Conditions: Breast Cancer
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 55
Healthy volunteers: no

Brief summary:
This is a multi-institution double-blind placebo-controlled trial whose main objective is to determine if 6 months of letrozole (2.5 mg daily) can reduce proliferation as assessed by Ki-67 in high risk postmenopausal women on systemic hormone replacement therapy who have random periareolar fine needle aspiration (RPFNA) evidence of hyperplasia with atypia or borderline atypia, and a minimum Ki-67 of \>1.5%.

The primary hypothesis is that proliferation and expression of other estrogen response genes will be favorably modulated by six months of letrozole relative to placebo without substantially increasing hot flashes or worsening overall quality of life.

Detailed description:
Subsequent to the 6 month RPFNA for assessment of biomarkers, toxicity and quality of life assessments, all women may receive optional open-label letrozole for an additional 6 months, followed by a third RPFNA and biomarker

Arms:
arm 1: Letrozole, 2.5 mg daily for 6 months arm 2: Placebo, daily for 6 months

Interventions: intervention 1: Letrozole intervention 2: Placebo
Intervention descriptions: intervention 1: Letrozole 2.5 mg tablet daily. Then optional open label letrozole for another 6 months. intervention 2: Placebo tablet daily for 6 months then optional open label letrozole for 6 months.

Protocol document:
Prot_SAP_000.pdf: 
 
 
PROTOCOL FACE SHEET 
 
Protocol Number:  
KUMC HSC# 10587  
 
Version/Date:  
Version 1.5 (28 June 2012) — Approval 05 July 2012 
 
 
 
 
Prior Versions: 
Version 1.4 (04 August 2011) — Approval 09 August 2011 
 
 
 
Version 1.3b (16 November 2010) — Approval 01 December 2010
 
 
 
Version 1.3a (18 August 2008) – Approval 28 August 2008 
Version 1.3 (19 May 2008) — Approval 5 August 2008 
 
 
 
Version 1.2 (6 July 2007) — Approval 30 July 2007 
  
 
 
Version 1.1 (28 September 2006) — Approval 13 October 2006 
 
Protocol Title:  
Breast Cancer Prevention by Letrozole in High Risk Women 
 
Protocol Chair: 
Carol J. Fabian, M.D. 
 
 
 
Professor of Medicine  
 
 
 
Phone: 913/588-7791 
 
 
 
Fax: 913/588-3679 
 
 
 
University of Kansas Medical Center (KUMC) 
 
 
 
3901 Rainbow Boulevard 
 
 
 
Kansas City, KS 66160-7820 
 
Institutions:  
University of Kansas Medical Center; Kansas City, KS 
Carol J. Fabian, M.D., PI 
 
Bruce F. Kimler, Ph.D., Co-PI 
Northwestern University, Chicago, IL 
Seema Khan, M.D., Site PI 
Ohio State University Medical Center, Columbus, OH 
Lisa D. Yee, M.D., Site PI 
University of California-San Francisco, San Francisco, CA 
Michael D. Alvarado, M.D., Site PI 
Laura J. Esserman, M.D., Co-PI 
University of Oklahoma Health Sciences Center,  
Oklahoma City, OK 
 
William C. Dooley, M.D., Site PI 
 
Protocol Statisticians: Matthew S. Mayo, Ph.D. and Henry Yeh, Ph.D.  
 University of Kansas Medical Center, Kansas City, KS 
 
KUMC Laboratory 
Brian Petroff, D.V.M., Ph.D., University of Kansas Medical Center,  
  
Director: 
 
Kansas City, KS 
 
 
Consultants: 
Carola M. Zalles, M.D. 
 
Drug:  
 
Letrozole (Femara) 
 
 
 
IND# 76,028 


Breast Cancer Prevention by Letrozole in High Risk Women 
KUMC HSC# 10587                 Version 1.5 – June 28, 2012                             Page 2 of 75 
 
SIGNATURE PAGE 
 
By signing this page, it is agreed that you will conduct the study as dictated by this 
protocol. 
 
 
 
 
 
 
 
________________________________ 
___________________________________ 
Site Principal Investigator Printed Name 
Site Co-Principal Investigator Printed Name 
 
 
 
 
________________________________ 
__________________________________ 
Site Principal Investigator Signature 
Site Co-Principal Investigator Signature 
 
 
 
________________________________ 
___________________________________ 
Date of Site Principal Investigator  
Date of Site Co-Principal Investigator Signature 
Signature 
 
 
 
 
 
 
 
 
 
 
 
Please sign and return this signature page to the Protocol Chair before initiating 
study.


Breast Cancer Prevention by Letrozole in High Risk Women 
KUMC HSC# 10587                 Version 1.5 – June 28, 2012                             Page 3 of 75 
 
TABLE OF CONTENTS 
 
Page 
COVER PAGE ......................................................................................................... 1 
SIGNATURE PAGE .................................................................................................. 2  
TABLE OF CONTENTS ............................................................................................. 3 
LIST OF ABBREVIATIONS ...................................................................................... 5  
SCHEMA ................................................................................................................ 6 
 
1. OBJECTIVES ....................................................................................................... 7 
 
2. BACKGROUND .................................................................................................. 8 
2.1 Need For Development of Prevention Intervention for Postmenopausal Women 
on HRT for Control of Menopausal Symptoms ................................................... 8 
2.2 Rationale for Use of Aromatase Inhibitors in High Risk Post-Menopausal Women  
 
Taking Hormone Replacement Therapy ........................................................... 8 
2.3 Preclinical Evidence Supporting Use of Letrozole in a High Estrogen Environment 13 
2.4 Biomarker Based KUMC Phase II Pilot Study Suggests Reduction in Proliferation 
in Benign Breast Disease in Postmenopausal Women Given Letrozole Despite 
Continuing HRT ......................................................................................... 13 
2.5 Use of Proliferation as a Primary Response Biomarker in Breast Cancer 
Treatment and Chemoprevention Trials ......................................................... 14 
2.6 Design of Pilot Study of Letrozole ................................................................. 15 
2.7 Methods for Pilot Study ............................................................................... 16 
2.8 Results of the Letrozole Pilot Study .............................................................. 18 
2.9 Summary ................................................................................................. 24 
 
3.  SUMMARY OF STUDY PLAN ............................................................................... 25 
 
4. PARTICIPANT SELECTION .............................................................................. 27 
4.1 Inclusion Criteria for Screening RPFNA .......................................................... 27 
4.2 Exclusion Criteria for Screening RPFNA ......................................................... 27 
4.3 Inclusion Criteria for Study Entry ................................................................. 28 
4.4 Exclusion Criteria for Study Entry ................................................................. 28 
4.5 Inclusion of Women and Minorities ............................................................... 29 
4.6 Recruitment and Retention Plan ................................................................... 29 
 
5. AGENT ADMINISTRATION ................................................................................ 30 
5.1 Dose Regimen and Dose Groups .................................................................. 30 
5.2 Study Agent Administration ......................................................................... 30   
5.3 Concomitant Medications ............................................................................ 30 
5.4 Dose Modification ...................................................................................... 30 
5.5 Interruption or Discontinuation of Treatment ................................................. 31 
5.6 Emergency procedure for unblinding ............................................................. 31  
5.7 Adherence/Compliance ............................................................................... 32  
5.8 Drug Records ............................................................................................ 32 
 
6. LETROZOLE INFORMATION ............................................................................. 33 
6.1 Letrozole IND Sponsor ................................................................................ 33 
6.2 Mechanism of Action .................................................................................. 33 
6.3 Pharmacokinetics ....................................................................................... 33  
6.4 Pharmacodynamics .................................................................................... 33 
6.5 Drug/Drug Interactions ............................................................................... 33 
6.6 Clinical Indications ..................................................................................... 34 


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6.7 Adverse Reactions ..................................................................................... 34 
6.8 Availabilty ................................................................................................ 34 
6.9 Agent Distribution ...................................................................................... 35 
6.10 Agent Accountability .................................................................................. 35 
6.11 Packaging and Labeling .............................................................................. 35 
6.12 Storage .................................................................................................... 35 
6.13 Registration/Randomization/Consent ............................................................ 35 
6.14 Blinding and Unblinding Methods .................................................................. 36 
6.15 Agent Destruction/Disposal ......................................................................... 36 
 
7. CLINICAL EVALUATIONS AND PROCEDURES .................................................... 37 
7.1   Schedule of Events  ................................................................................... 37 
7.2 Baseline Testing/Prestudy Evaluation ............................................................ 37 
7.3 Baseline Studies ........................................................................................ 39 
7.4 Evaluation during Study Intervention ............................................................ 40 
7.5 Evaluation at Completion of Randomized Portion of Study at 6 Months .............. 40 
7.6 Post-Intervention Follow-up Period ............................................................... 40 
 
8. BIOLOGIC SPECIMEN HANDLING, STORAGE AND SHIPPING ........................... 41 
 
9. SAFETY ASSESSMENTS ................................................................................... 42 
9.1 Definition of an Adverse Event ..................................................................... 42 
9.2 Adverse Event Reporting ............................................................................. 42 
9.3 Assessment of Relationship of AE to Treatment .............................................. 43 
9.4 Follow-up of AEs ........................................................................................ 43 
9.5 Serious Adverse Events .............................................................................. 43 
9.6 Reporting Serious Adverse Events ................................................................ 44 
 
10. BIOMARKER AND LABORATORY ASSESSMENTS .............................................. 46 
10.1 Ki-67  ...................................................................................................... 46 
10.2 Cytomorphology Assessment ....................................................................... 47 
10.3 Nuclear Morphometric Assessments .............................................................. 47 
10.4 RNA Extraction and qRTPCR Assessments ...................................................... 47 
 
10.5 Blood Assessments  ................................................................................... 48 
10.6 Breast Density Measurements ...................................................................... 50 
10.7 Quality of Life Evaluation ............................................................................ 50 
 
11. DATA MANAGEMENT ...................................................................................... 52 
11.1 
Source Documents .................................................................................. 52 
11.2 
Data and Safety Monitoring Plan ................................................................ 52 
11.3 
Sponsor or FDA Monitoring ....................................................................... 53 
11.4 
Record Retention .................................................................................... 54 
 
12. STATISTICAL METHODS.................................................................................. 55 
12.1 
Sample Size and Power   .......................................................................... 55 
12.2 
Statistical Analysis .................................................................................. 55 
 
13. REGULATORY ................................................................................................. 58 
 
14. REFERENCE LIST ........................................................................................... 59 


Breast Cancer Prevention by Letrozole in High Risk Women 
KUMC HSC# 10587                 Version 1.5 – June 28, 2012                             Page 5 of 75 
 
List of abbreviations 
 
AE 
AI 
Adverse Event 
Aromatase Inhibitor 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
b.i.d. 
BMD 
bis in diem/twice a day 
Bone mineral density 
CEE 
CI 
Conjugated equine estrogen 
Confidence interval 
CRF 
Case Report/Record Form 
CS&E 
Clinical Safety and Epidemiology 
CR 
Clinical Research 
CRO 
Contract Research Organization 
DCIS 
Ductal carcinoma in situ 
ECG 
Electrocardiogram 
ER 
HR 
Estrogen receptor 
Hazard ratio 
IEC 
Independent Ethics Committee 
i.v. 
intravenous(ly) 
IRB 
Institutional Review Board 
MPA 
Medroxyprogesterone acetate 
o.d. 
omnia die/once a day 
p.o. 
per os/by mouth/orally 
REB 
Research Ethics Board 
RPFNA 
Random periareolar fine needle aspiration 
SAE 
SHBG 
Serious Adverse Event 
Sex hormone binding globulin 
SOP 
Standard Operating Procedure 
WHI 
Women’s Health Initiative 
WHO 
World Health Organization 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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SCHEMA 
Prevention Visit 
Risk assessment of postmenopausal potential study subjects currently using  
hormones to control menopause symptoms.  
Gail 5-year risk ≥1.7% and/or a relative risk of ≥3X that for the average woman 
of the same age, as calculated by the NCI Breast Cancer Risk Assessment Tool; 
or a 10-year relative risk of > 3X that for the population, as calculated by the 
IBIS Breast Cancer Risk Evaluation Tool; 
or BRCA1/2 mutation carrier; or prior ADH, DCIS, LCIS,  
↓ 
Qualifying RPFNA visit 
Bone densitometry (DEXA) within two years prior to study entry 
No change in dose or route of hormones for six months 
Normal mammogram within three months of RPFNA.   
Biomarker Qualifiers for Trial Participation 
RPFNA = Epithelial hyperplasia +/- atypia with Ki-67 ≥1.5% 
RNA Extraction plus storage for later qRTPCR; History, breast and physical exam 
↓ 
Study Entry Visit (within three months of RPFNA) 
CBC chem. profile, fasting lipid profile, C-reactive protein,  
bioavailable testosterone and estradiol,  
serum bone turnover markers (osteocalcin and N-telopeptide),  
25-OH Vitamin D levels; Mammogram digitized for density measurement 
Complete hot flash scores, brief fatigue inventory, BCPT Symptom Checklist and 
HAQ Fibromylagia Questionnaire; History, breast and physical exam (if not 
previously done)  
Randomization to letrozole 2.5 mg/day or placebo;  
while continuing same dose of hormone replacement 
↓ 
        monthly phone contacts 
↓ 
Six Month Study Visit 
CBC chem. panel, fasting lipid profile, CRP, bioavaiable estradiol and 
testosterone, 
osteocalcin and N-telopeptide, 25-OH Vitamin D levels, reserve serum for 
letrozole levels 
Mammogram, density measurement 
Repeat questionnaires, history and physical,  
RPFNA and associated biomarkers; DEXA scan if not continuing on open label 
↓ 
Optional Open Label Letrozole 2.5 mg/d 
↓ 
monthly phone contacts 
↓ 
12 Month Study Visit (for women choosing open label letrozole) 
Repeat assessments performed at six month visit; DEXA scan at completion 
Goal:  96 evaluable subjects at six month visit 


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1.0 
Objectives 
 
Our primary hypothesis is that proliferation and expression of other estrogen 
response genes in hyperplastic breast tissue obtained by RPFNA from high risk 
women on chronic HRT will be favorably modulated by six months of letrozole 
relative to placebo without substantially increasing hot flashes or worsening 
overall quality of life. 
 
    
To test our hypothesis, we have formulated the following objectives: 
 
1.1 
To determine whether Ki-67 in hyperplastic foci of breast epithelium 
obtained by random periareolar fine needle aspiration from 
postmenopausal high-risk women on HRT is reduced by six months 
of letrozole. 
 
