Aging Mammary Stem Cells and Breast Cancer Prevention
Phase: PHASE2
Conditions: Cancer of Breast
Sponsor: LuZhe Sun

 
 
COVER PAGE FOR PROTOCOL AND STATISTICAL ANALYSIS PLAN 
 
 
 
 
 
Official Study Title:  
 AGING MAMMARY STEM CELLS AND BREAST CANCER PREVENTION 
 
NCT number: NCT02642094 
 
IRB Approval Date: 07.22.20 
 
Unique Protocol ID: CTMS 15-2096  
 

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PROJECT TITLE:  AGING MAMMARY STEM CELLS AND BREAST CANCER PREVENTION 
PARTICIPATING SITES 
UT Health San Antonio, Mays Cancer Center 
 
PRINICIPAL INVESTIGATOR 
 
 
 
CO-PRINCIPAL INVESTIGATOR 
Ismail Jatoi, MD 
 
 
 
 
LuZhe Sun, PhD 
UT Health San Antonio  
 
 
 
UT Health San Antonio 
Mays Cancer Center 
 
 
 
 
7703 Floyd Curl Drive 
Surgical Oncology  
 
 
 
 
San Antonio, TX 78229 
7979 Wurzbach Road 
sunl@uthscsa.edu 
 
 
 
San Antonio, TX  78229   
 
 
 
 
jatoi@uthscsa.edu  
 
 
 
 
 
SUB-INVESTIGATORS 
Boyce Oliver, MD 
Surgical Oncology 
7979 Wurzbach Rd                                               
San Antonio, TX. 78229   
oliverb@uthscsa.edu 
Virginia Kaklamani, MD 
Hematology/Oncology 
7979 Wurzbach Rd                                               
San Antonio, TX. 78229                                       
kaklamani@uthscsa.edu 
 
Andrew Brenner MD, PhD                         
Hematology/Oncology 
7979 Wurzbach Rd 
San Antonio, TX 78229 
Brenner@uthscsa.edu 
Kate Lathrop M.D. 
Hematology/Oncology 
7979 Wurzbach Road 
San Antonio, Texas, 78229 
Lathrop@Uthscsa.edu 
 
 
CTMS# 15-2096 
UT HEALTH SAN ANTONIO IRB# HSC20150556H 
IND#:  IND exempt 
PROTOCOL Version 9.0 
DATE: July 21, 2020 
 
 
CTMS# 15-2096
By Regulatory Affairs at 1:33 pm, Jul 22, 2020
CTMS# 15-2096
By Regulatory Affairs at 1:33 pm, Jul 22, 2020

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INVESTIGATOR’S AGREEMENT 
 
I have read and understand the contents of this clinical protocol, UT Health San Antonio Mays Cancer 
Center CTMS# 15-2096, and will adhere to the study requirements as presented, including all 
statements regarding confidentially.  In addition, I will conduct the study in accordance with current 
international conference on harmonization (ICH) guidance, Good Clinical Practice (GCP) guidance, the 
Declaration of Helsinki, US Food and Drug Administration (FDA) regulations and local IRB and legal 
requirements. 
 
Name of Clinical Investigator: Ismail Jatoi, MD 
 
Institution: UT Health San Antonio, May Cancer Center 
 
 
Investigator Signature 
Date 
 
 
 
CTMS# 15-2096

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ABBREVIATIONS  
 
3D-ECM 
 
 
3D Extracellular Matrix of Matrigel 
ADH 
 
 
 
Atypical Ductal Hyperplasia 
AE 
 
 
 
Adverse Event  
ALH 
 
 
 
Atypical Lobular Hyperplasia 
BP 
 
 
 
Biological Processes 
DAVID  
 
 
Database for Annotation, Visualization and Integrated Discovery 
DCIS 
 
 
 
Ductal Carcinoma In Situ 
DSM 
 
 
 
Data Safety Monitoring 
DSMB  
 
 
Data Safety Monitoring Board 
DSMC  
 
 
Data Safety Monitoring Committee 
 
DSMP  
 
 
Data Safety and Monitoring Plan 
DSO 
 
 
 
Data and Safety Officer 
DQA 
 
 
 
Director of Quality Assurance 
ECOG  
 
 
Eastern Cooperative Oncology Group – Performance Status 
ERα 
 
 
 
Estrogen Receptor Alpha 
eRapa  
 
 
Microencapsulated Rapamycin 
FDR 
 
 
 
False Discovery Rate 
GCP 
 
 
 
Good Clinical Practice 
GO 
 
 
 
Gene Ontology 
GSEA 
 
 
 
Gene Signature Enrichment Analysis 
IBC 
 
 
 
Invasive Breast Cancer 
IND 
 
 
 
Investigational New Drug 
LCIS 
 
 
 
Lobular Carcinoma In Situ 
MaSC  
 
 
Mammary Stem/Progenitor Cells 
MEC 
 
 
 
Mammary Epithelial Cells 
NIA 
 
 
 
National Institute of Aging 
 
QAD 
 
 
 
Quality Assurance Division 
PALS 
 
 
 
Priority of Audit Level Score 
PI 
 
 
 
Principal Investigator 
SAE 
 
 
 
Serious Adverse Event 
SASP 
 
 
 
Senescence Associated Secretory Phenotype 
SFD 
 
 
 
Sphere Formation and Differentiation 
SIR 
 
 
 
Senescence Associated Inflammatory Response 
UPIRSO  
 
 
Unanticipated Problem Involving Risks to Subjects or Others  

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TABLE OF CONTENTS 
1  SYNOPSIS .......................................................................................................................................6 
 
2  BACKGROUND AND SIGNIFICANCE ..................................................................................................7 
 
3  PRELIMINARY DATA .......................................................................................................................8 
 
4  OBJECTIVES .................................................................................................................................. 13 
 4.1  Primary Objective ............................................................................................................................. 13 
 4.2  Secondary Objectives ....................................................................................................................... 13 
 
5  PATIENT ELIGIBILITY ..................................................................................................................... 13 
 5.1  Inclusion Criteria .............................................................................................................................. 13 
 5.2  Exclusion Criteria .............................................................................................................................. 14 
 
6  TREATMENT PLAN ........................................................................................................................ 14 
 
7  STUDY DRUG ADMINISTRATION ................................................................................................... 15 
 
8  TOXICITIES AND ADVERSE REACTIONS ........................................................................................... 15 
 
9  DOSE MODIFICATIONS .................................................................................................................. 15 
 
10  CONCOMMITANT MEDICATIONS................................................................................................. 15 
 10.1  Not Permitted ................................................................................................................................ 15 
 10.2  Permitted ....................................................................................................................................... 15 
 
11  DURATION OF THERAPY .............................................................................................................. 16 
 
12  FOLLOW-UP POST-THERAPY ........................................................................................................ 16 
 
13  SCHEDULE OF EVENTS ................................................................................................................. 16 
 
14  DRUG FORMULATION AND PROCUREMENT ................................................................................ 16 
 14.1  Mechanism Of Action ..................................................................................................................... 17 
 14.2  PRECAUTIONS ................................................................................................................................ 17 
 14.2.1  General ........................................................................................................................................ 17 
 14.2.2  Lipids ........................................................................................................................................... 17 
 14.3  ADVERSE REACTIONS ..................................................................................................................... 18 
 14.4  Storage ........................................................................................................................................... 18 
 
15  STATISTICAL CONSIDERATIONS ................................................................................................... 18 
 15.1  Study Population ............................................................................................................................ 20 
 15.2  Statistical and Data Analysis .......................................................................................................... 20 
 
16  ADVERSE EVENTS REPORTING ..................................................................................................... 20 
 16.1  Definitions & Descriptions ............................................................................................................. 20 
 16.1.1  Adverse event (AE) ...................................................................................................................... 20 
 16.1.2  Severity of AEs ............................................................................................................................. 20 
 16.1.3  Serious Adverse events ............................................................................................................... 20 
 16.1.4  Exceptions to the Definition of SAE ............................................................................................ 21 

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TABLE OF CONTENTS (Cont.) 
 
