The Gut Microbiome and Gastrointestinal Toxicities as Determinants of the Response to Neoadjuvant Chemotherapies for Advanced Breast Cancer
Phase: NA
Conditions: Breast Cancer
Sponsor: University of Arkansas

The gut microbiome and gastrointestinal toxicities as determinants of the response to 
neoadjuvant chemotherapies for advanced breast cancer 
 
IRB # 204897 
Version: 5 
06/07/2019 
NCT02696759 
 
 
Principal Investigator: 
Issam Makhoul, MD  
 
Associate Professor and Director, Division of 
Hematology/Oncology 
 
Internal Medicine, College of Medicine, UAMS 
 
 
Sub-Investigators:   
 
Martin J. Cannon, PhD. 
Professor, Department of Microbiology and Immunology  
College of Medicine, UAMS 
 
Sharp Malak, M.D., M.P.H. 
Assistant Professor, Departments of Radiology and 
Epidemiology 
College of Medicine and COPH, UAMS 
 
Letycia Nunez-Argote, M.P.H. C.P.H., M.L.S. (A.S.C.P.) CM 
Assistant Professor, Department of Medical Technology  
College of Health Professions (CHP), UAMS 
 
Donna Dunn, APN 
Division of Hematology/Oncology 
Internal Medicine, College of Medicine, UAMS 
 
Yan Wang, B.S., M.S. 
Doctoral candidate, Bioinformatics 
University of Arkansas at Little Rock (UALR); UAMS 
 
 
Statistician:   
Bob Delongchamps, Ph.D. 
 
Professor, Department of Epidemiology 
 
Fay W. Boozman College of Public Health (COPH) 
 
 
 
 
 
 
  Study Location:   
  
 University of Arkansas for Medical Sciences  
4301 West Markham Street 

The gut microbiome and gastrointestinal toxicities as determinants of the response to neoadjuvant chemotherapies for 
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Little Rock, AR 72205 
 
ABSTRACT 
Many chemotherapeutic agents compromise the integrity of the mucosal barrier in the gut, 
allowing translocation of gram-positive bacteria in secondary lymphoid organs. While this has, 
until recently, been considered an undesirable side-effect, it may also represent one mechanism 
by which chemotherapy stimulates an effective anti-cancer immune response. It has been 
proposed that the priming of Th17 T-cell responses against commensal microbes following 
disturbance of the gut mucosal barrier facilitates the accumulation of Th1 helper T cells at the 
site of the tumor, and thus contributes to tumor regression. Gut microbes may also contribute 
to the efficacy of chemotherapeutic agents through metabolism of the chemotherapeutic agent 
to its active form. Our hypothesis is that gut microbial composition can influence immune 
response to the tumor, and therefore may account for variations among patients in the response 
to anti-cancer therapies.  In preparation for epidemiologic studies of this hypothesis, we propose 
a pilot study of as many as 50 women diagnosed with breast cancer and referred for 
neoadjuvant chemotherapies at the University of Arkansas for Medical Sciences.  Participants 
will provide blood and fecal samples prior to, and immediately following completion of 
neoadjuvant chemotherapies, and will authorize retrieval of archived breast tumor tissues 
collected prior to initiation of chemotherapy.  Microbial content of fecal samples will be 
evaluated based upon 454-pyrosequencing of 16s-rRNA.  Host immune response will be 
assessed by measuring relative abundances of immune cells in blood and in tumor tissue.  
Complete pathologic response will be assessed based upon findings at the time of cancer 
surgery.  The aims of the proposed study include demonstration of the feasibility of study 
measures, and estimation of key parameters including the occurrence of complete pathologic 
response in all participants and in specific subgroups, and the magnitude of associations 
between microbial and clinical measures.     
 
BACKGROUND 
The presence of immune cells in breast tumors was noted as early as the 1970’s and described 
using immunohistochemical staining of fixed tissues [1]. In the 1980’s, scientists at the National 
Institutes of Health observed, in patients whose disseminated breast cancer was treated using 
interleukin-2, that marked infiltration of breast tumors by T lymphocytes occurred following 
treatment and predicted tumor regression [2].  
An effective immune response to precancerous or malignant cells culminates in the destruction 
of abnormal cells by Natural Killer cells and cytotoxic T-lymphocytes [3]. In contrast, 
maladaptive immune responses may contribute to cancer development when chronic local 
inflammation stimulates intrinsic programs of tissue remodeling and angiogenesis [4]. Both 
chronic inflammation and impaired immune 
surveillance may facilitate processes of 
carcinogenesis, invasion and metastasis [5].  Current research suggests that immune factors 
may play important roles in cancer risk and cancer outcomes, by contributing to or preventing 
the pathogenesis of solid tumors [3] and by mediating the effects of diverse cancer therapies 
[6]. Immune markers also represent potentially useful biomarkers of treatment response and/or 
prognosis [7].  
Recent research has highlighted interactions of commensal microbes with the developing 
mucosal immune system as an important modulator of systemic immunity with implications for 