1.2 
To determine if letrozole causes an increase in hot flashes, fatigue, 
pain, stiffness; a deterioration in overall quality of life; as well as 
deleterious 
changes 
in 
bone 
turnover 
markers, 
total 
cholesterol:HDL ratio, and C-reactive protein.  
 
1.3 
To determine if morphologic characteristics as assessed by nuclear 
morphometric measurements and semiquantitative Masood index 
score improve with six months of letrozole.   
 
1.4 
To determine if six months of letrozole in women on a stable dose 
of hormone replacement results in consistent down modulation of 
estrogen responsive genes, particularly pS2 and GREB-1. 
 
At the conclusion of this proof of principle study, we will have established a firm 
clinical and biological basis for a large randomized study of letrozole versus 
placebo in high-risk postmenopausal women requiring hormone replacement for 
symptom control with development of breast cancer as an endpoint. If aromatase 
inhibitor treatment is found to reduce risk of breast cancer in women receiving 
hormone replacement, it would provide middle age women with tools to alleviate 
perimenopausal symptoms while minimizing breast cancer risk. 
 


Breast Cancer Prevention by Letrozole in High Risk Women 
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2.0  
Background  
 
2.1 
Need For Development of Prevention Intervention for        
           Postmenopausal women on HRT for Control of Menopausal   
           Symptoms  
 
It is estimated that one quarter of postmenopausal women in the United 
States take hormone replacement therapy [National Institutes of Health 
2005]. This is primarily because nothing else is as efficient in relieving 
perimenopausal symptoms [Barton 2002; Nelson 2004]. There is no 
identified primary prevention strategy for those women who are both at 
high risk for breast cancer because of family history or prior pre-cancerous 
biopsy and who need hormone replacement therapy to alleviate 
perimenopausal symptoms [Fabian 2005C; Wickerham 2006].   Standard 
drug prevention therapy for postmenopausal women is tamoixfen or 
raloxifene and major prevention Phase II clinical trials in postmenopausal 
women are investigating use of aromatase inhibitors [Cuzick 2005].  
Current expert opinion and ASCO guidelines suggest that hormone 
replacement not be used with tamoxifen [Vogel 2001; Chlebowski 2002] 
and clinical trials with aromatase inhibitors (AIs) or raloxifene exlude 
women who use hormone replacement therapy. Concerns about side 
effects and quality of life are major reasons why the majority of women 
have avoided primary prevention therapy with anti-estrogenic drugs in the 
past [Port 2001; Vogel 2002; Tchou 2004; Bober 2004]. Advice to a 
symptomatic high risk woman that she discontinue hormones in order to 
take anti-estrogens has a poor likelihood of being heeded as this would 
force her to both give up an effective therapy for her menopausal 
symptoms in addition to starting a medication which has a substantial 
chance of worsening them. An effective prevention strategy which would 
permit symptomatic high risk women to continue taking hormone 
replacement is needed and is the subject of this protocol. 
 
2.2 
Rationale for Use of Aromatase Inhibitors in High Risk Post-Menopausal 
Women Taking Hormone Replacement Therapy  
 
The rationale for use of aromatase inhibitors as primary prevention therapy 
in high risk women taking hormone replacement comes from six lines of 
evidence.   
 
1. Up-regulation of aromatase in areas of benign breast disease 
which is common in high risk women.  
 
2. Breast aromatase acitivity is responsible for the majority of 
breast tissue estrogen in postmenopausal.  
 
3. Aromatase inhibitors have been shown to be more effective than 
tamoxifen in adjuvant trials in preventing contra-lateral cancers in 
postmenopausal women. 
 
4. Pre-clinical data in aromatase over-expressing animals suggests 
the aromatase inhibitor, letrozole, is effective in a high estrogen 
environment.  
 


Breast Cancer Prevention by Letrozole in High Risk Women 
KUMC HSC# 10587                 Version 1.5 – June 28, 2012                             Page 9 of 75 
 
5. Low dose estrodiol may prevent some side effects associated with 
aromatase inhibitors. 
 
6. Our pilot study of 6 months of letrozole in high risk 
postmenopausal women on a stable dose of hormones showed a 
substantial reduction in proliferation in hyperplastic epithelium 
obtained by random periareolar fine needle aspiration without a 
significant chane in serum estradiol or increase in perimenopausal 
symptoms.  
 
These lines of evidence are explored in more detail below.  
 
2.2.1 Up-regulation of Aromatase in Benign Breast Disease as Well       
          As Breast Cancer 
 
Aromatase which converts androgens to extrogens is present not 
only in the ovary but in breast, muscle, brain, bone, uterus, blood 
vessels, and other organs especially adipose tissue. Aromatase 
enzyme promoter sensitivity differs by organ type. In the ovary the 
aromatase enzyme promoter is exquisitely sensitive to increases in 
follicle stimulating hormone (FSH). The aromatase promoter in the 
breast is minimally sensitive to FSH but very sensitive to 
inflammatory cytokines and growth factors such as IL6, TGFβ and 
TNFα and prostaglandin E2, most of which increase with age (e.g. 
IL6), or proliferative breast disease, and cancer [Santen 1999, 
Miller 2001; Yue 2001, Brueggemeier 2003]. Thus aromatase 
activity is often up-regulated in proliferative breast disease as well 
as breast cancer [Brueggemeier 2003]. 
 
2.2.2 Aromatase Activity Responsible for Much of Breast Tissue Estrogen 
in Postmenopausal Women 
 
The majority of breast estrogen is derived from local production not 
systemic uptake [Santen 1999]. The large amount of aromatase in 
breast tissue may be one of the reasons that breast tissue levels of 
estradiol are 10-50X that of serum levels in postmenopausal women 
who are not on HRT [Santen 1999].  Aromatase inhibitors have 
traditionally not been used for treatment of systemic metastases in 
premenopausal women as in this setting they produce only a partial 
blockade of estradiol synthesis by the ovary.  The reflex rise in LH 
and FSH, plus the large amount of the aromatase substrate 
androstenidione, allows the premenopausal ovary to continue 
estradiol synthesis at about 60% of baseline despite aromatase 
inhibitor treatment [Santen 1980].  Nonetheless, there have been 
anecdotal reports of responses in premenopausal women with 
metastatic breast cancer to the first generation AI, 
aminoglutethimide [Wander 1986; Santen 1989]. Clinical response 
to AIs in postmenopausal women with established breast cancer has 
been directly correlated with breast tumor aromatase activity, and 
this association seems to be stronger than for estrogen receptor 
levels [Miller 2001].  
 
 


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2.2.3 Aromatase Inhibitors More Effective Than Tamoxifen in  
Reducing Contra-Lateral Tumors in Adjuvant Trials  
 
Aromatase inhibitors are associated with superior disease free 
survival and reduced contralateral new primary cancers compared 
to tamoxifen in adjuvant trials. Adjuvant trials comparing an AI 
(anastrozole or letrozole) with tamoxifen as initial endocrine 
treatment or switching to an AI (exemestane) after 2-3 years of 
tamoxifen are associated with an approximate 50% reduction in 
contralateral breast cancer in women with hormone receptor 
positive tumors [Coombes 2004; Thurlimann 2005; Howell 2005]. 
Neither tamoxifen or raloxifene is effective in reducing ER negative 
cancer incidence [Fisher 1998; Wickerham 2006], and neither 
reduces estradiol levels.   
 
Estradiol activation of ERα increases production of several growth 
factors which may result in paracrine stimulation of growth of ER 
negative cells [Clarke 1997; Russo 1999; Shoker 1999]. Clinical 
evidence supporting estrogen depletion as a means of reducing the 
incidence of ER negative as well as ER positive tumors comes from 
studies of BrCa1 deleterious mutation carriers in whom 
oophorectomy substantially reduces the risk of breast cancer 
despite the fact that most BrCa1 mutation carriers develop ER- 
tumors [Rebbeck 1999, Eisen 2000; Rebbeck 2002]. Thus, AIs, by 
reducing breast estrogen synthesis, have the potential to reduce the 
incidence of ER negative as well as ER positive tumors [Santen 
1999].  
 
2.2.4 Low Dose Hormone Replacement May Prevent Some Side Effects 
Associated with Aromatase Inhibitors  
 
In adjuvant trials women randomized to aromatase inhibitors had 
fewer thromboembolic events, uterine cancers and vaginal bleeding 
compared to tamoxifen [Coombes 2004; Thurlimann 2005; Howell 
2005]. Women randomized to an AI had a similar incidence of hot 
flashes as tamoxifen and a higher incidence of arthralgias, bone 
mineral density loss, vaginal dryness, pain with intercourse, and 
loss of libido [Coombes 2004; Thurlimann 2005; Fallowfield 2004; 
Howell 2005].   
 
2.2.4.1 
Side effects of letrozole compared to placebo 
 
Compared with placebo, extended adjuvant therapy with the AI 
letrozole was associated with a higher incidence of hot flashes, 
arthritis, arthralgias and myalgias. The incidence of osteoporosis 
was borderline higher for letrozole (p=0.07). Quality of life studies 
(SF-36 and MENQOL) indicated worsening of sexual function, bodily 
pain, vitality, and vasomotor symptoms for women randomized to 
letrozole versus placebo. Most of these symptoms can be explained 
by estrogen deprivation at the target tissue level [Fallowfield 2004]. 
 
 
2.2.4.2 
AIs and Cardiovascular Disease 


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In adjuvant trials, there was an early non-significant trend toward a 
higher number of cardiovascular related events and deaths in 
women randomized to AIs compared to tamoxifen [Coombes 2004; 
Thurlimann 2005; Howell 2005], but generally, no increase in 
cardiovascular events when an aromatase inhibitor was compared to 
placebo [Goss 2003]. Tamoxifen’s partial estrogen agonist effects 
on the liver result in favorable modulation of total cholesterol:HDL 
ratio [Love 1990], an important predictor of cardiac events [Ridker 
2005]. In primary prevention trials the improved total/HDL 
cholesterol ratio observed with tamoxifen has not translated into a 
significant  overall protective effect on the cardiovascular system 
[Fisher 1998], possibly because this is partially offset by an increase 
in triglycerides and a number of pro-coagulant molecules [Hozumi 
1998; Brun 1986 Decensi 1998; Marttunen 2000]. Letrozole like 
other AIs has minimal effect on lipids when compared to placebo 
and when blood samples are obtained from fasting subjects 
[Harper-Wynne 2002; Goss 2003; Atalay 2004]. In a mature (5 
year) analysis of the Intergroup Exemestane Study (IES), in which 
women undergoing adjuvant hormonal therapy were randomized to 
exemestane or further tamoxifen after 2-3 years of tamoxifen, 
women randomized to exemestane had an overall survival 
advantage without a significant increase in ischemic heart disease 
or stroke [Coombes 2006]. 
 
Although the results of the IES trial are reassuring, it is possible 
that AI-induced severe estrogen depletion might have adverse 
effects on vascular endothelial function which might explain the 
non-significant trend in ischemic cardiovascular events compared 
with tamoxifen [Thurlimann 2005; Everson 1995; Elisaf 2001; 
Blumel 2003].  In preclinical models, activated ERα in blood vessels 
facilitates smooth muscle relaxation and vasodilatation in response 
to nitrous oxide [Bolego 2005]. This response is abolished by 
estrogen deprivation [Bolego 2005]. If AIs reduce available tissue 
estradiol in blood vessels the result could be accelerated 
atherosclerosis [Nakamura 2005]. It is theoretically possible that 
the use of low dose estradiol with an AI might prevent or blunt the 
effects of estrogen deprivation on vascular endothelium similar to 
the protective effects on bone turnover markers. 
 
2.2.4.3 
AIs and Osteoporosis 
 
While it is still unclear whether use of AIs increase risk of clinical 
heart disease, it is very clear they increase the risk of osteoporosis, 
bone fracture, arthralgias, and joint stiffness [Cuzick 2005].  
Increase in the risk of osteoporosis may in part be due to the 
unfavorable effect of estrogen depletion on vitamin D and joint 
physiology. Development of osteoporosis in women taking AIs may 
be due to unmasking of subclinical vitamin D insufficiency with 
estrogen depletion due to down regulation of the 1,25-α 
hydroxylase enzyme which converts circulating inactive provitamin 
D to the active form, and down regulation of the vitamin D receptor 


Breast Cancer Prevention by Letrozole in High Risk Women 
KUMC HSC# 10587                 Version 1.5 – June 28, 2012                             Page 12 of 75 
 
[Reichel 1989; Heaney 2005]. The result is reduced absorption of 
calcium and bone mineral loss due to elevation of parathormone.  
 
Kendall et al. from the Royal Marsden Hospital studied 10 women 
given letrozole for 6 weeks then letrozole plus a quarter of an  
estradiol patch for weeks 6-16. Serum estradiol levels increased to 
12 pmol with the patch. Serum crosslaps, a marker of bone 
turnover and predictor of bone mineral density loss, which had 
increased the first 6 weeks on estradiol alone returned towards 
normal with add back estradiol [Kendall 2006]. Arthralgias and 
morning stiffness are observed after menopause with increased 
frequency and have been presumed to be due to estrogen 
deprivation. Articular cartilage contains estrogen receptor α and β 
and physiologic changes have been observed in primate studies in 
proteoglycan 
synthesis 
and 
in 
IGFBP-2 
following 
estrogen 
administration [Richmond 2000]. 
 
2.2.4.4 
Risks and Benefits of Hormone Replacement Therapy  
 
Hormone replacement therapy, while associated with a reduction in 
menopausal symptoms, osteoporosis and hip fracture as well as 
colon cancer, is also associated with increased risk of breast cancer 
and venous and arterial thromboembolism [Rossouw 2002; 
Anderson 2004; Hays 2003; Peeyananjarassri 2005].  Risk varies 
with type, dose and route of drug administration as well as age of 
the individual.  For breast cancer, the increase in relative risk is 1-
2% per year of use for estrogen alone, and 5-10% per year for 
estrogens plus progestins [Chlebowski 2003; Anderson 2004; Beral 
2003; Fournier 2005]. No increase in risk of breast cancer has been 
demonstrated for women who began oral premarin alone in their 
50’s and continued for an average of eight years [Chlebowski 
2003].  Increase in risk of breast cancer applies to current users 
only and may be less for micronized progesterone than a synthetic 
progestin in women taking combination therapy [Fournier 2005].   
 
