Clinical trial NCT02010021
Conditions: Breast Cancer
Keywords: breast cancer postmenopausal letrozole RAD001 Everolimus
Sponsor: Dartmouth-Hitchcock Medical Center
Start date: 2014-01-01; Completion date: 2017-12-08
Enrollment: 17 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
Some tumors use estrogen in the body to assist with growth. Letrozole is a drug that is prevents cells from producing estrogens. This should assist with the slowing of growth of tumor cells. Letrozole also promotes cell destruction by inhibiting a cellular destruction pathway.

The objectives of this study will look at the differences between the cellular destruction pathway before and after letrozole use, and the differences in the cellular destruction pathway in participants that have received letrozole versus those who did not. The study will also look at a gene in all participants called Ki67. This gene is associated with the rate of tumor cell growth. The study will measure the levels of Ki67 and compare them to the amount of activation of the cellular destruction pathway.

Participants in this study will have undergone a diagnostic biopsy of their breast tissue.

In order to meet these objectives, one group of participants (Arm A) will not receive letrozole. Tissue leftover from their diagnostic biopsy will be treated with everolimus (RAD001) in the laboratory and the effects of this drug on the cellular destruction pathway will be studied.

The other group of participants (Arm B) will take letrozole for a minimum of 10 and maximum of 21 days. They will have a second tumor sample taken as part of their surgical procedure completed to remove the tumor tissue. Any differences in the cellular destruction pathway before and after exposure to letrozole will be measured.

Detailed description:

Arms: arm 1: Post-menopausal women with stage I-III breast cancer will have surgical resection of tumor and tumor tissue will be used to study cell growth signaling pathways ex-vivo. arm 2: Patients will receive Letrozole for 10-21 days prior to surgical resection of tumor tissue. This tissue will be used ex-vivo to study cell growth signaling pathway. The results will be compared to arm of the study with no intervention.
Interventions: intervention 1: Letrozole
Intervention descriptions: intervention 1: Patients will receive Letrozole for 10-21 days prior to surgical resection of tumor tissue. This tissue will be used ex-vivo to study cell growth signaling pathway. The results will be compared to arm of the study with no intervention.

Protocol documents:
Prot_SAP_000.pdf: 
Presurgical letrozole 
 
1 
 
 
 
Presurgical treatment with letrozole in patients with early-stage breast cancer 
 
 
 
 
Protocol Number: 
D13236 
 
Principal Investigator: 
Gary Schwartz, MD 
 
Co-Investigators: 
Todd Miller, PhD 
 
 
Kari Rosenkranz, MD 
 
 
Richard Barth, MD 
 
 
Christina Angeles, MD 
 
 
 
 
 
 
 
 
 
 
Date: 
 
November 17, 2015 
 


Presurgical letrozole 
 
2 
 
TABLE OF CONTENTS 
 
 
 
 
SCHEMA 
3  
 
 
ABBREVIATIONS 
4 
 
1.0 
INTRODUCTION 
5 
 
2.0 
OBJECTIVES 
9 
 
3.0 
ELIGIBILITY CRITERIA 
9 
 
4.0 
REGISTRATION 
10 
 
5.0 
TREATMENT PLAN 
11 
 
6.0 
DATA COLLECTION, HANDLING, AND RECORD KEEPING 
17 
 
7.0 
STUDY MONITORING, AUDITING, AND INSPECTING 
18 
 
8.0 
ADVERSE EVENT REPORTING 
19 
 
9.0                   STATISTICAL CONSIDERATIONS 
19 
 
 
10.0                 HUMAN SUBJECTS 
21 
 
11.0 
BIBLIOGRAPHY 
22 
 
 
 


 
Presurgica
 
l letrozole 
 
3 


Presurgical letrozole 
 
4 
 
 
ABBREVIATIONS 
 
CPHS 
Committee for the Protection of Human Subjects at Dartmouth College 
DMBA 
7,12-Dimethylbenz(a)anthracene 
DSMAC 
Data Safety Monitoring and Accrual Committee of the Norris Cotton Cancer Center 
ER 
estrogen receptor alpha 
FDA 
Food & Drug Administration (U.S.) 
FFPE 
formalin-fixed, paraffin-embedded 
HER2 
HER2 / ERBB2 proto-oncogene 
IGF-1R 
insulin-like growth factor receptor 
IHC 
immunohistochemistry  
InsR 
insulin receptor 
IRS1 
insulin receptor substrate 1 
IRS2 
insulin receptor substrate 2 
mRNA 
messenger RNA 
mTOR 
mammalian target of rapamycin 
mTORC1 
mTOR complex 1 
mTORC2 
mTOR complex 2 
PHI 
protected health information 
PI 
primary investigator 
PI3K 
phosphatidylinositol 3-kinase 
PR 
progesterone receptor 
OPRR 
Dept. of Health and Human Services Office for Protection from Research Risks (U.S.)  
 
 
 
 


Presurgical letrozole 
 
5 
 
 
1.0 
INTRODUCTION 
 
1.1. 
Breast cancer 
 
Most patients are diagnosed with Stage I-III breast cancer, which is localized to 
the breast (and adjacent lymph nodes), and is surgically resectable and potentially 
curable.  Seventy-five percent of primary breast tumors express estrogen receptor 
alpha (ER). Patients with Stage I-III ER-positive breast cancer are typically 
treated with adjuvant anti-estrogen therapy for 5 years after surgical removal of 
the primary tumor. Among the cases of ER+ breast cancer, ~10% harbor genomic 
amplification of the ERBB2 (HER2) proto-oncogene; patients with HER2+ cancer 
are treated with HER2-directed therapy, and will not be part of this study. This 
study focuses on patients with Stage I-III ER+/HER2- breast cancer. 
 
