ml plasma aliquots processed into cryovials labeled with Study ID, date, “plasma”. 
These may be temporarily stored at 4C but should be moved to -20C within 1 hour and to -80C 
within 2 hours of collection.   
 
Three 4 ml gold top gel clot tubes will be collected for estradiol, SHBG, testosterone and 
progesterone. Blood is allowed to clot for 30 minutes at room temperature and is then centrifuged 
at ~2200 x g for 15 minutes. A minimum of 0.5 ml to 1 ml of serum is placed in each of at least six 
cryovials pre-labeled with subject screening identifier, date, and type of specimen (e.g., serum). 
For non-menstruating women an additional 0.5 to 1.0 ml aliquot in one cryovial is required for 
confirmation of follicular phase. They are to be frozen at -20C until can be transported to a -80C 
freezer later that day for storage until they can be shipped to KUMC 
 
An additional lavender 4 ml EDTA tube and one 4 ml mint plasma separator tube are needed 
for CBC and fasting metabolic chemistry panel performed the day of off study visit. If all off study 
visit blood is performed this day. 
 


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The total tubes for this visit are two 4-ml red top, three 4-ml gold top, two 4-ml lavender top 
and one 4-ml mint top; except for non-menstruating women for whom an additional one or 
two 4-ml gold top tubes are required. 
 
6.5.3  Lignan intake questionnaire 
 
The lignan dietary/supplement questionnaire which queries intakes and amounts of ~125 foods 
containing significant amounts of lignans. This questionnaire, completed with a paper copy or with 
a web based tool at the same time plasma and urine are obtained for lignans, will help us interpret 
unexplained increases in these specimens in women not randomized to SDG.  
 
6.5.4  Quality of life questionnaires 
 
A symptom assessment will be made via the BCPT symptom checklist for overall measures of 
quality of life, the BPQ for breast pain and discomfort. The two questionnaires will be completed 
by subjects, with an estimated competition time of 30 minutes: 
 
 
BPQ breast pain questionnaire, a modified McGill Pain questionnaire [116],  
 
 
Breast Cancer Prevention Trial (BCPT) Symptom Checklist [117,118]  
 
6.5.5 Menstrual Diaries 
 
Menstrual Diaries will be collected to document days of menstrual bleeding or spotting throughout 
the trial. 
 
6.6  Post-intervention Follow-up Period 
 
Approximately two weeks after last consumption of study agent, subject will be contacted by 
telephone by the trial coordinator to assess adverse events and answer any remaining questions. 
 


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7.0 
BIOLOGIC SPECIMEN STORAGE AND SHIPPING 
 
All specimens are stored in polypropylene tubes or cryovials labeled with subject initials, study ID 
number, date of collection, and contents of tube (screening specimens should use the screening 
ID number instead of the study ID number). 
 
Tubes containing RPFNA specimens collected as in 6.2.3 should be placed on a rocker for 12-24 
hours to ensure thorough bathing of cells with fixative. Specimen should be shipped the day after 
collection but not if that involves shipment on a weekend (in that case, ship on Monday). Tubes 
containing RPFNA specimens should be overnight shipped with cool packs and using the KUMC 
Outside RPFNA Shipping Form. The shipping form should also be communicated to the KUMC 
Lab with notification of the shipment. A call or e-mail should be made to make certain the lab is 
aware of the shipment.  
 
Buccal cell specimens may be shipped in convenient batches at any time, using the same 
procedure as for RPFNA specimens: overnight, not on a weekend, with cool packs.   
 
All baseline and off-study blood (serum and plasma aliquots) and urine specimens for a subject 
should be sent together, overnight, on 5 lbs or more of dry ice. Notification of KUMC via e-mail, 
fax (913-588-3821) or phone (913-588-3917) must be made to make certain the lab is aware of 
the shipment.   
 