The gut microbiome and gastrointestinal toxicities as determinants of the response to neoadjuvant chemotherapies for 
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growth, development and health [8]. In two recent studies [9, 10] differences were observed in 
treatment efficacies of various therapies, including an immunotherapy and two commonly used 
therapeutic agents, platinum and cyclophosphamide, when they were employed in germ-free 
mice compared to counterparts with an intact gut microbiome.  Based upon this work, 
investigators have suggested that translocation of gram-positive bacteria following disruption of 
the intestinal barrier 
may elicit systemic a Th17-type response, indirectly facilitating 
accumulation of effector Th1 cells at the site of the tumor and contributing to tumor regression 
[9-11]. This has been demonstrated only for a few specific chemotherapeutic agents but many 
chemotherapeutic regimens have gastrointestinal side effects, suggesting that this mechanism 
of action may be generalizable across drug classes [12]. Thus, while GI toxicities have been 
considered as entirely undesirable, they may also represent one mechanism by which 
chemotherapy treats cancer. In addition, gut microbes are also known to play important roles in 
the metabolism of many chemotherapeutic agents to their active forms, which may also 
influence internal dose, tolerability, and outcomes of chemotherapeutic regimens [13, 14].   
Marked alterations of the gut microbiome have been observed in several studies of patients 
when the composition, diversity, and metabolic activities were compared in fecal samples 
collected prior to, and following completion of chemotherapy [15-17].   The changes in gut 
microbial composition may be influenced by the chemotherapeutic regimen, host factors, or 
the stability of the gut microbial population at baseline.  Changes in composition occurring over 
this interval may shed some light on which bacteria translocate and evoke immune responses 
and lasting changes in gut mucosal tolerance.   
Neoadjuvant chemotherapy, in the setting of breast cancer, is defined as a chemotherapeutic 
regimen provided prior to breast surgery.  The specific regimen may differ depending on 
prognostic and predictive factors that include tumor stage and histologic grade; pathologic 
characteristics of the primary tumor including hormone receptor status, HER2 status, and multi-
gene test results; and patient characteristics including age, comorbidities, and menopausal status.  
Neoadjuvant chemotherapy has generally been indicated for women with locally advanced 
invasive breast cancer (2-3 positive axillary nodes or involvement of skin or chest wall). metastatic 
breast cancer, or triple negative breast cancers (ER-/PR- and HER2-).    
More recently, neoadjuvant therapies have been applied in a wider range of breast cancer 
patients since randomized trials have demonstrated that pre- and post-surgical chemotherapy 
have comparable rates of both recurrence-free and overall survival [18].  The major benefit of 
neoadjuvant chemotherapy to patients is increased rates of breast conserving surgery [19].  In 
addition, tumor response to neoadjuvant chemotherapy, measured as achievement of pathologic 
complete response (pCR), provides a surrogate end-point for long-term survival [20-22].  A recent 
single-arm trial of a specific neo-adjuvant chemotherapy regimen included 40 breast cancer 
patients enrolled at UAMS and demonstrated heterogeneous outcomes by both tumor and host 
characteristics [23].    
Among women with breast cancer who undergo neoadjuvant chemotherapy, complete pathologic 
response is a predictor of long-term outcomes across and within many subgroups variously 
defined by intrinsic subtypes, stage, grade, and nodal status [24-26].  In a meta-analysis, Gluck 
and colleagues pointed out an exception; among women with early-stage luminal A breast 
cancers, prognosis was not predicted by pathological complete response [27].   
A recent meta-analysis gathered results from studies of immune infiltrates in breast tumors from 
patients who subsequently underwent neoadjuvant cancer therapies, and its findings confirm that 