1.2. 
The presurgical study model 
 
An initial diagnosis of breast cancer is typically made by histological 
confirmation of a tissue biopsy specimen. Patients with Stage I-III cancer will 
then typically undergo surgery within a few weeks to remove the primary 
tumor(s).  Therefore, it is common to have a paired tumor biopsy specimen and a 
surgical tumor specimen.  The availability of such paired specimens has prompted 
a large initiative in recent years to treat patients (e.g., with drug) during the 
intervening period between the time of diagnostic biopsy and the time of surgery. 
We can then compare the biopsy tissue (baseline, pre-treatment) to the surgical 
tissue (post-treatment) to assess biological changes (e.g., changes in mRNA and 
protein expression).  This study design has provided a wealth of information on 
the effects of systemic therapies on tumor biology [reviewed in ref. (1)], and it 
requires no additional biopsy procedure. 
 
1.3. 
Therapeutic inhibition of mTORC1 
 
mTORC1 is a molecular hub that integrates signaling inputs from an array of 
sources, including growth factor receptor signaling pathways, nutrient sensing 
pathways, and energy sensing pathways. Activated mTORC1 promotes cap-
dependent protein translation, and this pathway is hyperactivated in many cancer 
types. As such, mTORC1 is an attractive therapeutic target. The mTORC1 
inhibitor RAD001 (Afinitor, Everolimus) was recently approved for the treatment 
of patients with advanced/metastatic ER+/HER2- breast cancer in combination 
with the steroidal aromatase inhibitor Exemestane following progression on a 
non-steroidal aromatase inhibitor (Letrozole, Anastrozole). Everolimus is also 
approved for the treatment of other non-breast malignancies. While the phase 3 
BOLERO-2 
study 
demonstrated 
that 
Exemestane/Everolimus 
increased 
progression-free survival compared to Exemestane/Placebo (2), nearly all patients 
eventually progressed. Genetic analysis has not identified a clear, individual 
biomarker that predicted benefit from Everolimus (3). Furthermore, it is unclear 


Presurgica
 
 
 
 
l letrozole 
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6 
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Presurgica
 
 
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inhibition w
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uppress pla
(baseline, d
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ase inhibito
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Fig. 2). Pretr
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7 
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Presurgical letrozole 
 
8 
 
Fig. 2. Anti-estrogen-mediated inhibition of ER with fulvestrant abrogates PI3K/AKT activation induced 
by mTORC1 inhibition with RAD001. Left: MCF-7 cells were pre-treated with medium containing 10% 
FBS ± 1 M fulvestrant for 24 hours. Then cells were treated with medium ± 1 M fulvestrant ± 20 nM 
RAD001 for 24 hours before lysis. Lysates were analyzed by immunoblotting using the indicated 
antibodies. Right: ZR75-1 cells were pre-treated with medium containing 10% FBS ± 1 M fulvestrant for 
3 days. Then cells were treated with medium ± 1 M fulvestrant ± 20 nM RAD001 for 24 hours before 
lysis. Lysates were analyzed as in (A). Note that RAD001 treatment decreases P-S6 (marker of mTORC1 
activity) but increases P-AKT. Fulvestrant treatment decreases IRS-1 and IGF-1R levels, which are 
encoded by ER-inducible genes. 
 
1.5. 
Presurgical endocrine therapy 
 
Letrozole is appropriate neoadjuvant therapy for postmenopausal women with 
hormone receptor positive breast cancer.  Ellis et al randomized 250 post-
menopausal women with locally-advanced hormone receptor positive breast 
cancer requiring mastectomy, to four months of neoadjuvant endocrine therapy 
with either letrozole or tamoxifen.  Sixty percent of subjects receiving presurgical 
letrozole had an objective radiologic response and 48% were able to undergo 
breast-conserving surgery, while only 41% of subjects receiving tamoxifen had an 
objective response and 36% were able to undergo breast-conserving surgery (12).  
The Ki-67 proliferation biomarker can be assayed at diagnosis and again after a 
short run-in period of treatment with letrozole, and Ellis showed that suppression 
of Ki-67 with persistent expression of ER in a node negative patient with a 
primary tumor under 5 cm in size is predictive of such a low risk of recurrence 
that adjuvant chemotherapy is obviated (13).  377 post-menopausal women with 
clinical Stage II-III hormone receptor positive breast cancer were randomized to 
receive letrozole, anastrozole, or exemestane for 16 to 18 weeks prior to surgery.  
The clinical response rate to letrozole was 75% versus 69% for anastrozole and 
63% for exemestane.  51% of the patients who were judged to require a 
mastectomy at the time of study registration were able to achieve breast 
preservation (14).  Neoadjuvant endocrine therapy can therefore increase the 
likelihood of achieving breast preservation in a woman with a locally advanced 
hormone receptor positive breast cancer, and biopsies after a brief treatment 
interval have provided biomarkers which were predictive of prolonged disease-
free survival with endocrine therapy alone. Letrozole is an active agent in the 
neoadjuvant setting and can be considered an appropriate standard of care as 
neoadjuvant therapy for locally advanced breast cancer. 
 