No specimens should be shipped the day before a holiday or weekend. If it is uncertain whether 
the KUMC lab will be staffed, contact them before shipping. 
 


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8.0 
SAFETY ASSESSMENTS 
 
8.1  Definition of an Adverse Event 
 
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 study. 
 
Medical conditions/disease present before starting study treatment are only considered adverse 
events if they worsen after starting study treatment (any procedures specified in the protocol). For 
purposes of this study, only those events that occur after a subject is randomized are considered 
adverse events and should be noted as such. Abnormal laboratory values or test results 
constitute adverse events only if they induce clinical signs of symptoms or require therapy, and 
are recorded on the AE CRF under the signs, symptoms or diagnosis associated with them. 
 
8.2  Adverse Event Reporting 
 
8.2.1  Reportable adverse events 
 
For this study, only subjects that have signed the study consent and have been randomized to 
study agent will have adverse events recorded. 
 
8.2.2  Adverse event data elements 
 
o 
AE onset date 
o 
AE Verbatim Term 
o 
CTCAE Term (version 4.0) 
o 
AE end date 
o 
Severity grade 
o 
Attribution to study agent (relatedness) 
o 
Reported as a Serious Adverse Event (SAE)? 
o 
Action taken with study agent 
o 
Outcome of the event 
o 
Comments 
 
8.2.3 Severity of adverse events 
 
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: 
 
 
 
 
 


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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 
4 
Life 
threatening 
Immediate risk of death 
Life threatening or disabling 
5 
Fatal 
Causes death of the participant 
 
8.3  Assessment of Relationship of AE to Treatment 
 
The possibility that the adverse event is related to study agent will be classified as one of the 
following: not related, unlikely, possible, probable, definite. 
 
 
8.4  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, until such AEs 
resolve or become stable in the opinion of the investigator. 
 
 
8.5  Serious Adverse Events 
 
ICH Guidelines 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: 
 
o Results in death 
o 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). 
o Requires inpatient hospitalization or prolongation of existing hospitalization 
o Results in persistent or significant disability/incapacity 
o Is a congenital abnormality/birth defect 
o Events that may not meet these criteria, but which the investigator finds very unusual 
and/or potential serious, will also be report in the same manner. 
 
8.6  Reporting Serious Adverse Events 
 
8.6.1  Central Reporting 
 
KUMC will report all SAEs to the KUMC IRB and to the FDA using appropriate reporting 
procedures established by institutional and regulatory guidelines.   
 
All sub-contracted sites must report knowledge and information regarding an SAE to KUMC and 
the Protocol Chair or her designee within 72 hours of this occurrence via fax, email and/or 
telephone. A written report must be faxed to KUMC for review and possible submission to KUMC 
IRBI and the FDA, and to the local site IRB. 
 
 


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Attention: Carol Fabian, M.D. 
Protocol Chair  
cfabian@kumc.edu 
FAX: 913-588-3679  
Phone: 913-588-7791 
 
8.6.2  Required information 
 
Include the following information when contacting the Protocol Chair: 
 
o 
Date and time of the SAE 
o 
date and time of the SAE report 
o 
name of reporter 
o 
call back information 
o 
affiliation/institution conducting the study 
o 
protocol number 
o 
title of protocol 
o 
description of SAE, including attribution to study agent and expectedness 
o 
subject randomization number 
 
 
 8.6.3  Follow-up of SAE 
 
Site staff should send follow-up reports as requested when additional information is available. 
Additional information should be entered on the SAE form in the appropriate format. Follow-up 
information should be sent to the Protocol Chair at KUMC as soon as possible. 
 
All SAE’s will be followed until there is resolution or stability based on the PI’s judgment. 
 


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9.0 
BIOMARKER AND LABORATORY ASSESSMENTS 
 
All biomarker and laboratory assessments conducted at the central laboratory at KUMC are 
described in Appendix 1.   
 