 16.2  Adverse event reporting ................................................................................................................ 21 
 16.3  Expedited SAE Reporting ................................................................................................................ 22 
 16.4  Reporting to the UT Health San Antonio IRB ................................................................................. 22 
 16.5  Reporting to the FDA ..................................................................................................................... 22 
 16.6  Routine Reporting .......................................................................................................................... 23 
 
17  STUDY MANAGEMENT ................................................................................................................ 23 
 17.1  Institutional Review Board (IRB) Approval and Consent ............................................................... 23 
 17.2  Registration Procedures ................................................................................................................. 23 
 17.3  Access to REDCAP .......................................................................................................................... 24 
 17.4  Patient Referrals to the Study ........................................................................................................ 24 
 17.5  Data Management and Monitoring ............................................................................................... 24 
 17.6  Adherence to the Protocol ............................................................................................................. 24 
 17.7  Emergency Modifications .............................................................................................................. 24 
 17.8  Other Protocol Deviations .............................................................................................................. 24 
 17.9  Amendments to the Protocol ........................................................................................................ 25 
 17.10  Record Retention ......................................................................................................................... 25 
 17.11  Obligations of Investigators ......................................................................................................... 25 
 17.12  Publication Policy ......................................................................................................................... 25 
 17.13  Pathology Requirements .............................................................................................................. 25 
 
18  SAMPLES FOR BIOMARKER EVALUATION .................................................................................... 26 
 
APPENDIX 1 – Common Toxicity Criteria for Adverse Events..................................................................... 28 
 
APPENDIX 2 – Data Safety & Monitoring Plan............................................................................................ 29 
 
APPENDIX 3 – Data Collection & Submission ............................................................................................. 32 
 
APPENDIX 4 – List of prohibited strong CYP3A4 inducers and inhibitors .................................................. 33 
 
 

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1.  SYNOPSIS 
 
Title Aging Mammary Stem Cells and Breast Cancer Prevention 
Design Non-randomized, open-label, phase II, window of opportunity trial. 
Objectives 
The overall purpose of this study is to determine if rapamycin can reduce 
malignant markers and MaSC number in surgical specimens. 
 
The primary objectives are: 
1. Reduction of MaSC in DCIS, LCIS, ALH or ADH in patients receiving 
rapamycin 
2. Reduction of malignant markers in DCIS, LCIS, ALH or ADH in patients 
receiving rapamycin  
 
The secondary objectives are: 
1. Toxicity 
2. Surgical complications 
Patient Population Women (pre and post-menopausal) with DCIS, LCIS, ALH or ADH undergoing 
surgical resection.  
Treatment Plan 
Women who consent will be given rapamycin for 5-7 days treatment at 2 
mg/day. Patients will undergo mastectomy or lumpectomy 3-7 days after the 
last dose of rapamycin. Pathological and molecular biomarkers associated 
with breast cancer aggressiveness and recurrence will be assessed by the 
Breast Pathologist and the features of MaSCs will be determined by the PI’s 
lab for determining the effect of rapamycin.  
Efficacy Assessment Efficacy will be measured by measurement of MaSC and malignant markers in 
tissue from patients with DCIS, LCIS, ALH or ADH 
 
 
 

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2.  BACKGROUND AND SIGNIFICANCE 
 
Aging – the No. 1 risk factor for breast cancer. Cancer incidence rises exponentially during midlife in 
humans and in mice1. About 80% of all breast cancers arise in women over age 50 and age specific increase 
of invasive breast cancer results in a cumulative lifetime risk of 13.2% or 1 in 8 
(http://www.srab.cancer.gov/devcan). Even for women carrying BRCA1 and BRCA2 mutations with family 
history of familial breast cancer, aging is another important risk factor2. However, the knowledge 
regarding the cellular and molecular basis that links aging with clinical manifestation of breast cancer is 
indeed scanty3-5. Thus, understanding age-related changes, which promote breast cancer, should have a 
major impact on our ability to prevent and treat breast cancer for the high risk and the general aging 
population.  
 
Does breast cancer originate from mammary stem cells? During aging, stem cells responsible for lifelong 
tissue maintenance and repair are susceptible to changes in their niche and genomic integrity. Recent 
evidences indicated that they may be responsible for neoplastic transformation. However, there is little 
direct experimental evidence linking aging mammary stem/progenitor cells to spontaneous pre-
neoplastic transformation and tumorigenesis. Mammary stem cells (MaSCs) have been shown to have the 
potential to drive mammary gland development and to initiate neoplastic transformation when 
genetically altered6-8. Gene expression profiles of different subtypes of breast cancer have also been 
shown to correspond to the profiles of the basal or luminal mammary stem/progenitor cell-enriched 
epithelial cells, which suggests that MaSCs might be the origin of certain types of breast cancer. A recent 
study reported a shift in the balance between luminal and basal lineages in aging human breast cells and 
expansion of multipotent progenitor population with a basal differentiation bias9. These changes were 
postulated to contribute to aging-associated mammary tumorigenesis.  As shown in our preliminary data 
below, we have demonstrated that aging is associated with increased number of aberrant MaSCs, which 
can generate mammary glands with early neoplastic lesions. Our study is the first experimental 
demonstration that MaSCs are the precursors of spontaneous neoplastic lesions and aging MaSCs appear 
to be predisposed to neoplastic transformation.  
 
Role of aging/senescence-associated inflammatory response in tumorigenesis. Recent studies have 
suggested that senescent cells in the aged stroma might create a tissue microenvironment favoring cancer 
initiation and growth. Cellular senescence acts as a barrier to transformation, but it drives aging 
phenotypes and is also believed to induce a fertile ground for cancer development late in life10. Senescent 
cells increase with age in many mammalian tissues including human, primates and rodents11,12 and are 
found at sites of many age-related pathologies including benign prostatic hyperplasia13,14. Senescent 
human and mouse cells secrete pro-inflammatory cytokines, chemokines, extracellular matrix 
components, and growth factors. A phenotype termed as senescence associated secretory phenotype 
(SASP) disrupts local tissue homeostasis and is believed to promote tumorigenesis.  
 
Inoculation with premalignant or malignant epithelial cells together with senescent fibroblasts led to more 
and larger tumors in host mice than inoculation with the epithelial cells alone or with presenescent 
fibroblasts15. Using a Cdkn2aINK4A (p16)–luciferase reporter mouse model, one recent study found an 
exponential increase with age in senescence associated-expression of p16 and the induction of p16 in the 
benign stromal cells of the nascent neoplasm16. In our whole genome transcriptome analysis shown 
below, we found an inflammatory response both in the mammary stromal and MaSC-enriched cell 
compartments in association with senescent stromal cells. Although this senescence-associated 
inflammation response (SIR) has been shown in various tissues, it was only last year that SIR was shown 

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to contribute to colon tumorigenesis in genetically modified mouse models17. However, it is not known 
whether the SIR causes the transforming phenotype of MaSCs during progressive aging.  
 
3.  PRELIMINARY DATA 
 
Development of a novel in vitro assay for quantifying MaSCs. In order to quantitatively measure the 
effect of aging on the number and function of murine mammary stem/progenitor cells, we recently 
developed a novel in vitro method18. Previously, murine MaSCs were quantified with limited dilution in 
vivo transplantation of MaSC-containing mammary epithelial cells into de-epithelized inguinal mammary 
glands (also denoted as cleared mammary fat pads) of 21-day-old mice and subsequent examination of 
regeneration frequency of a fully functional mammary gland. This in vivo regeneration method requires 
large number of recipient mice for transplantation and a relatively long period (8 weeks) of experiment 
turnover, not to mention the associated expensive cost. Our in vitro method, termed sphere formation 
and differentiation (SFD) assay, involves suspension culture of mouse MaSC-enriched basal cells, which 
are FACS-sorted out with their cell surface immunophenotype of Lin-CD49fhighCD24med (Fig. 1A)19,20, for 
the formation of mammospheres (Fig. 1B). These spheres were verified to be of clonal origin of single 
MaSC18, and they can be differentiated to form morphologically distinct solid 3D spherical 
structures/organoids when embedded in 3D extracellular matrix of Matrigel (3D-ECM) (Fig. 1C). Upon in 
vivo transplantation, these single mammospheres or 3D solid structures can generate full ductal-lobular 
outgrowths (Fig. 1D). We were able to visualize ductal structures of the regenerated mammary ducts with 
green fluorescence imaging (Fig. 1D) as the mammospheres and 3D solid structures were formed by 
MaSCs isolated from GFP-transgenic mice. Thus, this SFD assay allows quantification of MaSC in vitro. 
 
 
 
Age-associated changes in MaSC frequency and gene expression. Using this SFD assay, we measured 
MaSC number as a function of aging. FACS analysis revealed that total basal epithelial cell frequency, 
which is percent of basal epithelial cells in total lineage negative (Lin-) mammary epithelial cells (MEC) 
(Fig. 1A), increased with progressive aging in C57Bl/6 mice (Fig. 2). This aging-associated increase of basal 
epithelial cells and decrease of luminal epithelial cells (data not shown) led to a significant decrease in the 
luminal to basal epithelial cell ratio (Fig. 3A) and a significant increase in MaSC frequency (Fig. 3B) in old 
(22-30 month-old) mice of both C57Bl/6 and Balb/c strains when compared to their respective young (4-
6 month-old) cohorts. However, the ability of the 3D structures to regenerated 3D structures during serial 
passage in 3D culture was much lower by the old MaSC-formed 3D structures than by the young MaSC-
formed 3D structures (data not shown). To elucidate molecular changes associated with aging MaSCs, we 

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used RNA-seq to profile whole transcriptome of FACS-sorted basal, luminal, stromal cells, and early stage 
mammospheres formed by basal or luminal epithelial cells, which are highly enriched in MaSCs or luminal 
progenitors respectively, from young and old C57Bl/6 mice.  
 