1.6. 
Study rationale 
 
We propose a presurgical study in patients with Stage I-III ER+/HER2- breast 
cancer to determine whether anti-estrogen therapy (Letrozole) suppresses 
mTORC1 inhibitor (RAD001)-induced activation of PI3K/AKT signaling. As the 
primary objective is to determine whether anti-estrogen therapy alters mTORC1 
inhibitor-induced signaling that can be assessed in a short time frame ex vivo, we 
will treat patients with or without Letrozole for 10-21 days prior to surgery; this 
time frame will allow sufficient time for maximal estrogen suppression and 
downstream changes in the levels of ER-regulated transcripts and encoded 


Presurgical letrozole 
 
9 
 
proteins. Viable core samples of surgically resected tumors will be used for ex 
vivo culture to assess the effects of RAD001 on signaling, to avoid exposing 
subjects to potentially adverse side effects of RAD001. Findings from this study 
will provide mechanistic insight into the effects of mTORC1 and ER inhibition on 
oncogenic signaling pathways in cancer cells. 
 
 
2.0 
OBJECTIVES 
2.1 
Primary Objectives 
1. Arm A: To determine whether mTORC1 inhibition with RAD001 increases 
IRS-1/PI3K/AKT pathway activation in ER+/HER2- breast tumors ex vivo. 
2. Arm B (comparison with Arm A): To determine whether presurgical treatment 
of patients with ER+/HER2- breast cancer with Letrozole suppresses IRS-
1/PI3K/AKT pathway activation compared to tumors from untreated patients. 
 
2.2 
Secondary objectives 
1. To determine whether presurgical treatment with Letrozole suppresses IRS-
1/PI3K/AKT pathway activation compared to baseline. 
2. To determine whether the level of IRS-1/PI3K/AKT pathway activation is 
correlated with rate of tumor cell proliferation as determined by Ki67 score. 
3. To determine whether RAD001-induced changes in IRS-1/PI3K/AKT 
signaling ex vivo are correlated with rate of tumor cell proliferation as 
determined by Ki67 score. 
4. To identify changes in mRNA levels of ER-regulated gene induced by 
presurgical treatment with Letrozole. 
 
 
3.0 
ELIGIBILITY CRITERIA 
 
   Inclusion Criteria: 
 
3.1. 
Histologic documentation of invasive breast cancer by core needle or incisional 
biopsy. Excess baseline biopsy tumor tissue sufficient to make three 5-micron 
sections must be available for molecular analyses as part of this study. 
 
3.2. 
The invasive cancer must be estrogen receptor alpha (ER)-positive, with ER 
staining present in greater than 50% of invasive cancer cells by IHC 
 
3.3.  
The invasive cancer must be HER2-negative (IHC 0-1+, or with a FISH ratio of 
<1.8 if IHC is 2+ or if IHC has not been done). 
 
3.4. 
Clinical Stage I-III invasive breast cancer with the intent to treat with surgical 
resection of the primary tumor. Tumor must be ≥2 cm to provide adequate tissue. 
 


Presurgical letrozole 
 
10 
 
3.5 
Patients with multicentric or bilateral disease are eligible if the target lesions meet 
the other eligibility criteria. Samples from all available tumors are requested for 
research purposes. 
 
3.6. 
Women ≥ age 18, for whom adjuvant treatment with an aromatase inhibitor would 
be clinically indicated. Women must be either post-menopausal, or pre-
menopausal having undergone oophorectomy. 
 
3.7. 
Patients must meet the following clinical laboratory criteria: 
 
Absolute neutrophil count (ANC)  1,000/mm3 and platelet count  
75,000/mm3. 
 
Total bilirubin  1.5  the upper limit of the normal range (ULN). 
 
Alanine aminotransferase (ALT) and aspartate aminotransferase 
(AST)  3  ULN. 
 
3.8. 
Ability to give informed consent. 
 
 
   Exclusion Criteria: 
 
 
3.8. 
Prior endocrine therapy for any histologically-confirmed cancer is not allowed. 
Prior endocrine therapy that was administered ≥5 years ago for the prevention of 
breast cancer in patients with no history of breast cancer is allowed. 
 
3.9. 
Systemic drug treatment to induce ovarian suppression if woman is pre-
menopausal. 
 
3.10. Any other neoadjuvant therapy for breast cancer [i.e., treatment with any other 
anti-cancer agent besides Letrozole (10-21 days) before surgical resection of the 
primary breast tumor]. 
 
 
4.0 
REGISTRATION 
Patients will be registered by the Clinical Research Associate through the Norris Cotton 
Cancer Center Clinical Trials office following completion of pre-treatment evaluations 
and informed consent. At the time of registration, the following information will be 
recorded: 
 
- 
Patient's name and subject identification number. 
 
- 
Patient's date of birth. 
 
- 
Date of treatment start. 
Eligibility criteria will be verified at the time of registration. A case number will be 
assigned following registration. 
  
 
 


Presurgical letrozole 
 
11 
 
5.0 
TREATMENT PLAN 
 
5.1 
Screening Period 
Subjects will provide written informed consent, be evaluated for eligibility 
criteria, and undergo standard baseline clinical and tumor assessments and 
staging. Subjects will undergo our standard pre-treatment surgical evaluation. To 
determine our ability to provide breast conservation, patients at DHMC have 
imaging of the breast prior to neoadjuvant chemotherapy to include 
mammography, ultrasound, and breast MRI, to determine the suitability of breast 
conservation, and to guide the surgical oncologist on surgical planning. This 
period will be up to 4 weeks prior to the start of presurgical treatment with 
Letrozole for subjects enrolled onto Arm B (this time period does not apply to 
Arm A). 
 