Increase in risk of cardiovascular events and deep venous 
thrombosis appear to be related to route of administration, type of 
preparation as well as a woman’s age.  Increase in deep venous 
thrombosis and cardiovascular events and dementia is probably due 
to the procoagulant effects of oral estrogen and progestins 
promoted by increased production of thrombophilic proteins as a 
result of a first pass effect of estrogen through the liver [Anderson 
2004].   
 
Younger women ages 50-60 on estrogen alone may actually 
experience a reduction in coronary disease risk due to the favorable 
properties of estrogen on vascular endothelium [Anderson 2004; 
Hsia 2006; Blumel 2003].  There appear to be fewer procoagulant 
effects [Modena 2005; Langer 2005] and thromboembolic events 
[Oger 2003; Scarabin 2003] with transdermal compared to oral 
preparations. C-reactive protein along with lipids are powerful 
predictive biomarkers for future cardiovascular events among 
healthy women [Ridker 1998, 2005].  Specifically the increase in C-


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KUMC HSC# 10587                 Version 1.5 – June 28, 2012                             Page 13 of 75 
 
reactive protein and other intermediate markers of inflammation 
observed with oral preparations are not observed after transdermal 
estradiol [Modena 2005; Langer 2005; Fournier 2005; Rossouw 
2002; Manson 2003; Lowe 2000, 2001; Post 2003].  Although there 
is increasing use of the more physiologic transdermal preparations 
[NIH 2005], it still accounts for a small minority of prescriptions and 
consequently it is difficult for us to mandate that women eligible for 
this study be on this type of hormone replacement.  However, if 
letrozole were found to reduce breast cancer risk in women on 
hormone replacement and the thromboembolic complications could 
be reduced by use of transdermal formulations, the potential impact 
on quality of life for a large number of menopausal high risk women 
could be substantial. 
 
Further, for those high risk women contemplating primary risk 
reduction with an AI the combination of the AI plus low dose 
hormones might be as effective as antihormonal primary prevention 
therapy, with fewer side effects than with AIs alone. 
 
2.3 
Preclinical Evidence Supporting Use of Letrozole in a High Estrogen 
Environment 
 
Aromatase 
over-producing 
transgenic 
mice 
spontaneously 
develop 
mammary hyperplasia and dysplasia which may progress to cancer if a 
carcinogen is also administered [Tekmal 1999]. Letrozole reduces ER 
expression, proliferative indices, and reverses dysplasia in hormonally 
intact animals [Kirma 2001; Luthra 2003]. Thus, in a preclinical model, 
letrozole appears effective in reversing precancerous changes in a 
moderately high estrogen environment. 
 
2.4 
Biomarker Based KUMC Phase II Pilot Study Suggests Reduction in 
Proliferation in Benign Breast Disease in Postmenopausal Women Given 
Letrozole Despite Continuing HRT 
 
2.4.1 Use of Risk Biomarkers as Response Endpoints in Phase II 
Chemoprevention Trials   
 
 
Several potentially reversible risk biomarkers are used as indicators 
of response in phase II prevention trials. These include intra-
epithelial neoplasia, and related molecular markers such as 
proliferation, mammographic breast density, serum bioavailable 
estradiol and testosterone, and serum insulin growth factor one 
(IGF-1) and its binding protein IGFBP3 [reviewed in Fabian 2005A]. 
Many of these biomarkers are interrelated such that pre-
menopausal women with high breast density may be more likely to 
have higher serum levels of IGF-1 and IGFBP3, and a greater 
chance of underlying proliferative breast disease [Byrne 2000; Boyd 
1995, 1992, 2002].   
 
 
Not all of these biomarkers are good risk or response indicators for 
postmenopausal women. For post-menopausal women, serum IGF-1 
and IGFBP3 do not appear to be good surrogates of breast tissue 
levels of these factors and are not associated with risk as in pre-


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menopausal women [Hankinson 1998]. Likewise breast density may 
not be a good response biomarker in postmenopausal women either 
because they have little density at baseline and/or little reduction 
has been observed with standard prevention therapy such as 
tamoxifen [Cuzick 2004]. For example, there was only a 1% median 
reduction of breast density observed with tamoxifen in women in 
the IBIS-1 trial over the age of 55 compared with marked reduction 
in density in women under age 45. Further, there may actually be 
an inverse relationship between some postmenopausal risk factors 
such as body mass and mammographic density [Boyd 2002]. Thus 
it is not clear that breast density is a useful response biomarker for 
postmenopausal women [Cuzick 2004]. 
 
Precancerous changes are common in high-risk women undergoing 
prophylactic mastectomy [Hoogerbrugge 2003], but the majority of 
high-risk women have never had a diagnostic biopsy. We have 
previously demonstrated that random periareolar fine needle 
aspiration (RPFNA) detected hyperplasia with atypia (Figure 1) 
increases the risk for DCIS or invasive breast cancer by 5 fold in 
high risk women and significantly stratifies risk assessments based 
on the Gail model alone [Fabian 2000; Mayo 2001]. Although 
atypical epithelial cells obtained by diagnostic biopsy, nipple 
aspirate fluid cytology, or random periareolar fine needle aspiration 
are associated with an approximate 5 fold increase in risk relative to 
those without atypia [Hartmann 2005; Wrensch 2001; Fabian 
2000], no improvement in benign breast disease morphology has 
been shown after 6-12 months of treatment with tamoxifen or other 
SERMS [Mohsin 2005, Fabian 2006].  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.5 
Use of Proliferation as a Primary Response Biomarker in Breast Cancer 
Treatment and Chemoprevention Trials 
 
Proliferation as measured by detection of mitotic index and/or detection by 
immunohistochemistry of nuclear associated proliferation antigens such as 
Ki-67 (MIB-l) progressively increases from normal to hyperplasia to 
atypical hyperplasia and DCIS [Shrestha 1992, Mommers 1999]. In 


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normal, non-proliferative breast tissue, Ki-67 is expressed in approximately 
0.2%-0.7% of cells in the terminal lobular duct unit but has been reported 
in 1-6% of cells in proliferative breast disease [Shaaban 2002, Pavelic 
1992, Allred 1998]. Shaaban et al. in a correlational cross-sectional study 
have reported that high Ki-67 in foci of hyperplasia (median 3.8%) is 
associated with a greater risk of breast cancer than those with low Ki-67 
(median 0.8%) [Shaaban 2002]. We have observed a median Ki-67 
labeling index of 1% in RPFNA samples from high-risk postmenopausal 
women exhibiting hyperplasia without atypia and a median Ki-67 of 2.8% 
in RPFNA samples containing hyperplasia with atypia [Khan 2005]. A 
number of antihormonal treatments effective in breast cancer treatment 
and/or prevention have been shown to reduce proliferation indices in 
benign and cancerous breast epithelial cells by 17-50% relative to baseline 
[Clarke 1993, Fernando 1994, Chang 2000, Dowsett 2000, Dowsett 2001, 
Dowsett 2005A, Decensi 2003]. Estrogen withdrawal has been directly 
correlated with a 5-fold reduction in Ki-67 in MCF-7 xenografts [Detre 
1999]. Reduction in proliferation (Ki-67) at two weeks and 12 weeks in the 
neoadjuvant IMPACT Trial (tamoxifen, anastrozole, combination) was 
positively associated with disease free survival [Dowsett 2005A]. 
Tamoxifen has been shown to reduce proliferation (Ki-67) in benign breast 
tissue of postmenopausal women [de Lima 2003]. For predictive value, Ki-
67 must be reduced by at least 35-50% by the intervention [Urruticoechea 
2005]. 
 
At the present time, change in proliferation as measured by Ki-67 is 
probably the most commonly used primary response indicator used in 
moderately sized proof-of-principal Phase I and II breast chemoprevention 
trials in high risk postmenopausal women [Fabian 2005A].   
 
2.6 
Design of Pilot Study of Letrozole 
We have completed a single arm pilot study of six months of letrozole in 
high-risk women who had been on a stable dose of hormone replacement 
therapy for at least six months prior to baseline random fine needle 
aspiration and continued their hormones throughout the study period.  
Change in proliferation as assessed by Ki-67 immunohistochemistry was 
the primary study endpoint. 
 
Eligibility 
Subjects were required to be at high risk as defined by one or more of the 
following criteria:  1) five year Gail risk >1.67%; 2) known BRCA1/2 
mutation carrier; 3) prior contralateral treated DCIS; 4) prior biopsy 
showing LCIS or atypical hyperplasia.  Postmenopausal status was defined 
as having either a prior bilateral oophorectomy, prior hysterectomy with 
ovaries intact and age greater than 50, or prior natural menopause with no 
period for at least 12 months prior to baseline RPFNA.  Potential subjects 
must have been in good health with an ECOG performance status of 0-1, 
reasonably normal organ function, a baseline serum estradiol of <150 
pg/ml, and a normal mammogram performed within six months prior to 
the baseline RPFNA. 
 
The subject’s baseline random RPFNA must have exhibited hyperplasia with 
atypia or borderline atypia with a Masood quantitative index score of ≥14.  


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In addition, there must have been ≥ 500 ductal cells on the 
cytomorphology slide, and ≥500 ductal cells on the slide designated for Ki-
67 staining with evidence of Ki-67 expression.  They must also have a third 
slide with at least 100 ductal cells evaluable for ER staining.   
 
2.7 
Methods for Pilot Study 
 
RPFNA and Cytomorphology 
RPFNA was performed under local anesthesia from two sites from both 
breasts and all cells pooled from both breasts (one breast in the case of 
prior DCIS or invasive cancer) into a single 15 cc tube containing modified 
Cytolyt™  (9 cc Cytolyt™ and 1 cc 10% neutral buffered formalin).  At least 
three slides were prepared by a standard ThinPrep 2000 (Cytyc) non-
gynecologic protocol. Slides for cytomorphology, Ki-67, and ER were first 
Pap-stained under RNase free conditions. Cytomorphology assessment was 
performed by a single cytopathologist and classified by three different 
methods traditional [Zalles 1995], Masood semiquantitative index score 
[Masood 1990], and the 1996 National Cancer Institute Consensus Panel 
Criteria [The Uniform Approach 1997].  Masood index scores between 6-10 
are 
generally 
associated 
with 
non-proliferative 
specimens, 
12-13 
hyperplasia and 15-18 with hyperplasia with atypia.  Scores of 11 are 
generally borderline between non-proliferative and hyperplasia and scores 
of 14 borderline between hyperplasia and atypia.  We have previously 
found that use of the Masood scoring system reduces intra-observer 
interpretive variance [Fabian 2002].  Consequently, the Masood score 
index was used to determine change in cytomorphology.  A change of 3 or 
more points is generally consistent with a descriptive category change and 
thus considered significant.   
 
Ki-67 Assessment 
Only baseline slides containing >500 epithelial cells were processed and 
stained for Ki-67.  Hyperplastic clusters were preferentially assessed and 
the number of cells with unequivocal nuclear staining out of 500 cells 
assessed was recorded.  Slides were hand scored by two readers and 
assessments recorded on case report forms.  In case of a disagreement 
between two readers, assessments were averaged for the final score; 
however, agreement for Ki-67 scores between the two readers is generally 
excellent with Cronbach’s α=0.99 [Khan 2005].   
 
Reliability and Reproducibility of Ki-67 
Change in Ki-67 between duplicate slides and over 6 months in a high-risk 
group not undergoing an intervention is being assessed in an AVON-NCI 
funded Breast SPORE grant. We have information on 25 subjects for which 
2 Ki-67 slides were made from different aliquots of the same specimen.  
There appears to be good reproducibility (Figure 2). 
 
 
 
 
 


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Estrogen Receptor Assessment 
Estrogen receptor expression was assessed with ID5 antibody from Dako 
diluted at 1:100 and applied for 30 minutes.  The ER protocol was 
optimized by us for fine needle aspirate specimens processed as ThinPrep 
slides [Petroff 2006A].  This protocol involved use of low temperature 
(90°C) retrieval for two minutes with a 0.2X nuclear decloaker (Biocare) 
and use of a 0.01% glucose oxidase blocking reagent for 30 minutes at 
37°C.  ER was hand scored by two readers utilizing 100 cells from a 
hyperplastic duct exhibiting ER staining.  The proportion of cells staining at 
each intensity (0-4+) was assessed.  The proportion of cells staining at 
each 
intensity 
was 
then 
multiplied 
by 
the 
intensity 
to 
achieve 
proportion/intensity sub-scores. The weighted intensity score (IS) was 
computed by adding together the proportion/intensity sub-scores.  Under 
this system scores can theoretically range from 0-4.  However, for benign 
hyperplastic cells which average 30% ER expression with 2+ staining,  our 
median intensity score was approximately 0.6. 
 
Hormones and Growth Factors 
In addition to cellular assessments, blood was obtained for estradiol, 
progesterone, testosterone, SHBG, and IGF-1/IGFBP-3 at baseline and at 
six months and frozen at -80°C until analysis.  Samples were not obtained 
under fasting conditions.  Estradiol and testosterone were measured in 
serum by EIA following ether extraction [England 1974; Pearce 1989]. 
Ether extraction is used to avoid false elevations of estradiol due to large 
circulating amounts of estrone sulfate. IGF-1 and IGFBP-3 were measured 
in serum by ELISA.  Baseline and six months samples were assessed in the 
same run.    
 
Mammographic Breast Density 
Mammograms were digitized for breast density assessment by the 
computer-assisted Cumulus program developed by Boyd and Yaffee [Boyd 
1995].  Baseline and six month films were assessed together by a single 
reviewer in a blinded fashion. 
 
qRT-PCR 
We have recently established the feasibility of quantitative real time 
reverse transcription PCR to allow the measurement of a panel of 
biomarker genes from Cytolyt-formalin fixed breast epithelial cells 


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processed per our normal RPFNA protocol and harvested by laser capture 
microdissection [Petroff 2006B].  Briefly, 50-500 breast epithelial cells are 
harvested from a recently Pap-stained slide using the Autopix platform and 
Paradise kits from Arcturus. mRNAs are selectively amplified through a 
single round of linear amplification with T7 polymerase, converted to cDNA, 
and then stored at -80°C until analysis by real time PCR using Taqman 
chemistry. 
 
Adverse Events 
Adverse events were collected using Common Toxicity Criteria.  In 
addition, a hot flash score index was assessed at baseline using 
methodology previously published by the Mayo Clinic in which the number 
of hot flashes/day is multiplied by the usual intensity [Loprinzi 2002].  
Subjects were contacted monthly for adverse event reporting.   
 
