Conditions: Breast Cancer
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 51
Healthy volunteers: no

Brief summary:
This is a pilot study evaluating the effect of hydroxytyrosol, a component of olive oil, on mammographic density in women at high risk of developing breast cancer.

Detailed description:
This is a pilot study evaluating the effect of hydroxytyrosol, a component of olive oil, on mammographic density in women at high risk of developing breast cancer. Participants will take hydroxytyrosol orally once daily for 1 year. Breast density as determined by mammography and the safety/toxicity of hydroxytyrosol will be assessed.

Arms:
arm 1: Hydroxytyrosol 25 mg orally once daily for 1 year.

Interventions: intervention 1: Hydroxytyrosol
Intervention descriptions: intervention 1: None

Protocol document:
Prot_SAP_000.pdf: 
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A PILOT STUDY OF HYDROXYTYROSOL, A COMPONENT OF OLIVE OIL FOR BREAST CANCER 
PREVENTION IN WOMEN AT HIGH RISK OF BREAST CANCER 
 
 
 
 
 
 
 
 
 
 
 
Authors 
Tejal Patel, M.D 
Sergio Granados, PhD. 
Jaime A. Mejia, M.D. 
Document type 
Olive Oil  Use Protocol  
HMRI IRB number 
Pro00009472 / 0613-0084 
Version number 
6 
Development  phase 
Pilot 
Document status 
Final 
Release date 
 
Amendment #:  
(05/06/2013) 
Amendment 6;  January 23, 2017 
IND 
118763 
Referenced Investigator Brochure Edition  
N/A  


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Table of contents 
Table of contents .................................................................................................................... 2 
List of figures .......................................................................................................................... 3 
List of tables ........................................................................................................................... 3 
1 
Background and Rationale ...................................................................................................... 4 
1.1 
Overview of pathogenesis, epidemiology and current treatment ...................................... 4 
1.2 
Introduction to the  investigational Product(s) ................................................................ 5 
1.2.1 
Preclinical Evidence for the Chemopreventive effects of HT ............................. 5 
2 
Mammographic Density as a Marker of Breast Cancer Risk ........................................................ 7 
2.1 
Methodology............................................................................................................... 7 
2.2 
Risk Assessment ........................................................................................................ 8 
3 
Objectives and endpoints ........................................................................................................ 8 
4 
Study design .......................................................................................................................... 9 
4.1 
Description of study design .......................................................................................... 9 
5 
Study Population .................................................................................................................. 10 
5.1 
Inclusion and Exclusion criteria .................................................................................. 10 
6 
Treatment ............................................................................................................................ 12 
6.1 
Study treatment ........................................................................................................ 12 
6.1.1 
Dosing regimen ............................................................................................... 12 
6.2 
Dose modifications .................................................................................................... 12 
6.2.1 
Dose modification and dose delay ................................................................... 12 
6.2.2 
Visits ................................................................................................................ 13 
7 
Visit schedule and assessments ............................................................................................ 15 
7.1 
Study flow and visit schedule ..................................................................................... 15 
7.2 
Assessment types ..................................................................................................... 18 
7.2.1 
Pregnancy and assessments of fertility ........................................................... 18 
7.2.2 
Drug levels and pharmacokinetic assessments N/A ........................................ 18 
8 
Safety monitoring and reporting ............................................................................................. 18 
8.1 
Adverse events ......................................................................................................... 18 
8.1.1 
Definitions and reporting .................................................................................. 18 
8.1.2 
Laboratory test abnormalities .......................................................................... 19 
8.2 
Serious Adverse Events ............................................................................................ 19 
8.2.1 
Definitions ........................................................................................................ 19 
8.2.2 
Reporting ......................................................................................................... 20 
9 
Statistical methods ................................................................................................................ 21 
10 References: ......................................................................................................................... 23 
11 Appendices ........................................................................................................................ 25 


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Appendix A: ECOG Performance Status Criteria ..................................................................... 25 
Appendix B: New York Heart Association (NYHA) Classifications .............................................. 26 
Appendix C: CTCAE Files ..................................................................................................... 27 
Appendix D:  Trial Logistics ................................................................................................... 28 
 
List of figures 
Figure 1-1 
Chemical structure of Hydroxytyrosol ................................................................ 6 
 
 
List of tables 
 
Table 7-1 
Visit evaluation schedule ................................................................................. 16 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1 
Background and Rationale 
1.1 
Overview of pathogenesis, epidemiology and current treatment 
Breast cancer is the most frequently diagnosed cancer in women in developed countries.  An estimated 
226,000 new cases of invasive breast cancer and 39,920 deaths are expected to occur in 2012 in the 
United States [1].  Although the average lifetime risk of breast cancer in an American woman is one in 
eight, the risk is increased in women with a strong family history of breast cancer, BRCA 1 and 2 gene 
mutations, atypical hyperplasia or lobular carcinoma in situ (LCIS), and history of mantle irradiation for 
Hodgkin’s disease[2]. Although new systemic treatments for established breast cancer has shown to 
reduce mortality[3],but the success rate is low. Major breakthroughs have come slowly, and greater 
attention is now being directed towards breast cancer prevention[4].  One particularly promising strategy 
has been to use medication to prevent breast cancer development.   
 
Selective estrogen receptor modulators (SERMs)/Aromatase Inhibitors: The SERM, tamoxifen has 
been used in the treatment of breast cancer for many years.  The observation that contralateral breast 
cancer was reduced in women treated with tamoxifen led to a number of chemoprevention trials with 
tamoxifen and eventually raloxifen.  Results from such breast cancer prevention trials summarized in an 
overview analysis showed that SERMs significantly reduced breast cancer incidence by 38%[5]. 
Exemestane, an aromatase inhibitor evaluated in a double-blind, placebo controlled phase III study in 
4560 postmenopausal women, demonstrated a 65% relative reduction in the annual incidence of 
invasive breast cancer as well as 53% decrease in the annual incidence of invasive plus noninvasive 
breast cancer[6]. Studies using selective estrogen receptor modulators, or SERMs, have shown that 
breast cancer prevention is feasible[5]. However, while breast cancer incidence is reduced by 
approximately 50% in these studies, not all cancers are prevented.  First, there is no reduction of 
estrogen receptor (ER)-negative tumors.  Secondly, even ER-positive tumors are only reduced by one-
half to two-thirds.  Because SERMs do not totally prevent breast cancer and because of the side effects 
of long-term SERM use, the majority of risk-eligible women are reluctant to take chronic antiestrogen 
therapy for primary prevention.  Chronic therapy is necessary because these drugs suppress 
proliferation, but do not remove abnormal, premalignant cells. Thus, there is an urgent need to develop 
more effective strategies to prevent breast cancer that are tolerable to women at risk. Several nutritional 
studies have shown inverse relationship between the consumption of olive oil and the incidence of 
breast cancer[7]. Many of the beneficial properties of olive oil are associated with minor components of 
which hydroxytyrosol (HT) is the most promising. 
 
The primary reasons for targeting pre and post-menopausal high-risk women are 1) epidemiological 
studies have suggested an association between increase in olive oil intake and reduction in breast 
cancer risk 2) Studies have shown a correlation between decrease in mammographic density and 
decrease in breast cancer risk in both pre and post menopausal women  3) there is a need for a 
compound that prevents all types of breast cancer and has less toxicity  
 
Imaging and tissue-based biomarkers have been incorporated into this trial and as new information 
becomes available during the course of this trial, this study will provide a useful biorepository to draw 
from for biomarker discovery in future studies.  
 