Assessments conducted at consulting laboratories (lignan assays, polymorphisms, etc.) will be 
performed according to Standard Operating Procedures developed by each responsible site. 
 


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10.0 DATA MANAGEMENT 
 
10.1 
Source Documents and CRFs 
 
Source documents will consist of CBC and chemistry profile clinical laboratory reports, and forms 
created specifically for this study that will capture information such as physical exam, phone 
contact information, adverse events, concomitant medications, demographics, etc. To the extent 
which possible, source documents (aside from reports) will be formatted to be similar to the CRF 
format, in order to make the transcription from source to CRF more reliable. Source document 
files/chart must be kept available at sites for monitoring visits during the conduct of the study 
through completion of data analysis.  
 
Information for data analyses will be entered by site trial coordinators from source document onto 
Case Report Forms (CRFs). Data items from the CRFs will then be entered into the web-based 
CRIS study database. 
 
Subsequently, the information entered into the database will be systematically checked by Data 
Management staff. Other errors or omissions will be entered on Data Query Forms, which will be 
returned to the site trial coordinators for resolution. Data queries must be addressed and returned 
to KUMC within 14 days of submission to site. 
 
Laboratory data from RPFNA and blood analyses performed at KUMC or consulting sites will be 
entered in Excel spread sheets which can be merged with the database held at KUMC. The 
spread sheets, with confirming printouts, will be considered as source documentation. 
 
All data collected for this trial will ultimately reside in a central database. There are appropriate 
checks (such as comparison of electronic files to validated paper printouts) on data accuracy and 
completeness for all sources of data. This is accomplished via inspection of summary reports 
prepared by the biostatistician for this project, Dr. Henry Yeh.  
 
When the database has been declared to be complete and accurate, the database will be locked. 
Any changes to the database after that time can only be made by joint written agreement between 
the Protocol Chair, the Trial Statistician and the Data Manager.  
 
10.2  Data and Safety Monitoring Plan 
 
The Data Safety and Monitoring Committee (DSMC) of the University of Kansas Cancer Center 
will provide annual review of this trial 
The DSMC monitors data management and subject safety. Subject accrual is reviewed annually 
by the Protocol Review and Monitoring Committee of the University of Kansas Cancer Center.  
The DSMC also provides advice on the overall conduct of the study. Meetings will be held on an 
annual basis.  Interval conference calls may be conducted as required.  The functions of the 
DSMC are primarily those of an overview nature in regard to both data and safety, as detailed 
below.   
 
All data collected for this trial will ultimately reside in a central trial specific database.  Data 
accuracy and completeness will be monitored via inspection of summary reports prepared for the 
DSMC by the biostatistician for this project.  
 
The subject cohort consists of essentially healthy women who are defined as being at high risk for 
the development of breast cancer, but do not have any clinical evidence of or suspicion for an 


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existing malignancy.  As such, side effects that might be due to the study intervention are 
expected to be minimal but should be easily identified and monitored. 
 
In regard to adverse events (AEs) or serious adverse events (SAEs), it is not deemed necessary 
for the DSMC to address these for individual subjects.  Rather, all grade 4 toxicities are reported 
as an SAE to the institutional review board immediately. 
 
In addition to the functions performed by the DSMC, an External Advisory Board (EAB) will serve 
in a general advisory role in regards to assessing the progress that is being made on the study.  
Subject accrual rates, and specifically problems with accrual that might impact on the ability of the 
trial to be completed, can be addressed by the EAB.  Likewise, the EAB may make suggestions 
on other aspects that would improve the quality of the trial.  The EAB consists of two individuals 
with experience in clinical prevention trial: Sandhya Pruthi, M.D., of Mayo Clinic, Rochester, MN; 
and Kala Visvanathan, MBBS, of Johns Hopkins University, Baltimore, MD. 
 
10.3  FDA Monitoring 
 
The FDA may monitor/audit various aspects of the study. These monitors will be given access to 
facilities, databases, supplies and records to review and verify data pertinent to the study.  
 