Accrual 
We planned to enter 45 subjects over a period of 24 months in order to 
obtain 40 subjects evaluable for change in Ki-67.  With this accrual, we 
anticipated being able to detect an effect size for change in Ki-67 of 0.48 
SD with 80% power at a type I error rate of 5%. 
2.8 
Results of the Letrozole Pilot Study 
 
Forty-two subjects were accrued in 23 months.  Accrual was stopped early 
when it was apparent that the early dropout rate was negligible and that 
we would have sufficient subjects with Ki-67 to meet our goal. Forty-two 
subjects have completed trial, with one subject discontinuing letrozole at 4 
months but she did undergo repeat RPFNA and is therefore evaluable.   
 
Table 2 summarizes results for the 42 subjects enrolled on the study.  
Median age at baseline was 50 (range 39 to 68), with 7% of subjects over 
60.  The median five year Gail risk was 2.2%.  Thirty-five of 42 subjects 
were taking estrogen alone (various preparations) and seven estrogen plus 
a progestin.  Fifty-nine percent were on transdermal preparations.  The 
median duration of hormone replacement therapy was two years (range 
0.5-21 years).  Median baseline estradiol was 57 pg/ml and median Ki-67 
4.0% (range 0.3 to 23.6%). Median ER weighted intensity score (IS) was 
0.66; 36/42 subjects had >10% cells which exhibited ER expression in 
hyperplastic foci.  Median baseline Masood score was 14 (range 14 to 16).  
 


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A
fter six months of letrozole, a reduction in Ki-67 was observed in 31/42 
(74%) subjects, while continuing with baseline hormone replacement.  The 
median pre- and post- study Ki-67 was 4.0% and 1.5%, respectively 
(Table 1). The mean absolute change in Ki-67 was –2.3%, with 95% 
confidence intervals (-4.2% to -0.8%) which did not encompass zero.  The 
median relative change was -66% 
of the baseline values.  Reduction 
in Ki-67 was observed for 27 of 
the 35 subjects taking estrogen 
alone and for 4 of the 7 subjects 
taking estrogen plus a progestin.  
As an example of the robust 
response possible with 6 months 
of 
letrozole, 
Figure 
3 
shows 
cytomorphology 
and 
Ki-67 
expression for the pre-study and 
post-study RPFNA specimens.  For 
this 
subject, 
not 
only 
was 
cytology 
changed 
from 
hyperplasia with atypia to simple 
hyperplasia, but Ki-67 was reduced from 13.2% to almost 0.   
 
Our results differ from those published by Harper-Wynne et al. for a small 
pilot study of letrozole [Harper-Wynne 2002].  They found no significant 
decrease in Ki-67 after 12 weeks of letrozole in core biopsy specimens of 
benign breast tissue from high risk postmenopausal women.  However, 
their median baseline Ki-67 was only 1.25%.  Seven of our subjects had a 
Ki-67 <1.5%.  Six of these seven exhibited no decrease after six months of 
letrozole. This underscores the inability to detect change at very low levels 
of Ki-67 as has been reported by others [Harper-Wynne 2002; Dowsett 
2005A].   
 
Six subjects had essentially no baseline staining for ER in areas of 
hyperplasia (ER IS score <0.10).  Baseline Ki-67 ranged from 0.8 to 
Table 1. Pilot Trial Results for 42 Women Enrolled on Study 
 
Median Values 
Variable/Biomarker 
Baseline
Post- 
Study 
Change 
(relative) 
Age, years  
50 
 
 
5-Year Gail Risk, % 
2.2 
 
 
Cytomorphology, Masood Score 
14 
14 
-1 
Ki-67, % 
4.0 
1.5 
-2.3 (-0.66) 
ER, % 
40.5 
50.0 
0.0 (-0.07) 
ER, IS 
0.66 
0.60 
0.0 (-0.09) 
Mammographic Density, % area 
31.5 
29.4 
-0.4 
Estradiol, pg/ml 
57.4 
47.6 
-7.5 
Testosterone, ng/ml 
0.58 
0.56 
-0.07 
Progesterone, ng/ml 
3.22 
2.6 
-0.28 
SHBG, nM 
55.8 
51.5 
0.48 
IGF-1, ng/ml 
172 
181 
6.3 
IGFBP-3, ng/ml 
4281 
4396 
79 
IGF-1:IGFBP-3 molar ratio 
0.15 
0.15 
0.002 


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23.6%, and Masood scores from 14 to 16 in these subjects.  All subjects 
without baseline ER expression, except one who also had a low baseline Ki-
67 of 0.8%, exhibited a dramatic reduction in Ki-67 at six months.  These 
findings in benign breast tissue are similar to those of Miller et al. [Miller 
1987] and Yue et al. [Yue 2001] who found that aromatase expression is 
similar in ER- and ER+ breast cancer; and that aromatase activity is a 
stronger predictor of clinical response to AIs than is ER [Miller 1987, 2001].   
 
There were no significant changes in cytomophology, serum estradiol 
(Figure 4), testosterone, progesterone, SHBG, ER intensity score, or 
mammographic 
density 
although small reductions 
were 
seen 
for 
Masood 
score, 
estradiol, 
testosterone, 
and 
progesterone (see Table 
1).  There was no change 
observed 
in 
IGF-1.  
Results for IGF-1 and ER 
are 
similar 
to 
those 
reported by Harper-Wynne 
et 
al. 
[Harper-Wynne 
2002] 
for 
high 
risk 
postmenopausal 
women 
not taking hormones.   
 
Reported side effects included hot flashes, arthralgias, fatigue and 
temporary hair thinning. Median change in hot flash score was 0 (–8 to 
+9).  Seventeen percent reported an increase, 14% a decrease, and 69% 
reported no change in hot flashes over the duration of the study.  Thirty-
eight percent reported arthralgias and 41% fatigue when questioned on a 
monthly basis.  The regimen was well-tolerated and only one subject 
discontinued study prematurely (after 4 months).  This is in comparison to 
the ongoing National Cancer Institute of Canada MAP.2 trial of exemestane 
vs placebo, where 5 of our accrued 32 subjects discontinued study within 
the first few months primarily due to hot flashes and related symptoms.  
 
We performed a sub-analysis utilizing the 33 subjects whose baseline Ki-67 
was ≥2% and who were taking estrogen with or without a progestin, i.e., 
those who meet the eligibility criteria for the proposed Phase II trial.  The 
baseline, post-study, and change values (Table 2) were similar to those of 
the entire group of 42 subjects. 


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Table 2. Pilot Trial Results for 33 Women Taking E or E+P, with 
Baseline Ki-67 ≥2.0% 
 
Median Values 
Variable/Biomarker 
Baseline 
Post- 
Study 
Change 
(relative) 
Age, years  
49 
 
 
5-Year Gail Risk, % 
1.9 
 
 
Cytomorphology, Masood 
Score 
14 
14 
-1 
Ki-67, % 
5.0 
1.6 
-3.0 (-0.83) 
ER, % 
41.0 
50.0 
-7.0 (-0.11) 
ER, IS 
0.70 
0.53 
-0.04 (-0.12)
Mammographic Density, % 
area 
32.8 
32.6 
0.9 
Estradiol, pg/ml 
59.9 
46.7 
-7.4 
Testosterone, ng/ml 
0.60 
0.57 
-0.07 
Progesterone, ng/ml 
3.5 
2.5 
-0.3 
SHBG, nM 
57.1 
51.5 
0.3 
IGF-1, ng/ml 
185 
187 
6.6 
IGFBP-3, ng/ml 
4291 
4409 
81 
IGF-1:IGFBP-3 molar ratio 
0.16 
0.16 
0.005 
 
Median baseline Ki-67 was 5.0% and median six month Ki-67 was 1.6%.  
The median absolute change was a reduction of 3.0% (95% CI, -5.6% to -
1.7%).  The relative change was an 83% reduction (95% CI, -75% to -
10%).  A decrease in Ki-67 was exhibited by 28 of the 33 subjects (Figure 
5).  Median change in Masood cytomorphology index was –1 (range -5 to 
+1).   
 


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Most critically, there was no correlation between the change in serum 
levels of estradiol and changes observed for Ki-67 expression in RPFNA 
specimens (Figure 6). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
qRT-PCR Analysis of Gene Expression 
 
For the pilot study, primer probes with a 3’ bias were constructed for ER, 
aromatase, pS2, PCNA, MCM2, Cyclin D1, survivin, and VEGF. The quantity 
of RNA is expressed relative to the level of that for a housekeeping gene. 
The housekeeping gene differed by type of marker to be detected. Markers 
expected to be in relatively high abundance (e.g., ER, Cyclin D1) were 
tested against actin which is highly expressed.  Markers expected to be 
expressed in relatively low levels (e.g., proliferation markers, pS2, 
aromatase) were tested against TATA box binding protein (TBP), a 
housekeeping gene that is expressed at low levels.  PCR reactions were run 
on an Applied Biosystems Prism 7000 Sequence Detection System.  PCR 
values obtained with crossing threshold (CT) values if fewer than 30 cycles 
were considered valid for analysis [Giulietti 2001; Feldman 2002; Gjerdrum 
2004; Goldworthy 1999; Liss 2002; Al-Taher 2000; Liviak 2001; 
DeCremoux  2003].  
 
We began harvesting RNA for qRT-PCR midway through the trial using an 
IRB approved ancillary protocol.  Only 21 subjects had baseline and 6-
month material harvested on cells remaining after 3 slides had been 
prepared, as per the main protocol.  Initially, there were several technical 
challenges in modifying the standard Arcturus protocol to maximize good 
quality RNA from formalin/Cytolyt fixed RPFNA samples.  Microdissection 
and isolation of RNA should be performed within a few days of H&E 
staining. Further, with ThinPrep liquid processing, most of the desirable 
larger clumps of cells float to the edge of slide viewing area where it is not 
possible to employ laser-assisted microdissection.  Nonetheless, in this 
exploratory substudy, we were able to identify 14 subjects with good 
quality RNA as confirmed by QC Metric following a 1:10 dilution of RNA.  
Eight genes plus two housekeeping genes were assessed on this material.   
 
In general, ER was detected in all 14 subjects at both baseline and 6 
months in fairly abundant amounts, aromatase at very low levels, and pS2 
in low levels.  The most informative marker appeared to be pS2 where CTs 


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were below 30 both at baseline and 6 months for 8/14 cases, and these 8 
cases correlated well with change in Ki-67. Figure 7 plots the relative 
expression for pS2 (6 month value/baseline) vs. change in Ki-67 
expression (6 months – baseline).  It is encouraging that pS2 (an estrogen 
responsive gene) was generally reduced (values less than 1) when Ki-67 
was decreased (negative values), supporting the hypothesis that we are 
lowering estrogen at the tissue level despite continuing systemic 
administration of hormone replacement therapy. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Nuclear Morphometry 
 
A developmental study in collaboration with the University of Arizona was 
conducted to examine quantitative nuclear morphometry (karyometry) as a 
means of describing the proportion of abnormal cells before and after 
prevention intervention. Papanicolaou-stained ThinPrep slides of RPFNA 
samples from 11 subjects were digitally recorded at high resolution with 
200 cells measured in each slide, at baseline and at 6 months. The nuclear 
chromatin pattern was assessed for its characteristics by multivariate 
analytic techniques; a determination of nuclear abnormalities was carried 
out and procedures were applied that allowed the identification of a 
subpopulation on cells which showed expression of abnormality. This 
provided an estimate of 6.6 % of abnormal cells at base line, and of 1.6 % 
at 6 months, indicating a decrease in the frequency of abnormal cells 
following treatment with letrozole.  The slides from the remaining 31 
subjects are being analyzed for a validation of this preliminary result.  
These results will provide the algorithm for blinded (sequence of slides and 
study agent assignment) analysis of specimens from the proposed trial. 


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2.9 
Summary 
 
Based on these encouraging pilot study results, we propose a multi-
institutional, randomized placebo-controlled proof-of-principal trial (Figure 
8) in postmenopausal high risk women taking hormone replacement 
therapy, with reduction in Ki-67 as the primary study endpoint.   
 
 


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3.0    Summary of Study Plan 
 
This is a multi-institution double-blind placebo-controlled trial to whose main 
endpoint is to determine if 6 months of letrozole (2.5 mg daily) can reduce 
proliferation as assessed by Ki-67 in high risk postmenopausal women on systemic 
hormone replacement therapy who have RPFNA evidence of hyperplasia with 
atypia or borderline atypia, and a minimum Ki-67 in a hyperplastic foci of > 1.5%. 
Secondary efficacy endpoints include assessment of change in morphology by 
nuclear morphometry and the semi-quantitative Masood score index and change 
in qRT-PCR assessed estrogen response genes (GREB-1, pS2). Mammographic 
density and bioavialable estradiol and testosterone will be assessed as well. No 
difference in change between letrozole versus placebo is expected for 
mammographic breast density, Masood Index score, or serum hormones, but 
significant reductions are expected in Ki-67, expression of estrogen response 
genes by qRTPCR, and the proportion of cells deemed abnormal by nuclear 
morphometry. Other secondary endpoints include change in biomarkers 
associated with bone and cardiovascular health, toxicity measurements using the 
NCI CTCAE version 3.0, BCPT Symptom Check List (Mayo Clinic) hot flash score, 
general fatigue inventory the Fibromyalgia Impact Questionnaire from the Health 
Assiessment Questionnaire, (which assessed joint stiffness and functional impact). 
These are validated questionnaires and it should take no more than 10-15 minutes 
to complete all three questionnaires. We predict there will be little difference in 
most parameters between placebo and letrozole at 6 months in these women 
continuing their HRT. Exceptions may be arthralgais, fatigue in a minority of 
women, particularly those with sub-optimal 25-OH vitamin D levels. We will be 
assessing baseline and six month 25-OH vitamin D levels to determine if women 
with an increase in bone turnover markers, fatigue and arthralgias are likely to 
have underlying vitamin D insufficiency which is unmasked by use of letrozole. 
Subsequent to the 6-month RPFNA for assessment of biomarkers, toxicity and 
quality of life assessments, all women may receive otional open-label letrozole for 
an additional 6 months, followed by a third RPFNA and biomarker assessment. The 
primary purpose of the subsequent 6 months open label letrozole is to facilitate 
recruitment, but also to assess biomarker and toxicity endpoint changes with 
longer treatment.  A DEXA scan will be performed at completion of study to 
document any effects on bone health.  
 