Using pairwise comparison among young mammary cells, we obtained a set of basal or luminal cell specific 
genes/markers, named Basal Signature or Luminal Signature respectively and performed gene signature 
enrichment analysis (GSEA) using the basal or luminal markers we identified as well as those previously 
reported by Lim et al.21. As shown in Fig. 4, the old basal (CD49fhighCD24med) cells and their 
mammospheres showed significantly reduced expression of basal markers, but significantly increased 
luminal markers. In contrast, GSEA did not show a significant change of the basal or luminal signatures in 
old luminal (CD49flowCD24high) cells and their mammospheres (data not shown). These findings indicate 
aberrant changes of old MaSCs towards a more luminal phenotype, which may contribute to their reduced 
self-renewal capacity and transforming property as described below. 
 
 
 

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Old MaSC-mediated development of spontaneous neoplastic lesions. Significantly, in vivo 
transplantation of single 3D solid structures and subsequent histology analysis by a breast pathologist 
revealed that the ducts of the old MaSC-regenerated mammary glands were similar to the ducts of the 
old primary mammary glands with significantly higher number of hyperplastic and dysplastic foci of 
atypical ductal hyperplasia (ADH, indicated by abnormal glandular proliferation) and ductal carcinoma in 
situ (DCIS, indicated by numerous mitotic figures, nuclear enlargement, and coarse chromatin pattern) 
than those from the young primary glands and young MaSC-regenerated glands (Fig. 5A-C). We define the 
ability of generating the early neoplastic lesions in the MaSC-regenerated glands as the transforming 
phenotype/property of MaSCs. Interestingly, the cells in the dysplastic lesions of the old MaSC-
regenerated glands express various luminal epithelial markers such as estrogen receptor alpha (ERα) and 
Her2 (Fig. 6) indicating their resemblance to human breast adenocarcinoma. 
 
 

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Increased inflammation and immune responses in old mammary cells. As the initial step in elucidating 
cellular pathways that may contribute to the aging-associated transforming phenotype of MaSCs, we 
compared gene expression profiles between young and old mammary stromal cells and found that the 
Cdkn2a locus, containing p16Ink4a and p19Arf, was the most highly up-regulated transcript in the old 
stromal cells among all significantly (adjusted P value (aka FDR, false discovery rate )<0.05) and 
differentially regulated genes with a 19-fold difference (see the heatmap in Fig. 10). The significant 
increase of both p16Ink4a and p19Arf in the old stromal cells was confirmed with real time RT-PCR (Fig. 
7) suggesting a senescence phenotype of the old stromal cells. This is consistent with the old mammary 
gland expressing higher senescence-associated β-gal activity (Fig. 8). We found 66 differentially expressed 
genes between young and old stromal cells with an adjusted P value of <0.05 (Fig. 9).  
 
Functional annotation of these differentially expressed genes with the Database for Annotation, 
Visualization and Integrated Discovery (DAVID)22 platform identified many significantly (P<0.05) altered 
Biological Processes (BPs) depicted as gene ontology (GO) terms with the top ten listed in Fig. 9. While 
over 200 genes were found to be differentially expressed between young and old luminal progenitor-
enriched spheres, DAVID analysis yielded top enriched BPs mostly related to cell adhesion, biological 
adhesion, and extracellular matrix organization (data not shown) suggesting that aging-associated luminal 
cell functions are more involved in epithelium structure remodeling with a limited role in the early 
neoplastic transformation by the old MaSCs. Consequently, we have focused our attention on the altered 
gene expression in aging stromal cells and MaSCs. Although there are only 7 genes that are differentially 
regulated during aging in both basal cell-formed mammospheres and stromal cells (Fig. 9), the functional 
annotation revealed that the top enriched Biological Processes in both sets of the genes are related to 
immune, inflammatory, defense, and wounding responses (Fig. 9).  
 
These processes and responses are mostly performed by a shared set of genes in each cell type, which are 
shown in the heatmaps in Fig. 10. Some of the genes associated with the Oxidation Reduction, the top GO 
term for the stromal gene set, are involved in biosynthesis/production of eicosanoids, which may also 
contribute to inflammation (Fig. 10). We have also observed a significant increase of Ptgs2 transcript in 
our RNA-seq data (pointed by the arrow in Fig. 10) and its protein COX2 in old mammary gland with 
immunostaining (Fig. 14 shown in Aim 2.2), and an increased number of infiltrating macrophages in the 
old mammary gland (data not shown). Thus, there is an increased senescence-associated inflammation 
response (SIR) in the old mammary glands.  
 

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Rapamycin treatment mitigated the aberrant phenotypes of old MaSCs. To investigate whether the 
increased SIR in the old mammary gland contributes to the increased number and transforming 
phenotype of the old MaSCs, we tested the efficacy of rapamycin in inhibiting the aging-associated 
mammary gland dysfunction because it is an anti-inflammatory and immune modulatory drug and its 
target mTOR is known to be up-regulated in senescent cells23. Rapamycin was shown to significantly 
extend lifespan and reduce tumor incidence in mice by investigators in our university as well as by 
others24. It was also shown recently to inhibit senescent mammary stroma-promoted growth of 

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xenografted breast cancer cells in a premature aging mouse model with Cav-1 knockout25. As shown in 
Fig. 3B, old mammary glands have higher percent of MaSCs. Both long-term (2 years) and short-term (5-
10 days) treatment with a diet containing eRapa (microencapsulated rapamycin) significantly reduced the 
frequency of MaSCs in old mice (Fig. 11A). More significantly, both long- and short-term treatment also 
reduced hyperplastic and dysplastic foci not only in the primary old mammary glands but also in the 
regenerated glands by old MaSCs (Fig. 11B and C).  
 
 
 
These exciting novel observations prompted us to propose the following research aims. 
 
4.  OBJECTIVES 
 
The overall purpose of the study is to evaluate the efficacy of short-term rapamycin treatment on 
malignant markers and MaSC in patients with DCIS, LCIS, ALH or ADH. 
 
4.1  Primary Objective 
1. Reduction of MaSC in DCIS, LCIS, ALH or ADH in patients receiving rapamycin 
2. Reduction of malignant markers in DCIS, LCIS, ALH or ADH in patients receiving rapamycin  
 
4.2  Secondary Objectives 
1. Toxicity 
2. Surgical complications 
 
5.  PATIENT ELIGIBILITY 
 
5.1  Inclusion Criteria 
Subjects must meet all of the following criteria to be eligible for the trial: 
1. Women with confirmed menopausal status.  All patients who have NOT had a prior bilateral 
oophorectomy and/or are younger than age 60, will require menopausal status verified by FSH 
and estradiol local labs. 
3. Women diagnosed with DCIS, LCIS, ALH or ADH lesions detected by pathology 
4. Women scheduled for mastectomy or lumpectomy after DCIS, LCIS, ALH or ADH diagnosis 
5. Women consented to the tissue biorepository (HSC20070684H/CTRC# 07-32) 

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6. Women of child-bearing potential willing to practice 2 forms of contraception, one of which must 
be a barrier method until at least 30 days after the last dose of rapamycin. 
7. Women of child-bearing potential must have a negative serum pregnancy test at time of 
enrollment. 
8. Patients must be able to swallow and retain oral medication. 
9. All patients must have given signed, informed consent prior to registration on study. 
10. Patients must have normal organ and marrow function as defined below: 
a. Leukocytes ≥ 3,000/µL 
b. Absolute neutrophil count ≥ 1,500/µL 
c. Platelets ≥ 100,000/µL 
d. AST ≤ 2.5 X ULN  
e. ALT ≤ 2.5 X ULN  
f. Total bili ≤ 1.5 X ULN or Direct bili ≤ 1 X ULN 
 
5.2  Exclusion Criteria 
1. Women who are pregnant. 
2. Women who are receiving any other concomitant treatment for their DCIS, LCIS, ALH or ADH 
3. Women who are taking rapamycin for another diagnosis. 
4. Women with an allergy to rapamycin or its derivatives. 
5. Active infection requiring systemic therapy. 
6. Patients who are taking any pills containing herbal (alternative) medicines are NOT eligible for 
participation.  Patients must be off any such medications by the time of registration. 
7. Immunocompromised subjects, including patients with human immunodeficiency virus  
8. Women currently taking strong CYP3A4 inducers or inhibitors. Drugs that cannot be coadministered 
with rapamycin include but are not limited to: Calcium channel blockers: nicardipine, antifungal 
agents: clotrimazole, fluconazole, antibiotics: troleandomycin, rifapentine, gastrointestinal prokinetic 
agents: cisapride, metoclopramide, Other drugs: bromocriptine, cimetidine, danazol, HIV-protease 
inhibitors (e.g., ritonavir, indinavir), anticonvulsants: carbamazepine, phenobarbital, phenytoin.  
Appendix 4 has a complete list of these medications. 
9. Patients with any of the following conditions or complications are NOT eligible for participation: 
a. GI tract disease resulting in an inability to take oral medication 
b. Malabsorption syndrome 
c. Require IV alimentation 
d. History of prior surgical procedures affecting absorption 
e. Uncontrolled inflammatory GI disease (e.g., Crohn’s, ulcerative colitis) 
 
6.  TREATMENT PLAN 
 
This is a phase II window of opportunity trial in which women with a diagnosis of DCIS, LCIS, ALH or ADH 
will be treated with rapamycin at 2 mg/day for 5-7 days.  Surgery will be performed 3-7 days after the last 
dose of rapamycin.  An evaluable patient for toxicity is any patient who has received at least 1 dose of 
rapamycin.  An evaluable patient for efficacy is any patient who has completed at least 5 days of rapamycin 
therapy and has had surgery within the 7-day window.  
 