5.2 
Treatment Period 
Patients will receive either A) no presurgical drug treatment, or B) presurgical 
treatment with Letrozole for 10-21 days prior to surgery. Arm A will enroll 
patients first until accrual is full (n=10). Following successful completion of 
molecular analyses to support the first primary objective (To determine whether 
mTORC1 inhibition with RAD001 increases IRS-1/PI3K/AKT pathway 
activation in ER+/HER2- breast tumors ex vivo), we will proceed to enroll 
patients in Arm B (presurgical Letrozole, as below in Section 5.2.2).  An 
estimated 10 subjects will be enrolled into Arm B to obtain the necessary 
evaluable number of subjects (n=10).  
 
5.2.1. Surgery and tumor specimens 
The patient will undergo definitive surgical resection of the primary 
tumor. 
Management of the axilla will be left to the judgment of the surgical 
oncologist and the patient. 
 
It is strongly preferred that surgery be scheduled between Monday and 
Thursday for optimal timing of tissue fixation and processing. 
 
The Pathologist will promptly dissect the surgical primary tumor 
specimen. ASCO-CAP guidelines require initiation of dissection of a 
surgical specimen within 1 hour of removal from the patient; for this 
study, minimal “cold ischemia time” is preferred.  
 
The Pathologist will assess whether the tumor specimen is large enough to 
procure: 
   A. tissue for routine diagnostics 
   B. ≥8 core punch biopsies that are 1 mm in diameter and ≥0.5 cm long 
for research 


Presurgical letrozole 
 
12 
 
   C. ≥4 core punch biopsies that are 3 mm in diameter and ≥0.25 cm long 
for research 
 
A 2-cm tumor is expected to provide sufficient material for all needs. 
 
When the tumor is sufficiently large to provide adequate tissue (e.g., >3 
cm), the Pathologist will first remove core punch biopsies for this research 
study using 1-mm and 3-mm punch biopsy devices.  All research samples 
must be acquired within 1.5 hours of tissue removal from the patient; 
a shorter lag time is strongly preferred to maximize tissue viability. 
The Pathologist will then acquire tissue samples for routine diagnostics. 
 
If the tumor specimen is approximately 2 cm, the Pathologist may opt to 
first procure tissue for routine diagnostics. Then if adequate tumor tissue 
remains to provide punch biopsies for research purposes, these samples 
will be taken from the tumor. If the tumor is too small to provide adequate 
tissue for research, the tumor will be considered non-evaluable and the 
patient will need to be replaced. 
 
Punch biopsy tumor specimens required for this research study are listed 
below in order of priority. 
 
A) Fresh tissue (n=8) - Using a 1-mm punch biopsy device with plunger 
(provided by Dr. Miller), remove 8 tumor samples ≥0.5 cm long. These 
samples will be immediately placed into cold (0-4°C) tissue culture 
medium (serum-free DMEM + Pen/Strep; provided by Dr. Miller to be 
kept on-hand in Pathology lab) and kept on ice or refrigerated (0-4°C). 
These tissues will be transported within 30 minutes of dissection to Dr. 
Miller’s lab on the 6th floor of Rubin Research Building. Jennifer Bean, 
Wei Yang, and Todd Miller (members of the Miller lab) will be 
responsible for transporting tissues, and need to be contacted early on the 
date of surgery with notification that samples will be available the same 
day. They will be paged when tumor tissues are available for pick-up. 
 
B) Frozen tissue (n=2) - Using a 3-mm punch biopsy device with plunger 
(provided by Dr. Miller), remove 2 tumor samples ≥ 0.25 cm long, put 
into cryotubes (1 core per tube), and snap-freeze in liquid nitrogen. Liquid 
nitrogen can be provided in a small container by the Miller lab, if they are 
notified early on the day of surgery. 
 
C) Formalin-fixed (FFPE) tissue (n=2) - Using the same 3-mm punch 
biopsy device, remove 2 tumor samples ≥ 0.25 cm long, fix in formalin (3 
hours to overnight), and paraffin-embed the next day as per standard tissue 
processing procedures in Pathology lab. 
Alternatively, if Pathology will be processing diagnostic FFPE tumor 
samples that will provide sufficient excess tissue for molecular analyses, 


Presurgical letrozole 
 
13 
 
and tissues are fixed for less than 24 hours, then such samples may be 
used in lieu of punch biopsies. 
 
D) Extra tumor tissue- If tissue requirements for categories A, B, and C 
are filled and excess tumor tissue is available, more 1-mm cores of fresh 
viable tissue for tissue culture are requested (up to 20 more). Such extra 
cores should be placed into tissue culture medium as in section A (above). 
 
Histological qualification of tumor samples- The Pathologist will perform 
routine histological analysis of the diagnostic FFPE surgical tumor 
specimen. If the diagnostic  tumor sample is found to have <50% tumor 
cellularity, the tumor samples procured for research will be considered 
non-evaluable, and this patient will need to be replaced. 
 
