Comprehensive Single-Cell Transcriptional Analysis of Aromatase Inhibitor-Resistant Breast Cancer
Conditions: Breast Adenocarcinoma, Invasive Breast Carcinoma, Resectable Breast Carcinoma
Sponsor: Mayo Clinic

 
 
 
 
 
21-003046 
 
 
Comprehensive Single-Cell Transcriptional Analysis of Aromatase 
Inhibitor-Resistant Breast Cancer 
 
 
NCT05447910 
 
 
Document Date: 12/01/2022 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 1 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
 
 
Comprehensive Single-Cell Transcriptional Analysis of Aromatase Inhibitor-Resistant 
Breast Cancer 
 
Regulatory Sponsor: 
Saranya Chumsri, M.D. 
Mayo Clinic Florida 
4500 San Pablo Rd 
Jacksonville, FL 32224 
904/953-0707 
Chumsri.saranya@mayo.edu  
 
Co-investigators 
Sunil Krishnan, M.D. √ 
Study Product: 
Aromatase inhibitor, Letrozole 
Protocol Number: (IRBe) 
 
IRB 21-003046 
 
 
 
 
 
 
√ Study contributor(s) not responsible for patient care. 
* Grant holder 
 
 
Initial version: 22 April 2022 Version (1.0) 
Revised: 
01 December 2022 Version (1.1) 
 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 2 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
Table of Contents 
 
STUDY SUMMARY ...................................................................................................................................... 4 
1 
INTRODUCTION ................................................................................................................................. 5 
1.1 
BACKGROUND .................................................................................................................................... 5 
1.2 
INVESTIGATIONAL AGENT .................................................................................................................... 7 
2 
STUDY OBJECTIVES .......................................................................................................................... 8 
3 
STUDY DESIGN ................................................................................................................................... 8 
3.1 
GENERAL DESCRIPTION ....................................................................................................................... 8 
3.2 
NUMBER OF SUBJECTS ......................................................................................................................... 9 
3.3 
DURATION OF PARTICIPATION .............................................................................................................. 9 
3.4 
PRIMARY STUDY ENDPOINTS ................................................................................................................ 9 
3.5 
SECONDARY STUDY ENDPOINTS ........................................................................................................... 9 
4 
SUBJECT SELECTION ENROLLMENT AND WITHDRAWAL ........................................................ 9 
4.1 
INCLUSION CRITERIA ........................................................................................................................... 9 
4.2 
EXCLUSION CRITERIA ........................................................................................................................ 10 
4.3 
SUBJECT RECRUITMENT, ENROLLMENT AND SCREENING ....................................................................... 10 
4.4 
EARLY WITHDRAWAL OF SUBJECTS .................................................................................................... 10 
4.4.1 
When and How to Withdraw Subjects ........................................................................................ 10 
4.4.2 
Data Collection and Follow-up for Withdrawn Subjects .............................................................. 11 
5 
STUDY DRUG .................................................................................................................................... 11 
5.1 
DESCRIPTION .................................................................................................................................... 11 
5.2 
TREATMENT REGIMEN ....................................................................................................................... 11 
5.3 
DRUG ACCOUNTABILITY .................................................................................................................... 11 
6 
STUDY PROCEDURES ...................................................................................................................... 11 
6.1 
TEST SCHEDULE ................................................................................................................................ 12 
6.2 
SAMPLE PROCESSING ......................................................................................................................... 12 
6.3 
SAMPLE SIZE DETERMINATION ........................................................................................................... 13 
7 
SAFETY AND ADVERSE EVENTS ................................................................................................... 13 
7.1 
DEFINITIONS ..................................................................................................................................... 13 
7.2 
RECORDING OF ADVERSE EVENTS ....................................................................................................... 15 
7.3 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS .......................................... 15 
7.3.1 
Sponsor-Investigator reporting: notifying the Mayo IRB ............................................................. 15 
8 
DATA HANDLING AND RECORD KEEPING .................................................................................. 17 
8.1 
CONFIDENTIALITY ............................................................................................................................. 17 
8.2 
SOURCE DOCUMENTS ........................................................................................................................ 17 
8.3 
DATA COLLECTION ........................................................................................................................... 17 
9 
STUDY MONITORING ...................................................................................................................... 17 
10 
ETHICAL CONSIDERATIONS ......................................................................................................... 17 
11 
BUDGET ............................................................................................................................................ 18 
11.1 
COSTS CHARGED TO PATIENT ......................................................................................................... 18 
11.2 
TEST TO BE RESEARCH FUNDED ...................................................................................................... 18 
12 
REFERENCES ................................................................................................................................... 18 
 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 3 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
 