The purpose of this trial is to evaluate the effect of hydroxytyrosol on mammographic density in women 
at high risk of breast cancer, and secondarily to gain information concerning the ability of hyroxytyrosol 
to modulate the expression of breast tissue markers and serum markers.  
 


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1.2 
Introduction to the  investigational Product(s)  
Hydroxytyrosol is a product of hydrolysis of oleuropein and due to its polar character it is found in great 
quantities in the remains from oil processing such as pomace olive oil, olive-mill waste water, or the 
rinse waters[8].  Preclinical studies have demonstrated HT to have anti-atherogenic, cardioprotective, 
anti-inflammatory, platelet aggregation inhibitor, and antimicrobial properties. This natural compound 
has been reported to regulate transcription factors (Nrf2, NFĸB), cytokines (IL-6), prostaglandins 
(PGE2), COX-2, myeloperoxidase and response to stress through Wnt pathway[8-13].  Preclinical in 
vivo studies demonstrated the potent antioxidant HT to have an anti-neoplastic role by interacting with 
Wnt/sFRP signaling pathway. 
Wnt/ β-catenin in Breast Cancer: Wnt/β-catenin signaling pathway is an important regulator of the 
stem cell pathway controlling the development of the mammary gland during embryogenesis. This 
pathway is also frequently found in invasive breast cancer and leads to a more aggressive phenotype 
and poor clinical outcome through the regulation of important cell events like proliferation, migration, 
differentiation, and apoptosis[14]. The sFRP family consists of five secreted glycoproteins (sFRP1-5) 
that function as Wnt pathway inhibitor by directly interacting with both Wnt ligands and FRP receptors. 
sFRP family are silenced in several types of cancers including breast cancer[14, 15].  The Wnt/ β-
catenin pathway has also been implicated in the control of stem cells[16]. Recently, it has been 
demonstrated that sFRP4 generate ROS (Reactive Oxygen Species)[17] by blocking Wnt/β-catenin and 
resulting in oxidative stress. Effects of Hydroxytyrosol on Wnt pathway: The in vivo antitumor activity 
of HT was evaluated in an animal model of breast carcinogenesis[18], where the authors treated breast 
tumor-bearing rats with 0.5mg/kg HT for 5 days/week for 6weeks by oral gavage. They reported HT 
decreased tumor volumes in rat models to the same degree as treatment with adriamycin.  HT also 
caused a marked decrease in tumor cell proliferation, and differential expression of genes related to 
apoptosis, cell proliferation, survival, and response to stress through inhibition of Wnt. Also a dose 
dependent decrease of β-catenin and cyclin D1 protein levels were observed.  Similar findings were 
seen in epithelial to mesenchymal markers, a decrease in both Snail and Slug transcription factors and 
an increase in protein levels of epithelial marker E-Cadherin.  
 
1.2.1 
Preclinical Evidence for the Chemopreventive effects of HT 
Antitumor in vitro studies have reported antiproliferative action due to overexpression of p21 and p27, 
and inhibition of CDK6, and pro-apoptotic events through activation of caspase-3. Moreover, regulation 
of NFĸB, COX-2, and PGE2 are also involved in regulation of carcinogenesis attributed to HT[8-13, 18].  
An in vitro study assessing HT effects on human promyelocytic leukemia cells and colon 
adenocarcinoma demonstrated that HT arrested cell cycle and induced apoptosis in tumor cells by 
arresting the cells in G0/G1 phase[19].  An in vitro study in MCF-7 human breast cancer cells 
demonstrated a similar finding of cell cycle arrest in G0/G1 phase resulting in a dose dependent growth 
inhibition[10].  
 
 
 
 
 
 
 
 
 


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Figure 1-1 
Chemical structure of Hydroxytyrosol 
 
 
 
1.2.1.1 
Epidemiological Evidence for the Chemopreventive effects of Olive oil 
In comparison with Northern European or other Western countries, Mediterranean countries have lower 
rates of mortality from cardiovascular disease and cancer. This is partially attributed to the so-called 
Mediterranean diet. Historically, scientific efforts have been focused on healthful benefits of olive oil as 
key element of a Mediterranean diet. Numerous preclinical, animal studies and at least six case-control 
and one cohort study has been conducted to test the health benefits of olive oil.  A meta-analysis 
evaluating relative risk (RR) estimates for high as opposed to low olive oil consumption that 
demonstrated reduction of the incidence of breast cancer by 38% (RR 0.62 95% CI 0.44-0.88).  One of 
the limitations for the meta-analysis was different definitions of the high olive oil consumption which 
ranged from two teaspoons a day, more than once a day, consuming 40.7g/day, or more than 8.8g/day.  
Despite that, it supports an inverse association between olive oil consumption and breast cancer risk[7]. 
Traditionally, many of the healthful properties associated with this edible oil have been ascribed to its 
high content MUFAs (monounsaturated fatty acids), such as oleic acid. Today, it is clear that many of 
the beneficial properties derived from the consumption of virgin olive oil are due to some of its minor 
compounds. The bulk of the research has focused on one of those compounds phenol hydroxytyrosol 
(HT). 
 
1.2.1.2 
Toxicology studies 
Oral administration of a single gavage dose of solid olive-pulp extract at levels of 0, 1,000, 1,500 or 
2,000 mg caused no adverse effects, except for soft or liquid feces.  The rats were administered 
5000mg/kg of olive pulp extract for 29 consecutive days and no mortality or clinical signs of toxicity were 
noted.  The authors reported that the LD50 of solid olive-pulp extract was greater than 5g/kg (equivalent 
to 2.5-3.5g/kg of HT) suggesting that the extract is non toxic[20]. 
 
1.2.1.3 
Clinical Studies in HT 
In a multicenter, randomized, crossover, controlled trial of 200 healthy male volunteers were randomized 
to olive oils with high (hydroxytyrosol content 63.5mg/L, tyrosol 24.4mg/L, and oleuropein derivaties 
327.2mg/L), moderate(hydroxytyrosol content 28.5mg/L) and low (no hydroxytyrosol) [21].  There was a 
linear increase in high-density lipoprotein (HDL) cholesterol levels with increasing in phenolic 
(hydroxytyrosol) content with the mean change of 0.025, 0.032 and 0.045mmol/L respectively.  
Triglyceride levels decreased by an average of 0.05mmol/L for all three levels of olive oil.  Oxidative 
stress markers also decreased with increasing phenolic content [21, 22].  Oxidative damae to lipids was 
assessed by measuring plasma-circulating oxidized LDL, plasma total F2α-isoprostanes, plasma C18 
hydroxy fatty acids (GC-MS) and serum LDL cholesterol un-induced conjugated dienes.  There was a 


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significant linear decrease in biomarkers of lipid peroxidation (conjugated dienes, hydroxyl fatty acids 
and oxidated LDL) with increasing phenolic content of olive oils.  Based on these data, the European 
Food Safety Authority (EFSA) felt that a cause and effect relationship has been established and 
approved 5mg hydroxytyrosol and its derivatives in olive oil should be consumed daily [23]. 
 