7.  STUDY DRUG ADMINISTRATION 
 
Study drug will be given orally once a day in the form of tablets. Patient may take drug consistently with 
or without food.  

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8.  TOXICITIES AND ADVERSE REACTIONS 
 
Rapamune® 
The incidence of adverse reactions was determined in two randomized,  double-blind, multicenter 
controlled trials in which 499 renal transplant patients received Rapamune Oral Solution 2 mg/day, 477 
received Rapamune Or l Solution 5 mg/day, 160 received azathioprine, and 124 received placebo. All 
patients were treated with cyclosporine and corticosteroids. Data (≥12 months post-transplant) presented 
in the table below show the adverse reactions that occurred in any treatment group with an incidence of 
≥20%.  
 
Specific adverse reactions associated with the administration of Rapamune (sirolimus) Oral Solution 
occurred at a significantly higher frequency than in the respective control group. For both Rapamune Oral 
Solution 2 mg/day and 5 mg/day these include hypercholesterolemia, hyperlipemia, hypertension, and 
rash; for Rapamune Oral Solution 2 mg/day acne; and for Rapamune Oral Solution 5 mg/day anemia, 
arthralgia, diarrhea, hypokalemia, and thrombocytopenia. The elevations of triglycerides and cholesterol 
and decreases in platelets and hemoglobin occurred in a dose-related manner in patients receiving 
Rapamune.  
 
Patients maintained on Rapamune Oral Solution 5 mg/day, when compared with patients on Rapamune 
Oral Solution 2 mg/day, demonstrated an increased incidence of the following adverse events: anemia, 
leukopenia, thrombocytopenia, hypokalemia, hyperlipemia, fever, and diarrhea. In general, adverse 
events related to the administration of Rapamune were dependent on dose/concentration.  
 
9.  DOSE MODIFICATIONS 
 
No dose modifications will be allowed. 
 
10.  CONCOMMITANT MEDICATIONS 
 
All concomitant treatments, including blood and blood products, must be reported on the appropriate 
electronic case report form (eCRF). 
 
10.1  Not Permitted 
Patients who are taking any herbal (alternative) medicines are NOT eligible for participation.  Patients 
must be off any such medications before the first dose of study treatment.  Patients who are undergoing 
concomitant radiotherapy or chemotherapy are NOT eligible for participation.  
 
10.2  Permitted 
Supportive care, including but not limited to anti-emetic medications, may be administered at the 
discretion of the treating physician.   
 
11.  DURATION OF THERAPY 
 
Treatment duration is 5-7 days 
 

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12.  FOLLOW-UP POST-THERAPY 
 
Patients will be followed 3 months after surgery at the time of routine clinic visit or via phone call with 
study personnel, See APPENDIX 3 –subsection titled Research Chart (case report form needed for data 
collection during this 3-month follow-up visit). 
 
Treatment after study completion will be left to the discretion of the treating physician(s) and will not be 
considered part of this study.  However, patients should still be followed for surgical complications as part 
of their standard of care. 
 
13.  SCHEDULE OF EVENTS 
 
Procedures 
Screening & 
Baseline 
Day 1-74 
Day before 
Surgery 
Surgery2 
3 month 
Follow-Up 
Tissue procurement1 
 
 
 
X 
 
Informed consent 
X 
 
 
 
 
Rapamycin administration 
 
X 
 
 
 
Cancer history3 
X 
 
X 
 
 
Physical exam3 
X 
 
X 
 
 
CBC w/ diff 
X3 
 
X 
 
 
Comprehensive metabolic 
panel 
X3 
 
X 
 
 
Lipid panel 
X 
 
X 
 
 
Estradiol5 
X 
 
X 
 
 
FSH5 
X 
 
X 
 
 
Toxicity (AE) assessment 
 
 
X 
 
X 
1 Fresh tissue procurement will be required for study purposes.  
2 Surgery can take place 3-7 days after completion of rapamycin treatment 
3 Pre-study laboratory tests, cancer history, and physical exam (including review of systems) must be 
completed within 28 days prior to registration. At the discretion of the treating physician and only if 
clinically indicated as part of their standard of care, the pre-study CBC and CMP will be repeated one 
(1) day prior to treatment administration. 
4 Rapamycin will be administered for 5-7 days 
5 Estradiol and FSH are required only if patient is under 60 years of age and has not had a bilateral 
oophorectomy. 
 
14. DRUG FORMULATION AND PROCUREMENT 
 
Rapamune® (sirolimus) is an immunosuppressive agent. Sirolimus is a macrocyclic lactone produced by 
Streptomyces hygroscopicus. The chemical name of sirolimus (also known as rapamycin) is 
(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32, 
33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-
methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4] 

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oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone. Its molecular formula is C51H79NO13 and 
its molecular weight is 914.2. The structural formula of sirolimus is shown below. The inactive ingredients 
in Rapamune® Tablets include sucrose, lactose, polyethylene glycol 8000, calcium sulfate, microcrystalline 
cellulose, pharmaceutical glaze, talc, titanium dioxide, magnesium stearate, povidone, poloxamer 188, 
polyethylene glycol 20,000, glyceryl monooleate, carnauba wax, dl-alpha tocopherol, and other 
ingredients. The 2 mg dosage strength also contains iron oxide yellow 10 and iron oxide brown 70. 
 
14.1  Mechanism Of Action 
Rapamycin inhibits T lymphocyte activation and proliferation that occurs in response to antigenic and 
cytokine (Interleukin [IL]-2, IL-4, and IL-15) stimulation by a mechanism that is distinct from that of other 
immunosuppressants. Rapamycin also inhibits antibody production. In cells, rapamycin binds to the 
immunophilin, FK Binding Protein-12 (FKBP-12), to generate an immunosuppressive complex. The 
rapamycin:FKBP-12 complex has no effect on calcineurin activity. This complex binds to and inhibits the 
activation of the mammalian Target Of Rapamycin (mTOR), a key regulatory kinase. This inhibition 
suppresses cytokine-driven T-cell proliferation, inhibiting the progression from the G1 to the S phase of 
the cell cycle. Studies in experimental models show that rapamycin prolongs allograft (kidney, heart, skin, 
islet, small bowel, pancreatico-duodenal, and bone marrow) survival in mice, rats, pigs, and/or primates. 
Rapamycin reverses acute rejection of heart and kidney allografts in rats and prolongs the graft survival 
in presensitized rats. In some studies, the immunosuppressive effect of rapamycin lasts up to 6 months 
after discontinuation of therapy. This tolerization effect is alloantigen specific. In rodent models of 
autoimmune disease, rapamycin suppresses immune-mediated events associated with systemic lupus 
erythematosus, collagen-induced arthritis, autoimmune type I diabetes, autoimmune myocarditis, 
experimental allergic encephalomyelitis, graft-versus-host disease, and autoimmune uveoretinitis. 
 
14.2  PRECAUTIONS 
14.2.1  General 
Rapamune® is intended for oral administration only. 
Lymphocele, a known surgical complication of renal transplantation, occurred significantly more often in 
a dose-related fashion in patients treated with Rapamune®. Appropriate operative measures should be 
considered to minimize this complication. 
 
14.2.2  Lipids 
The use of Rapamune® in renal transplant patients was associated with increased serum cholesterol and 
triglycerides that may require treatment. In Studies 1 and 2, in de novo renal transplant recipients who 
began the study with normal, fasting,  total serum cholesterol (<200 mg/dL) or normal, fasting, total serum 
triglycerides (<200 mg/dL), there was an increased incidence of hypercholesterolemia (fasting serum 
cholesterol >240 mg/dL) or hypertriglyceridemia (fasting serum triglycerides >500 mg/dL), respectively, 
in patients receiving both Rapamune® 2 mg and Rapamune® 5 mg compared with azathioprine and 
placebo controls. Treatment of new-onset hypercholesterolemia with lipid-lowering agents was required 
in 42 - 52% of patients enrolled in the Rapamune® arms of Studies 1 and 2 compared with 16% of patients 
in the placebo arm and 22% of patients in the azathioprine arm. 
 