List of Abbreviations 
 
 
LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
DSMB 
Data and Safety Monitoring Board 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
 
 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
PHI 
Protected Health Information 
PI 
Principal Investigator 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOP 
Standard Operating Procedure 
 
 
 
 
 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 4 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
Study Summary 
 
Title 
Comprehensive Single-Cell Transcriptional Analysis of Aromatase 
Inhibitor-Resistant Breast Cancer 
Running Title 
Single cell sequencing for aromatase inhibitor-resistant breast cancer. 
Phase 
Pilot 
Subject Participation 
Duration 
2-8 weeks 
Single or Multi-Site  
Single site 
Objectives 
To comprehensively evaluate differences in tumor microenvironment 
subpopulations in AI-sensitive vs. AI-resistant HR+ breast cancer. 
Number of Subjects 
50 patients 
Diagnosis and Main 
Inclusion Criteria 
1. Female ≥ 18 years. 
2. Postmenopausal and suitable to receive aromatase inhibitor as per 
physician’s discretion.  
3. Histologically confirmed un-resected operable invasive 
adenocarcinoma of the breast ≥ 0.5 cm with estrogen receptor (ER) 
and/or progesterone receptor (PR) positive ≥ 10%, and no human 
epidermal growth factor receptor 2 (HER2) amplification or 
overexpression. 
4. Patients must not have received any prior chemotherapy, radiation 
therapy, or endocrine therapy for their current breast cancer. 
Patients who received tamoxifen or raloxifene or another agent for 
prevention of breast cancer may be included. 
Study Product, 
Dose, Route, 
Regimen 
Letrozole 2.5 mg oral daily (standard of care) 
Duration of 
Administration 
2-8 weeks 
Reference therapy 
N/A 
Statistical 
Methodology 
This is a pilot study to evaluate the difference in tumor 
microenvironment subpopulation in AI-sensitive vs. AI-resistant HR+ 
breast cancer. 
 
 
 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 5 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
1 
Introduction 
 
This document is a protocol for a human research study. This study will be carried out in 
accordance with the applicable United States government regulations and Mayo Clinic research 
policies and procedures.  
 
Aromatase inhibitor is currently the cornerstone standard of care endocrine therapy for patients 
with hormone receptor positive breast cancer.  This is a pilot study to evaluate in-depth 
transcriptomic analysis using single cell sequencing technology to evaluate the effects of 
aromatase inhibitor not only in cancer breast cell but also noncancerous cells in the tumor 
microenvironment. 
1.1 
Background 
 
In-depth transcriptomic analysis for each subpopulation in the tumor microenvironment of pre-
treatment and post-treatment tumor samples obtained from AI-sensitive vs. AI-resistant patients 
will be performed using single-nucleus RNA-sequencing (snRNA-seq) (Figure 1). Re-clustering 
of stromal cells into sub-clusters will be undertaken to uncover hidden transcriptomic 
heterogeneity and to identify transcription factor regulation signatures that define these sub-
clusters. Validation of transcriptome data will be performed using qPCR and immunofluorescence 
staining of cultured flow-sorted cells from human tissue sections and immunohistochemistry of 
these sections. We also seek to study the interactions between spatially distinct stromal sub-
clusters and other cells (tumor, immune and endothelial) that will be studied by annotating our 
dataset with curated human ligand-receptor pairs. In turn, this will shed light on the role of stromal 
sub-clusters on modulating immune activation vs. tolerance/evasion via recruitment/exclusion 
and/or (dys)regulation (exhaustion/activation) of specific immune subpopulations including T-
regs, cytotoxic T cells, and tumor-associated macrophages. Furthermore, peripheral blood will also 
be collected at baseline and after AI treatment to evaluate systemic changes of immune response 
after AI in responders vs. nonresponders.  
We had experience working with patient 
samples with triple-negative breast cancer 
(TNBC). The purpose of the study is to show 
the impact of different combinations of 
chemotherapeutics on tumor microenvironment 
remodeling in bad responders (chemotherapy-
resistant). Trichrome staining (a staining 
method to detect total collagen) was performed 
on both biopsies (before treatment) and surgical 
specimens (after treatment), which were 
obtained from the same patients, to compare the 
collagen expression level. Our findings showed 
that 
chemotherapeutics 
cause 
fibrosis, 
represented by higher collagen expression in 
the surgical specimens in comparison to 
collagen expression in biopsies (Figure 2) (1,2).  
Figure 1. The schematic representations of the 
study: The collection of pre-treated biopsies from 
breast cancer patients with estrogen receptor positive 
(ER+) and human epidermal growth receptor 2 negative 
(HER2-) is shown. The obtained surgical specimens 
from good responders and bad responders (progressors) 
will be analyzed and compared with pre-treated 
samples. All analyses will be carried out through single 
nucleus RNA-sequencing. 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 6 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
 