Two studies have evaluated the absorption of HT in healthy volunteers. The first study administered 
pure HT as a supplement in an aqueous solution (single oral dose of 2.5 mg/kg) in the plasma and urine 
of 10 healthy volunteers[24]. They found that the absorption of HT is rapid, with maximal plama 
concentration detected in 13 to 16 minutes and the levels were undetectable in 2 hours after 
administration. Another study administered 50ml olive oil in six healthy volunteers and demonstrated 
absorption of HT. The absorption was increased with sustained doses of olive oil [25]. An update in 2013 
concluded a no observed adverse effects level (NOAEL) of 500mg/kg/day [26]. 
 
A dose of 25mg/day of HT was chosen due to following reasons 1) EFSA approved hydroxytyrosol as a healthy dietary 
compound up to 15mg/day.  There is a previous unpublished study of using hydroxytyrosol 10mg BID in healthy volunteers 
with class I obesity (communication with PI).  With this dose they did not see any changes in LDL oxidation levels and did not 
have any adverse effect.  We used 25mg of hydroxytyrosol as there is demonstrated safety in previous studies and higher 
than usual healthy dietary compound.   
 
 
2 
Mammographic Density as a Marker of Breast Cancer Risk 
Studies have consistently demonstrated a 4-6 fold increase in breast cancer risk for women with 
increased mammographic density for up to 8 years following measurement [25, 27, 28]. Breast density 
as assessed qualitatively by BI-RADS category (1= ―almost entirely fat, 2 = ―scattered fibroglandular 
densities, 3 = ―heterogeneously dense, 4 = ―extremely dense).  Randomized studies have 
demonstrated that tamoxifen lowers mammographic breast density up to 4.3% yearly [29]. Given that 
tamoxifen has been shown to decrease the risk of breast cancer and does decrease breast density, 
density change may be a useful surrogate endpoint biomarker for the effect of tamoxifen and other 
chemopreventive agents on breast cancer incidence. A decrease in density of 1% could potentially 
translate into a nearly 2% lower risk of developing breast cancer [27]. Effects of HT on Breast Density: 
Data from epidemiological studies demonstrated that increase in olive oil intake was associated with 
decrease in breast densities[30]. A series of 2,000 women who had undergone routine regular 
mammography and who had detailed information regarding dietary and lifestyle available were 
evaluated. The authors reported that women in the highest tertile of olive oil consumption (>30.5 g/day) 
were associated with 30% reduction in breast density[30]. 
2.1 
Methodology  
The mammographic density will be classified by two different methods.  Firstly, according to the 
American College of Radiology’s Breast Imaging—Reporting and Data System (BIRADS), and secondly, 
by using the Cumulus software package developed by the Ontario Cancer Institute to assess percent 
density as a continuous measure (0-100% scale). The mammograms will be read for research purposes 
only. They will be read for clinical purposes at the time of the mammogram. The mammograms will be  
independently reviewed by two radiologists specializing in breast imaging. Quantitative classiﬁcation of 
mammographic parenchyma will be based on radiological assessment after digitizing mammograms and 
measuring percentage density[28, 31].  
 


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2.2 
Risk Assessment 
• Breast Risk Calculation using the modified Gail risk score [32, 33].  This model is an 
interactive tool designed to estimate a woman’s risk of developing invasive breast 
cancer in her lifetime and the next 5 years.   
 http://www.cancer.gov/bcrisktool/ 
 This model can only be used if there is no known diagnosis of DCIS or LCIS 
• A patient with a diagnosis of lobular carcinoma in situ (LCIS) or atypical lobular or 
atypical ductal hyperplasia. Germline mutations of BRCA 1 or BRCA 2 genes.  
Germline mutations in the PTEN (cowden syndrome) and TP53(Li-Fraumeni syndrome) 
• History of unilateral breast cancer 5 years ago 
• At least 10% probability of carrying BRCA mutation 
 
3 
Objectives and endpoints 
 
Primary 
• To conduct a pilot breast cancer prevention study of hydroxytyrosol in women at 
increased risk of breast cancer.   
• To assess whether mammographic density is reduced in pre or post menopausal 
women at high risk of breast cancer taking hydroxytyrosol for 1 year compared with 
baseline. 
Secondary 
• To assess the toxicity of hydroxytyrosol 
• To evaluate FACT ES score baseline, 6 months and 1 year.  
•  
Exploratory objectives (Mandatory biopsy) 
• To assess whether proliferation (Ki67 staining, Cdkn2a tumor suppressor, cyclin D1), is 
reduced in women at high risk of breast cancer after taking hydroxytyrsol for 1 year 
compared with baseline. 
• To explore the difference in the expression of other biomarkers of apoptosis (Fas 
receptor, cleaved caspase-3, caspase-8, cytochrome c), enzymes related to oxidative 
stress (Fas receptor, cleaved caspase-3, caspase-8, cytochrome c), DNA damage by 
immunohistochemistry, Wnt signaling(LRP6, β-catenin, Sfrp proteins 1 to 5, DKK1, c-
jun, c-myc)  and stem cell pathway before and after drug therapy.  
• To collect and bank breast tissue from women at high risk of breast cancer prior to and 
after treatment with hydroxytyrosol for future biomarker analysis. 
 
Exploratory endpoint (MRI Breast Imaging) 
• To assess breast MRI density pretreatment and 12 months post hydroxytyrosol therapy 
in subjects with available breast MRIs.   


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4 
Study design 
4.1 
Description of study design 
 
 
  
One hundred (100) pre and post-menopausal women at high risk of breast cancer who have declined 
tamoxifen, raloxifene or aromatase inhibitor (standard of care) will receive 25mg HT.  Eligible women will 
have a 5 yr risk by Gail Model  > 1.7, or 10% probability of BRCA mutation by BRCAPRO or similar 
model, or have a previous breast biopsy showing LCIS, atypical lobular or ductal hyperplasia, or have a 
known germline mutation in BRCA1 or BRCA2. Patients with unilateral breast cancer 5 or more years 
prior to registration are also allowed. The main exclusion criteria include women who are pregnant or 
breast feeding, with diagnosis of any malignant disease, or with inability to take oral medication.  The 
women will undergo a mammogram as well as a biopsy of normal breast tissue before and after taking 
study drug. This biopsy will be directed to the upper outer quadrant, which is typically a dense region. 
The primary endpoint of this study is change in mammographic density after treatment with HT. The 
Secondary endpoint will assess toxicity, apoptosis (Fas receptor, cleaved caspase-3, caspase-8, 
cytochrome c) and cell proliferation (Ki 67 staining, Cdkn2a tumor suppressor, cyclin D1), enzymes 
related to oxidative stress (ROS and related enzymes), DNA damage by immunohistochemistry, stem 
cell markers (CD44, CD24, and ALDH), and affectation of Wnt signaling (LRP6, β-catenin, Sfrp proteins 
1 to 5, DKK1, c-jun, c-myc) before and after drug therapy. If patients undergo a breast MRI before 
and/or after the completion of the drug, the images will be collected. The study drug will be supplied by 
PLThomas & Co..  The patients will have a study visit at3, 6 and 12 months. The study visit will include a 
general history and physical exam, an assessment for safety and concomitant medications and pill 


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count. After completion of therapy, the patient will undergo a mammogram as well as a second breast 
biopsy. 
 