5.2.2. Presurgical Letrozole 
Patients enrolled on Arm B will be treated with Letrozole (2.5 mg/day, 
administered orally) for 10-21 days prior to surgery. This time period has 
been and continues to be used as a standard time frame in presurgical 
studies with aromatase inhibitors in patients with ER+ breast cancer. This 
drug and dose are given as standard adjuvant therapy for up to 5 years 
following surgical removal of a primary ER+/HER2- breast tumor in 
patients with Stage I-III disease.  The final dose of Letrozole should be 
taken on the morning of surgery. 
 
It is strongly preferred that surgery be scheduled between Monday and 
Thursday for optimal timing of tissue fixation and processing. 
 
The primary tumor will be surgically resected as in Section 5.2.1. The 
Pathologist will promptly dissect the surgical tumor specimen, and obtain 
punch biopsies for this study as in Section 5.2.1. 
 
5.2.3. Letrozole drug information 
Letrozole (4,4'-(1H-1,2,4-Triazol-1-ylmethylene)bis-benzonitrile) is a syn-
thetic achiral benzydryltrizole derivative. It is an orally active highly 
selective, non-steroidal competitive inhibitor of the aromatase enzyme 
system (15, 16). Aromatase inhibitors block the aromatase enzyme, 
consequently lowering estrogen levels and thereby deprive the tumor of its 
growth stimulus. Letrozole effectively inhibits the conversion of 
androgens to estrogens in both in vitro and in vivo (15).  This property 
makes it in particular suitable for postmenopausal women whose main 
source of estrogen is via peripheral aromatization of androgen precursors. 
 
Preclinical and pharmacodynamic data in humans 
In vivo, in female rats, the aromatase inhibition manifests itself in a 
reduction in uterine weight, an increase in body weight, a decrease in 
serum estradiol concentration, an increase in plasma LH concentration 


Presurgical letrozole 
 
14 
 
(16, 17) and suppression of the growth of DMBA-induced rat mammary 
tumors (15, 18). The ED50 values for some of these effects lie in the range 
of 0.001 to 0.03 mg/kg with maximal endocrine and anti-tumor efficacy in 
the dose range of 0.3 to 1.0 mg/kg po (18). 
 
Comparison to Aminoglutethimide (AG) 
Letrozole is up to 150-250 times more potent than the first generation 
aromatase inhibitor AG, in vitro and more than 10,000 times as potent as 
AG in inhibiting aromatase in vivo (15). The high potency of letrozole is 
not accompanied by any significant effect on adrenal steroidogenesis in 
vitro or in vivo over its maximally effective dose range (16, 17). Inhibition 
of adrenal steroidogenesis resulting in adrenal hypertrophy does occur 
with therapeutic doses of AG. The high potency and selectivity of 
letrozole explains its pharmacological profile and high therapeutic index. 
 
Activity in breast cancer patients 
In postmenopausal patients with advanced breast cancer, daily doses of 0.1 
to 5 mg letrozole suppressed plasma levels of estradiol, estrone and 
estrone sulfate to 75-95% from baseline (15, 19).  Estrogen suppression 
was maintained throughout the treatment period of 28 days in all patients. 
 
Selectivity of letrozole 
Letrozole is a highly selective inhibitor of the aromatase enzyme. No 
clinically relevant changes in the plasma levels of cortisol, aldosterone, 
11-deoxycortisol, 17-hydroxyprogesterone, ACTH or plasma renin 
activity were found in postmenopausal patients treated with a daily dose of 
letrozole ranging from 0.1 to 5 mg (15, 20, 21). Synacthen7 tests 
performed after 6 weeks of treatment with daily doses of 0.1, 0.25, 0.5, 1, 
2.5 and 5 mg letrozole indicated no attenuation of aldosterone or cortisol 
production (22). 
 
Toxicities of letrozole 
Most adverse reactions to letrozole in the literature are mild to moderate in 
severity and are indistinguishable from the consequences of the patient’s 
metastatic breast cancer, the effects of estrogen deprivation, or other 
concurrent illness. 
 
Reported events in greater than 5% of subjects include chest pain, 
hypertension, edema, hyperlipidemia, cough, dyspnea, nausea, vomiting, 
abdominal pain, anorexia, increased appetite, constipation, diarrhea, 
weight gain, weight loss, bone fractures, arthralgias, arthritis, back pain, 
bone pain, breast pain, myalgias, weakness, asthenia, dizziness, 
depression, fatigue, headache, insomnia, lethargy, malaise, rash, hot 
flashes, sweats, vaginal bleeding, vaginal dryness, and infections. 
 


Presurgical letrozole 
 
15 
 
Letrozole will be prescribed by the subject’s oncology provider and the 
subject will be asked to fill the prescription at her commercial pharmacy. 
 
5.3 
Study exit 
Following surgery, the patient’s role in the study will end. The patient will 
then be treated as per standard-of-care by her treating oncologist. Subjects 
who begin letrozole in the pre-surgical setting may receive chemotherapy 
followed by resumption of letrozole or letrozole alone following the 
definitive surgical resection.  
 
5.4. 
Evaluable patients 
Subjects who proceed to a definitive surgical procedure with A) tumor tissue 
sufficient to provide punch biopsy specimens for research purposes, and B) 
histologically-confirmed tumor tissue in the surgical specimen with ≥50% tumor 
cellularity, will be considered evaluable. Subjects whose surgical specimens do 
not contain adequate tumor tissue to assess the primary endpoint will not be 
considered evaluable, and will need to be replaced. 
 