In 
another 
study, 
we aimed 
to 
analyze 
the 
fibroblast 
subpopulations in doxorubicin (dox) sensitive vs. resistant murine TNBC models. For this study, 
doxorubicin-resistant murine triple-negative 4T1 cells were developed and inoculated into a mouse 
mammary fat pad. Tumors were taken, fibroblasts were isolated and analyzed with 10x genomics 
single-cell RNA-sequencing. Our findings showed that the fibroblasts which were isolated from 
dox-resistant tumors activated overexpression of collagen-related genes (Figure 3). 
 
There have been several studies addressing the issues on the impact of chemotherapeutics on 
different cell populations in breast tumors (3, 4). The amount of infiltrated immune cell 
populations into breast tumors, for example, has been strongly related to the response to 
chemotherapy (5). However, not much is known about the impact of AIs on the cell populations 
of breast tumors. A very appreciated effort from Miller and colleagues reported their findings on 
patients who developed letrozole resistance (6). This study, however, was unfortunately limited 
by bulk RNA-based gene expression profiling.  
 
In our project, by the help of single nuclei RNA-sequencing, we will be able to analyze the 
expression profile of the different type of tumor cell populations before and after letrozole 
treatment. This will provide an intensive analysis identifying novel markers predicting for response 
and understanding molecular mechanisms of resistance.     
 
Figure 2. Trichrome staining of samples obtained from disease progressor patients 
with TNBC: Representative images show that different combination chemotherapy causes 
an increase in collagen content in the tumor microenvironment (*p<0.01).    
 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 7 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
 
Figure 3. Development of doxorubicin resistant 4T1 cells and the gene expression profile of fibroblasts isolated 
from resistant 4T1 tumor: The development of doxorubicin resistant 4T1 cells from sensitive cells through stepwise 
dose increase and schematic explanation of resistant cancer cells are shown (a). Representative microscopy images 
demonstrate p-glycoprotein (red) expression and intracellular uptake of doxorubicin (red) in sensitive and resistant 
4T1 cells (b). The schematic representation shows inoculation of sensitive and resistant 4T1 cells into mammary fat 
pad  (c). Similar growth rate of sensitive and resistant tumors is shown (d). The analysis of different cell populations 
in sensitive and resistant tumors is shown (e). Four clusters, 0; epithelial cells, 1; fibroblasts, 2; macrophages and 3; 
endothelial cells are shown (f). Selected top 10 genes in cell clusters are shown (f). The heat-map analysis of top 10 
genes for each cluster is shown (h)These results show that the fibroblasts overexpress extracellular matrix related 
protein, such as collagens, which can cause fibrosis (i).     
1.2 
Investigational Agent 
 
Letrozole is an aromatase inhibitor, which is a commonly used endocrine therapy for breast 
cancer as per standard of care.  Letrozole is an oral agent commonly supplied as a tablet. 
 
 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 8 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
2 Study Objectives 
 
Our approach is to study the impact of AIs on not only cancerous cells but also on noncancerous 
cells in the tumor microenvironment. We seek to characterize factors discriminating non-
responders from responders to AI early during treatment by analyzing different cells. We also aim 
to work on the impact of AIs on the immune landscape, as a noncancerous cell subpopulation, of 
AI-sensitive and AI-resistant tumors. This, we believe, will contribute to the current knowledge to 
improve the therapeutic efficacy of current treatment modalities, including chemotherapy and 
immunotherapy. Accordingly, we hypothesize that snRNA-seq can uncover hidden transcriptomic 
heterogeneity and underlying preexisting mechanisms driving AI resistance when analyzed at the 
single-cell level. Moreover, our research will also shed more light on the impact of estrogen 
deprivation on the immune landscape with AI treatment. 
Primary Objective 
 
1. To comprehensively evaluate differences in tumor microenvironment subpopulations in 
AI-sensitive vs. AI-resistant HR+ breast cancer. 
 
Secondary Objective 
 
1. To assess the effects of estrogen deprivation on the systemic immune response. 
2. To assess the effects of estrogen deprivation on tumor immune microenvironment. 
 