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immune infiltrates in pre-treatment breast tissue, and CD8+ infiltrates in particular, are predictive 
of pathologic complete response in many, but not all of the distinct breast cancer subtypes, 
including luminal A breast cancers ; her2+ breast cancers [28]; and basal breast cancers [24].  
CD8+ immune infiltrates have been also observed to predict recurrence-free survival, breast 
cancer specific survival, and overall survival in breast cancer patients treated surgically prior to 
hormonal or chemotherapies [24].  Similar associations have been observed in studies looking at 
early stage breast cancers [25] and advanced breast cancers ; in patients receiving different anti-
cancer therapies, including first-line chemotherapies [26], rescue chemotherapies, immune 
therapies and endocrine therapies; and in studies with different endpoints, including recurrence-
free survival, overall survival and breast cancer specific-mortality.   
We hypothesize that some of the heterogeneity in treatment outcomes may be explained by 
variations in systemic immune response and its determinants, including the gut microbiome.   
Therefore we are proposing a pilot study to evaluate feasibility of the proposed measures and to 
estimate parameters that will aid in planning a larger study designed to test the hypotheses that the 
gut microbiome can modulate the efficacy of neoadjuvant chemotherapies to treat breast cancer. 
 
SPECIFIC AIMS 
Many chemotherapeutic agents compromise the integrity of the mucosal barrier in the gut, 
allowing translocation of gram-positive bacteria in secondary lymphoid organs. While this has, 
until recently, been considered an undesirable side-effect, it may also represent one mechanism 
by which chemotherapy stimulates an effective anti-cancer immune response. It has been 
proposed that the priming of Th17 T-cell responses against commensal microbes following 
disturbance of the gut mucosal barrier facilitates the accumulation of Th1 helper T cells at the 
site of the tumor, and thus contributes to tumor regression. Gut microbes may also contribute 
to the efficacy of chemotherapeutic agents through metabolism of the chemotherapeutic agent 
to its active form. We will test the hypothesis that gut microbial composition can influence 
immune response to the tumor, resulting in inter-individual differences in the response to anti-
cancer therapies.  We will also explore how host factors and microenvironmental factors 
influence the local immune response to the tumor.   
 
We propose the following specific aims: 
1. To demonstrate feasibility and acceptability of protocol procedures and measures, 
including in-home collection of a fecal sample, collection of a blood sample by 
venipuncture, and collection of breast tissue from newly diagnosed breast cancer 
patients prior to initiation of neoadjuvant breast cancer therapy. 
2. To estimate rates of gastrointestinal side effects and pathological complete response in 
women with breast cancer treated with neoadjuvant chemotherapies, by breast cancer 
subtypes, tumor characteristics, cancer treatment, and by categorical measures of gut 
microbial composition, diversity, and metabolic activity. 
3. To estimate the associations of pre-treatment gut microbial composition, diversity, 
and metabolic activity with anticancer immune responses, measured as relative 
abundance of immune infiltrates in pre-treatment FFPE breast tumor tissues, and as 
relative abundance of circulating lymphocytic subtypes (Th1, Th2, Th17, and Treg) 
in blood samples collected prior to initiation and following completion of neoadjuvant 

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chemotherapy. 
4. To estimate associations of local and systemic immune responses with host factors 
such as age, BMI, and smoking status, and with measures of the breast 
microenvironment including mammographic density and benign parenchymal 
enhancement on breast MRI.   
 
Study Population 
We anticipate identification of 60 eligible women among patients diagnosed with breast cancer 
at UAMS and recommended for neoadjuvant therapy over the course of a year and estimate that, 
of these, 40 will consent to participate and complete study assessments.  
Potentially eligible participants will be identified by their treating oncologist in the Medical 
Oncology Clinic.  The study will be presented to potential participants prior to initiation of 
chemotherapy.  We will allow the patient as much time as she needs to think about participation, 
and discuss her concerns with friends, relatives, and professional staff before making a decision. 
Written consent will be requested from patients who meet eligibility criteria. The informed 
consent process will be conducted by study staff. We will not advertise for study participants. 
No compensation will be provided to study participants. 
 
Inclusion Criteria 
Eligible participants are women aged 18 years or older, who are: 
• 
Diagnosed with invasive breast cancer and prescribed a regimen that includes neo-
adjuvant therapy prior to breast surgery. 
• 
Able to provide informed consent. 
 
Exclusion Criteria 
• 
History of any previous malignancy, other than non-melanoma skin cancers 
• 
Inability to tolerate phlebotomy 
• 
Immunosuppressive therapy for any other condition 
• 
Fever or active uncontrolled infection in the last 4 weeks 
• 
Inflammatory bowel disease 
• 
Surgery of the stomach, small or large intestines, appendectomy, gastric bypass or 
gastric banding in the past 6 months.   
• 
Active autoimmune disease, including, but not limited to, SLE, MS, ankylosing spondylitis 
 
Study Design 
We propose a small longitudinal study to demonstrate the feasibility of measuring the gut 
microbiome and immune infiltrates in pre-treatment tumor tissue from newly diagnosed breast 
cancer patients who will subsequently undergo neoadjuvant therapies. This study will provide 
preliminary data in support of a proposal for an outcomes study of the roles of the gut microbiome 
in response to cancer therapies. 