In Study 4 (cyclosporine withdrawal study) during the prerandomization period, mean fasting serum 
cholesterol and triglyceride values rapidly increased, and peaked at 2 months with mean cholesterol 
values > 240 mg/dL and triglycerides > 250 mg/dL. After randomization mean cholesterol and triglyceride 
values remained higher in the cyclosporine withdrawal arm compared to the Rapamune® and 
cyclosporine combination. Renal transplant patients have a higher prevalence of clinically significant 

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hyperlipidemia. Accordingly, the risk/benefit should be carefully considered in patients with established 
hyperlipidemia before initiating an immunosuppressive regimen including Rapamune®. 
 
Any patient who is administered Rapamune® should be monitored for hyperlipidemia using laboratory 
tests and if hyperlipidemia is detected, subsequent interventions such as diet, exercise, and lipid lowering 
agents, as outlined by the National Cholesterol Education Program guidelines, should be initiated. In 
clinical trials, the concomitant administration of Rapamune® and HMG-CoA reductase inhibitors and/or 
fibrates appeared to be well tolerated. During Rapamune® therapy with cyclosporine, patients 
administered an HMG-CoA reductase inhibitor and/or fibrate should be monitored for the possible 
development of rhabdomyolysis and other adverse effects as described in the respective labeling for these 
agents. 
 
14.3  ADVERSE REACTIONS 
Rapamune® Oral Solution: The incidence of adverse reactions was determined in two randomized, 
double-blind, multicenter controlled trials in which 499 renal transplant patients received Rapamune® 
Oral Solution 2 mg/day, 477 received Rapamune® Oral Solution 5 mg/day, 160 received azathioprine, and 
124 received placebo. All patients were treated with cyclosporine and corticosteroids. Data (≥ 12 months 
post-transplant) presented in the table below show the adverse reactions that occurred in any treatment 
group with an incidence of ≥ 20%. Specific adverse reactions associated with the administration of 
Rapamune® (sirolimus) Oral Solution occurred at a significantly higher frequency than in the respective 
control group. For both Rapamune® Oral Solution 2 mg/day and 5 mg/day these include 
hypercholesterolemia, hyperlipemia, hypertension, and rash; for Rapamune® Oral Solution 2 mg/day 
acne; and for Rapamune® Oral Solution 5 mg/day anemia, arthralgia, diarrhea, hypokalemia, and 
thrombocytopenia. The elevations of triglycerides and cholesterol and decreases in platelets and 
hemoglobin occurred in a dose-related manner in patients receiving Rapamune®. Patients maintained on 
Rapamune® Oral Solution 5 mg/day, when compared with patients on Rapamune® Oral Solution 2 
mg/day, demonstrated an increased incidence of the following adverse events: anemia, leukopenia, 
thrombocytopenia, hypokalemia, hyperlipemia, fever, and diarrhea. In general, adverse events related to 
the administration of Rapamune® were dependent on dose/concentration. 
 
14.4  Storage 
Rapamune® Oral Solution bottles should be stored protected from light and refrigerated at 2°C to 8°C 
(36°F to 46°F). Once the bottle is opened, the contents should be used within one month. If necessary, 
the patient may store the bottles at room temperatures up to 25°C (77°F) for a short period of time (e.g., 
not more than 15 days for the bottles). An amber syringe and cap are provided for dosing and the product 
may be kept in the syringe for a maximum of 24 hours at room temperatures up to 25°C (77°F) or 
refrigerated at 2°C to 8°C (36°F to 46°F). The syringe should be discarded after one use. After dilution, the 
preparation should be used immediately. 
 
Rapamune® Oral Solution provided in bottles may develop a slight haze when refrigerated. If such a haze 
occurs allow the product to stand at room temperature and shake gently until the haze disappears. The 
presence of this haze does not affect the quality of the product.  Rapamune® tablets should be stored at 
20° to 25°C (USP Controlled Room Temperature) (68° to 77°F). Use cartons to protect blister cards and 
strips from light. Dispense in a tight, light-resistant container as defined in the USP. 
 
15.  STATISTICAL CONSIDERATIONS 
 
15.1  Study Population 

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This study proposes recruitment of women with DCIS, LCIS, ALH or ADH cases.  The population in the San 
Antonio area are mostly Hispanics (~55%) and Caucasian (~45%).  
 
15.2  Statistical and Data Analysis 
Availability of case and justification of sample size. According to our co-investigator and breast 
pathologist, the surgeons at our cancer center operated 24 cases of DCIS in 2013. 
 
Ages 
# of patients at UT Health San 
Antonio Mays Cancer Center 
< 50 
7 
50-59 
10 
> 60 
7 
 
We consider about 60-70% enrollment in 5-year recruitment duration, we shall be able to recruit about 
50-60 patients with menopausal status clinically confirmed.  Tissue from this study will be compared to 
DCIS specimens from patients not treated with rapamycin. These specimens will be collected through the 
ongoing biorepository.  
 
Statistically significant differences of the histopathological parameters and the molecular markers 
between the core biopsy and surgical specimens due to the rapamycin treatment will be tested by Mantel-
Haenszel test for categorical data with scores ranging from 0 to 4 (or higher) as shown in Figure 2. To 
reach statistical power >70%, a patient group size of 24 or more is required for us to detect the difference 
of a score of 0.3 between untreated biopsy and treated surgical specimens with the P value targeted at 
0.05 for one-sided Mantel-Haenszel test. 
 
The Mantel-Haenszel test (between Rapamycin treatment & non-participant patients) will be set up as 
follows. 
 
 
    Test between treatment & non-participant patients 
Study 
 
IHC score ≤ 2 
IHC score > 2 
Percentage 
UT Health San Antonio Rapamycin 
# of patients (a) # of patients (b) a/(a+b) 
 
No Rapamycin # of patients (c) 
# of patients (d) c/(c+d) 
 
Mantel-Haenszel test will allow us to extend IHC score to full range (instead of dichotomizing the IHC 
score), as well as other sites if more patients will be enrolled at different sites (we will not lump all patients 
together into one group, in case the underlying population demographic is a concern at different 
enrollment sites). Significance will be determined at p-value < 0.05 (Chi-square distributed with one 
degree of freedom). During the recruitment period, we will monitor the IHC scores different from two 
groups (treated/untreated) with simple t test on average IHC score (across all patients within each group) 
for each marker, in order to assess the IHC effectiveness, enrollment progress and patient demographic 
parameters and their association to final study outcomes. 
 
For statistical test within either treatment or control arm, we will use McNemar’s test to examine IHC 
score differences between biopsy and surgical tissues (at significance level of 0.05). The 2x2 table set up 
as follows. 
 
 Test within treatment or no treatment 
 
 
After treatment 

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IHC score ≤ 2 
IHC score > 2 
Before treatment IHC score ≤ 2 # of patients with 
lower IHC scores before & after 
# of patients with  
higher IHC score after treatment 
 
IHC score > 2 # of patients with  
lower IHC score after treatment 
# of patients with  
higher IHC scores before & after 
 
Similar to between arm test, during the recruitment period, we will monitor the IHC scores different within 
each arm with simple paired-t test on average IHC score difference for each marker, in order to assess the 
IHC effectiveness, enrollment progress and patient demographic parameters and their association to final 
study outcomes. 
 
16.  ADVERSE EVENTS REPORTING 
 
Adverse event data collection and reporting, which are required as part of every clinical trial, are done to 
ensure the safety of subjects enrolled in the studies as well as those who will enroll in future studies using 
similar agents.  Subjects must be carefully monitored for AEs. Adverse events should be assessed in terms 
of their seriousness, intensity, and relationship to the study drug. Adverse events are reported in a routine 
manner at scheduled times during a trial.  Additionally, certain adverse events must be reported in an 
expedited manner to allow for optimal monitoring of patient safety and care. 
 
All patients experiencing an adverse event, regardless of its relationship to study drug, will be monitored 
until:  
 
1. the adverse event resolves or the symptoms or signs that constitute the adverse event return to 
baseline;  
2. there is a satisfactory explanation other than the study drug for the changes observed; or 
3. death occurs. 
 
16.1  Definitions & Descriptions 
 
16.1.1  Adverse event (AE) 
An adverse event (AE) is any untoward medical occurrence in a patient receiving study treatment and 
which does not necessarily have a causal relationship with this treatment. An AE can, therefore, be any 
unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of an experimental intervention, whether or not related to the 
intervention. A “drug-related toxicity" includes as events, any toxicity considered related, probably 
related, or possibly related to study drugs. Toxicities clearly not related to the drug, such as disease 
progression, environmental, unrelated trauma may not be considered “drug related toxicity”. 
 