3 Study Design 
 
This is a pilot study to evaluate the effects of aromatase inhibitor using single cell sequencing 
technology to evaluate both cancerous cells and tumor microenvironment. 
3.1 
General Description 
 
This is a prospective study to collect tissue samples from patients with early-stage hormone 
receptor-positive HER2-negative breast cancer.  Patients with newly diagnosed hormone receptor-
positive HER2-negative invasive breast cancer will be approached by the study team prior to 
surgery. If there is no indication for neoadjuvant therapy and the patient can proceed with surgery, 
the patient will be registered and start on aromatase inhibitor with letrozole 2.5 mg oral daily for 
2-8 weeks until surgery as part of their standard of care. Both archival biopsy and surgical 
specimens will also be collected for single-cell sequencing.  For patients who screen fail and do 
not consent to future research, tissue and blood will be discarded. 
 
Peripheral blood prior to and after letrozole treatment will also be collected. 
 
Estrogen receptor, progesterone receptor, HER2, and Ki-67 will be evaluated in both biopsy and 
surgical specimens as per standard of care. Tumors with persistent elevation of Ki-67 ≥ 10% after 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 9 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
2-8 weeks of letrozole will be classified as non-responders, and tumors with Ki-67 < 10% after 
treatment will be classified as responders. 
 
 
Registration 
Histologically confirmed 
ER/PR+ HER2- breast cancer 
 
 
↓ 
 
 
Treatment 
Letrozole 2-8 weeks 
 
 
↓ 
 
 
Surgery 
 
 
↓ 
 
 
At any time: 
Disease Progression 
Intolerable adverse effects 
Patient refusal 
 
 
↓ 
 
 
Off study 
 
3.2 
Number of Subjects 
  
Up to 50 patients 
3.3 
Duration of Participation 
 
2-8 weeks 
3.4 
Primary Study Endpoints 
 
To comprehensively evaluate differences in tumor microenvironment subpopulations in AI-
sensitive vs. AI-resistant HR+ breast cancer. 
3.5 
Secondary Study Endpoints 
 
1. To assess the effects of estrogen deprivation on the systemic immune response. 
2. To assess the effects of estrogen deprivation on tumor immune microenvironment. 
 
4 Subject Selection Enrollment and Withdrawal 
4.1 
Inclusion Criteria 
 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 10 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
Screening inclusion criteria 
1. Female ≥ 18 years. 
2. Postmenopausal and suitable to receive aromatase inhibitor as per physician’s 
discretion. 
3. Histologically confirmed un-resected operable invasive adenocarcinoma of the breast 
≥ 0.5 cm with estrogen receptor (ER) and/or progesterone receptor (PR) positive ≥ 10%, 
and no human epidermal growth factor receptor 2 (HER2) amplification or 
overexpression. 
4. Patients must not have received any prior chemotherapy, radiation therapy, or 
endocrine therapy for their current breast cancer. Patients who received tamoxifen or 
raloxifene or another agent for prevention of breast cancer may be included. 
5. Willing and able to provide research tissue samples. 
6. Willing and able to provide research blood samples. 
4.2 
Exclusion Criteria 
1. Immunocompromised patients including patients known to be HIV positive or those on 
chronic steroids. 
NOTE: Must be off systemic steroids at least 14 days prior to pre-registration. However, 
topical steroids, inhalants or steroid eye drops are permitted. 
2. Known history of active autoimmune disease that has required systemic treatment 
within ≤ 30 days (i.e., with use of disease modifying agents, corticosteroids, or 
immunosuppressive drugs) prior to pre-registration.  
NOTE: Replacement therapy (e.g., thyroxine, insulin, or physiologic corticosteroid 
replacement therapy for adrenal or pituitary insufficiency) is not considered a form of 
systemic treatment. Patients with vitiligo, Graves disease, or psoriasis not requiring 
systemic treatment within the past 30 days are not excluded. Patients with Celiac disease 
controlled with diet modification are not excluded. 
4.3 
Subject Recruitment, Enrollment and Screening 
 
This is a prospective study to collect tissue samples from patients with early-stage hormone 
receptor-positive HER2-negative breast cancer. Patients with newly diagnosed hormone receptor-
positive HER2-negative invasive breast cancer will be approached by the study team prior to 
surgery. Patients will be screened by a study team member through provider list or schedule list. 
Patients will be contacted preferably via a phone call prior to their visit or in person at the time of 
their visit. Patients will be consented either electronically remotely via email or in person at the 
time of their visit by a study team member.  
4.4 
Early Withdrawal of Subjects 
4.4.1 When and How to Withdraw Subjects 
 
Patients will be withdrawn from the study prior to completing all study related procedures if: 
- 
Disease progression 
- 
Intolerable adverse effects 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 11 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
- 
Patient decision to withdraw from the study 
- 
Failure to adhere to protocol requirements, including refusal of surgery and 
noncompliance to letrozole ≥ 5 consecutive days.  
4.4.2 Data Collection and Follow-up for Withdrawn Subjects 
 
A patient is deemed a withdrawal if he/she is removed from the study for any reason before any 
study treatment is given. On-study material and the End of Active Treatment/Cancel Notification 
Form must be submitted. No further data submission is necessary. 
 