5 
Study Population 
Each of the criteria in the following section must be met in order for a participant to be considered 
eligible for registration.  
 
Study Duration:   
The duration of patient participation in the study treatment will be a total of 12 months, which is counted 
from the start of treatment.  Study Follow-up for all patients will include medical history update (30 days 
after the last dose of the study drug)   
 
Safety Criteria:   
All participants will be assessed by a physician for pre-existing medical conditions and baseline physical 
abnormalities prior to the initiation of investigational therapy. Patients presenting with any medical 
history, physical exam, or laboratory abnormality that, in the in the opinion of the treating physician, 
would put the subject’s safety at risk will be excluded. Baseline signs and symptoms are to be recorded 
and followed throughout the trial.  These will be monitored throughout the study and recorded if they 
increase in severity or frequency during treatment or within the follow up period. Participants will be 
assessed for adverse events by a physician or designated midlevel provider while the subject is on 
study. Vital signs including blood pressure, heart rate and temperature should be performed at each 
physical exam.  Assessments may be performed more frequently if clinically indicated. In addition, 
hematology and serum chemistry profiles will be drawn prior to the initiation of treatment to determine 
whether the study drug combination affects hematologic values, electrolytes or liver function tests. 
Laboratory assessments will be performed more frequently if clinically indicated. This clinical and 
laboratory data will be used to determine whether these women in the study with Hydroxytyrosol have 
any symptoms or side effects associated with the study medications. Subjects will be followed for 
adverse events for a period of 30 days after the completion of investigational therapy.  Patients with 
abnormal laboratory or clinical findings that are believed to be treatment related will be followed until the 
condition resolves or stabilizes, or until the laboratory values are no longer considered clinically 
significant.   
 
CTC Version 4.0 will be used to grade toxicities.  Laboratory tests may be done more frequently if 
medically indicated. If a participant develops any grade 3 or grade 4 toxicity possibly, probably or 
definitely related to the study drug, the study drug will be discontinued. If the participant develops grade 
1 or 2 toxicities that are felt to be possibly, probably or definitely related to the study drug, the participant 
may go on a drug holiday (for no longer than 12 consecutive weeks) and restart the drug at the same 
dose. In each of these cases, the follow-up mammograms, breast biopsies will still be collected as per 
protocol one year after the initial start date of the intervention.   
 
If CTC Grade 3 or 4 hematologic toxicity is seen, CBC + differential + platelets should be repeated 
weekly until recovery. 
 
5.1 
Inclusion and Exclusion criteria 
Patients must have baseline evaluations performed prior to the first dose of study drug and must meet 
all inclusion and exclusion criteria. Results of all baseline evaluations, assuring that all inclusion and 
exclusion criteria have been satisfied, must be reviewed by the Principal Investigator or his/her designee 


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prior to enrollment of that patient. In addition, the patient must be thoroughly informed about all aspects 
of the study, including the study visit schedule and required evaluations and all regulatory requirements 
for informed consent. The written informed consent must be obtained from the patient prior to 
enrollment. The following criteria apply to all patients enrolled onto the study unless otherwise specified. 
1. Participants must have ≥18 years of age. 
2. Participants must have an elevated risk of breast cancer as defined by at least one of the 
following categories and have declined tamoxifen, raloxifene and/or aromatase inhibitor therapy: 
a. Diagnosis of LCIS, atypical ductal or lobular hyperplasia.   
b. A known deleterious mutation in BRCA1, BRCA2, PTEN or TP53. (Note: The participant 
must be a documented carrier to meet this criterion. If there is a known mutation in a 
hereditary breast cancer susceptibility gene in a participant’s family member, the participant 
herself must have undergone genetic testing as per NCCN clinical guidelines to be eligible 
per this criterion.) Known mutation in PALB2 or CHEK2 del 100C or any germline mutation. 
c. Modified Gail/CARE model risk at 5 years ≥ 1.67%. (Note: Risk models are to be used only 
if there is no known previous diagnosis of resected DCIS or LCIS and there is no known 
deleterious mutation in BRCA1, BRCA2, PTEN or TP53). 
d. 10% or more probability of BRCA mutation by BRCAPRO or similar model 
3. Participants must have at least one breast available for imaging and biopsy. A previously 
irradiated breast (i.e., for resected DCIS) is not evaluable for breast imaging or biopsy. 
a. _____ Participants must not have a recent diagnosis of invasive breast cancer, however, 
women with breast cancer who have been disease-free for more than 5 years are eligible.  
They will undergo standard follow-up evaluations appropriate to their recurrent cancer risk. 
4. Participants must allow submission of core needle breast material (obtained per Section 7.3) for 
future use  
5. Participants must have a baseline mammogram performed within 90 days prior to study entry, 
done on a digital mammography machine that shows either normal or benign findings.  
Participants with mammograms that are reported as suspicious for malignancy are eligible as 
long as the biopsy is negative.  
6. Participants must have baseline mammographic density > 10% based upon the classification 
system (2 = 11-50%, “scattered fibroglandular densities”; 3 = 51-75%, “heterogeneously dense”; 
4 = >75%, “extremely dense”). Women with a baseline mammographic density of ≤ 10% (1 = 
≤10%, breasts are almost entirely fat) will not be eligible.  Women with a BIRADs score of 4 will 
be considered eligible as long as the biopsy is benign.   
7. Prior tamoxifen, raloxifene or aromatase inhibitor use is allowed provided treatment is 
completed at least 1 year prior to registration. 
8. Participants must not have bilateral breast implants, but prior breast reduction surgery is 
allowed. (Breast implants are not allowed as they affect density measurements and because of 
the risk of rupturing the implant with biopsy). 
9. Participants must have a ECOG Performance Status of 0 – 1 (see Section 11). 
10. Prior anticoagulant therapy use is allowed provided therapy is discontinued at least 7 days prior 
to the breast biopsy in order to reduce the risk of bleeding.  For subjects who have taken an 
anticoagulation within the past 7 days, INR (International Normalized Ratio) must be ≤ 1.5 x 
institutional upper limit of normal and Prothrombin Time and Partial Thromboplastin Time ≤ 
IULN prior to the breast biopsy. 
11. No prior malignancy is allowed within the past five years except for the following: adequately 
treated skin cancer and in situ cervical cancer .  