Number of subjects:  The primary endpoint will be the change in Ki-67 expression 
at 6 months. Specifically, we will compare this change using a two-sample t-test 
between subjects randomized to letrozole versus those randomized to placebo.  
From our pilot study, the average percentage of cells expressing Ki-67 at baseline 
was 6.4% with an average change of -3.7% with a common standard deviation of 
5.5% in subjects meeting the eligibility criteria for this trial.  Using a 1 to 1 
randomization scheme and presuming mean change for placebo is 0, we will have 
90% power to detect a difference of 3.7% in Ki-67 in the letrozole group with a 
type I error rate of 5% with a total of 96 evaluable subjects. We intend to accrue 
108 subjects in order to attain 96 evaluable subjects.  


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The clinical flow design we will use to satisfy the Specific Aims is as follows: 
 
*Baseline history, breast exam and physical may be done at any point within 3 
months prior to randomization and starting study agent. If done prior to Visit 2, 
any new conditions or changes will be evaluated and documented before 
randomization and starting study agent. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
←Screening period→ 
-3 to 0 months 
←⎯⎯⎯⎯⎯⎯⎯ Treatment period ⎯⎯⎯⎯⎯⎯⎯→ 
0-12 months 
VISIT 1 
High Risk 
Postmenopausal 
Subjects on ERT for 
> 6 months with 
stable dose and 
route 
DEXA within 2 years
Breast Exam, 
Mammogram 
 (for breast density) 
 RPFNA (cytology,  
 Ki-67) Hyperplasia  
 +/- atypia and Ki-67
 >1.5%  
CBC, chem, 25-OH-D
Baseline history/ 
Breast exam, 
Physical* 
 
VISIT 2 
Day 0 of drug/ 
placebo dosing
Monthly 
phone 
contacts 
VISIT 3 
Month 6 
Monthly 
phone 
contacts 
VISIT 4 
Month 12
OFF STUDY 
PHONE 
CONTACT
Enter study 
Baseline 
history/ Breast 
exam,  
Physical* 
bioavailable 
estradiol, 
testosterone 
25-OH-D 
Fasting lipid 
profile 
Osteocalcin  
N-teleopeptide
Surveys 
Questionnaires 
 
Trial 
coordinator 
contact 
subject via 
telephone 
evaluate 
AE’s, 
compliance 
 
Repeat 
baseline 
studies, 
return 
unused study 
agent or 
placebo, 
evaluate 
AE’s, 
compliance, 
dispense 
open label 
drug  
DEXA scan if 
completing 
study 
Trial 
coordinator 
contact 
subject via 
telephone 
evaluate 
AE’s, 
compliance 
Repeat 
baseline 
studies, 
return 
unused 
study agent, 
evaluate 
AE’s, 
compliance 
DEXA scan 
 
Trial 
coordinator 
contact 
subject via 
telephone 
evaluate 
AE’s 


Breast Cancer Prevention by Letrozole in High Risk Women 
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4.0   PARTICIPANT SELECTION 
 
4.1 
 Inclusion Criteria for Screening RPFNA 
 
Note that the screening RPFNA is conducted under a separate IRB-aproved 
research protocol with its own consent form.  Potential subjects are not 
required to make a decision regarding participation in the letrozole trial 
until after receipt of the results of their RPFNA. 
 
4.1.1 Menopause Status 
 
4.1.1.1 
Postmenopausal women at increased risk for breast 
cancer, age 30-69, on a stable dose of systemic HRT for six or more 
months. 
 
Postmenopausal status defined as having either a prior bilateral 
oophoerctomy, prior hysterectomy with ovaries intact and age 
greater than 50, or prior natural menopause with no period for at 
least 12 month prior to baseline RPFNA 
 
4.1.2 
Hormone Replacement 
 
On a well-absorbed systemic estrogen or estrogen and progestin 
preparation at least six months (women receiving only poorly 
absorbed vaginal estrogen such as vagifem or estring, or those 
receiving a testosterone plus estriol vaginal gel do not qualify). 
Dose and type of hormone replacement must be stable for at least 
six months. 
 
4.1.3 
Minimum Risk Criteria 
 
• 
Gail 5-year Risk >1.7% and/or relative risk of ≥3X that for the 
average woman of the same age group, as calculated by the NCI 
Breast Cancer Risk Assessment Tool 
(http://www.cancer.gov/bcrisktool/Default.aspx); or a 10-year 
relative risk of > 3X that for the population, as calculated by the 
IBIS Breast Cancer Risk Evaluation Tool (http://www.ems-
trials.org/riskevaluator/); 
• 
Known to carry BRCA1/2 mutation characterized as deleterious or 
of uncertain significance;or family history consistent with 
hereditary breast cancer 
• 
Prior ADH/DCIS/LCIS; or hyperplasia with atypia on RPFNA.  
 
4.1.4 
Subjects must have had a baseline mammogram performed within 
three months prior to screening baseline RPFNA, interpreted as not 
suspicious for breast cancer, with any supplementary imaging 
required performed and negative prior to RPFNA.  
 
4.1.5 
Subjects must have had bone densitometry (DEXA) within two years 
of entry onto the study that demonstrated a normal bone mineral 
density (BMD).  T-score must be greater than -1.0; or -1.5 to -1.0 if 
on active osteoporosis treatment. 


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4.2 
Exclusion Criteria for Screening RPFNA 
 
o 
Started or stopped or changed dose of hormone replacement within 6 
months; 
o 
Receipt of any aromatase inhibitor or selective estrogen receptor 
modulator (tamoxifen, raloxifene, arzoxifene, etc.) within the prior 12 
months. 
o 
Participation on any chemoprevention trial within the past 6 months 
o 
Bilateral breast implants  
o 
Current anticoagulant use 
o 
Any other condition or intercurrent illness that in the opinion of the 
investigator makes the subject a poor candidate for RPFNA 
o 
Invasive breast cancer or other invasive diagnosis within five years 
o 
Metastatic malignancy of any kind 
o 
NSAIDS use within prior three weeks 
 
4.3 
Inclusion Criteria for Study Entry 
 
o 
RPFNA hyperplasia +/- atypia (Masood score of ≥13), Ki-67 ≥1.5% 
positivity (≥500 cells), extra cells for qRTPCR studies (estimated by 
lab technician from remaining amount in Preservcyt Container). 
o 
Willing to continue the type, dose and route of their hormonal regimen 
at baseline aspiration throughout the duration of the study 
participation (6 months for randomization portion, 12 months if 
participates in the optional open label period); 
o 
Have reasonable normal organ function before being dispensed study 
agent/placebo, as defined as: 
Platelets 
 
 
 
>100,000/dL 
Hb 
 
 
 
 
>10 g/dl 
Absolute granulocyte count 
>1000/ul 
Creatinine 
 
 
 
<1.5 mg/dl 
Albumin 
 
 
 
>3.0 g/dl 
Bilirubin 
 
 
 
<2.0 mg/dl 
AST  
 
 
 
<2 times upper limit of normal 
Alkaline Phosphatase 
 
<2 times upper limit of normal 
 
o 
Have serum level of 25-OH vitamin D of at least 30 ng/ml prior to 
study entry. 
o 
Willing to have a repeat RPFNA and mammogram at 6 months and 12 
months (if participates in the open label portion of the study) following 
initiation of study drug. 
o 
Willing to undergo a history, breast and physical exam prior to 
randomization/starting study agent and at 6 months and be contacted 
monthly by the trial coordinator during the 6 month study period (12 
months if participates in the open label portion of the study); 
o 
Willing to have approximately six tubes of blood drawn at baseline and 
six months (12 months if participates in open label portion of the 
study);  
o 
Willing to sign and able to understand separate consents for the 
RPFNA’s and study participation; 
 
4.4   
Exclusion Criteria for Study Entry 


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o 
Prior history of osteoperosis or osteoperotic fracture 
o 
Prior History of Invasive Breast Cancer or other invasive cancer within 
five years from date of study entry 
o 
Current and chronic use of Cox-2 Specific Inhibitors or NSAIDs, 
defined as seven consecutive days (1 week) for greater than three 
weeks during the six months prior to RPFNA  
o 
Receiving treatment for rheumatoid arthritis or fibromyalgia 
o 
Current history of poorly controlled migraines or perimenopausal 
symptoms-  
o 
Currently receiving other investigational agents. 
o 
Receipt of more than 6 months of an aromatase inhibitor (anastrozole, 
exemestane, letrozole, etc.) at any time in the past. 
 
4.5 
Inclusion of Women and Minorities  
 
All women who meet eligibility criteria and members of all races and ethnic 
groups are eligible for this trial.   
 
4.6    Recruitment and Retention Plan 
 
 
Potential subjects will be screened in the Breast Cancer Prevention Center at 
the University of Kansas Medical Center (KUMC), Kansas City, Kansas. Or in 
similar clinics at the other participating institutions. Accrual duration is 
expected to take 42 months, accruing 2.5 subjects/month. It is expected 
that for every 4 postmenopausal subjects taking hormone replacement 
therapy screened by RPFNA, 1 of those will be eligible for participation.  
 
Subjects will be contacted by phone during the months between clinic visits 
in order to insure active participation and to collect adverse event, dosing 
and other pertinent information. It is expected that the dropout rate will be 
less than 5%.  
 


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5.0  
AGENT ADMINISTRATION 
 
Intervention will be administered on an outpatient basis. Reported adverse events 
and potential risks are described in Section 9.2.   
 
5.1   
Dose Regimen and Dose Groups 
 
 
 
Letrozole 2.5 mg or letrozole placebo will be given to all trial participants 
for the first 7 months and open label letrozole the second 6 months. All 
subjects will be given eight months (one month of overage) of drug at the 
baseline and 6 months visit to allow for scheduling study procedures. All 
subjects should have off-study procedures no later than within 36 hours of 
the last dose of letrozole or placebo. However, if a participant stops study 
drug prematurely in a unscheduled situation, she should come in for 
biomarker re-assessment as early as possible. 
 
5.2 
 Study Agent Administration 
 
The Institutional Investigational Pharmacy will be responsible for receiving, 
inventory and dispensing of study agent.  
 
A study number will be assigned to each participant and provided to the 
investigational pharmacy. Study agent will be dispensed with the study 
number of each subject. Seven months of study agent will be dispensed. 
The study coordinator will then 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. 
 
5.3   
Concomitant Medications 
Subjects are required to maintain any hormonal use throughout the 
duration of study participation. 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. Subjects are not allowed to start tamoxifen, 
raloxifene, anastrozole, letrozole or any other SERM or aromatase inhibitor 
(or other chemopreventive) while participating on this trial.  
 
All medications (prescription and over-the-counter), vitamin and mineral 
supplements, and/or herbs taken by the participant will be documented on 
the concomitant medication CRF and will include: 1) start and stop date, 
dose and route of administration, and indication. Medications taken for a 
procedure (e.g., biopsy) should also be included. 
 
5.4 
Dose Modification 
 
Dose modifications will not be allowed on this study. Because study agent 
is only available in a single dose 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 letrozole, this must 
be noted in the CRF on the dose suspension page. At the discretion of the 


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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.  If the investigator is certain that the toxicity is not due to the 
agent, the agent may be re-instituted after resolution of the adverse event, 
providing: 
 
1) the subject will have had a minimum of three months of study 
agent prior to biomarker assessment at six or twelve months;   
  
2) the investigator feels relatively certain taking letrozole or 
participating in the study will not affect recovery from the condition 
 
5.5 
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. 
 
 
 
 


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5.6 
Emergency procedure for unblinding 
 
Any adverse event or situation that requires immediate unblinding is 
subject to the mutual consideration of the Protocol Chair and the Trial 
Statistician. 
 
 
5.7 
Adherence/Compliance 
 
 
Each subject will be given a seven month supply of drug/placebo at trial 
entry and instructed to return all excess drug/placebo to investigator at the 
six month visit. Six months of dosing is the expected amount of dosing, 
however, seven months of drug/placebo will be given to each subject to 
accommodate scheduling issues. At the end of treatment the actual 
quantity of unused drug/placebo will be compared to the anticipated 
amount of unused drug. At the six month visit, all subjects have the option 
to receive seven months of open label drug supply, expected to cover six 
months of dosing and allow some flexability with scheduling the repeat 
RPFNA while on study drug. 
 
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 thenumber 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.  
 
5.8  
Drug Records 
 
Drug records regarding receipt, distribution and disposition of study drug 
will be maintained by the institutional investigational pharmacist. The 
packing slip will indicate from and to whom study drug/placebo was 
shipped, date, quantity, and batch or lot number. The dispensing record 
will indicate quantity and dates that study drug was dispensed to and 
return by each subject. At the completion of the study, all unused study 
drug/placebo will either be returned to Novartis or destroyed per Novartis 
instructions. The record documenting study drug return will include date, 
quantity, batch or code number, and the name of person/department to 
whom the drug was returned. 
 
          
 
 
 
 
 
 
 
 


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6.0  
LETROZOLE INFORMATION 
 
6.1 
Letrozole IND Sponsor 
 
Letrozole is an approved agent for treatment of breast cancer and will 
shortly begin evaluation in The NSABP4 trial as an agent for the primary 
prevention of breast cancer in postmenopausal women not on hormone 
replacement therapy. Since in this instance letrozole is being used in an 
experimental manner, we will file for an IND exemption, as we did for the 
pilot study. 
 
6.2 
Mechanism of Action 
 
Letrozole is a non-steroidal aromatase inhibitor (4, 4’-1H-1,2,4-Triazol-1 
ymethylene dibenzonitrile) with a molecular weight of 285.31. It is supplied 
by Novartis in 2.5 mg tablets for oral administration.  
 
6.3 
Pharmacokinetics 
 
Letrozole is rapidly absorbed from the GI tract, and absorption is not 
affected by food. Letrozole is slowly metabolized to an inactive metabolite 
whose glucouride metabolite is renally excreted. About 90% of letrozole is 
recovered in urine.  Plasma concentrations at steady state are 1.5 - 2 
times higher than after a single dose, but after steady state is reached 
there is no accumulation over time. Letrozole is weakly protein bound and 
has a large volume of distribution. No change in letrozole pharmacokinetic 
parameters have been noted with age or moderate renal impairment. 
Subjects with moderate hepatic dysfunction had 37% higher mean AUC 
levels than those without impaired function. 
 
6.4 
Pharmacodynamics 
 
Letrozole inhibits aromatase by competitively binding to the heme moiety 
of cytochrome p450 resulting in a reduction of estradiol and estrone 
synthesis in all tissues.  
 