5 Study Drug  
5.1 
Description 
 
Letrozole is a standard of care aromatase inhibitor used to treat hormone receptor positive breast 
cancer. 
5.2 
Treatment Regimen 
 
Letrozole 2.5 mg oral daily dose will be used for 2-8 weeks prior to surgery.  
5.3 
Drug Accountability 
 
Drug accountability will be performed via patient pill diaries and patient appointments.  
 
 
6 Study Procedures 
 
This is a prospective study to collect tissue samples from patients with early-stage hormone 
receptor-positive HER2-negative breast cancer.  Patients with newly diagnosed hormone receptor-
positive HER2-negative invasive breast cancer will be approached by the study team prior to 
surgery.  Once pathology confirms the diagnosis for hormone receptor-positive HER2 negative 
invasive breast cancer, the patient will be evaluated by breast surgeons. If there is no indication 
for neoadjuvant therapy and the patient can proceed with surgery within 2-8 weeks, the patient 
will be considered registered and start on aromatase inhibitor with letrozole 2.5 mg oral daily for 
2-8 weeks until surgery as part of their standard of care. Patients will take letrozole until within 48 
hours prior to surgery. Both archival biopsy and surgical specimens will also be collected for 
single-cell sequencing.  
 
Peripheral blood will be drawn at two timepoints: 1.) prior to letrozole treatment and 2.) at least 
two weeks after the start of letrozole treatment, but prior to surgery. 
 
Estrogen receptor, progesterone receptor, HER2, and Ki-67 will be evaluated in both biopsy and 
surgical specimen as per standard of care. Tumors with persistent elevation of Ki-67 ≥ 10% after 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 12 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
2-8 weeks of letrozole will be classified as non-responders, and tumors with Ki-67 < 10% after 
treatment will be classified as responders. 
 
This study is not utilizing the Cancer Center Registration Application. Patients will be considered 
enrolled upon completion of the ICF in PTrax. However, patients will not be considered registered 
within the Cancer Center until they have met all eligibility criteria and have a status of accrued in 
PTrax. 
6.1 
Test Schedule 
 
Tests and Procedures 
Screening 
Baseline 
(Day 1 of 
Letrozole) 
After 2-8 
weeks of 
Letrozole 
Surgery2 
Visit Window 
-30 days  
from 
baseline 
-7 days 
 
 
Standard of Care Procedures 
Consent 
X 
 
 
 
History and Physical 
 
X 
 X5 
 
 
Adverse event assessment 
 
X 
 X5 
 
 
Tissue collection 
X1 
 
 
X1 
Research Procedures 
Blood samples 
- Sodium heparin (green top) 
10 mL x 6 tubes 
- Serum (red top) 10 mL x 1 
tube 
 
X3 
X3 
 
 
 
1 Archival sample collection from the standard of care biopsy and surgical resection at least 5 x 5 uM slides will be 
collected.  
2 Surgery should occur less than 48 hours after the last dose of letrozole.  
3 Completed at two timepoints: 1.) prior to letrozole treatment and 2.) at least two weeks after the start of letrozole 
treatment, but prior to surgery. 
 
6.2 
Sample Processing 
 
All biopsy and surgical specimens will be snap frozen by BAP/PRC or CRC team. Samples will 
be kept at -80oC and held for pick up.  
 
Blood samples will be processed by BAP/PRC. PBMC, serum, and plasma will be aliquoted, kept 
at -800C, and held for pick up.   
 
 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 13 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
Statistical Plan 
6.3 
Sample Size Determination 
 
This is a pilot study to evaluate the difference in tumor microenvironment subpopulation in AI-
sensitive vs. AI-resistant HR+ breast cancer. Therefore, power calculation will not be required.  
Statistical Methods 
 
Secondary analysis: Sequenced data will go through secondary analysis in department of 
bioinformatics in Mayo Clinic Rochester campus. The estimated duration is 3 weeks for this 
analysis. 
 
Tertiary analysis: The last step of data analysis will be performed by myself and our collaboration 
partners of Dr. Daniel Wickland and Dr. Xue Wang. 
 
Endpoints 
 
Specific Aim 1: To comprehensively evaluate differences in tumor microenvironment 
subpopulations in AI-sensitive vs. AI-resistant HR+ breast cancer.  
 