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12. Participant must not be pregnant or nursing and must agree to use effective contraception. 
Hormone-based birth control (pills, patches or shots) are allowed, but switching birth control 
methods is discouraged while on-study as hormonal changes can affect mammographic 
density.  Hormone replacement therapy is not allowed for post-menopausal female.  
13. Individuals must not participate in any other clinical trial for the treatment or prevention of 
cancer unless they are no longer receiving the intervention and are in the follow-up phase only. 
Participants must also agree not to join such a trial while participating in this study. 
14. All participants must be informed of the investigational nature of this study and must sign and 
give written informed consent in accordance with institutional and federal guidelines. 
Exclusion Criteria 
1. No prior malignancy within the past five years except for the following: adequately treated basal 
cell or squamous cell skin cancer and in situ cervical cancer.  .  
2. Prior Tamoxifen or Raloxifene or aromatase inhibitor or hormone replacement therapy use in 
the past 1 year 
 
 
6 
Treatment 
6.1 
Study treatment 
Hydroxytyrosol  will be administered at 25 mg oral daily. 
All dosages prescribed and dispensed to the patient and all dose changes during the study must be 
recorded. 
Each bottle will contain 90 capsules.  Each capsule will contain 250 mg of Hytolive powder (containing 
25 mg of hydroxytyrosol). Medication labels will comply with US legal requirements and be printed in 
English. They will supply no information about the patient. The storage conditions for Hydrotyrosol will 
be described on the medication label. 
Hydrotyrosol is supplied by PLThomas & Co, Inc. Hydrotyrosol is formulated as capsules for oral 
administration of 25mg,  Capsules  
 
6.1.1 
Dosing regimen 
The investigator should promote compliance by instructing the patient to take the study drug exactly as 
prescribed and by stating that compliance is necessary for the patient’s safety and the validity of the 
study. The patient should be instructed to contact the investigator if he/she is unable for any reason to 
take the study drug as prescribed. 
Hydroxytyrosol should be administered orally once daily at the same time every day, either consistently 
with or consistently without food 
Capsules 
The capsules should be swallowed whole with a glass of water and should not be chewed or crushed.  
If vomiting occurs, no attempt should be made to replace the vomited dose. Patients should be 
instructed that if they miss a dose on one day, they must not take any extra dose the next day, but 
instead to immediately contact the study center as soon as possible to ask for advice.  
6.2 
Dose modifications 
 
6.2.1 
Dose modification and dose delay  
If the participant develops grade 1 or 2 toxicities that are felt to be possibly, probably or definitely related 
to the study drug, the participant may go on a drug holiday (for no longer than 12 consecutive weeks) 
and restart the drug at the same dose. In each of these cases, the follow-up mammograms, breast 
biopsies will still be collected as per protocol one year after the initial start date of the intervention.   


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6.2.2 
Visits 
Before the drug start, the participants will have a general physical exam including a clinical breast exam, 
(with consent), height, weight, medical history, abdominal circumference, blood tests (CBC, CMP, 
fasting lipid panel), and a bilateral mammogram (if one not available within 90 days of registration).  
Breast MRI images will be collected and reviewed if available.  If the mammogram is read as normal or 
benign , participants will undergo a non-directed core needle biopsy within 30 days of signing consent.  
For subjects who have taken an anticoagulation within the past 7 days, INR (International Normalized 
Ratio) must be ≤ 1.5 x institutional upper limit of normal and Prothrombin Time and Partial 
Thromboplastin Time ≤ IULN prior to the breast biopsy. If the patient has had a bone density (within the 
past 1 year) than it will be collected. Participants will then receive oral hydroxytyrosol for 1 year.  A six 
month supply of study drug will be dispensed at Day 1 and another 6 months sample on month 6 visit.  
Participants will complete the self-administered FACT ES score at baseline, 6 months as well as at 12 
months (Form ES).  
 
6.2.2.1 
Follow Up(s) 
For follow-up visits, the participant will be seen at the study site at Months 3, 6 and 12. Follow-up 
assessments are based from the date of treatment start. Clinic visit should occur on Days 90, 180 and 
360 (+/- 14 days for each assessment) to allow flexibility in scheduling. During these visits, counts of 
capsules will be recorded on the Treatment Form  
and in the participant’s medical record. At month 13, the patient will receive a phone call for toxicity 
check.  
Reported side-effects will also be assessed during these visits. Physical and clinical breast exam will be 
done at Months 3, 6 and 12. In addition, blood (CBC, CMP, fasting lipid panel) will be collected to 
monitor for toxicity at baseline, 3,6 and 12 months (see Study Calendar). Pill dispensing will be done at 
baseline and the Month 6 visit.     
 
6.2.2.2 
Drug Completion Visit (Month 12; Day 360 +/- 14 days) 
After the 12 month intervention, all participants will have a complete physical exam including clinical 
breast exam. Once the participant completes the study drug, she should have the bilateral mammogram  
and biopsy scheduled within 4 weeks of stopping the intervention.  If a subject has a breast MRI, the 
images will be collected.  Participants will complete the self-administered Patient Questionnaire (FACT 
ES) at the completion of the treatment.  
 
6.2.2.3 
Compliance 
The investigator/designee will count remaining unused hydroxytrosol at each visit to assess compliance.  
A study medication log is provided in the CRF to capture subject compliance.  If the subject forgets to 
return unused hydroxytrosol at a scheduled visit, compliance will be confirmed by direct questioning of 
the subject.  Subjects should be instructed to record any missed doses of hydroxytrosol. 
Compliance with hydroxytrosol will be calculated by the ratio of number of pills actually taken to the 
number of pills that should have been taken.  Compliance will be defined as a ratio ≥ 0.75.  Subjects 
with ratios between 1.00 and 0.75 will be allowed to continue on study.  The importance of compliance 
with study medication should be reinforced to subjects with a ratio of less than 1.00.  Subjects who have 
ratios < 0.75 will be taken off study for non-compliance.   


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Compliance with hydroxytrosol will be evaluated by a physical pill count.  In the event that the patient 
forgets to bring unused drug/empty drug bottles to the study visit, patients will be queried by study staff 
regarding compliance.   
 
6.2.2.4 
Post Intervention Follow Up Phone Call (Month 13; Day 390 +/- 7 days): 
Participants will be called for assessment and/or resolution of adverse events.  
 
6.2.2.5 
Biopsy Procedures  
Participants will undergo core needle biopsy of normal breast tissue and the entire specimen will be 
submitted in total to research.  The patient will not receive any results of the biopsy. Which breast to 
sample for biopsy will be at the discretion of the treating physician. This biopsy will not be directed at a 
particular lesion, but instead will be directed to the upper outer quadrant of the breast, which is typically 
a dense region of the breast. Core needle biopsy of the breast will be performed using a 14-gauge 
needle.  After providing local anesthesia, a small skin incision is made in the breast and the core needle 
inserted. Up to four core biopsies are taken through this single incision, and after completing the 
biopsies, the wound is steri-stripped, dressed, and ice is placed on the wound to minimize bruising and 
scarring. The participant is encouraged to keep ice on the biopsy site for up to 2 hours. Part of the 
breast biopsy specimen will be saved as frozen tissue and other part will be placed in fixative for H & E 
and immunohistochemical staining for biomarkers. Potential complications include bleeding, pain, 
hematoma formation and bruising, or infection. The first biopsy will be before beginning hydroxytyrosol 
and the second biopsy will be done after treatment with hydroxytyrosol for one year. There will be no 
attempt to biopsy the exact same area of the breast or the same on the second breast biopsy. Instead, 
the upper outer quadrant of the breast will again be sampled.  
 
Breast tissue can be collected from a scheduled biopsy or surgery and used in place of the breast 
biopsy.     
 
Rationale for Mandatory Biopsy before and after the treatment with hydroxytyrosol. 
 
Identifying risk biomarkers in benign breast tissue may provide direct measure of the effect of treatment 
on target tissue beyond any information imaging can provide.  Ki-67 expression in benign breast tissue 
has been included into many early phase chemoprevention trials[34,35].  Unfortunately there are many 
limitations of using Ki67 expression in benign tissue (due to very low baseline Ki67 in these patients) 
and other tissue biomarkers are necessary.   Therefore biopsy including markers for apoptosis, stem cell 
as well as Wnt Pathway (primary action of hydroxytyrosol) is proposed.  Ki-67 expression does not have 
correlation with mammographic density therefore it is possible for the patient to have tissue changes but 
not able to detect it in the mammogram[36]. 
 