10.4  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 by the Protocol 
Lead Investigator (Dr. Carol Fabian) in a secure storage facility in compliance with HIPAA, OHRP, 
FDA regulations and guidances, and NCI/DCP requirements unless the standard at the site is 
more stringent. The records will be maintained, at a minimum, for two (2) years after completion of 
the study. The records should be accessible for inspection and copying by authorized persons of 
the Food and Drug Administration.   


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11.0 
STATISTICAL METHODS 
 
The study is a Phase II, double-blind, randomized, placebo-controlled clinical trial to assess the 
potential efficacy of SDG as a chemopreventive agent, as measured by changes in proliferation 
index (Ki-67) between baseline and 12 months. Secondarily, the study will evaluate other 
pharmacodynamic properties of the agent and assess which biomarkers, if any, are modulated.  
Side effects, adverse events, and quality of life will also be assessed. 
 
Standard randomization strategies will be employed. Institutional site and whether the woman is 
on or not on oral contraceptives will be the only stratification factors, so that each site can be 
provided with a unique randomization schedule at initiation. All eligible subjects will be randomly 
assigned to SDG or placebo in a planned 2:1 ratio. 
 
11.1  Sample Size and Power 
 
The primary outcome of interest is efficacy of SDG relative to the placebo, thus the pre- to post-
treatment change in a proliferation index (percent of cells expressing Ki-67) is the basis of the 
power analyses.  From our pilot study, the average percentage of cells expressing Ki-67 at 
baseline was 4%, and the average change in percent of cells expressing Ki-67 was a 2.4% 
reduction, for subjects meeting similar eligibility requirements as  used in thistrial.  
 
A 2:1 randomization of 220 evaluable subjects will provide 83% power to detect a 2.5% reduction 
in Ki-67 for the treatment group compared to no reduction in the control group; this will be our 
primary endpoint. This is based on an assumption of a common standard deviation of 6% for 
change in Ki-67 in both groups and a Type I error rate of 5%.  Within the target of 220 evaluable 
subjects, we expect to recruit 72 women taking oral contraceptives (OC) and 148 women not 
taking OCs. Because the efficacy of SDG is unknown for OC users, we considered different 
scenarios and evaluated the average effect in the SDG arm and the corresponding power.  For 0-
70% efficacy of SDG among OC users, the respective average Ki-67 reduction range from 1.7% 
to 2.3% will result in a statistical power ranging from 50% to 76%. 
 
Ki-67 reduction in 
SDG arm (non-OC) 
Efficacy of 
SDG in OC 
Ki-67 reduction in 
SDG arm (OC) 
Ki-67 reduction in SDG 
arm (OC & non-OC avg.) 
Power 
2.5% 
0% 
0% 
1.7% 
50% 
2.5% 
20% 
0.5% 
1.8% 
55% 
2.5% 
50% 
1.3% 
2.1% 
68% 
2.5% 
70% 
1.8% 
2.3% 
76% 
2.5% 
100% 
2.5% 
2.5% 
83% 
 
11.2  Statistical Analysis 
 
All analyses will be intent-to-treat.  All subjects randomized will be evaluable for safety and all 
subjects that have both a pre and post specimen are evaluable for efficacy.  Primary analysis of 
efficacy will be performed on all evaluable subjects and secondary analysis will be done on those 
subjects who received at least 6 months of study medication and were confirmed as being in the 
follicular phase for both RPFNA procedures.  From our experience we have very little missing 
data; however, if the missing data rate is above 5%, we will use multiple imputation to impute 
these values to perform the primary and secondary analyses. 
 