16.1.2  Severity of AEs 
All adverse events will be graded according to the NCI Common Terminology Criteria for Adverse Events 
(CTCAE) version 4.03. The CTCAE v. 4.03 is available at:  
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf 
 
If no CTCAE grading is available, the severity of an AE is graded as follows: 
 
• 
Mild (grade 1): the event causes discomfort without disruption of normal daily activities. 

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• 
Moderate (grade 2): the event causes discomfort that affects normal daily activities. 
• 
Severe (grade 3): the event makes the patient unable to perform normal daily activities or significantly 
affects his/her clinical status. 
• 
Life-threatening (grade 4): the patient was at risk of death at the time of the event. 
• 
Fatal (grade 5): the event caused death. 
 
16.1.3  Serious Adverse events 
All SAEs, regardless of attribution, occurring during the study or within 30 days of the last administration 
of study drug must be reported upon discovery or occurrence.  Additional expedited or routine reporting 
may be required, depending on the nature of the SAE. A “serious” adverse event is defined in regulatory 
terminology as any untoward medical occurrence that: 
 
1. Results in death.  If death results from (progression of) the disease, the disease should be reported as 
the event (SAE) itself. 
2. Is life-threatening. The patient was at risk of death at the time of the event; it does not refer to an 
event that hypothetically might have caused death if it were more severe. 
3. Requires in-patient hospitalization or prolongation of existing hospitalization for ≥ 24hours. 
4. Results in persistent or significant disability or incapacity. 
5. Is a congenital anomaly/birth defect. 
6. Is an important medical event. 
7. Any event that does not meet the above criteria, but that in the judgment of the investigator 
jeopardizes the patient, may be considered for reporting as a serious adverse event. The event may 
require medical or surgical intervention to prevent one of the outcomes listed in the definition of 
“Serious Adverse Event“. Examples: allergic bronchospasm requiring intensive treatment in an 
emergency room or at home; convulsions that may not result in hospitalization; development of drug 
abuse or drug dependency. 
 
16.1.4  Exceptions to the Definition of SAE 
Certain hospitalizations or prolongation of hospitalizations should not be considered SAEs, including those 
that meeting the following criteria: 
 
1. An admission resulting in a hospital stay of less than 24 hours. 
2. An admission that is pre-planned (i.e. elective or scheduled surgery arranged prior to the start of the 
study). 
3. An admission that is not associated with an AE (i.e. social hospitalization for purposes of respite care). 
 
16.1.5  Unanticipated Problems Involving Risks to Subjects or Others (UPIRSO) 
A UPIRSO includes events that meet ALL of the following criteria: 
 
1. Are unanticipated in terms of nature, severity, or frequency;  
2. Place the research subject or others at a different or greater risk of harm; AND 
3. Are deemed to be related or possibly related to participation in the study.   
 
16.2  Adverse event reporting 
Steps to determine if expedited reporting is required include all events that occur within 30 days of the 
last dose of protocol treatment.  Any event that occurs more than 30 days after the last dose of treatment 

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and is attributed (possibly, probably, or definitely) to the agent, must also be reported accordingly.  You 
are required to: 
 
1. Identify the type of adverse event using the NCI CTCAE v 4.03. 
2. Grade the adverse event using the NCI CTCAE v 4.03. 
3. Determine whether the adverse event is related to the protocol therapy.   
 
Attribution categories are as follows: 
Definite:  AE is clearly related to the study treatment (attribution should be separated by each study drug) 
Probable:  AE is likely related to the study treatment (attribution should be separated by each study drug) 
Possible:  AE may be related to the study treatment (attribution should be separated by each study drug) 
Unrelated:  AE is clearly NOT related to the study treatment (attribution should be separated by each 
study drug 
 
Determine the prior experience of the adverse event.  Expected events are those that have been 
previously identified as resulting from administration of the specific agent.  An adverse event is considered 
unexpected, for expedited reporting purposes only, when either the type of event or the severity of the 
event is not listed in the current protocol and/or the drug package insert. 
 
16.3  Expedited SAE Reporting 
All SAEs must be reported following the DSMP outlined in Appendix 2 of this protocol within 24 hours of 
becoming aware of the event. Completion of the UT Health San Antonio Mays Cancer Center SAE Form is 
required.   The completed form should assess whether or not the event qualifies as a UPIRSO.  The report 
should also include: 
 
1. Protocol description and number 
2. Patient’s Identification Number 
3. Description of event, severity, treatment, and outcome if known 
4. Supportive laboratory results and diagnostics 
5. Hospital Discharge Summary (if available) 
 
All SAEs will be reported to, and reviewed by, the DSMB at their next meeting.   
 
16.4  Reporting to the UT Health San Antonio IRB 
Any death of a subject that is unanticipated in nature and at least possibly related to study participation 
will be promptly reported to the UT Health San Antonio Mays Cancer Center Regulatory Affairs Division 
for reporting within 24 hours of notification. Other SAE’s that are life threatening, but not fatal, should be 
reported to UT Health San Antonio Mays Cancer Center Regulatory Affairs within 7 days of notification. 
 
The following SAEs will be reported to the UT Health San Antonio Mays Cancer Center Regulatory Affairs 
for reporting to the UT Health San Antonio IRB at the time of continuing review: 
 
1. All deaths of UT Health San Antonio Mays Cancer Center subjects that were not previously reported 
2. All deaths of non-UT Health San Antonio subjects that are deemed to be unanticipated in nature and 
unrelated to participation 
3. Other UPIRSO’s 
4. All other SAEs not previously reported to the UT Health San Antonio IRB as UPIRSOs 
 

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In addition, participating sites should follow local guidelines for reporting of SAEs to their IRB as required. 
 
16.5  Reporting to the FDA 
The UT Health San Antonio Mays Cancer Center Regulatory Affairs division will notify FDA within 7 
calendar days of any SAE that is associated with study treatment, is unexpected, and is fatal or life-
threatening. The UT Health San Antonio Mays Cancer Center Regulatory Affairs Division will notify the 
FDA within 15 calendar days of any SAE that is associated with the study treatment, unexpected, and 
serious but not fatal or life-threatening. This includes any previous SAEs that were not initially deemed 
reportable, but are later determined to meet the criteria for reporting by the DSMB.  In these instances, 
an FDA Med Watch Form will be completed. 
 
16.6  Routine Reporting 
All other adverse events, such as those that are expected, or are unlikely or definitely not related to the 
study participation, are to be reported on the appropriate eCRF according to the time intervals noted in 
the Schedule of Events.  Routine AEs will be reviewed by the assigned DSMB for the study in accordance 
with the Mays Cancer Center’s Multi-site DSMP Procedures. These will be reviewed by the DSMB on an 
on-going basis.  A summary of all these events will be reported annually to the UT Health San Antonio IRB 
as part of the continuing review process. 
 
17.  STUDY MANAGEMENT 
 
17.1  Institutional Review Board (IRB) Approval and Consent  
It is expected that the IRB will have the proper representation and function in accordance with federally 
mandated regulations. The IRB should approve the consent form and protocol. 
 
In obtaining and documenting informed consent, the investigator should comply with the applicable 
regulatory requirement(s), and should adhere to Good Clinical Practice (GCP) and to ethical principles that 
have their origin in the Declaration of Helsinki. 
 
Before recruitment and enrollment onto this study, the patient will be given a full explanation of the study 
and will be given the opportunity to review the consent form. Each consent form must include all the 
relevant elements currently required by the FDA Regulations and local or state regulations. Once this 
essential information has been provided to the patient and the investigator is assured that the patient 
understands the implications of participating in the study, the patient will be asked to give consent to 
participate in the study by signing an IRB approved consent form. 
 
Prior to a patient’s participation in the trial, the written informed consent form should be signed and 
personally dated by the patient and by the person who conducted the informed consent discussion. 
 
17.2  Registration Procedures 
Patients may not begin protocol treatment prior to registration. All patient registrations will be centralized 
through the Research/Nurse Coordinator of this study at the Institute for Drug Development at the Mays 
Cancer Center of the UT Health San Antonio. Please contact the assigned UT Health San Antonio Mays 
Cancer Center Research/Nurse Coordinator for questions regarding patient registration procedures.   
 
17.3  Access to REDCAP  

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Enrolled study patients will be recorded in the web-based application REDCAP. Please note that a 
username and password is required to use this program and will be provided prior to training on the 
REDCAP system.   
 
17.4  Patient Referrals to the Study 
For potential patients please email the Nurse/Research Coordinator prior to consenting a patient to 
determine whether a slot is available on the study.  In order for patient referrals to be processed 
efficiently, study teams are asked to inform the Nurse/Research Coordinator of any enrollment deadline 
date, if any. 
 