The samples will be stored at: 
Houston Methodist Research Institute Biorepository Core 
6565 Fannin St. Ste. F774 (Foundren Building) 
Houston Texas, 77030 
 
6.2.2.6 
FACT – ES survey 
The current standard of care in patients with increased risk of developing breast cancer is anti-estrogen 
therapy.  The FACT-ES score has been validated in patients receiving endocrine therapy.  We are using 


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the FACT-ES score at baseline, 6 months and 1 year so that we can historically compare the side 
effects in patients on hydroxytyrosol to known endocrine side effects.  
 
6.2.2.7 
Breast MRI 
Breast MRI images will be collected if available and are not a required test for study participation.  The 
images available will be used to calculate MRI breast density.  The axial view T1-weighted images 
without fat suppression will be used for the analysis of breast density in this study. The images will be 
acquired using a 2D turbo spin-echo pulse sequence with TR=800 ms, TE=8.6 ms, flip angle=90°, 
matrix size=480x480, FOV=31–38 cm, and slice thickness=2 mm. The breast and fibroglandular tissue 
segmentation will be performed using a modified published method. [31-33] Before the segmentation, 
the operator will view the whole axial T1W images dataset and determined the superior and inferior 
boundaries of the breast (the beginning and ending slices) by comparing the thickness of breast fat with 
the body fat. The breast segmentation procedures consisted of: 1) Perform an initial horizontal line cut 
along the posterior margin of each individual subject’s sternum to exclude thoracic region. 2) Apply 
Fuzzy-C-Means (FCM) clustering and b-spline curve fitting to obtain the breast-chest boundary. 3) A 
bias field correction method based on nonparametric nonuniformity normalization (N3) and adaptive 
FCM algorithm [33] will be used to remove the strong intensity non-uniformity for segmentation of 
fibroglandular tissue and fatty tissue. 4) Apply dynamic searching to exclude the skin along the breast 
boundary. 5) The standard FCM algorithm is applied to classify all pixels on the image. The default 
setting is to use a total of 6 clusters, 3 for fibroglandular tissue and 3 for fatty tissues. After completing 
the segmentation processes in all image slices, the quantitative breast volume, fibroglandular tissue 
volume, and the percent density (calculated as the ratio of the fibroglandular tissue volume over the 
breast volume x100%), will be calculated.  
 
6.2.2.8 
Criteria for Removal from Protocol Treatment  
a. Evidence of any cancer at any time, including DCIS.  If the pathologic review of the baseline core 
needle breast biopsy confirms invasive breast cancer, the participant must be removed from protocol 
treatment. 
b. Unacceptable toxicity (as defined in Section 8.0). 
c. A participant who becomes pregnant while on treatment will be removed from protocol treatment 
since hormonal changes affect mammographic density. 
d. Delay of study intervention > 12 consecutive weeks due to any reason. 
e. Completion of twelve months of study intervention. 
f. The participant may withdraw from the study at any time for any reason. 
g. All reasons for discontinuation of the intervention must be documented in the Off Treatment Notice – 
Prevention Studies. 
8. All participants will be followed for 13 months after registration. After 13 months, no further follow-up 
is required. 
9.  Non-compliance with study drug.   
 
6.2.2.9 
Withdrawal from Protocol Treatment 
Follow-up visits will continue even if the participant goes off study intervention early unless there is a 
withdrawal of consent.   
 
 
7 
Visit schedule and assessments 
7.1 
Study flow and visit schedule


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Table 7-1 
Visit evaluation schedule 
Required Studies 
Prestudy 
Evaluation 
Day 1 
Month 1-2 
(Day 30 to 
60) 
Month 3 
(day 90)1 
Month 6 
(day 180)1 
Month 9 
(day 270)1 
Month 12 
(day 360)1 
Month 13 (day 390)1 
Clinic Visit 
 
 
 
 
 
 
 
 
Breast Cancer Risk Assessment2 
X 
 
 
 
 
 
 
 
Baseline and Pre-existing conditions 
X 
 
 
 
 
 
 
 
History and Physical Exam13 
X 
 
 
X 
X 
 
X 
 
Height and Weight 
X 
 
 
 
 
 
X 
 
Vital Signs 
X 
 
 
X 
X 
 
X 
 
Performance Status 
X 
 
 
 
 
 
X 
 
Toxicity Notation3 
 
 
 
X 
X 
 
X 
X12 
Review of Intake Calendar4 
 
 
 
X 
X 
 
X 
 
Laboratory 
 
 
 
 
 
 
 
 
Serum liver function tests 
X 
 
 
X 
X 
 
X 
 
Serum lipid panel (fasting) 
X 
 
 
X 
X 
 
X 
 
PT/PTT/INR5 
X 
 
 
 
 
 
X 
 
FSH (if necessary)6 
 
 
 
 
 
 
 
 
CBC and CMP 
X 
 
 
X 
X 
 
X 
 
Serum Pregnancy14 
X 
 
 
 
 
 
 
 
 
Radiographs 
 
 
 
 
 
 
 
 
Bilateral Mammogram7 
X 
 
 
 
 
 
X 
 
Bilateral Breast MRI image collection7 
X 
 
 
 
 
 
X 
 
Bone Density8 
X 
 
 
 
 
 
X8 
 
Procedures 
 
 
 
 
 
 
 
 


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Random core biopsy9 
X 
 
 
 
 
 
X 
 
Intervention 
 
 
 
 
 
 
 
 
Dispense study drug10 
 
X 
 
 
X 
 
 
 
Count Pills 
 
 
 
X 
X 
 
X 
 
Questionnaire11 
X 
 
 
 
X 
 
X 
 
 
 
 
 
1. 
Followup visits can occur (+/- 14 days for each assessment) to allow flexibility in scheduling. Follow-up visits will continue even if the participant goes off study intervention early 
unless there is a withdrawal of consent. 
2. 
Risk assessment using Gail risk model if no known history of DCIS or LCIS and no deleterious mutation in BRCA 1 or 2, PTEN or P53 
3. 
On months 3, 6,  12 (Days 90, 180,  360  (+/- 14 days for each assessment)) adverse events will be noted 
4. 
On months 3, 6,  and 12 (90, 180,  360 +/- 14 days for each assessment), participants will be seen at the study sites to review the intake calendar which will document drug 
compliance and date of last menses.  
5. 
PT/PTT/INR required if the patient has been on anticoagulation therapy within 7 days prior to breast core biopsy.  
6. 
FSH only to be performed if menopausal status needs to be determined (e.g. in patient with hysterectomy with at least one ovary intact – see section 7.2.2 ) 
7. 
Mammogram within 90 days of registration. Imaging to be done prior to breast biopsy.  Bilateral Breast MRI images to be collected if conducted within 90 days of registration at 
baseline   End of treatment Breast MRI images to be collected if available.    
8. 
This test information will be collected if available. Bone density not to be ordered specifically for the study..   
9. 
The random core needle biopsy is mandatory for study participation. All new patients on the study will undergo biopsy before and after the study drug. No attempt will be made to 
have the patients already on the study to undergo mandatory biopsy.  All imaging is to be done prior to breast biopsy.  If a patient is scheduled for a breast biopsy or surgery, tissue 
can be collected at that time.      
10. A six month supply of the study drug will be dispensed at Day 1 visit and at 6 months. Compliance to be verified via pill count and pill diary.   
11. The Fact ES questionnaire will be given at baseline, 6 months and at 12 months 
12. Phone call to assess adverse events. 
13. Physical exam to include abdominal circumference at baseline, 3, 6 and 12 months 
14. Serum pregnancy test if premenopausal and has not had tubal ligation.  
 