The primary outcome will be change in Ki-67 (percent of cells expressing Ki-67) from baseline to 
12 months.  The two-sample t-test will be used to compare the change in Ki-67 between the SDG 


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and placebo groups.  Any difference in baseline values of Ki-67 between the two groups would be 
due to chance, however, if there is a significant difference, then analysis of covariance adjusting 
for baseline values will be performed.  Due to potential skewness in these variables we will 
assess whether or not the Central Limit Theorem may be assumed.  Given the sample size the 
existence of an extremely high degree of skewness would require the use of the Wilcoxon Signed 
Rank test.   
 
For the secondary endpoints, similar methods as above will be used to compare the treatment 
and control groups at two time points for all markers, i.e., serum hormones and clinical laboratory 
tests.  We will also correlate change in the primary biomarker Ki-67 with change in other markers 
(e.g., bioavailable estradiol, cytokines, RTqPCR results) using Pearson’s correlation coefficient.  
Again, if there exists a high degree of skewness in these variables, Spearman’s correlation will be 
utilized. 
 
Multiple regression analysis will also be used to investigate whether changes in Ki-67 are due to 
variables other than treatment effect (e.g., age, cytomorphology, Gail risk; as well as the 
predictive effect of other biomarkers on modulation of Ki-67). Best subsets and stepwise 
approaches will be employed while controlling for treatment group.  
 
For comparison of moderate or worse toxicities we will consider grade 3 toxicity or premature trial 
discontinuation as the critical events.  The prevalence of events in the SDG vs. the placebo arm 
will be examined by Fisher’s exact test.  
 
Evaluation of agent-induced hormonal changes - We will examine whether SDG has any effects 
on serum levels of bioavailable estradiol, and testosterone, determined at baseline and after 12 
months of study medication. We will determine whether addition of SDG alters the frequency or 
length of menstrual cycles. Effects on follicular reserve will be evaluated with anti-mullerian 
hormone in a sub-cohort of pre-menopausal women between ages 21 and 35 at study entry. 
Antimullerian hormone (AMH) is one of the earliest and most sensitive measures of follicular 
reserve declines for approximately 10 years before the cessation of menses. A distinct advantage 
for trial purposes is that it does not vary with day of menstrual cycle [119].   
 
Adverse Events and Quality of Life – Change from baseline to 12 months will be assessed for 
mastalgia (expected in 30-50% of subjects at baseline), menstrual cycle and follicular reserve, 
and quality of life, as a function of SDG intake, plasma lignan levels and urinary lignan excretion. 
We will use the breast pain questionnaire (BPQ), which is a modified short form of the McGill Pain 
questionnaire (SF-MPQ) to assess breast discomfort and the Breast Cancer Prevention Trials 
(BCPT) Symptom Checklist to monitor quality of life 
 
Correlative analyses will be performed with plasma and urine ENL and total lignans as the 
independent variable, with modifying variables of baseline BMI age and specific SNPs such as 
CYP17. 
 
Since this is a phase IIB trial, the power and sample size for the primary endpoint was determined 
with a two-sided type I error rate of 5%.  All subsequent secondary, tertiary and exploratory 
analyses will also be tested with a two-sided type I error rate of 5%.  This may result in an 
elevated false positive rate for these secondary and tertiary analyses, however, since this is a 
Phase IIB study, we felt to control the type I error rate for the secondary, tertiary and exploratory 
analyses would be too conservative. 
 


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12.0 REGULATORY 
 
Any changes made to the protocol will be initiated by the Protocol Chair, and submitted to KUMC 
IRB (HSC) for approval. After KUMC IRB approval, collaborating sites will submit revised protocol 
changes to local IRBs for review. 
 
The determination of the necessity to cease enrollment until revisions are approved will be 
determined by the Protocol Chair. 
 


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Appendix 1 – Biomarkers and Testing Procedures 
 
1. Measurement of Serum Hormones  
 
Serum will be stored at -80°C until assay. Pre and post study samples will be run together to avoid batch 
variation. 
 