In premenopausal animals, letrozole decreases ovarian steroidogenesis, 
reduces uterine weight, increases LH but not FSH, results in regression of 
estrogen dependent tumors but does not alter adrenal mineral corticoid or 
glucocorticoid synthesis. 
In postmenopausal women letrozole suppresses conversion of testosterone 
to estradiol and androstenedione to estrone in the fat, adrenal glands and 
other tissues. Treatment of postmenopausal women reduces serum 
estrone, estradiol and estrone sulfate but does not alter adrenal 
corticosteroid, aldosterone, or thyroid hormone synthesis. 
 
6.5 
Drug/Drug Interactions 
 
No drug/drug interactions have been noted with warfarin or cimetidine.  
Concomitant use of tamoxifen and letrozole may decrease letrozole levels.   
 
 
 


Breast Cancer Prevention by Letrozole in High Risk Women 
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6.6. 
Clinical Indications 
 
Letrozole is currently approved as first and second line therapy of 
metastatic breast cancer, and for use in the adjuvant setting instead of 
tamoxifen or after 5 years of tamoxifen in a dose of 2.5 mg per day. In the 
adjuvant setting, letrozole is given for 5 years  
 
6.7  
Adverse Reactions 
 
6.7.1 Compared to placebo in adjuvant setting 
 
Adverse events which occurred more significantly more frequently in 
the MA.17 trial of extended adjuvant letrozole versus placebo 
following 5 years of tamoxifen were: hot flashes (net differerce + 
4%), loss of appetite (net difference + 2%), myalgias ( net 
difference +  3%), arthralgias  (net difference + 4%), alopecia (net 
difference +2%) [Goss 2003]. 
 
6.7.2 Compared to tamoxifen in adjuvant setting 
 
In adjuvant trials women randomized to aromatase inhibitors had 
fewer events, uterine cancers and thromboembolic phenomena 
compared to tamoxifen [Coombes 2004; Thurlimann 2005; Howell 
2005]. However, women randomized to an AI had a similar 
incidence of hotflashes and a higher incidence of arthralgias, bone 
mineral density loss, vaginal dryness, pain with intercourse, and 
loss of libido compared to those taking tamoxifen [Coombes 2004; 
Thurlimann 2005; Fallowfield 2004; Howell 2005].   
 
6.7.3 Compared to tamoxifen in metastatic setting 
 
In a study of 455 women with metastatic breast cancer with a 
median exposure time of 11 months the incidence of adverse 
experiences was similar for Femara (letrozole) (n=455) and 
tamoxifen (n=455). The most frequent adverse events were nausea 
(15%), hot flushes (18%), fatigue (11%), dyspnea (14%) and 
arthralgias (14%).  Constipation was noted in 9%, headache 8%, 
diarrhea in 7%, vomiting in 7%, decreased appetite 4%, weight 
decrease in 6%, alopecia 5%, lower limb edema in 5%, and 
hypertension 5% (package insert). Discontinuation of drug for 
adverse experiences was performed for 2% of patients taking 
Femara (letrozole) and 3% of those taking tamoxifen. A similar 
distribution of adverse events has been reported for subjects 
randomized to letrozole vs placebo in the MA17 trial 
 
6.8  
Availability 
 
Both letrozole and matched placebo are to be provided by Novartis to the 
investigational pharmacy at each participating Institution. 
 
 
 
Both letrozole and letrozole placebo supplied in bottles of 100 tablets. 
 
 


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6.9 
Agent Distribution 
 
Agent will only be released after documentation of IRB approval of the 
KUMC-approved protocol and consent.  
 
200 tablets will be dispensed at entry of the subject onto the study so as to 
provide an adequate number of capsules for 7 months.  The 7 months is 
based on the planned 6 months of administration, plus approximately one 
additional month if required for scheduling of procedures or travel.  
 
6.10  Agent Accountability 
 
The Site Principal Investigator, or a responsible party designated by the 
Site Principal Investigator, must maintain a careful record of the inventory 
and disposition of all agents using a Drug Accountability Record Form 
(DARF). The Site Principal Investigator is required to maintain adequate 
records of receipt, dispensing and final disposition of study agent. This 
responsibility has been delegated to the Investigational Pharmacy at each 
participating site. 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.  
 
6.11 
Packaging and Labels 
 
Letrozole and letrozole placebo will be supplied by Novartis in a form ready 
for dispensing. Each bottle will be labeled with a one-part label identifying 
study specific information, such as Study title, HSC protocol number, 
dosing instructions, recommended storage conditions, the name and 
address of the distributor, 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. 
 
6.12 
Storage 
 
Storage should be in a locked cabinet with limited access, at room 
temperature. 
 
 
6.13 
Registration/Randomization/Consent 
 
Potential subjects will be registered for the screening portion of the study 
by site study coordinator and principal investigator on a site screening log. 
Potential subjects will be given a unique screening number by the site staff 
and this will be affixed to all eligibility specimens and documents that will 
be sent to KUMC for review. No PHI should be attached to these items.  
 
Site staff will not randomize a subject until verification by KUMC Trial 
Coordinator by means of a faxed request (standard form provided for this 
purpose which will include an eligibility checklist and notification of 
acceptable consent) for a particular potential subject. The designated 
KUMC Trial Coordinator will fax back to the Site Coordinator an approval to 
randomize the subject based on the review of the eligibility checklist 
provided by the site.  


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Approved eligibile subjects will be randomized by a randomization schedule 
at each site, prepared and provided by the Study Statistician. The site staff 
and subjects will be blinded as to the assignment to drug or matched 
placebo.   
 
The subject will be assigned a randomization number, after stratification by 
site and type of hormone replacement therapy. The randomization 
schedule will be stratified by site (each site will be provided a unique list 
for use only at that site), and whether the subject is on an estrogen 
preparation alone, or estrogen plus a progestin preparation. Randomization 
will be in sequential order according to the randomization list provided. 
Subjects approved for randomization will be assigned a site specific 
randomization number, consisting of four digits. The first digit will identify 
the site (to be determined later) and three digits identifying the specific 
potential subject.  
 
Sub-contracted sites other than KUMC may use the study consent for the 
screening RPFNA, or use a research/clinical consent for that purposes. 
Subjects are not officially on study until they have made it through the 
entire eligibility process. KUMC will use the High Risk Protocol Consent 
(KUMC HSC#4601) for the purposes of the screening RPFNA.  
 
6.14  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 Protocol Investigator at KUMC and the 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.  
 
6.15 
Agent Destruction/Disposal 
 
At the completion of investigation, all unused study agent will be returned 
to Novartis or destroyed in accordance with each site’s investigational  
pharmacy regulations. 
 


Breast Cancer Prevention by Letrozole in High Risk Women 
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7.0  
CLINICAL EVALUATIONS AND PROCEEDURES  
 
 
 
7.1  
Schedule of Events 
 
Study Procedure 
Eligibility 
Period 
Treatment Period 
 
 
Visit 1 
Baseline 
Visit 2 
On-
Study 
Monthly 
Phone 
Contacts 
Visit 3 
6 month 
Visit 
Monthly 
Phone 
Contacts 
Visit 4 
12 month visit 
(optional 
participation) 
4 week 
follow-up 
phone 
contact 
Eligibility Checklist 
X 
 
 
 
 
 
 
Informed Consent* 
X 
X 
 
X 
 
X 
 
Medical History 
X˚ 
X˚ 
 
 
 
 
 
Breast Exam 
X˚ 
X˚ 
 
X 
 
X 
 
Physical Exam 
X˚ 
X˚ 
 
X 
 
X 
 
Mammogram 
X 
 
 
X 
 
X 
 
Ht/Wt/Vital Signs 
X 
X 
 
X 
 
X 
 
RPFNA 
X 
 
 
X 
 
X 
 
CBC, Chemistry, 25-OH 
vitamin D +  
X 
X 
 
X 
 
X 
 
#Fasting blood for: 
Bioavailable estradiol, 
testosterone, SHBG, 
osteocalcin, N-
telopeptide, CRP, lipid 
panel 
X 
 
 
X 
 
X 
 
#Trough blood (serum) 
sample 
 
 
 
X 
 
X 
 
Hot flash assessment 
X 
X 
X 
X 
X 
X 
X 
Fatigue inventory, BCPT 
Symptom Check List, 
HAQ Fibromylagia 
Impact Questionnaire  
 
X 
 
X 
 
X 
 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
Dispense Study Agent/ 
Evaluate Compliance 
 
X 
 
X 
 
X 
 
Concomitant Medication 
X 
X 
X 
X 
X 
X 
X 
Return Study Agent 
 
 
 
X 
 
X 
 
Off-Study DEXA scan 
 
 
 
    X                or              X 
 
Off-Study evaluation 
 
 
 
 
 
 
X 
*Subjects will sign a consent each time an RPFNA procedure is performed (at KUMC HSC#4601). 
Only subjects qualifying for the drug/placebo dispensation will sign the study consent document. 
For sites other than KUMC, they may determine to use the study consent for the screening RPFNA 
per the site IRB requirements. Subjects will not be officially “on study” until eligibility is 
determined. 
 
#After a 10-12 hour fast, four gel-clot 7 ml tubes drawn, allowed to clot at room temperature for 
30 minutes, spun at 3500 rpm for 15 minutes, and aliquots of serum into 8 polypropylene storage 
tubes. Tubes to be labeled with subject initials, randomization number and date of draw. Tubes 
frozen at -80◦C and then shipped to KUMC on > 5 lbs of dry ice, with pre/post specimens shipped 
together. Notify KUMC by fax and email prior to shipment. 
 
+CBC, Chemistry, and 25-OH vitamin D to be performed by local laboratory at the expense of site 
PI (to be included in budget/subject costs) 


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˚ Study medical history, breast and physical exams may be done at any point within 3 months 
prior to randomization and starting study agent. If done prior to Visit 2, any new conditions or 
changes will be evaluated and documented before randomization and starting study agent. 
 
 
7.2 
Pre-Study Procedures and Evaluations 
 
 
 
7.2.1 Pre-study Eligibility RPFNA Procedure Details 
 
 
 
RPFNA will be performed, under local anesthesia from both breasts 
(two sites on each breast) and all cells pooled from both breasts 
(one breast in the case of prior DCIS or invasive cancer) into a 
single 15 cc tube containing modified Cytolyt™ (9 cc Cytolyt™ and 1 
cc 10% neutral buffered formalin). After cleansing the breast with 
betadine and alcohol swabs, the breast is anesthetized with 
epinephrine and lidocaine using a tuberculin syringe. After the 
surface has been anesthetized, epinephrine and lidocaine are 
injected deeper into the breast at two sites, approximately at the 10 
and 2 o’clock locations. Then, 5-6 needle passes with sterile RPMI 
prewetted 10-12 cc syringes/with 21 gauge 1 ½ inch needle, within 
the anesthetic puncture sites to withdraw epithelial cells lining the 
ductal tree. Clinician will preferentially sample areas in which 
resistance (density) is appreciated with the tip of the needle. 
Sample is immediately expelled into a 15 cc plastic tube containing 
9 cc of Cytolyt™ (Cytyc Corp) and 1 cc of 10% neutral buffered 
formalin. Fluid from the Cytolyt™ tube should be drawn into the 
needle/syringe hub approximately 4x when expelling material into 
tube solution, and subject 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 mailing 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 are offered Ativan 1 mg prior to the procedure for anxiety, 
and analgesics after the procedure in order to control any pain that 
may occur. These medications are optional. Subjects should also be 
instructed to eat a full breakfast the morning of the RPFNA, to 
abstain from vitamin E, NSAIDS and fish oil products for three 
weeks prior to RPFNA, and if planning on taking Ativan, to have 
someone with them to drive them to and from the clinic. 
 
Subjects meeting the eligibility criteria from the RPFNA procedure 
and completing other pre-study procedures will be offered 
participation in the trial. 
 
7.2.2 Other pre-study eligibility procedures (see Eligibility in section 4.0) 
 
• 
Risk assessment 


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• 
Mammogram not suspicious for cancer, and any follow-up 
imaging recommended completed and not suspicious for 
cancer 
 
 
7.3 
Baseline Studies 
 
 
7.3.1 Brief Physical and Medical History 
 
Subjects will have a brief physical (including clinical breast exam) 
performed by a study-associated clinician. All abnormal findings will 
be recorded on the physical source document, including duration 
and severity. Vital signs, height, and weight. Percent body fat will 
be recorded for those institutions with appropriate scales in the 
clinic. 
 
Subject will have a medical history collected by study personnel, 
including event dates, duration, and severity.  
 
Physical (including clinical breast exam) and Medical History can be 
performed up to 3 months prior to randomization and starting study 
agent, but will be confirmed at on-study visit and evaluated. 
 
 
 
 
 
7.3.2 Bloodwork  
 
Standard CBC/Chemistry (and 25-OH vitamin D for determination of 
eligibility) will be collected and analyzed in a local CLIA approved 
lab at the expense of the site investigator. All materials will need to 
be obtained by individual study site. CBC/Chemistry/25-OH vitamin 
D may be done at any time up to six weeks prior to day of 
randomization. Results must be known prior to subject 
randomization.  
 
Fasting blood (10-12 hour fast) will be collected in four gel-clot 
serum tubes for a lipid panel, bioavailable estradiol and 
testosterone, bone turnover markers (osteocalcin and N-telopeptide 
crosslinks), high sensitivity C-reactive protein, 25-OH Vitamin D and 
processed, stored and shipped per section 7.0 and 8.0. Assays will 
be performed centrally by KUMC. Kits and explicit instructions will 
be provided for this bloodwork by KUMC to individual sites.  
 
An additional 3 cc specimen will be collected for possible letrozole 
blood levels. See section 7.0 and 8.0 for processing, storage and 
shipping instructions. Assays will be performed centrally after 
shipment to KUMC. Kits and explicit instructions will be provided for 
this bloodwork by KUMC to individual sites. 
 
7.3.3 Baseline symptom assessment 
 
A baseline symptom assessment will be made via the BCPT 
symptom checklist, including such relevant conditions as hot 
flashes, menstrual abnormalities, pelvic pain, muscle and joint pain, 
and fatigue. 