 
 


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7.2 
Assessment types 
Followup visits can occur (+/- 14 days for each assessment) to allow flexibility in scheduling. Follow-up 
visits will continue even if the participant goes off study intervention early. Risk assessment using Gail 
risk model if no known history of DCIS or LCIS and no deleterious mutation in BRCA 1 or 2, PTEN or 
P53. On months 3, 6, and 12 and 13 (Days 90, 180,  360 and 390 (+/- 14 days for each assessment)) 
adverse events will be noted. On months 3, 6,  and 12 (90, 180, 360 +/- 14 days for each assessment), 
participants will be seen at the study sites to review the intake calendar which will document drug 
compliance and date of last menses.  PT/PTT/INR required if the patient has been on anticoagulation 
during the past 7 days.  FSH only to be performed if menopausal status needs to be determined (e.g. in 
patient with hysterectomy with at least one ovary intact). Mammogram within 90 days of registration. 
Bilateral Breast MRI will not be performed specifically for this study but if the images available within the 
past 90 days, they will be collected.   Bone density not to be ordered specifically for the study and if not 
available does not exclude the patient from participating. The random core needle biopsy is mandatory 
for study participation.   A six month supply of the study drug will be dispensed at Day 1 and at 6 
months.  The Fact ES questionnaire will be given at baseline, 6 months and at 12 months.   
 
7.2.1 
Pregnancy and assessments of fertility 
Pregnancy testing is required at screening for premenopausal patients that have not had tubal ligation or 
whenever pregnancy is suspected.  Serum pregnancy testing should be performed at screening 
7.2.2 
Determination of Menopausal Status 
 
The following criteria will be used to define postmenopausal: 
• Age 56 or older with no spontaneous menses for at least 12 months prior to study 
entry; or 
• Age 55 or younger with no spontaneous menses for at least 12 months prior to study 
entry (e.g., spontaneous or secondary to hysterectomy) and with a documented 
estradiol level in the postmenopausal range according to local institutional/laboratory 
standard; or 
• Documented bilateral oophorectomy. 
 
Women failing to meet one of these criteria will be classified as pre-menopausal. 
 
 
7.2.2 
Drug levels and pharmacokinetic assessments N/A 
 
8 
Safety monitoring and reporting 
Information about all adverse events, whether volunteered by the subject, discovered by investigator 
questioning, or detected through physical examination, laboratory test or other means, will be collected 
and recorded and followed as appropriate. 
 
8.1 
Adverse events 
8.1.1 
Definitions and reporting 
Adverse events that begin or worsen after informed consent should be recorded in the Adverse Events 
CRF. Conditions that were already present at the time of informed consent should be recorded in the 
Medical History page of the patient’s CRF. Adverse event monitoring should be continued for at least 30 
days (or 5 half-lives, whichever is longer) following the last dose of study treatment. Adverse events 


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(including lab abnormalities that constitute AEs) should be described using a diagnosis whenever 
possible, rather than individual underlying signs and symptoms. When a clear diagnosis cannot be 
identified, each sign or symptom should be reported as a separate Adverse Event. 
The occurrence of adverse events should be sought by non-directive questioning of the patient at each 
visit during the study. Adverse events also may be detected when they are volunteered by the patient 
during or between visits or through physical examination, laboratory test, or other assessments. As far 
as possible, each adverse event should be evaluated to determine: 
1. The severity grade (CTCAE Grade 1-4) 
2. Its duration (Start and end dates or if continuing at the Safety Follow-up Visit) 
3. Its relationship to the study treatment (Reasonable possibility that AE is related: No, Yes)  
4. Action taken with respect to study or investigational treatment (none, dose adjusted, temporarily 
interrupted, permanently discontinued, hospitalized, unknown, not applicable) 
5. Whether medication or therapy was given (no concomitant medication/non-drug therapy, 
concomitant medication/non-drug therapy) 
6. Outcome (not recovered/not resolved, recovered/resolved, recovering/resolving, recovered/resolved 
with sequalae, fatal, unknown) 
7. Whether it is serious, where a serious adverse event (SAE) is defined as in Section 8.2.1 
All adverse events should be treated appropriately. Such treatment may include changes in study drug 
treatment including possible interruption or discontinuation, starting or stopping concomitant treatments, 
changes in the frequency or nature of assessments, hospitalization, or any other medically required 
intervention. Once an adverse event is detected, it should be followed until its resolution, and 
assessment should be made at each visit (or more frequently, if necessary) of any changes in severity, 
the suspected relationship to the study drug, the interventions required to treat it, and the outcome. 
Information about common side effects already known about the investigational drug can be found in the 
[Investigators’ Brochure]. This information should be included in the patient informed consent and 
should be discussed with the patient during the study as needed. 
Adverse event monitoring should be continued for at least 30 days following the last dose of study 
treatment 
 
8.1.2 
Laboratory test abnormalities 
Laboratory abnormalities that constitute an Adverse event in their own right (are considered clinically 
significant, induce clinical signs or symptoms, require concomitant therapy or require changes in study 
treatment), should be recorded on the Adverse Events CRF. Whenever possible, a diagnosis, rather 
than a symptom should be provided (e.g. anemia instead of low hemoglobin). Laboratory abnormalities 
that meet the criteria for Adverse Events should be followed until they have returned to normal or an 
adequate explanation of the abnormality is found. When an abnormal laboratory or test result 
corresponds to a sign/symptom of an already reported adverse event, it is not necessary to separately 
record the lab/test result as an additional event. 
Laboratory abnormalities, that do not meet the definition of an adverse event, should not be reported as 
adverse events. A Grade 3 or 4 event (severe) as per CTCAE does not automatically indicate a SAE 
unless it meets the definition of serious as defined below and/or as per investigator’s discretion. A dose 
hold or medication for the lab abnormality may be required by the protocol and is still, by definition, an 
adverse event. 
  