Estradiol and SHBG will be assayed directly in serum by commercial kits from Diagnostics Biochem 
Canada Inc. The estradiol kit # CAN-E-430 (direct EIA); serum only) has a limit of detection of 10 pg/ml; 
intra-assay precision (CV) of 4.6-9.3%; inter-assay precision (CV) of 6.2-10.1%. SHBG (Direct ELISA) 
Diagnostics Biochem Canada Inc.  kit # CAN-SHBG-4010 has a limit of detection of 0.1 nmol/L; intra-assay 
CV of 3.0-8.6%; inter-assay CV of 7.2-11.6%. 
 
2. Measurement of Anti-Mullerian Hormone 
 
Anti-Mullerian hormone will be assayed using commercial kits available from Beckman-Coulter (Brea, CA).  
Serum will be directly assayed using sandwich ELISA kits (#A16507); limit of detection is 1 pM (1 ng/ml = 
7.14 pM), intra-assay CV ≤12.3%, inter-assay CV≤14.2%. 
 
3. Measurement of Serum Adipokines, Cytokines,  
 
Serum is collected in clinic, divided into 1-2 ml portions in cryovials and immediately frozen at -80C  prior to 
transfer to Dr. Hursting’s Lab at University of Texas at Austin. Luminex Multiplex Cytokine Assays, 
adipokine Luminex Multiplex Endocrine Assay kits will be used according to kit directions. 
 
4.  Measurement of Serum IGF-I and IGFBP-3  
 
IGF-I and IGFBP-3 will be performed from serum which has been stored frozen in aliquots at -80°C until 
analysis. Pre and post study samples will be run together. IGF-I and IGFBP-3 will be assayed by ELISA 
with the standard kits from R&D Systems (Minneapolis, MN).  Aliquots of pooled sera will be used to assess 
inter and intra batch variation. Molar ratio of IGF-I/IGFBP-3 will be calculated as [.130 X IGF-I (mg/ml)]/[.036 
X IGFBP-3 ng/ml].  
 
The IGF-I  kit (#DG100) is a sandwich ELISA kit with intra-assay CV 3.5-4.3%; inter-assay CV 7.5-8.3%; 
limit of detection 0.026 ng/ml. 
 
The IGFBP-3 kit (# DGB300) is a sandwich ELISA kit with intra-assay CV 2.3-5.0%; inter-assay CV 5.4-
8.0%; limit of detection 0.05 ng/ml. 
 
5.  Breast Tissue Assays   
 
Fixed tissue will be stored at room temperature in the original collection tube of CytoLyt® for a minimum of 
24 hours prior to transfer to PreservCyt®.in the Breast Cancer Prevention Laboratory. Cells may remain in 
PreservCyt® for up to 1 year before processing to micro-dissection for RNA harvest.   
 
Frozen tissue in the cryovials will be pooled and aliquoted in the Breast Cancer Prevention Laboratory for 
later assessment of tissue, adipokines and cytokines via luminex, and phosphorylated intermediates of the 
PI3 kinase and AKT/mTOR using reverse phase proteomics. Cryovials are thawed in a cold tap water bath 
and immediately placed on ice.  A 1000 µl pipettor is used to mix sample in each cryovial and pool all 
samples for 1 patient in a sterile snap-cap polypropylene tube on ice.  An aliquot of a 10X cocktail of 
PhosSTOP phosphatase inhibitor (Roche Applied Science, Indianapolis, IN) and complete protease 
inhibitor (Roche) is added to bring the combined inhibitors to 1X. 250 µl samples are aliquoted into 4 or 
more cryovials and refrozen for retention or shipping to the responsible assay laboratory. 
 