Specific Aim 2: To assess the effects of estrogen deprivation on the immune microenvironment.  
• Sub Aim 2A: To assess the effects of estrogen deprivation on the systemic immune 
response. 
• Sub Aim 2B: To assess the effects of estrogen deprivation on tumor immune 
microenvironment. 
 
7 Safety and Adverse Events 
7.1 
Definitions 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
Any unanticipated problem or adverse event that meets the following three criteria:  
• Serious: Serious problems or events that results in significant harm, (which may be physical, 
psychological, financial, social, economic, or legal) or increased risk for the subject or 
others (including individuals who are not research subjects). These include: (1) death; (2) 
life threatening adverse experience; (3) hospitalization - inpatient, new, or prolonged; (4) 
disability/incapacity - persistent or significant; (5) birth defect/anomaly; (6) breach of 
confidentiality and (7) other problems, events, or new information (i.e. publications, DSMB 
reports, interim findings, product labeling change) that in the opinion of the local 
investigator may adversely affect the rights, safety, or welfare of the subjects or others, or 
substantially compromise the research data, AND 
• Unanticipated: (i.e. unexpected) problems or events are those that are not already described 
as potential risks in the protocol, consent document, not listed in the Investigator’s 
Brochure, or not part of an underlying disease. A problem or event is "unanticipated" when 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 14 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
it was unforeseeable at the time of its occurrence. A problem or event is "unanticipated" 
when it occurs at an increased frequency or at an increased severity than expected, AND 
• Related: A problem or event is "related" if it is possibly related to the research procedures. 
 
Adverse Event 
An untoward or undesirable experience associated with the use of a medical product (i.e. drug, 
device, biologic) in a patient or research subject. 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.  Serious problems/events can be well 
defined and include; 
• death 
• life threatening adverse experience 
• inpatient hospitalization or prolongation of existing hospitalization 
• persistent or significant disability or incapacity 
• substantial disruption of the ability to conduct normal life functions 
• birth defect/congenital anomaly 
 
and/or per protocol may be problems/events that in the opinion of the sponsor-investigator 
may have adversely affected the rights, safety, or welfare of the subjects or others, or 
substantially compromised the research data. 
 
All adverse events that do not meet any of the criteria for serious, should be regarded as non-
serious adverse events.  
 
Adverse Event Reporting Period 
For this study, the study treatment follow-up period is defined as 2-8 weeks while on letrozole up 
until surgery, or until early discontinuation of letrozole. The study period during which adverse 
events must be reported is normally defined as the period from the initiation of any study 
procedures to the end of the study treatment follow-up.   
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization at least possibly 
related to the study should be documented and reported as a serious adverse event unless 
specifically instructed otherwise in this protocol.  Any condition responsible for surgery should be 
documented as an adverse event if the condition meets the criteria for an adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an 
adverse event and are not considered to be SAEs in the following circumstances: 
• Planned hospitalizations required by the protocol, including breast biopsy and surgery. 
• Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures 
for a preexisting condition.  Surgery should not be reported as an outcome of an adverse 
event if the purpose of the surgery was elective or diagnostic and the outcome was 
uneventful 
• Hospitalization for elective procedures unrelated to the current disease and/or treatment on 
this trial 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 15 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
• Hospitalization for insertion of access for administration of a drug 
• Hospitalization for routine maintenance of a device (e.g., battery replacement) that 
was in place before study entry 
• Hospitalization, or other serious outcomes for signs and symptoms of progression of cancer. 
7.2 
Recording of Adverse Events 
Adverse events will be assessed at baseline and after at least 2-8 weeks of Letrozole.  The patient 
will be contacted by the coordinator via phone call, if not seen in clinic, to assess adverse events, 
particularly: hot flashes and arthralgia (joint pain).  If patient reports ≥ grade 3 adverse events, the 
study coordinator will notify the provider to follow-up with the patient within 2 business days to 
provide further guidance.  In case of an emergency, patient will be instructed to seek immediate 
medical attention and go to the emergency room, and the study coordinator will notify the provider 
immediately. 
 
 
All adverse events at least possibly related occurring during the study period must be recorded.  
The clinical course of each event should be followed until resolution, stabilization, or until it has 
been ultimately determined that the study treatment or participation is not the probable cause.  
Serious adverse events at least possibly related that are still ongoing at the end of the study period 
must be followed up by the coordinator via phone call, if not seen in clinic, to determine the final 
outcome or 30 days after last dose of letrozole.  Any serious adverse event that occurs during the 
Adverse Event Reporting Period and is considered to be at least possibly related to the study 
treatment or study participation should be recorded and reported according to the Mayo IRB Policy 
and Procedures. 
 