5.5.  
Analyses 
  5.5.1. Ex vivo analysis of RAD001 effects on IRS-1/PI3K/AKT signaling. 
Fresh viable tumor cores in tissue culture medium (serum-free DMEM + 
Pen/Strep) will be placed into medium containing 20 nM RAD001 (4 
cores) or DMSO control (4 cores) and cultured at 37°C for 1 hour. If extra 
tissue cores are available, we will treat +/- 20 nM RAD001 for 2-8 hours 
to evaluate delayed changes in signaling, and treat +/- 1 uM OSI-906 
(IGF-1R/InsR inhibitor) for 1-8 hours to determine whether PI3K/AKT 
signaling is IGF-1R/InsR-dependent in these tumors. Tissue cores will 
then be snap-frozen in liquid nitrogen and stored at -80°C. 
 
Immunoblotting- Two cores each of DMSO- and RAD001-treated tumor 
will be homogenized and lysed on ice in RIPA buffer containing protease 
and phosphatase inhibitors. Samples will be sonicated and centrifuged, 
and supernatant will be used to quantify protein concentration. Protein 
lysates for immunoblotting will then be mixed with reducing sample 
buffer and stored at -80°C.  Lysates will be heated at 95°C for 1 min. just 
before SDS-PAGE following by immunoblot analysis using antibodies 
against: 
 
IRS-1 
phospho-IRS-1-Ser636/639 (marker of p70S6K activity) 
IRS-2 
AKT 
phospho-AKT-S473 (marker of mTORC2 activity) 
phospho-AKT-T308 (marker of PIP3 levels and PI3K/PDK1 activity) 
4E-BP1 
phospho-4E-BP1-Thr37/46 (marker of mTORC1 activity) 


Presurgical letrozole 
 
16 
 
p70S6K 
phospho-p70S6K-Thr389 (marker of mTORC1 activity) 
S6 
phospho-S6-Ser240/244 (marker of p70S6K activity) 
phospho-S6-Ser235/236 (marker of p70S6K and RSK activity) 
Erk1/2 
phospho-Erk1/2-Thr202/Tyr204 (marker of MEK activity) 
IGF-1Rb 
InsRb 
 
If excess protein lysate is available, we will immunoprecipitate the p85 
subunit of PI3K, and immunoblot for IRS-1 and IRS-2 to assess drug 
induced changes in IRS-1/2-induced PI3K activation.  We will also 
immunoprecipitate using phospho-Tyrosine antibody, and immunoblot for 
IGF-1Rb, InsRb, IRS-1, and IRS-2 to assess protein activation. 
 
The primary goal of this analysis is to determine whether RAD001 
treatment ex vivo globally decreases mTORC1 signaling (indicated by a 
decrease in levels of phospho-p70S6K, phospho-S6, and phospho-4E-
BP1), increases IRS-1/2 levels and phosphorylation, and increases AKT 
phosphorylation. Secondary goals are to assess changes in IGF-1R and 
InsR activation, and compensatory signaling through the MEK/Erk 
pathway. 
 
Immunohistochemistry (IHC) on frozen tissues- Two cores each of 
DMSO- and RAD001-treated tumor will be sectioned and processed for 
IHC in the Pathology Translational Research Shared Resource. Tissue 
sections will be immunostained using antibodies against phospho-S6-
Ser240/244, phospho-AKT-S473, phospho-AKT-T308 and phospho-IGF-
1Rb/InsR- Tyr1131/Tyr1146. Samples will be scored using a Histoscore 
scale that incorporates staining intensity and frequency (12). 
 
The primary goal of this analysis is to determine whether RAD001 
treatment ex vivo decreases mTORC1 signaling (indicated by a decrease in 
levels of phospho-S6) and increases AKT phosphorylation uniformly 
throughout tumor cores, and whether drug treatment alters IGF-1R/InsR 
phosphorylation (which cannot be assessed by immunoblot analysis of 
protein lysates). Staining intensities in the center of each core will be 
compared to the outer edges to assess uniformity of signal; non-uniformity 
may be due to time of exposure of cells to drug or medium. 
 
 
5.5.2.  Immunohistochemical analysis of pre- vs. post-Letrozole tumors. 
Baseline (diagnostic core biopsy specimen) and post-Letrozole (surgical 
specimen) tumor samples will be sectioned and processed for IHC in the 
Pathology Translational Research Shared Resource. Tissue sections will 
be immunostained using antibodies against ER, PR, and Ki67. ER and PR 


Presurgical letrozole 
 
17 
 
will be scored using a Histoscore scale (23); we expect Letrozole 
treatment to decrease PR levels, while ER may be unaffected. Ki67 will be 
scored by counting the number of positively-stained cells in 10 high-
power (400x magnification) microscopic fields (24, 25). Pre- and post-
Letrozole Ki67+ cell counts will be compared by t-test; Letrozole 
treatment should induce an overall decrease in Ki67 score. 
 
The primary goal of this analysis is to determine whether changes in 
signaling induced by RAD001 treatment ex vivo are associated with Ki67 
score. A higher post-anti-estrogen Ki67 score, and a smaller decrease in 
Ki67 score in response to presurgical treatment with an anti-estrogen, are 
associated with shorter recurrence-free survival (26). Hence, it is thought 
that a higher level of residual tumor cell proliferation (higher Ki67 score) 
following anti-estrogen treatment, or a weaker biological response (change 
in Ki67 score) to anti-estrogen treatment, are indicative of less dependence 
of tumor cells on ER for growth and survival. Our preclinical data indicate 
that ER drives IRS-1 and IGF-1R expression, which facilitate RAD001-
induced PI3K/AKT activation. Therefore, in tumors that are less 
dependent upon ER, Letrozole treatment is expected to have less of an 
effect on IRS-1/PI3K/AKT signaling. Such findings would support the use 
of combinations of anti-estrogens and mTORC1 inhibitors in patients with 
ER-driven tumors. 
 