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This protocol involves the collection of three types of biospecimens from each participant. These 
collections 
would occur as follows: fecal samples will be collected prior to, and following 
completion of neoadjuvant chemotherapy by the participant, in her home, using a standard 
collection kit; venipuncture-collected blood samples will be collected prior to, and following 
completion of neoadjuvant chemotherapy.  In addition, we will retrieve archival breast tissue 
samples, if available, from a clinically-indicated pre-treatment biopsy.  Participants will be asked 
to complete a baseline questionnaire which includes items querying demographic and lifestyle 
factors and medical history which may be related to breast health and disease prognosis. 
Participants will be asked to complete FACT-G7 (Quality of life) questionnaire and a 
questionnaire on gastrointestinal side effects while at routine chemotherapy-related visits.  
All study procedures are considered to be of minimal risk. 
 
Schedule of Study Events 
Assessment 
Screening 
Baseline 
(prior to 
chemo) 
Interim 
(during chemo 
at the time of 
routine visits) 
Prior to 
Surgery 
After 
Surgery 
Informed consent 
X 
 
 
 
 
Eligibility  
X 
 
 
 
 
Archival tumor tissue from pre-
treatment biopsy for IHC 
 
If available 
 
 
 
Data from clinically indicated 
CBC with diff 
 
If available 
 
 
 
In home collection of fecal 
sample 
 
X 
 
X 
 
Study blood sample (<30mL) 
 
X 
 
X 
 
General questionnaire 
(demographic information, 
health history and lifestyle) 
 
X 
 
 
 
Pretreatment mammographic 
evaluations and breast MRI  
 
If available 
 
 
 
FACT-G7 (Health-related quality 
of life) 
 
X 
Every 2 
weeks 
 
 
Gastrointestinal symptom 
checklist  
 
 
Every 2 
weeks 
 
 
Assessment of pathologic 
complete response during 
routine clinical care 
 
 
 
 
X 
 
 

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Outcome definition 
The study outcome would be assessed at the time of surgery. Complete pathologic response 
will be defined as the absence of any residual invasive cancer on hematoxylin and eosin (H&E) 
evaluation of the resected breast specimen and all sampled ipsilateral lymph nodes following 
completion of neoadjuvant systemic therapy (ypT0 ypN0 in the current AJCC staging system). 
 
Microbial DNA assays 
Fecal collection: The first fecal sample should be collected prior to initiation of neoadjuvant 
therapy; a second fecal collection will be collected after chemotherapy and prior to surgery. In 
each case, the sample will be collected by the participant at home using an OMNIgene GUT 
sample collection kit. The samples, labeled with a code and no other patient identifiers, can be 
brought to the clinic or can be mailed by participants to University of Arkansas for Medical Sciences, 
Cancer Clinical Trials Office, Re: IRB # 204897, 4301 West Markham Street, Slot 724, Little Rock, 
AR 72205. If mailed, the postage date must be on or before the first day of chemotherapy 
treatment to be considered eligible to continue.  It is vital that the first fecal sample be collected 
prior to the first dose of chemotherapy. Following receipt, samples will be aliquoted into cryovials 
and stored in a -80 C freezer in the Cannon lab, until the complete batch can be sent to Dr. Jacques 
Ravel, c/o Mike Humphrys / Melissa Nandy, at the Insitute for Genome Sciences, University of 
Maryland School of Medicine, BioPark - 6th floor, 801 W. Baltimore Street, Baltimore, MD 21201. 
Samples will be labeled with a unique code and no other identifiers prior to sending to the 
University of Maryland.  
DNA extraction and sequencing: DNA will be extracted from fecal samples at the Ravel lab.   
Microbial 16S rRNA genes will be amplified using indexed primers and sequenced using 454-
pyrosequencing, as described in the paper by Fuhrman et al 2013 [29].  The DNA analysis that will 
be performed will focus on the identification and study of microbes in the sample rather than 
characterization of human genes.     
Bioinformatics: Reads will be filtered through the Institute of Genome Sciences bioinformatics 
pipeline [15]. Operational taxonomic units (OTUs) will be defined using the Quantitative Insights 
Into Microbial Ecology (QIIME) pipeline as sequences with at least 97% identity, rarified 
(randomly sampled with replacement) to the minimum number of observed OTUs. Using the 
Ribosomal Data Project Bayesian classifier in QIIME, OTUs will be assigned to phylum, class, 
order, family, and genus level-taxa, and their relative abundances will be calculated [16]. 
Additional microbial measures of interest include alpha diversity (Shannon index, Inverse 
Shannon), beta diversity (UNIFRAC distances), and phylogenetic diversity (whole tree 
phylogenetic diversity), as measured using QIIME after using rarefaction to account for 
differences in amplification across fecal samples [16]. In addition, we would like to consider as 
exposures phylogenetic composition, assessed down to the levels of phylum, family, and genus. 
Finally, we will use the programs PICRUSt and Lefse to generate metagenomic data and 
measures of KEGG pathway abundances from phylogenetic profiles [17]. 
 