8.2 
Serious Adverse Events 
8.2.1 
Definitions 
A serious adverse event is an undesirable sign, symptom or medical condition which: 


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• is fatal or life-threatening 
• results in persistent or significant disability/incapacity 
• constitutes a congenital anomaly/birth defect 
• requires inpatient hospitalization or prolongation of existing hospitalization, unless hospitalization is 
for: 
• routine treatment or monitoring of the studied indication, not associated with any deterioration in 
condition (specify what this includes) 
• elective or pre-planned treatment for a pre-existing condition that is unrelated to the indication 
under study and has not worsened since the start of study drug 
• treatment on an emergency outpatient basis for an event not fulfilling any of the definitions of a 
SAE given above and not resulting in hospital admission 
• social reasons and respite care in the absence of any deterioration in the patient’s general 
condition 
• is medically significant, i.e., defined as an event that jeopardizes the patient or may require medical 
or surgical intervention to prevent one of the outcomes listed above 
 
8.2.2 
Reporting 
The principal investigator has the obligation to report all serious adverse events to the FDA, and 
institutional IRB. 
All events reported to the FDA by the investigator are to be filed utilizing the Form FDA 3500A 
(MedWatch Form).  
To ensure patient safety, every SAE, regardless of suspected causality, occurring 
• after the patient has provided informed consent and until at least 30 days after the patient has 
stopped study treatment/participation 
• after protocol-specified procedures begin (e.g., placebo run-in, washout period, double-blind 
treatment, etc.) and 30 days after the patient has stopped study treatment 
• after the start of any period in which the study protocol interferes with the standard medical 
treatment given to a patient (e.g., treatment withdrawal during washout period, change in treatment 
to a fixed dose of concomitant medication) and until 30 days after the patient has stopped study 
treatment 
must be reported to The Methodist hospital research Institute and The Methodist hospital Cancer Center  
within 24 hours of learning of its occurrence (fax: 713-793-1642). This includes serious, related, labeled 
(expected) and serious, related, unlabeled (unexpected) adverse experiences. All deaths during 
treatment or within 30 days following completion of active protocol therapy must be reported within 5 
working days. 
Any SAEs experienced after this 30 days period should only be reported to TMHS and TMHCC if the 
investigator suspects a causal relationship to the study drug. Recurrent episodes, complications, or 
progression of the initial SAE must be reported as follow-up to the original episode within 24 hours of the 
investigator receiving the follow-up information. A SAE occurring at a different time interval or otherwise 
considered completely unrelated to a previously reported one should be reported separately as a new 
event.  The end date of the first event must be provided. 
The original copy of the SAE Report and the fax confirmation sheet must be kept within the Trial Master 
File at the study site. 
Follow-up information is sent to the same fax number as the original SAE Report Form was sent, using 
a new fax cover sheet, stating that this is a follow-up to the previously reported SAE, and giving the date 
of the original report. Each re-occurrence, complication, or progression of the original event should be 


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reported as a follow-up to that event regardless of when it occurs. The follow-up information should 
describe whether the event has resolved or continues, if and how it was treated, whether the blind was 
broken or not (if applicable), and whether the patient continued or withdrew from study participation. 
If the SAE is not previously documented in the Hydroxityrosol  Investigator Brochure or Package Insert 
(new occurrence) and is thought to be related to the study drug, may urgently require further information 
from the investigator for Health Authority reporting. TMHCC and TMHRI may need to issue an 
Investigator Notification (IN), to inform all investigators involved in any study with the same drug that this 
SAE has been reported. Suspected Unexpected Serious Adverse Reactions (SUSARs) will be collected 
and reported to the competent authorities and relevant ethics committees in accordance with Directive 
2001/20/EC or as per national regulatory requirements in participating countries. 
For Comparator Drugs/Secondary Suspects (Concomitant Medications), all serious adverse 
experiences will be forwarded to the comapartor drug company by the investigator. 
 
9 
Statistical methods 
Study Design and Sample Size.  This is a single-arm, pilot breast cancer prevention study of 
hydroxytyrosol in women at increased risk of breast cancer.  Our primary study endpoint will be defined 
as proportion of women who exhibit a 10% absolute reduction in mammographic density at one-year of 
follow-up (Cuzick JNCI 2011).  For each of the post and pre-menopausal group of women, we will 
assume under the null hypothesis that 25% of women will exhibit a response (at least a 25% reduction 
from baseline) versus an alternative hypothesis equal to 45% based on data presented from Cuzick 
(JNCI 2011).  Under these assumptions, a sample of 50 pre-menopausal and 50 post-menopausal 
women will provide 85% power based on a two-sided test for a single proportion with 5% significance 
level for each group of women.  
Statistical Analysis.  Demographics and pre-treatment characteristics will be summarized for  50 
premenopausal & 50 postmenopausal women using descriptive statistics. Mammographic density levels 
at baseline and follow-up time-points as well as categorized levels based on BIRAD criteria will also be 
summarized using means, standard deviations and proportions.  Change from baseline levels of breast 
density will be analyzed using paired tests while the proportion of women who exhibit at least a 10% 
absolute reduction from baseline will be calculated along with 95% binomial confidence intervals for 
each group of women.   
Correlative endpoints including Ki-67, apoptosis, Wnt signaling and stem cell pathway markers will 
likewise be summarized descriptively at each time point of follow-up.  Correlations between these 
markers and with mammographic density levels will be assessed using Spearman or Pearson’s 
correlation coefficients.  Change from baseline levels will be analyzed using paired t-test or Wilcoxon-
signed rank test.  
Multivariate analysis to adjust for demographic/clinical characteristics, specifically age or menopausal 
status (pre, peri, and post) will likewise be considered using logistic regression for analyzing proportion 
of women with response or using linear regression models for analysis of quantitative levels of 
mammographic density.  Given the pilot nature of this study, these multivariate analyses will be 
considered exploratory.  
Expected outcome: We expect a decrease in apoptosis, cell proliferation, in post-treatment breast tissue 
in comparison to pre-treatment. Our previous data reveal an inhibition of Wnt signaling by HT, thus we 
expect a change in this signaling in this prenvention study.   
Potential pitfalls, and resolutions: If we do not have enough breast tissue for all determinations, it will be 
difficult to measure mRNA levels. In such a scenario, we can determine the expression of Sfrp proteins 
only by multiplex nucleic acid in situ hybridization technology (RNAscope) in paraffin embedded tissue 
slides.  


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Fabian CJ, Kimler BF, Zalles CM, Klemp JR, Petroff BK, Khan QJ, Sharma P, Setchell KD, 
Zhao X, Phillips TA et al: Reduction in Ki-67 in benign breast tissue of high-risk women with the lignan 
secoisolariciresinol diglycoside. Cancer Prev Res (Phila) 2010, 3(10):1342-1350. 
 
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Khan QJ, Kimler BF, O'Dea AP, Zalles CM, Sharma P, Fabian CJ: Mammographic density does 
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9(3):R35. 
 
 


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11 
Appendices  
Appendix A: ECOG Performance Status Criteria 
 
 
 
 
 
 


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Appendix B: New York Heart Association (NYHA) Classifications 
 
This table is an excerpt from the Oxford Textbook of Medicine, 2nd ed. Oxford; New York: Oxford University Press, 1987, p. 2228. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix C: CTCAE Files 
Version 4.0 (dated June-14-2010) 
NCI Common Terminology Criteria for Adverse Events (CTCAE) v.4 data files and related documents 
are published here. The most current release files appear in this directory:  
Files: Booklet 
 
Content 
CTCAE_4.03_2010-06-
14_QuickReference_5x7.pdf 
Most recent release of core terminology: PDF document, 
traditional small booklet format. 
 
 
http://evs.nci.nih.gov/ftp1/CTCAE/About.html 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix D:  Trial Logistics 
No. of Centers:   
Key Milestones: 
FPFV / First Dose: With documented ERligibility for a high risk Study.  
LPLV / Last Subject completed:    
Database Lock:  
Final Report: - 
 
Sponsoring Department: 
TMHCC ________TMHRI ________TMHS________ 
 
Prepared by: _________________________________ Date ___________