 
5.1  Breast Tissue Cytomorphology 


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Two slides are made from each pooled sample by a liquid-based technique (ThinPrep®): two for 
cytomorphology under RNAse-free conditions one of which will undergo microdissection, processing to 
RNA and then cDNA and frozen at -80°C until later use for RT-qPCR. Remaining cells will be left in 
PreservCyt® for later use for RT-qPCR.  Initial cytomorphologic assessment is performed by the cytotech 
and then the slide is photographed and sent to the cytopathologist, Dr. Carola Zalles for final assessment. 
Slides will be given both a categorical classifier (non-proliferative or normal, hyperplasia, hyperplasia 
borderline atypia, atypical hyperplasia, or suspicious for cancer) and a Masood Index score [106]. The 
Masood score is the result of 1-4 points given for 6 different morphologic characteristics: cellular 
arrangement (monolayer, overlapping nuclei, clustering, loss of cohesion),  pleomorphism, myoepithelial 
cells (many, moderate, few, absent); anisonucleosis (absent, mild, moderate, conspicuous); nucleoli 
(absent, micronucleoli, micro and rare macronucleoli, predominately macronuclei); and chromatin clumping 
(absent, rare, occasional, frequent). Change in Masood score will be assessed by subtracting the off-study 
score from the baseline score  
 
5.2  Processing of Fixed Breast Tissue for RT-qPCR  
 
Good quality RNA is obtained from Cytolyt® fixed cells when they are processed and Pap-stained under 
RNAse-free conditions if >500 epithelial cells are used and RNA is harvested within 72 hours of Pap-
staining, or after a year or more if they are stored in the refrigerator unstained in PreservCyt® fixative 
[Petroff, 2006].  Following RNA isolation, the RNA undergoes one round of linear amplification, is converted 
to cDNA and stored at -80°C until assayed by RT-qPCR [122].  Currently 83% of LCM epithelial samples 
yield good quality RNA/cDNA using the above methods and we can reliably measure gene expression by 
RT-qPCR for a minimum of 25 genes from approximately one-quarter to one-third of the formalin-fixed 
aspirate. Specimens from PreservCyt® vials are first processed and Pap-stained under RNAse-free 
conditions and then checked by light microscopy to assure that similar types of cells are present on the 
slides for LCM as for cytomorphology and Ki-67. At least 500-1000 cells from the epithelial clusters on the 
ThinPrep® Slides are harvested by LCM within 72 hours of Pap-staining using an AutoPix™ Automated 
Laser Capture Microdissection System (MDS Analytical Technologies; Sunnyvale, CA).  RNA is isolated 
using column based chromatography and treated with DNase to eliminate any residual DNA (RecoverAll™ 
Total Nucleic Acid Isolation kit, Ambion/Applied Biosystems).  RNA is then linearly amplified using 
MessageAmp™ II RNA Amplification kit (Ambion/Applied Biosystems). The amplified aRNA is reverse-
transcribed to cDNA using SMARTScribe Reverse Transcriptase (Clontech Laboratories, Inc., Mountain 
View, CA) primed with random nonamers.  Quality of RNA/cDNA is determined by the QC Metric Assay 
[Petroff, 2006].  Primer/probe sets are designed, using Primer Express™ v 2.0, as close as possible (ideally 
within 300 bp) to the 3’ end of each transcript, because the amplified RNA is 3’-biased. 
 
5.3 Ki-67 Immunocytochemistry 
 
For Ki-67 assessments, an extra slide will be made from PreservCyt® material and total epithelial cells will 
be delineated with Mayer’s hematoxylin counterstain and proliferating cells with a MIB-1 (DAKO M7240) 
antibody and citrate buffer antigen retrieval. A DAKO automatic stainer is used for all stains to insure 
staining consistency. Manual counting is performed by two laboratory technicians. Hyperplastic ducts (if 
present) with the highest concentration of MIB-1 positively staining cells are selected for analysis and a 
minimum of 500 total ductal cells are scored. The consensus score is utilized for analysis. 
 