6.0 
DATA COLLECTION, HANDLING AND RECORD KEEPING 
6.1. 
Confidentiality 
Information about study subjects will be kept confidential and managed according 
to the requirements of the Health Insurance Portability and Accountability Act of 
1996 (HIPAA). Participants will sign an authorization that includes the following:  
• 
What protected health information (PHI) will be collected from subjects in 
this study 
• 
Who will have access to that information and why 
• 
Who will use or disclose that information 
• 
The rights of a research subject to revoke their authorization for use of 
their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the 
investigator, by regulation, retains the ability to use all information collected prior 
to the revocation of subject authorization. 
 
Loss of patient confidentiality is a risk of participation.  Study participant 
identities will be kept confidential except as required by law.  Subjects’ samples 
will be identified by code only (i.e., linked, but de-identified).  Patient samples 
will be de-identified at the time and site of collection.  Participant and study 
information will be kept in the Velos eResearch password-protected database or 
its equivalent.  Additionally, documents containing participant identifiers, such as 
those from the medical record to confirm eligibility, will be filed in binders and 


Presurgical letrozole 
 
18 
 
kept in a locked, secure location in the Office of Clinical Research at the Norris 
Cotton Cancer Center. 
 
6.2. 
Data retention 
Following closure of the study, the investigator will maintain all site study records 
in a safe and secure location.  The records are maintained to allow easy and timely 
retrieval, when needed (e.g., audit or inspection) and, whenever feasible, to allow 
any subsequent review of data in conjunction with assessment of the facility, 
supporting systems, and staff.  Upon completion of study analysis, research 
information is stored in Dartmouth College Records Management off-site storage 
located at 6218 Etna Road, Hanover, NH  03755.  Documents are shredded on site 
after 50 years of storage. 
 
Participant and study information will be kept in the Velos eResearch password 
protected database (or its equivalent) indefinitely. 
 
7.0 
STUDY MONITORING, AUDITING, AND INSPECTING 
 
7.1 
Safety and Data Monitoring 
 
This study will be monitored by the Data Safety Monitoring and Accrual 
Committee (DSMAC) of the Norris Cotton Cancer Center.  The Committee meets 
quarterly to review accrual rates and information of all studies that have accrued 
participants.  The DSMAC has the authority to suspend or terminate all research 
activities that fall within its jurisdiction.  In the event that a study is suspended or 
terminated by the DSMAC, that information will be forwarded to the CPHS 
office.   
 
7.2 
On-Site Monitoring 
 
Clinical research monitoring will be conducted by appropriately trained staff of 
Dartmouth-Hitchcock Medical Center Clinical Trials Office. This monitoring will 
include periodic assessment of the regulatory compliance, data quality, and study 
integrity. Study records will be reviewed and directly compared to source 
documents and the conduct of the study will be discussed with the investigator, 
Monitors may request access to all regulatory documents, source documents, 
CRFs, and other study documentation for on-site inspection. Direct access to 
these documents is guaranteed by the investigator, who must provide support at 
all times for these activities. 
 
7.3 
Auditing and Inspecting 
 
Participation as an investigator in this study implies acceptance of potential 
inspection by government regulatory authorities and applicable Dartmouth 
compliance and quality assurance offices. The investigator will permit study-


Presurgical letrozole 
 
19 
 
related audits and inspections by the Dartmouth CPHS, government regulatory 
bodies, and Dartmouth compliance and quality assurance groups of all study 
related documents (e.g. source documents, regulatory documents, data collection 
instruments, study data etc.) The investigator will ensure the capability for 
inspections of applicable study-related facilities (e.g. pharmacy, diagnostic 
laboratory, etc.) 
 
 
8.0 
ADVERSE EVENT REPORTING 
 
Patients on Arm B will be instructed to report the occurrence of any adverse event (this 
will not apply to participants on Arm A). An adverse event is any undesirable event 
associated with the administration of letrozole. Adverse events will be graded according 
to the NCI Common Toxicity Criteria Version 4.0.  A copy of the CTC version 4.0 can be 
downloaded from the CTEP home page (http://ctep.info.nih.gov).  All appropriate 
treatment areas should have access to a copy of the CTC version 4.0. 
 
This trial will be independently monitored by the Norris Cotton Cancer Center Data 
Safety and Accrual Monitoring Committee.  
 
The following definitions will be used to assess causality: 
No: 
The clinical adverse event is definitely unrelated to the study 
medications (e.g., does not follow a reasonable temporal sequence 
from study medication administration, present prior to receiving study 
medication, etc.) 
Unlikely: The study medications do not have any reasonable association with the 
observed experience; however, relationship cannot be definitely 
excluded. 
Possibly: The connection with study medication administration appears feasible, 
but cannot be excluded with certainty (e.g., follows a reasonable 
temporal sequence from medication administration, but may also be 
related to other known factors). 
Probably: The clinical experience appears related to the study medications with a 
high degree of certainty (e.g., follows a reasonable temporal sequence 
from medication administration and abates upon discontinuation of the 
medication, cannot be reasonably explained by known characteristics 
of the patient’s clinical state or other modes of therapy administered to 
the patient, etc.) 
  