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We will utilize the hot flash scoring system developed by Dr. Charles 
Loprinzi at Mayo Clinic. For muscle and joint symptoms and function 
we will use the validated HAQ Fibromyalgia Impact Questionnaire 
from the Health Assessment Questionnaire (Stanford), and for 
fatigue the brief fatigue inventory [Wolfe 2004; Mendoza 1999; 
Loprinzi 2002]. Adverse events will be recorded using NCI common 
terminology criteria for adverse events version CTCAE 3.0. 
 
7.4 
Evaluations During Study Intervention 
 
Monthly Telephone Assessment 
 
Subjects will be contacted by the study coordinator on a monthly basis to 
review dosing and adverse events. Subjects who are difficult to reach or do 
not respnd 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.  
 
7.5   
Evaluation at Completion of Randomized Portion of Study at 6 Months        
(See section 7.1 for Study Calendar) 
 
If necessary for synchronizing scheduling of RPFNA with continued use of 
letrozole, study agent may be continued for up to one additional month, 
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 six months. 
 
Subjects should NOT take the study drug the day of the procedure and 
should have blood drawn after a 10-12 hour fast.  
 
Subjects will have a mammogram, RPFNA, CBC/Chem and fasting study 
bloodwork collected, and a brief physical (including clinical breast exam) 
performed by a study-associated clinician (see the study calendar, section 
7.1 for a comprehensive list). All abnormal findings will be recorded on the 
physical source document, including duration and severity. Vital signs, 
height, and weight will be recorded. Percent body fat will also be and 
percent body fat will also be recorded. 
 
Bloodwork for CBC/Chem (sent to local CLIA lab), fasting (10-12 hour fast) 
bloodwork and drug trough serum sample will be collected. 
 
Subjects wishing to participate an additional six months in the open label 
portion of the study will be given an additional 7 month supply and then 
asked to return for a repeat RPFNA, breast density and other biomarker 
assays (see study calendar section 7.1).  
 
When the subject has completed taking study agent, any remaining supply 
will be 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. 
 


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When the subject has completed taking study agent, she will receive a 
DEXA scan to document any effects on bone health.  
 
7.6   
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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8.0 
BIOLOGIC SPECIMEN HANDLING, STORAGE AND SHIPPING 
 
 
All specimens will be placed in polypropylene storage tubes labeled with subject 
initials and study/randomization number, date of collection and contents of tube. 
 
 
RPFNA specimens are to be collected as in 7.1.1. Tubes should be placed on a 
rocker for 12-24 hours to ensure thorough bathing of cells. Tubes containing 
RPFNA specimens should be overnight shipped with cool packs and using the 
KUMC Outside RPFNA Shipping Form within 36 hours of collection. The shipping 
form should also be faxed to 1-913-588-3821 in order to notify the KUMC Lab of 
the shipment. A call to 913-588-3917 should also be made to make certain the lab 
is aware of the shipment.  
 
 
CBC/Chem specimens and assays of 25,OH-vitamin D for eligibility will be sent to 
the local CLIA approved lab and the costs covered by the local PI budget. 
 
 
Study bloodwork should be collected fasting in four gel-clot tubes, allowed to clot 
at room temperature for 30 minutes, spun for 15 minutes at 3500 rpm, and the 
serum poured into four polypropylene storage tubes and labeled as in 8.1. These 
specimens should be immediately frozen and stored at -80◦C until shipment to 
KUMC. All specimens for each subject should be sent together, overnight, on 5 lbs 
or more of dry ice, in order to be run together. Notification of KUMC via fax (913-
588-3821) and 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 open, contact them before shipping. 


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9.0 
SAFETY ASSESSMENTS 
 
9.1 
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. 
 
 
9.2  
Adverse Event Reporting 
 
 9.2.1 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. 
 
 9.2.2 Adverse Event data elements 
 
o AE onset date 
o AE Verbatim Term 
o CTCAE Term (version 3.0) 
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 
 
9.2.3 Severity of Adverse Events 
 
Identify the adverse event using the NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 3.0. 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 
 
9.3 
Assessment of Relationship of AE to Treatment 
 
The possibility that the adverse event is related to study drug will be 
classified as one of the following: not related, unlikely, possible, probable, 
definite. 
 
 
9.4 
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. 
 
 
9.5 
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. 
 
 
 


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9.6 
Reporting Serious Adverse Events 
 
 9.6.1 Central Reporting 
 
KUMC will report all SAEs to Novartis, Pharmaceuticals Clinical 
Safety and Epidemiology Department (CS&E). NCI and to the FDA 
per GCP guidelines.  These reports are to be filed utilizing the Form 
FDA 3500A (MedWatch Form). Documentation of this submission 
should also be forwarded to Novartis CS&E in a timely manner. The 
investigator must also notify Novartis CS&E department which of 
the SAEs has been expedited (15-day submissions) to the FDA, 
within 24 hours of submission to the FDA.  
 
Novartis CS&E department must be copied on any study related 
safety information sent to the IRB.   
 
In addition, any serious adverse event regardless of relationship to 
the study drug, including pregnancy, occurring in a subject after 
providing informed consent, while receiving study drug, and until 
four weeks after stopping study drug, must be reported to Novartis 
Pharmaceuticals CS&E Department by FAX (888-299-4565) within 
24 hours of learning of it's occurrence.  It is requested that SAEs 
are reported to Novartis using the Novartis PIND Serious Adverse 
Event Report form, with clear documentation in the narrative that 
the PI is the holder of a private IND. The period after discontinuing 
study drug may be extended if there is a strong suspicion that the 
drug has not yet been eliminated. All serious adverse events must 
also be reported for the period in which the study protocol interferes 
with the standard medical treatment given to a subject (e.g., 
treatment withdrawal during washout period, change in treatment 
to a fixed dose of concomitant medication). 
 
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 Novartis, NCI and/or the FDA, and to the local site 
IRB. 
 
KUMC FAX: 913-588-3679  
KUMC Phone: 913-588-7791 
Attention: Carol Fabian, MD 
cfabian@kumc.edu 
Protocol Chair 
 
 
 
 
9.6.2  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 


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o title of protocol 
o description of SAE, including attribution to drug and 
expectedness 
o subject randomization number 
 
 
  9.6.3  
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 NCI DCP 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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10.0 
BIOMARKER AND LABORATORY ASSESSMENTS 
 
The RPFNA specimen will be analyzed in the KUMC Breast Cancer Prevention 
Center Laboratory, under the direction of Brian Petroff, DVM, PhD. The Breast 
Cancer Prevention Laboratory and Research Administrative Unit are located in the 
Lied Research Building. It consists of three laboratory rooms with a total of 1597 
square feet plus 1200 square feet of office space. The laboratories are fully 
equipped for cell culture, the processing and assessment of cytology and 
immunocytochemistry slides, assay of serum hormones and IGF-1/IGFBP-3, 
(ELISA’s RIAs), laser microdissection, qRT-PCR, and various other molecular 
biology techniques. Staff located in the administrative/laboratory unit include two 
cytology/immunochemistry technicians, and an additional technician experienced 
in performing ELISAs RIA’s for hormone assays as well as RTPCR and other 
molecular assays. 
 
10.1 
Ki-67  
 
Breast Cells from RPFNA are pooled in modified cytolyt and sent to KUMC 
by express mail within 72 hours of RPFNA and all will be processed to slides 
within 96 hours. Cells are processed to thin prep slides via standard 
manufacturer’s specifications.  
 
A minimum of three slides will be made by a liquid-based cytologic 
technique: at least one each for cytomorphology, Ki-67 and microdissected 
prior to RNA extraction. Total epithelial cells will be delineated with Mayer’s 
hematoxylin counterstain and proliferating cells with a MIB-1 antibody for 
Ki-7. Only baseline slides containing > 500 epithelial cells are processed 
and stained for Ki-7. Antigen retrieval is performed with 10 nmol/L citrate 
buffer (pH 6) in a Biocare (Walnut Creek, CA) decloaking chamber for two 
minutes at 120 degrees C. Slides are then stained with a MIB-1 monoclonal 
antibody (M7240 Dako Cytomation, Carpinteria, CA) at a 1:20 dilution in a 
Dako Autostainer [Khan 2005]. Hyperplastic clusters are preferentially 
assessed and the number of cells with unequivocal nuclear staining out of 
500 cells assessed and recorded. Manual counting will be employed and 
scoring assessed by two laboratory technicians. The scores will consist of 
number of cells assessed, and number of positive cells for Ki-67. A 
separate score for each technician and a consensus score will be recorded. 
The consensus score will be utilized for analysis of the primary endpoint. 
Representative cell clusters are archived with a digital imaging system. 
 
In the event that fewer that 500 epithelial cells are present in the Ki-67 
slide on the follow-up RPFNA, the cytology slide will be destained and used 
for Ki-67 staining. If there are still less than 500 evaluable cells present 
when all slide material is combined the post-treatment value for Ki-67 will 
be considered as the proportion of epithelial cells staining positive for Ki-67 
relative to the total number of epithelial cells present. If no staining for Ki-
67 is observed and there are less than 500 evaluable cells, the value will 
be imputed as the reciprocal of the total number of evaluable cells present. 
If there is no staining and greater than 500 cells, the value will be assumed 
to be zero. Subjects with less than 100 evaluable cells will be considered 
not evaluable for Ki-67.  
 
 


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10.2 
Cytomorphologic Assessment 
 
 
Cytomorphologic assessment will be performed as in the pilot study by Dr. 
Carola Zalles. She will provide both a traditional characterization (QNS, 
normal, apocrine metaplasia, epithelial hyperplasia, or hyperplasia with 
atypia) [Zalles et al 1995] and a semiquantitative cytology index score 
(modified Masood score) [Masood et al 1990] and a consensus panel (QNS, 
benign, intermediate/atypia, atypia/suspicious) description [The Uniform 
Approach to Breast Fine-Needle Aspiration Biopsy 1997]. The Masood 
semiquantitative index score will be the primary measure determining 
morphologic hange as we have observed that interpretive variance (> 3 
points) is less using this system [Fabian 2002].  
 
10.3 
Nuclear Morphometric Assessments  
 
Nuclear Morphometric Assessments will be performed at the University of 
Arizona by Dr. Bartels and associates. Briefly, the cytomorphlogy slide is 
imaged, individual nuclei are acquired and stored in libraries.  
Morphometric analysis of approximately 40 features is performed to 
generate a cumulative score that reflects deviation from normality.    
 
10.4 
RNA Extraction and qRTPCR Assessments 
 
RNA extraction will be performed after microdissection of the third slide 
using an Acturus Autopix and Macro LCM caps (Arcturus, Mountain View, 
California).  RNA from microdissected epithelial cells and residual whole 
slide scraping is obtained using a commercial protocol (Paradise Reagent 
Solution, Arcturus) optimized for formalin-fixed specimens [Petroff 2006B].  
Material is heated in an overnight incubation with proteinase K to lyse cells 
and dissociate nucleic acids prior to harvesting. RNA is isolated using 
column based chromatography and treated with DNase to eliminate any 
residual DNA.  Purified RNA is amplified with a single round of T7 based 
linear amplification (Paradise Reagent System, Arcturus) and converted to 
cDNA and stored at -80°C until all specimens from a subject can be run in a 
single batch.  Quality of RNA is checked by PCR (QC Metric, Arcturus).  The 
quantity of RNA is expressed relative to the level of that for a 
housekeeping gene. The housekeeping gene differed by type of marker to 
be detected. Markers expected to be in relatively high abundance (e.g., ER, 
Cyclin D1) were tested against actin which is highly expressed.  Markers 
expected to be expressed in relatively low levels (e.g., proliferation 
markers, pS2, aromatase) were tested against TATA box binding protein 
(TBP), a housekeeping gene that is expressed at low levels.  PCR reactions 
were run on an Applied Biosystems Prism 7000 Sequence Detection 
System.  PCR values obtained with crossing threshold (CT) values if fewer 
than 30 cycles were considered valid for analysis [Giulietti 2001; Feldman 
2002; Gjerdrum 2004; Goldworthy 1999; Liss 2002; Al-Taher 2000; Liviak 
2001; DeCremoux 2003]. 
The primary purpose of qRT-PCR assessments on estracted RNA is to a) 
confirm presence of the targets for letrozole (aromatase) and estradiol 
(ER); b) provide indirect evidence of reduction in breast tissue estradiol 
through assessment of the estrogen response genes pS2 (trefoil factor-1) 
and GREB-1 [Rae 2005]. 


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10.5 
Blood Assessments 
 
 
 
10.5.1 Complete Blood Count and Metabolic Chemistry Profile will be 
performed at the participating sites at on-study, intermediate, and 
off-study visits.  Results will be used to confirm absence of serious 
liver or renal dysfunction or blood dyscrasia.  
 
 
 
Blood for all biomarker assays will be collected at the participating 
institutions in gel clot tubes, with serum separated according to 
standard procedures, 1-2 ml of serum aliquoted into six plastic 
tubes.  These tubes will be labeled with the date and a study 
subject specific number, and then frozen and stored at -80°C until 
shipment on dry ice to KUMC.  Assays on baseline, 6-month, and 
12-month samples will be performed together in the same run to 
avoid batch variation. 
 
10.5.2 Bioavailable Estradiol and Bioavailable Testosterone 
 
We have extensive experience with use of serum hormones as 
biomarkers in clinical chemoprevention trials [Fabian 2002, 2004].  
Serum levels of estradiol will be measured by sensitive 
radioimmunoassay at Ligand Assay and Core Analysis Laboratory at 
the University of Virginia following diethyl ether extraction at KUMC.  
The lower limit of detection at the Ligand Lab is 1.5 pg/ml and the 
intra-assay variation is 4.3%.  Extraction prior to RIA avoids 
contamination by estrone sulfate and is often necessary to obtain 
accurate values in postmenopausal women on HRT [Dowsett 
2005B]. Testosterone after diethyl ether extraction is also measured 
by a sensitive RIA with a lower limit of detection of 5 ng/dl and the 
intra-assay variation is 5%.  SHBG will be assayed by an 
immunometric assay using the Immulite analyzer (Diagnostic 
Products Corp), with a lower limit of detection 0.2 nmol/L and intra-
assay variation of 2.4%.   
 
Bioavailable estradiol or testosterone is calculated from the fraction 
that is unbound to SHBG [Stumpf 1981].  Tracer amounts of 
tritiated estradiol or testosterone are added to aliquots of serum, 
ammonium sulfate is added to precipitate the SHBG with bound 
steroid, the sample is centrifuged, and then activity in the 
supernatant and the precipitate are measured in parallel by liquid 
scintillation counting. The percent activity in the supernatant is 
calculated to yield the fraction of bioavailable (non-SHBG bound) 
hormone. Sensitive chemiluminescent assays are being developed 
by the core analysis lab at the University of Virginia and may be 
used instead of RIA for analysis of estradiol and tesosterone 
[Dowsett 2005B]. 
 