Once a patient is referred for possible study enrollment, the treating physician will be asked to review, 
sign and date the study Eligibility Checklist as verification that the patient has met all inclusion/exclusion 
criteria.  It will be the responsibility of the treating physician to issue informed consent to the patient.    
 
17.5  Data Management and Monitoring 
This study will be monitored as an Investigational Initiated study, which requires auditing after the first 
patient has completed 1 cycle of treatment at each site and at least an annual audit, as outlined in the 
Mays Cancer Center’s DSMP.  Please refer to Appendix 2 for more specific details on the monitoring plan.  
Data submission requirements can be found in Appendix 3. 
 
17.6  Adherence to the Protocol 
Except for an emergency situation in which proper care for the protection, safety, and well-being of the 
study patient requires alternative treatment, the study shall be conducted exactly as described in the 
approved protocol.  
 
17.7  Emergency Modifications 
Investigators may implement a deviation from, or a change of, the protocol to eliminate an immediate 
hazard(s) to trial subjects without prior IRB approval. 
  
For any such emergency modification implemented, the coordinating site must be notified of the event 
within five business days of making the change.  
 
17.8  Other Protocol Deviations 
According to the IRB, a protocol deviation is any unplanned variance from an IRB approved protocol that:  
1. Is generally noted or recognized after it occurs 
2. Has no substantive effect on the risks to research participants 
3. Has no substantive effect on the scientific integrity of the research plan or the value of the data 
collected  
4. Did not result from willful or knowing misconduct on the part of the investigator(s).  
 
A protocol deviation is considered an instance of promptly reportable event if the occurrence: 
1. Has harmed or increased the risk of harm to one or more research participants. 
2. Has damaged the scientific integrity of the data collected for the study. 
3. Results from willful or knowing misconduct on the part of the investigator(s). 
4. Demonstrates serious or continuing noncompliance with federal regulations, State laws, or University 
policies. 
 
17.9  Amendments to the Protocol  

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Should amendments to the protocol be required, the amendments will be originated and documented by 
the Principal Investigator. It should also be noted that when an amendment to the protocol substantially 
alters the study design or the potential risk to the patient, a revised consent form might be required.  The 
written amendment, and if required the amended consent form, must be sent to the IRB for approval 
prior to implementation.  
 
17.10  Record Retention 
Study documentation includes all Case Report Forms, data correction forms or queries, source documents, 
Sponsor-Investigator correspondence, monitoring logs/letters, and regulatory documents (e.g., protocol 
and amendments, IRB correspondence and approval, signed patient consent forms). 
 
Source documents include all recordings of observations or notations of clinical activities and all reports 
and records necessary for the evaluation and reconstruction of the clinical research study. 
 
Government agency regulations and directives require that the study investigator must retain all study 
documentation pertaining to the conduct of a clinical trial.  
 
17.11  Obligations of Investigators 
The Principal Investigator is responsible for the conduct of the clinical trial at the site in accordance with 
Title 21 of the Code of Federal Regulations and/or the Declaration of Helsinki. The Principal Investigator 
is responsible for personally overseeing the treatment of all study patients. The Principal Investigator must 
assure that all study site personnel, including sub-investigators and other study staff members, adhere to 
the study protocol and all FDA/GCP/NCI regulations and guidelines regarding clinical trials both during and 
after study completion. 
 
The Principal Investigator at each institution or site will be responsible for assuring that all the required 
data will be collected and entered onto the Case Report Forms. Periodically, monitoring visits will be 
conducted and the Principal Investigator will provide access to his/her original records or de identified 
documents to permit verification of proper entry of data. At the completion of the study, all case report 
forms will be reviewed by the Principal Investigator at each site and will require his/her final signature to 
verify the accuracy of the data. 
 
17.12  Publication Policy 
All potential publications and/or data for potential publications (e.g. manuscripts, abstracts, posters, 
clinicaltrials.gov releases) must be approved in by the PI of this study prior to release. If the investigator’s 
wish to obtain monitored data prior to this point (or prior to the point dictated by study design), the 
investigator must send a written request for data to the PI of this study that includes justification. If the 
request is approved, data will be provided no later than 4 weeks after this request approval. The 
investigators are expected to use only monitored, accurate and approved data in all publications. The 
investigators should submit a copy of the manuscript to the biostatistician to confirm that approved data 
are used appropriately.  Once the biostatistician gives final approval, the manuscript may be submitted to 
external publishers. 
 
17.13  Pathology Requirements 
Biopsy proven disease by confirmation of biopsy specimen at the treating institution pathology 
department is required for enrollment to the study. Biopsy material will be reviewed by the pathology 
department at the treating institution.   
 

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18.  SAMPLES FOR BIOMARKER EVALUATION 
 
Fresh tissue will be collected from patients with DCIS, LCIS, ALH or ADH treated with rapamycin. Tissue 
will be processed for IHC staining of p16, COX2, and Ki-67 in the core biopsy following the procedures 
described by Kerlikowske et al (66). We will also do IHC for autophagy markers, Beclin1 and LC3, and 
TUNEL assay to determine how rapamycin treatment may alter autophagy and apoptosis in the DCIS 
lesions. 
 
To determine the effect of short-term rapamycin treatment on histopathological biomarkers in DCIS. A 
pathologist will quantify histopathological parameters including lesion size, nuclear grade, and presence 
of necrosis in each patient’s surgical specimens, as well as IHC for the markers including p16, COX2, and 
Ki-67. 
 
Three specific parameters will be evaluated:  
 
1. Luminal-to-basal epithelial ratio, which will examine the MaSC in both luminal and basal cells, and 
their ratio, in order to address whether this ratio will change during aging; 
2. Basal and luminal stem/progenitor cell frequency, which will examine the percentage of cells that are 
capable forming spheres, and whether these frequencies will change during aging; and 
3. 3D structure regeneration frequency in serial passages, which will examine the self-
renewal/regeneration capacity of the stem and progenitor cells that are capable of forming spheres 
in suspension culture and in 3D-ECM culture, and can re-form the spheres when the sphere-forming 
cells are dissociated and allowed to regenerate spheres. 
 
All fresh tissue specimens will be delivered to Dr LuZhe Sun’s laboratory located at: 
 
UT Health San Antonio 
7703 Floyd Curl Drive, Mail Code 7762 
San Antonio, TX  78229-3900  
Phone: 210-567-5746 
 
 
 

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APPENDIX 1 – Common Toxicity Criteria for Adverse Events 
 
Toxicity will be graded according to the NCI’s Common Toxicity Criteria for Adverse Events (CTCAE) version 
4.03.  The CTCAE version 4.03 can be accessed at the following link: 
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf 
 
 
 

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APPENDIX 2 – Data Safety & Monitoring Plan 
 
a.  Data and Safety Monitoring Oversight 
A Data and Safety Monitoring Plan is required for all individual protocols conducted at UT Health San 
Antonio Mays Cancer Center. All protocols conducted at UT Health San Antonio Mays Cancer Center are 
covered under the auspices of the UT Health San Antonio Mays Cancer Center Institutional Data Safety 
Monitoring Plan (DSMP).   
 
The UT Health San Antonio Mays Cancer Center Institutional DSMP global policies provide individual 
trials with:  
1. institutional policies and procedures for institutional data safety and monitoring,  
2. an institutional guide to follow, 
3. monitoring of protocol accrual by the UT Health San Antonio Mays Cancer Center Protocol Review 
Committee, 
4. review of study forms and orders by the Forms Committee, 
5. tools for monitoring safety events, 
6. independent monitoring and source data verification by the UT Health San Antonio Mays Cancer 
Center QA Monitor/Auditor 
7. monitoring of UPIRSO’s by the Director of Quality Assurance and DSMC, 
8. determining level of risk (Priority of Audit Level Score – PALS),  
9. oversight by the Data Safety Monitoring Committee (DSMC), and 
10. Verification of protocol adherence via annual audit for all Investigator Initiated Studies by the UT 
Health San Antonio Mays Cancer Center Quality Assurance Division. 
 
b.  Monitoring Progress and Safety 
Due to the risks associated with participation in this protocol, the UT Health San Antonio Mays Cancer 
Center DSMB #2 in conjunction with the Principal Investigator will perform assessment of adverse events, 
adverse event trends and treatment effects on this study.  The UT Health San Antonio Mays Cancer Center 
DSMB #2 acts as an independent Data Safety Monitoring Board (DSMB) for IIS conducted at UT Health San 
Antonio Mays Cancer Center.  The UT Health San Antonio Mays Cancer Center DSMB #2 will monitor data 
throughout the duration of a study to determine if continuation of the study is appropriate scientifically 
and ethically.  An additional layer of review is provided by the UT Health San Antonio Mays Cancer Center 
Data Safety Monitoring Committee (DSMC) who will review DSMB quarterly reports. Monitoring will be 
conducted every 12 months from the initiation of the study until the last enrolled patient has had the end 
of study visit.  
 