9.0. 
STATISTICAL CONSIDERATIONS 
9.1 
PRIMARY ENDPOINT – The primary endpoint of this trial is the level of IRS-
1/PI3K/AKT pathway activation in primary tumors, measured by densitometric 
analysis of immunoblot signals of markers of pathway activation (IRS-1, 
phospho-IRS-1-Ser636/639, phospho-AKT-T308, phospho-AKT-S473). 
9.2  
SECONDARY ENDPOINT – The secondary endpoint is Ki67 score, as 
determined by the percentage of Ki67+ tumor cells by IHC. 


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Presurgical letrozole 
 
21 
 
alter IRS-1/PI3K/AKT pathway activation in breast tumors. RAD001-induced 
pathway activation will be defined as a ≥50% increase in phospho-AKT 
densitometric signal value compared to DMSO-treated control; this scoring 
system provides a binary metric for sample size determination for Arm B (below). 
Given that this is a pilot study, we do not know what fraction of ER+/HER2- 
breast tumors will exhibit RAD001-induced IRS-1/PI3K/AKT activation. Hence, 
tumors from 10 untreated patients in Arm A will be used to determine the 
frequency with which RAD001 induces AKT activation. 
The fraction of tumors in Arm A (untreated patients) that exhibit RAD001-
induced AKT activation will be used to determine the number of patients that 
need to be enrolled in Arm B (presurgical Letrozole treatment) to detect a 
significant effect of presurgical Letrozole treatment on RAD001-induced AKT 
activation. We estimate that ≤15% of Letrozole-treated tumors will exhibit 
RAD001-induced AKT activation. If we observe that RAD001 increases 
phospho-AKT in _X_ of 10 tumors in Arm A, we will enroll _Y_ patients in Arm 
B (presurgical Letrozole) to provide a sufficient number of tumors to detect a 
significant effect (p≤0.05) of presurgical Letrozole on RAD001-induced changes 
in phospho-AKT levels with 80% power (calculated using uncorrected chi-
squared statistic). If we observe that ≤3 tumors from untreated patients in Arm A 
exhibit RAD001-induced AKT activation, the study will be terminated at this 
point, and Arm B will not enroll patients. To account for interpatient/intertumor 
variability, we will enroll a minimum of 10 patients in Arm B. 
 
 
 
 
 
 
 
 
 
10.  
10. 
HUMAN SUBJECTS 
10.1 
Subject patient population 
X 
(n of tumors  from untreated patients that exhibit 
a ≥50% increase in P-AKT levels in response 
to RAD001 treatment ex vivo)  
Y 
(n of patients to be enrolled in 
Arm B, presurgical Letrozole) 
0-3 
Terminate study 
4-6 
30 
7-8 
14 
9-10 
10 


Presurgical letrozole 
 
22 
 
Patients with potentially resectable Stage I-III biopsy-proven ER+/HER2- breast 
cancer. 
 
10.2 
Consent Procedures 
Patients must give a statement of informed consent.  The informed consent must 
be approved by the Committee for the Protection of Human Subjects at Dart-
mouth College, the Dartmouth-Hitchcock Medical Center IRB.  Before initiating 
a trial, the investigator will have written and dated approval from the CPHS of 
Dartmouth-Hitchcock Medical Center for the trial protocol, amendment(s), 
written informed consent form, consent form updates, subject recruitment 
procedures (e.g., advertisements) and written information to be provided to 
subjects.  The investigators, or a person designated by the investigators, will 
explain the benefits and risks of participation in the study to each subject (and  
impartial witness when applicable) and obtain written informed consent prior to 
the subject entering the study (before initiation of non-routine tests and 
administration of study drug).  The final form must be agreed to by the CPHS of 
Dartmouth-Hitchcock Medical Center and must contain all elements in the sample 
form, in language readily understood by the subject.  Each subject’s original 
consent form, signed and dated by the subject and by the person who conducted 
the informed consent discussion, will be retained by the investigator.  In addition, 
a copy of the signed informed consent will be given to the subject. 
 
10.3  Potential Risks and Benefits 
The physical risk of study participation is low. Subjects in Arm A are receiving 
no study-related treatment. Subjects in Arm B are receiving 10-21 days of 
treatment with 2.5 mg/day Letrozole prior to surgery; adjuvant therapy with this 
drug and dose are standard-of-care for this patient population, and will be 
administered for up to 5 years after surgery. The results of this investigation may 
provide novel information on ER and PI3K/mTOR signaling pathways in breast 
cancer, which may shape the development of future treatment regimens to benefit 
future patients with breast cancer. Loss of patient confidentiality is a risk of 
participation.  Participation in this study is of low overall risk and has no potential 
for direct clinical benefit. 
 
10.4  Confidentiality 
The pathology specimens from the patient biopsies will be labeled only with the 
patient case number.  The key matching patient identification with the patient case 
number will be kept in a locked drawer in the PI’s office.  Only the PI and his 
designated representatives will have access to this key.  No one will have access 
to the study records but for the Investigators and CRA’s, the Dartmouth-
Hitchcock CPHS, the FDA, and OPRR. 
 
 
11.  
BIBLIOGRAPHY 
 
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