We do not anticipate any significant difference in change in estradiol 
or testosterone between the letrozole and placebo groups. 
 
 
 


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 10.5.3 Cardiovascular and Bone Fracture Risk Biomarkers 
 
 
The combination of high sensitivity C-reactive protein and total 
cholesterol:HDL ratios provide excellent risk biomarkers for 
cardiovascular disease [Ridker 2005]. Lipid panel with total 
cholesterol:HDL ratio is performed on serum by an enzymatic assay 
after a 12 hour fast.  High sensitivity C-reactive protein, an indicator 
of inflammation, will be performed by an immunochemiluminometric 
assay. 
 
N-telopeptide crosslinks correlate with the rate of bone reabsorption 
and bone mineral density in osteoperotic women and can be used to 
monitor the efficacy of anti-reabsorptive therapy with estrogen and 
calcium [Greenspan 2000].  The test is performed on serum by EIA.  
Mean values for women are 12.6 nM BCE/L (6.2 – 19.0 nM BCE/L).   
 
Osteocalcin is a measure of bone/reabsorption.  It together with the 
N-telopeptide is helpful in determining reduction in bone turnover in 
response to anti-reabsorptive agents or predicting early bone 
mineral loss after hormone therapy is stopped.  The test is 
performed on serum by an immunochemiluminometric (ICMA) assay 
[Slovik 1984]. It is best performed fasting as it cannot be 
performed on a lipemic specimen. 
 
The above assays will be performed at LabCorp Inc. Research 
Division or a similar CLIA-approved laboratory.   
 
Estrogen supplementation increases high density lipoproteins and 
thus it is possible that administration of letrozole will result in a 
higher total cholesterol:HDL ratio.  We do not expect any difference 
in change in C-reactive protein, osteocalcin, or N-telopeptide 
between letrozole and placebo groups. 
 
10.5.4 Serum 25-OH Vitamin D Levels  
 
The cause of the increase and/or induction of joint stiffness and 
arthralgias in women taking aromatase inhibitors is not clear but 
may be due to estrogen deprivation in combination with suboptimal 
vitamin D levels.  Blood levels of 25-OH vitamin D are a good 
measure of vitamin D adequacy and for optimum bone and general 
health should be in the 32-50 ng/ml range [Vieth 1999; Holick 
2003; Heaney 2004].  Suboptimal or deficient levels of 25-OH 
vitamin D are common in postmenopausal women, ranging from 25 
to 50% of those tested when 37 ng/ml is used as the lower cutoff 
[LeBoff 1999; Aguado 2000; Glowacki 2003].  Persistent pain, 
particularly in shoulders, wrists, hips, knees, legs, and ankles, as 
well as joint stiffness has been reported in 93% of individuals with 
25-OH vitamin D levels <20 ng/ml [Plotnikoff 2003].  Taylor et al. 
has recently reported in abstract form that a high proportion of 
postmenopausal women on AIs with musculoskeletal complaints had 
vitamin D deficiency [Taylor 2004].  We will assess the effect of 
baseline and follow-up 25-OH vitamin D levels on subsequent 
development of arthralgias.   


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All samples will be run in in KUMC’s clinical laboratory by a widely 
used RIA kit [Gunter 1988-1994]. The assay limit of detection is 1.5 
ng/ml.  Assay precision varies only slightly with vitamin D level from 
8.6 to 12.5% for run-to-run variation and 8.2 to 11% for between-
run variation.  
 
We do not expect any increase or decrease in 25-OH vitamin D 
levels other than that expected from seasonal variations.  However, 
low 25-OH vitamin D levels may explain development of arthralgias 
or an increase in bone turnover if encountered in women 
randomized to letrozole. 
 
 
10.6 
Breast Density Measurements 
 
Mammograms will be performed within three months of the baseline 
aspiration and at six and twelve months. An attempt will be made to 
use the same machine at baseline and at 6 and 12 months. However, it 
is imperative that a woman has breast imaging performed on either 
analogue or digital for all three imaging procedures. Either copies of the 
mammograms or direct digital files will be sent to KUMC from 
collaborating sites for assessment of breast density. For assessment of 
mammographic breast density from analog films, the right and left 
cranial caudal views will be digitized using a Lumiscan 85 film digitizer.  
 
These films will be used for a blinded assessment of breast density 
parameters using the Cumulus software program [Boyd 1998; Boyd 
1997]. Both Dr. Fabian and Dr. Kimler have been trained by Dr. Boyd in 
Toronto in the use of this method. Specifically, Dr. Fabian achieved 
>85% agreement with Dr. Boyd on training.  
 
Prior to the analaysis, images will be checked to make sure cropping 
(total breast volume) is similar. For the analysis, dates will be removed 
so the reader will not know which is the baseline and which is the 6 or 
12 month film, but the three will be analyzed in the same batch. Stone 
et al have recently reported that the method of analysis with the least 
intraobserver variance is for the reviewer to view the initial and the 
follow-up mammograms together, but with the reviewer blinded as to 
which is the baseline and which is the follow-up study [Stone 2003] 
Both the absolute area of increased density and proportional density 
(area of increased density divided by total area of breast) will be 
assessed. 
 
 
10.7 
Quality of Life Evaluations 
 
The most frequent adverse events in our pilot trial were arthralgias, 
fatigue and hot flashes.  Consequently, in addition to utilizing the 
Breast Cancer Prevention Trial (BCPT) symptom scale (used as part of 
NSABP prevention trials) [Ganz 1995; Day 1999], we will use specific 
tools geared to the most prevalent symptoms in the pilot study.   
 
The BCPT symptom checklist is a 42 item list of problems common to 
peri and postmenopausal women and those undergoing anti-hormonal 


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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 [Stanton 2005].  
  
The Mayo Clinic Hot Flash scoring system was developed by Loprinzi et 
al.  Subjects are asked about their daily number of hot flashes and rate 
usual severity from 1 to 4.  The frequency is multiplied by usual 
severity to obtain a total score [Loprinzi 2002].  
 
We do not expect an increase in symptoms or hot flash scores for 
women randomized to letrozole. 
 
The Fibromyalgia Impact Questionnaire (HAQ) is a validated tool 
routinely used by rheumatologists to evaluate pain, stiffness, and 
physical functioning.  Individual domains or a total score can be utilized 
in assessment [Wolfe 2004].   
 
The brief fatigue inventory asks the woman to rate her current as well 
as worst and usual fatigue in the past 24 hours.  It also asks her to rate 
how the fatigue has affected mood, general activity, walking, work 
relations, and enjoyment of life [Mendoza 1999].   
 
We do not expect an increase in joint pain, stiffness or fatigue for 
women randomized to letrozole, unless a woman has suboptimal levels 
of 25-OH vitamin D. 


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11.0 
DATA MANAGEMENT 
 
Participant data will be collected using protocol-specific case report forms (CRF) 
developed by KUMC. A copy of all documents up to the month six visit will be 
mailed to KUMC as soon as they are completed, and the remainder as soon as the 
subject completes study.  
 
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 be 
entered into the the web-based CRIS study database by KUMC. 
 
Source document files/chart must be kept available at sites for monitoring visits 
during the conduct of the study through data analysis.  
 
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 analysis perform at KUMC will be entered 
on source documentation, entered into a database and reviewed by accuracy by 
KUMC study staff.  
 
Co-existent disease and adverse events will be coded using the NCI CTCAE, 
version 3.0. 
 
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 by made by 
joint written agreement between the Protocol Chair, the Trial Statistician and the 
Data Manager. 
 
11.1 
Source Documents 
 
Source documents will consist of reports (mammogram reports, laboratory 
reports, etc.), and forms created specifically for this study that will capture 
information such as physical exam, phone contact information, adverse 
events, concomitant medications, demographics, etc. 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. Special laboratory CRF’s will be created to capture 
biomarker laboratory data including cytomorphology, nuclear morphometry 
mammographic density, Ki-67, serum hormone levels, osteocalcin, C-
reactive protein, fasting lipid profile and 25-OH vitamin D levels.  Data will 
be entered in an Excel spread sheet which can be merged with the 
database held at KUMC. 
 
11.2    Data and Safety Monitoring Plan 
 
Prior to activation of the clinical trial, a Data Safety and Monitoring 
Committee (DSMC) will be established. The DSMC will consist of four 
members that will be identified later: a clinical faculty member at KUMC, a 
member of the Clinical Trials Division of the Kansas Cancer Institute, and 


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two external members with experience in clinical prevention trials. One 
member will be selected by the Committee to serve as Chairman for 
administrative purposes. 
 
The DSMC monitors data management, subject safety, and subject accrual.  
The DSMC also provides advice on the overall conduct of the study. 
Meetings will be held by phone 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 database.  
The sources of data are three-fold: (1) All clinical data that originate at 
KUMC or collaborating sites are entered onto specific Case Report Forms 
(CRFs).  The CRFs will be audited and source documents reviewed by a 
central multi-site monitor, after which the CRFs will be sent to KUMC.  The 
data are entered into the central database and audited/validated.  
Periodically, these data are output to individual subject summary sheets so 
as to allow periodic assessment by the Protocol Chair (Dr. Carol Fabian) 
and staff.  (2) Biomarker data from the assessments at KUMC of fine 
needle aspiration specimens (cytologic characterization, cytology score, 
and Ki-67 and ER immunocytochemistry score) and mammographic breast 
density are entered onto trial specific CRFs.  Data are then entered into a 
trial specific 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 for the DSMC by the biostatistician 
for this project, Dr. Matt Mayo.  
 
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 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 listed above, the DSMC may 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 DSMC.  Likewise, the committee may make suggestions 
on other aspects that would improve the quality of the trial. 
 
11.3  Sponsor or FDA Monitoring 
 
The NCI, DCP (or their designee), pharmaceutical collaborator (or their 
designee), or 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.  


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11.4 
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 for all studies performed under an IND will be maintained, at a 
minimum, for two (2) years after the approval of a New Drug Application 
(NDA). The records should be accessible for inspection and copying by 
authorized persons of the Food and Drug Administration.  If the study is 
done outside of the United States, applicable regulatory requirements for 
the specific country participating in the study also apply. 


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12.0 
STATISTICAL METHODS 
 
The study is a Phase II, double-blind, randomized, placebo-controlled clinical trial 
to assess the potential efficacy of letrozole as a chemopreventive agent, as 
measured by changes in proliferation index (Ki-67) between baseline and 6-
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 with type of HRT (E vs E+P) 
as the only stratification factor other than institutional site so that each site can be 
provided with a unique randomization schedule at initiation.  All eligible subjects 
will be randomly assigned to letrozole or placebo in a 1:1 ratio. 
 
12.1 
Sample Size and Power 
 
The primary outcome of interest is efficacy of letrozole 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 6.4%, and the average change in percent of cells expressing 
Ki-67 was a 3.7% reduction, for subjects meeting the same eligibility 
requirements as will be used in this proposed trial.  
 
A 1:1 randomization of 96 evaluable subjects will provide 90% power to 
detect an effect size of 0.67 standard deviations with a Type I error rate of 
5% using a two-sample, two-sided t-test. In other words, if one assumes a 
common standard deviation of change of 5.5% (as observed in the pilot 
study for the 33 subjects matching eligibility criteria for the proposed trial) 
for both placebo and letrozole groups and if mean change for placebo is 
zero, then there will be 90% power to detect an average change of 3.7% in 
Ki-67 in the letrozole group with a Type 1 error rate of 5%.  Note that the 
3.7% reduction is what was seen in the pilot study.  Using a conservative 
drop-out rate of 10% (it was 2% in the pilot study), enrollment of 108 
subjects should provide the required 96 evaluable subjects for the primary 
endpoint. 
 
12.2 
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 3 months of study medication.  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 6 months.  The two-sample t-test will be used to 
compare the change in Ki-67 between the letrozole and placebo groups.  
Any difference in baseline values of Ki-67 between the two groups would 


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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, mammographic breast density, and clinical 
laboratory tests.  We will also correlate change in the primary biomarker 
Ki-67 with change in other markers (e.g., bioavailable estradiol, pS2, 
GREB-1) 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 letrozole vs. the placebo arm will be examined 
by Fisher’s exact test.  
 
Evaluation of drug-induced hormonal changes - We will examine whether 
Letrozole has any effects on serum levels of bioavailable estradiol, and 
testosterone, determined at baseline and after six months of study 
medication. We will determine whether addition of letrozole alters the 
frequency of menopausal symptoms. 
 
Adverse Events and Quality of Life – Change from baseline to six months 
for hot flash score (Mayo Clinic), fatigue (brief fatigue inventory), overall 
quality of life (BCPCT), bone turnover markers, HDL/total cholesterol, and 
C-reactive protein will be compared between the two groups using the two-
sample t-test. Similarly, demographic variables (e.g., age, education) and 
hormonal levels will be tested for use as covariates on each of these 
endpoints.  We will also assess the effects of change in serum hormones (if 
any) and baseline 25-OH vitamin D levels on incidence and severity of 
arthralgias and fatigue. 
 
Within Letrozole Comparisons at baseline, 6 and 12 months - On all of our 
quantitative continuous measures we will perform a repeated measures 
analysis comparing the 6 and 12 month values to baseline and also with 
each other.  This will give us some preliminary indication whether there is 
any benefit or detriment of 12 months of treatment.  For all the above 
analyses of exploratory endpoints, as well as for changes in proliferation, 
results from the 12-month timepoint (following six months of open-label 
treatment with active agent) will also be analyzed and compared to the 
baseline and six month values with caution since the double-blind, 
randomized nature of the comparison is no longer present. However, since 


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every effort is made to conduct the assessment and quantification of 
biomarkers without knowledge of treatment arm, the results could be 
informative. 
 
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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13.0 
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, other sites 
will submit revised protocol changes to local IRB for review. 
 
The determination of the necessity to cease enrollment until revisions are 
approved will be determined by the Protocol Chair. 
 
Novartis and NCI, DCP as the funder of the study, have the right to request 
discontinuation of the study at any time. A frequency of greater than grade 2 
adverse events (exclusive of hot flashes and menstrual irregularities) that exceeds 
20% will be cause for consideration of stopping the trial; unless there is adequate 
information to suggest that the adverse events are not related to the study agent. 


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