CTCAE Version 5.0 (v5.0: November 27, 2017) 
https://ctep.cancer.gov/protocoldevelopment/electronic_applications/ctc.htm will be used.     
7.3 
Reporting of Serious Adverse Events and Unanticipated Problems 
When an adverse event has been identified, the study team will take appropriate action necessary 
to protect the study participant and then complete the Study Adverse Event Worksheet and log.  
The sponsor-investigator will evaluate the event and determine the necessary follow-up and 
reporting required. 
7.3.1 Sponsor-Investigator reporting: notifying the Mayo IRB 
The sponsor-investigator will report to the Mayo IRB any UPIRTSOs and Non-UPIRTSOs at least 
possibly related to the study according to the Mayo IRB Policy and Procedures. 
 
 
Information below will be collected:  
• Subject’s name:  
• Medical record number:  
• Disease/histology (if applicable):  
• The date the adverse event occurred:  
• Description of the adverse event:  
• Relationship of the adverse event to the research (drug, procedure, or intervention*):  

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 16 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
• If the adverse event was expected:  
• The severity of the adverse event: (use a table to define severity scale 1-5**) 
• If any intervention was necessary:  
• Resolution: (was the incident resolved spontaneously, or after discontinuing treatment) 
• Date of Resolution:  
 
The sponsor-investigator will review all adverse event reports to determine if specific reports 
need to be made to the IRB .  The sponsor-investigator will sign and date the adverse event 
report when it is reviewed.  For this protocol, only SAEs/UPIRTSOs at least possibly related to 
the study will be reported to the IRB. 
 
The relationship of an AE to the Investigational Drug is a clinical decision by the sponsor-
investigator (S-I) based on all available information at the time of the completion of the form and 
is graded as follows: 
 
1. Not related: a reaction for which sufficient information exists to indicate the etiology is 
unrelated to the study drug; the subject did not receive the study medication or the temporal 
sequence of the AE onset relative to administration of the study medication is not reasonable or 
the event is c1early related to other factors such as the subject' s clinical state, therapeutic 
intervention or concomitant therapy. 
 
2. Unlikely: a clinical event, including laboratory test abnormality, with a temporal relationship 
to drug administration which makes a causal relationship improbable and in which other drugs, 
chemicals, or underlying disease provide plausible explanations. 
 
3. Possible: a clinical event, including laboratory test abnormality, with a reasonable time 
sequence to administration of the drug but which could also be explained by concurrent disease 
or other drugs or chemicals; information on drug withdrawals may be lacking are unclear. 
 
4. Probable: a clinical event including laboratory test abnormality, with a reasonable time 
sequence to administration of the drug, unlikely to be attributed to concurrent disease or other 
drugs or chemicals and which follows a clinically reasonable response on withdrawal (de-
challenge): re-challenge information is not required to fulfill this definition. 
 
5. Definite: a reaction that follows a reasonable temporal sequence from administration of the 
drug, or in which the drug level has been established in body fluids or tissues, that follows a 
known or expected response pattern to the suspected drug, and that is confirmed by improvement 
on stopping or reducing the dosage of the drug, and reappearance of the reaction on repeated 
exposure (re-challenge). 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 17 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
 
8 Data Handling and Record Keeping 
8.1 
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).  Those 
regulations require a signed subject authorization informing the subject of 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.  For subjects that have revoked authorization to collect or use PHI, attempts should 
be made to obtain permission to collect at least vital status (long term survival status that the 
subject is alive) at the end of their scheduled study period. 
8.2 
Source Documents 
Source data is all information, original records of clinical findings, observations, or other activities 
in a clinical trial necessary for the reconstruction and evaluation of the trial.  Source data are 
contained in source documents.  Examples of these original documents and data records include: 
hospital records, clinical and office charts, laboratory notes, memoranda, subjects’ diaries or 
evaluation checklists, pharmacy dispensing records, recorded data from automated instruments, 
copies or transcriptions certified after verification as being accurate and complete, microfiches, 
photographic negatives, microfilm or magnetic media, x-rays, subject files, and records kept at the 
pharmacy, at the laboratories, and at medico-technical departments involved in the clinical trial. 
8.3 
Data Collection 
This study will use REDCap for data collection.  
 
9 Study Monitoring 
The principal investigator(s) and the study statistician will review the study monthly to identify 
accrual, adverse event, and any endpoint problems that might be developing. 
 