5.4 Processing of Frozen Breast tissue for RT-qPCR 
 
RNA isolation can be performed on fresh frozen tissue which has not undergone LCM. This is most useful 
for high abundance genes expressed in stroma and fat +/- epithelium such as adiponectin and leptin. A 250 
μl aliquot of pooled thawed RPFNA specimen is removed and mixed with 750 μl TRIzol® LS (Invitrogen 
Corporation, Carlsbad, CA) at room temperature.  After at least 5 minute incubation at room temperature 
the TRIzol® LS-sample mixture can be stored at -80°C for later RNA extraction.  RNA is extracted from the 
TRIzol® LS-sample mixture according to the manufacturer's protocol, including glycogen (Invitrogen) as a 
carrier prior to RNA precipitation.  RNA amplification, reverse transcription, and PCR analysis are 
performed in the same way as for formalin-fixed RPFNA samples [122]. 
 


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5.5 Frozen Tissue Adipokine and Cytokine, Analysis 
 
250 μl aliquots of frozen RPFNA will be labeled with date, HRBC #, and Study # and stored at -80C until 
shipment to Dr. Stephen Hursting at the University of Texas at Austin for analysis via kits supplied by 
Luminex [120,121].   
 
5.6 Frozen Tissue Reverse Phase Proteomics:  
 
250 μl aliquots of frozen RPFNA will be labeled with date HRBC # and Study # and stored at -80C until 
shipment to Dr. Gordon Mills at MD Anderson Cancer Center for immunoblotting with specific antibodies 
and analysis via ImageQuant with Viagene Software [123-125]. 
 
6. Assessment of Single Nucleotide Polymorphisms 
 
Samples of buccal cells collected in Scope® mouthwash will be used to assay for single nucleotide 
polymorphisms,(SNPs) thought to be associated with influencing risk for developing breast cancer by virtue 
of modulating a variety of cellular and metabolic processes. Specifically, variants of CYP17 will be assayed 
since it has been hypothesized to affect lignin levels.  Samples are stable in the mouthwash for months at 
room temperature and no special handling. Specimens will be shipped to Dr. Eldon Jupe at Intergenetics, in 
Oklahoma City, OK, for assessment [114,115], at an alternate laboratory if necessary, or will be assayed at 
KUMC. 
 
7. Lignans  
 
7.1  Measurement of Plasma Lignans 
 
Fasting plasma will be shipped to Cincinnati Children’s Hospital where it will be assessed by HPLC analysis 
with Waters Quattro Micro UPLC system coupled to electrospray tandem mass spectrometry (ESI-MS/MS) 
by Dr. Kenneth Setchell [111]. 
 
7.2 Measurement of Urine Lignans 
 
Spot and overnight (12 hour) collections of urine will be shipped to Fred Hutchinson Cancer Research 
Center for measurement by Dr. Johanna Lampe using isotope dilution gas chromatography-mass 
spectrometry in SIM mode [41,112,113], with normalization by creatinine level. 
 
8. Questionnaires  
 
8.1 The BCPT symptom checklist is a 42 item list of problems common to peri and postmenopausal women 
and those undergoing anti-hormonal treatment in prevention trials.  Women are asked if they have had the 
symptom in the past four weeks.  If they answer yes, they are asked to grade the severity (0-4).  This tool 
can be used to compute a total score or individual or related items of interest (e.g., three items relate to joint 
stiffness and arthralgias) may be scored [117,118].  
 
8.2 The Breast Pain Questionnaire (BPQ), a modified short form of the McGill Pain questionnaire (SF-
MPQ), will be used to assess breast discomfort and a possible effect of SDG to ameliorate symptoms of 
mastalgia [116]. 
 
8.3 Changes in menstrual cycle length will be assessed by having subjects complete a menstrual diary for 
each month they are on study. 
 
8.4 Lignan intake will be assessed at study entry using a questionnaire modified after that developed by 
Thompson et al. [16,42] which queries intakes and amounts of ~125  foods containing significant amounts 
of SECO, pinoresinol, matairesinol, and sesamin, all of which can be converted to the mammalian lignans 
ENL and EDL [16].