Baseline adverse events and serious adverse events will be captured using the UT Health San Antonio 
Mays Cancer Center Master Adverse Events Document (or proper AE tracking mechanism as per Clinical 
Trial Office policy) for each patient using CTCAE V. 4.03 for the grading and attribution of adverse events 
(Appendix 1).  
 
Usage of the UT Health San Antonio Mays Cancer Center Master Adverse Events Document (or proper AE 
tracking mechanism as per Clinical Trial Office policy) centrally documents:  
 
1. the event and grades the seriousness of the event,  
2. if the event was a change from baseline,   
3. the determination of the relationship between the event and study intervention,  
4. if the event was part of the normal disease process, and  

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5. What actions were taken as a result of the event.  
 
c.  Safety Definitions 
 
For this study, the following safety definitions will be applicable: 
Adverse Event Definition:  An adverse event (AE) is defined as any untoward or unfavorable medical 
occurrence in a human subject, including any abnormal sign (for example, abnormal physical exam or 
laboratory finding), symptom, or disease, temporally associated with the subject’s participation in the 
research, whether or not considered related to the subject’s participation in the research  For this study, 
all adverse events will be documented starting with day 7 and ending 30 days after the last dose of study 
drug is received.  
 
Serious Adverse Event Definition:  A serious adverse event (SAE) is defined as any adverse event that: 
 
1. results in death; 
2. is life-threatening (places the subject at immediate risk of death from the event as it occurred); 
3. results in inpatient hospitalization or prolongation of existing hospitalization; 
4. results in a persistent or significant disability/incapacity; 
5. results in a congenital anomaly/birth defect; or 
6. based upon appropriate medical judgment, may jeopardize the subject’s health and may require 
medical or surgical intervention to prevent one of the other outcomes listed in this definition  
 
Unanticipated Problems Involving Risks to Subjects or Others Definition: Unanticipated problem involving 
risk to subjects or others includes any incident, experience or outcome that meets all of the following 
criteria: 
 
unexpected (in terms of nature, severity, or frequency) given (a) the research procedures that are 
described in the protocol-related documents, such as the IRB-approved research protocol and informed 
consent document; and (b) the characteristics of the subject population being studied (note: the 
unfounded classification of a serious adverse event as “anticipated” constitutes serious non-compliance); 
definitely related or probably related to participation in the research; and suggests that the research 
places subjects or others at a greater risk of harm (including physical, psychological, economic, or social 
harm) than was previously known or recognized 
 
d.  Reporting Requirements 
For this study, the Master Adverse Events Documents collected on patients for this protocol will be 
reviewed by the PI on a monthly basis to determine if a serious safety problem has emerged that result in 
a change or early termination of a protocol such as: 
 
1. dose modification, 
2. suspending enrollment due to safety or efficacy, or 
3. termination of the study due to a significant change in risks or benefits.   
 
AE’s and SAE events that occur during clinical trials with or without an Investigational New Drug (IND) 
application are mandatory reports submitted to FDA via Medwatch FDA F3500A within 15 days for events 
that have at least a possible relationship with the drug. 
 
e.  Assuring Compliance with Protocol and Data Accuracy 

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As with all studies conducted at UT Health San Antonio Mays Cancer Center, the PI has ultimate 
responsibility for ensuring protocol compliance, data accuracy/integrity and responding to 
recommendations that emanate from monitoring activities.  The research site will be audited for accuracy 
at the end of the treatment cycle for the first patient enrolled.  Protocol compliance, data accuracy and 
reporting of events is further ensured by an annual audit conducted by the Data Safety Officer, whose 
audit report is shared with the PI, the research team, and will be reviewed by the UT Health San Antonio 
Mays Cancer Center DSMB.  
 
f.  UT Health San Antonio Mays Cancer Center DSMB Membership 
The UT Health San Antonio Mays Cancer Center has two DSMB’s with a primary set of members specific 
to the histology of the study consisting of UT Health San Antonio faculty and staff.  This Protocol is under 
the jurisdiction of DSMB#2 for Solid Tumor Studies.  
 
As per NCI guidelines and to eliminate conflict of interest (financial, intellectual, professional, or 
regulatory in nature), the UT Health San Antonio Mays Cancer Center DSMB specific to this study will not 
treat patients on this protocol.  Usage of the DSMB specific to the histology has been created to ensure 
that experts in that histology are represented on the DSMB assembled for this protocol, but may be 
expanded, at the PI’s discretion, to include other members which may include: 
 
1. experts in the fields of medicine and science that are applicable to the study (if not currently 
represented on the DSMB),  
2. statistical experts,  
3. lay representatives,  
4. multidisciplinary representation, from relevant specialties including experts such as bioethicists, 
biostatisticians and basic scientists, and 
5. others who can offer an unbiased assessment of the study progress.  
 
Additional or alternate membership of in the DSMB is selected by the DSMC chair, in conjunction with the 
PI of this protocol.   
 
g.  UT Health San Antonio Mays Cancer Center DSMB Charter and Responsibilities 
The UT Health San Antonio Mays Cancer Center DSMB will provide information on the membership 
composition, including qualifications and experience to both the UT Health San Antonio IRB and UT Health 
San Antonio Mays Cancer Center PRC for review.  The UT Health San Antonio Mays Cancer Center DSMB 
for this study will act as an independent advisory board to the PI and will report its findings and 
recommendations to the PI, the UT Health San Antonio IRB and the UT Health San Antonio Mays Cancer 
Center DSMC. UT Health San Antonio Mays Cancer Center DSMB reports will utilize the Investigator 
Initiated Study Quarterly DSMB Report Form and meetings will occur on a monthly basis to review any 
updates from the prior meeting. 
 
Once the protocol is activated, if not already established elsewhere in the protocol the UT Health San 
Antonio Mays Cancer Center DSMB will establish and provide:  
1. procedures for maintaining confidentiality;  
2. statistical procedures including monitoring guidelines, which will be used to monitor the identified 
primary, secondary, and safety outcome variables; 
3. consider factors external to the study when relevant information becomes available, such as scientific 
or therapeutic developments that may have an impact on the safety of the participants or the ethics 
of the study;  

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4. plans for changing frequency of interim analysis as well as procedures for recommending protocol 
changes; 
5. recommendation of dose escalation, MTD recommendation of early termination based on efficacy 
results;  
6. recommendation of termination due to unfavorable benefit-to-risk or inability to answer study 
questions;  
7. recommendation of continuation of ongoing studies;  
8. recommend modification of sample sizes based on ongoing assessment of event rates; and 
9. review of the final results and publications. 
 

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APPENDIX 3 – Data Collection & Submission 
 
Study-specific instructions regarding the entry and submission of data using paper CRFs or eCRFs using an 
electronic data system (e.g. REDCap), will be provided at the time of training prior to study activation.  
UT Health San Antonio Mays Cancer Center compliance policies of the Cancer Therapy & Research Center 
at the UT Health San Antonio DSMP regarding data collection and submission will be strictly enforced. 
 
a.  Research Chart 
In addition to the regular hospital chart, a separate patient research folder will be kept which includes the 
patient's signed, dated informed consent document. 
Case Report Forms to be submitted within 5 days of patient’s first dose of study treatment: 
• 
Master Adverse Event Form (or proper AE tracking mechanism as per Clinical Trial Office policy): 
Update to reflect the adverse events that are present at baseline. 
 
Case Report Forms to be submitted no later than 1 week after End of Treatment Visit: 
• 
Master Adverse Events Form (or proper AE tracking mechanism as per Clinical Trial Office policy): All 
AEs should be finalized within 30 days of last dose 
 
Case Report Forms to be completed during follow-up: 
• 
Master Adverse Events Form (or proper AE tracking mechanism as per Clinical Trial Office policy): 
Reflecting 3-month toxicity status 
 
 
 

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APPENDIX 4 – List of prohibited strong CYP3A4 inducers and inhibitors  
 
Category  
Drug Name 
Strong CYP3A Inhibitors 
Boceprevir, Clarithromycin, Cobicistat, Conivaptan, Danoprevir, 
Grapefruit juice (citrus paradisi fruit juice, 240 mL TID), Elvitegravir, 
Idealisib, Indinavir, Itraconazole, Ketoconazole, LCL161, Lopinavir, 
Mibefradil, Nefazodone, Nelfinavir, Posaconazole, Ritonavir, Saquinavir, 
Telaprevir, Telithromycin, Tipranavir, Troleandomycin, 
Voriconazole 
Strong CYP3A Inducers 
Avasimibe1,2, Carbamazepine, Enzalutamide, Mitotane, Phenobarbital, 
Phenytoin, Rifabutin, Rifampin (Rifampicin)2, St. John's wort (hypericum 
perforatum)2 
 
 
 

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