10 Ethical Considerations 
This study is to be conducted according to United States government regulations and Institutional 
research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted local Institutional 
Review Board (IRB), in agreement with local legal prescriptions, for formal approval of the study.  
The decision of the IRB concerning the conduct of the study will be made in writing to the sponsor-
investigator before the commencement of this study. 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 18 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
 
All subjects for this study will be provided a consent form describing this study and providing 
sufficient information for subjects to make an informed decision about their participation in this 
study.  This consent form will be submitted with the protocol for review and approval by the IRB 
for the study.  The formal consent of a subject, using the approved IRB consent form, must be 
obtained before that subject undergoes any study procedure.  The consent form must be signed by 
the subject or the subject’s legally authorized representative, and the individual obtaining the 
informed consent. 
 
11 Budget 
This study is funded through a grant from Mayo Clinic.  
11.1 Costs charged to patient 
- 
Routine clinical care 
- 
Biopsy 
- 
Surgery 
- 
Letrozole 2.5 mg  
11.2 Test to be research funded 
- 
Research blood draw 
- 
Tumor tissue sequencing  
 
12 References 
 
1. Echeverria, G.V., Ge, Z., Seth, S., Zhang, X., Jeter-Jones, S., Zhou, X., Cai, S., Tu, Y., 
McCoy, A., Peoples, M. and Sun, Y., 2019. Resistance to neoadjuvant chemotherapy in 
triple-negative breast cancer mediated by a reversible drug-tolerant state. Science 
translational medicine, 11(488). 
2. Saatci, O., Kaymak, A., Raza, U., Ersan, P.G., Akbulut, O., Banister, C.E., Sikirzhytski, 
V., Tokat, U.M., Aykut, G., Ansari, S.A. and Dogan, H.T., 2020. Targeting lysyl oxidase 
(LOX) overcomes chemotherapy resistance in triple negative breast cancer. Nature 
communications, 11(1), pp.1-17. 
3. Nguyen, Q.H., Pervolarakis, N., Blake, K., Ma, D., Davis, R.T., James, N., Phung, A.T., 
Willey, E., Kumar, R., Jabart, E. and Driver, I., 2018. Profiling human breast epithelial 
cells using single cell RNA sequencing identifies cell diversity. Nature communications, 
9(1), pp.1-12. 
4. Kim, C., Gao, R., Sei, E., Brandt, R., Hartman, J., Hatschek, T., Crosetto, N., Foukakis, T. 
and Navin, N.E., 2018. Chemoresistance evolution in triple-negative breast cancer 
delineated by single-cell sequencing. Cell, 173(4), pp.879-893. 
5. Denkert, C., von Minckwitz, G., Darb-Esfahani, S., Lederer, B., Heppner, B.I., Weber, 
K.E., Budczies, J., Huober, J., Klauschen, F., Furlanetto, J. and Schmitt, W.D., 2018. 
Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a 

 
 
Version 1.1 
 
 
Revised 12/1/22 
Page 19 of 19 
Mayo Clinic-Chumsri 
 
CONFIDENTIAL 
pooled analysis of 3771 patients treated with neoadjuvant therapy. The lancet oncology, 
19(1), pp.40-50. 
6. Miller, W.R., Larionov, A., Renshaw, L., Anderson, T.J., Walker, J.R., Krause, A., Sing, 
T., Evans, D.B. and Dixon, J.M., 2009. Gene expression profiles differentiating between 
breast cancers clinically responsive or resistant to letrozole. J Clin Oncol, 27(9), pp.1382-
1387. 
7. Zheng GXY, Terry JM, Belgrader P, Ryvkin P, Bent ZW, Wilson R, et al. Massively 
parallel digital transcriptional profiling of single cells. Nature Communications. 
2017;8(1):14049 
8. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast 
universal RNA-seq aligner. Bioinformatics (Oxford, England). 2013;29(1):15-21. Epub 
2012/10/25.  
9. Lun ATL, Riesenfeld S, Andrews T, Dao TP, Gomes T, Marioni JC, et al. EmptyDrops: 
distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing 
data. Genome Biol. 2019;20(1):63.  
10. Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM, 3rd, et al. 
Comprehensive Integration of Single-Cell Data. Cell. 2019;177(7):1888-902.e21. Epub 
2019/06/11.  
11. Finak G, McDavid A, Yajima M, Deng J, Gersuk V, Shalek AK, et al. MAST: a flexible 
statistical framework for assessing transcriptional changes and characterizing 
heterogeneity in single-cell RNA sequencing data. Genome Biol. 2015;16:278. 
12. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdóttir H, Tamayo P, Mesirov JP. 
Molecular signatures database (MSigDB) 3.0. Bioinformatics (Oxford, England). 
2011;27(12):1739-40. Epub 2011/05/05.