Characterization of immune response 
Breast tissue collection: Tissue-based measures of immune response will require formalin-
fixed paraffin-embedded tissues from primary tumor samples.  After informed consent is 

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obtained, archival tissue from a pre-treatment biopsy of primary breast tumor, in the form of a 
FFPE tissue block or pre-cut slides will be requested either from the diagnosing center or from 
the Tissue Biorepository.  Four slides will be received or cut from formalin-fixed paraffin-
embedded tissues from primary tumor samples collected prior to chemotherapy.  Slides will be 
prepared from an FFPE tumor tissue block of at least 5-μm thickness. An H&E slide will be 
scanned using an Aperio slide scanner and will be used to characterize the sample in terms of 
tissue composition (cell density, relative abundance of stromal, epithelial, and adipose cells). We 
will stain slides for T-lymphocytes and microenvironmental cues using antibodies for CD8, CD4, 
FoxP3, and PDLM1 using standard methods for cell surface markers and intracellular markers 
as described in the following reference [30]. 
Blood sample collection: A sample of not more than 30 mL or approximately 6 teaspoons 
(three 10-mL yellow ACD tubes) will be taken by venipuncture in order to obtain peripheral blood 
leukocytes. This will be done twice, prior to the initiation, and after completion, of chemotherapy.  
A trained phlebotomist or registered nurse will draw the blood required for clinical and research 
purposes. This blood sample will be collected in coordination with clinical care to minimize the 
number of blood draws experienced by participants.  It is important that the first sample be 
obtained prior to the first dose of chemotherapy.  For logistical reasons, blood draws should be 
completed before 2:30 pm Monday-Friday so that the sample can be processed the same day.  
After collection, the sample should be kept and transported at room temperature.   
Delivery to the laboratory:  Research staff will pick up specimens from the Cancer Institute 
Blood Draw or Infusion Centers.  Research staff will re-label specimens with a patient identifier, 
date of the specimen, and name of visit.  When a research specimen is collected, designated 
research staff will be notified to coordinate delivery and processing of the sample at the Cannon 
Laboratory, in Room B532 of Biomedical Building 1, in the Department of Microbiology and 
Immunology at the University of Arkansas for Medical Sciences.   
Processing:  Samples will be processed within 24 hours of the blood draw.  Peripheral blood 
mononuclear cells (PBMCs) will be freshly isolated (within 8 h after the blood draw) from EDTA-
treated samples using Ficoll-Hypaque gradient separation (Lymphoprep, Axis Shield, Oslo 
Norway) and washed, following a standard protocol.    
Storage:  One of the three aliquots will be stored in the Tissue Biorepository at -80ºC or lower. 
Samples will be kept indefinitely. If a sample is deemed unviable at the time of use, the sample 
will be mixed with bleach and disposed in a sink. All specimen containers (tubes and containers) 
will be disposed into biohazard red plastic bags according to UAMS Occupational Health & 
Safety policy. Red bag waste will be picked up by Occupational Health & Safety for final 
disposal.  
Flow cytometry:  Leukocytes will be isolated and tagged using monoclonal antibodies directed 
against CD4, CD8.  Following permeabilization, cells will be incubated with anti-FOXP3 mAb 
(eBioscience, USA).  Samples will be analyzed using a FACS Canto flow cytometer (BD 
Biosciences, USA). Number of events will be acquired in list mode.  The FACS Diva software 
package (BD Biosciences) will be used. 
 
 
Measures of the breast microenvironment: mammographic density and benign 
parenchymal enhancement 

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Available mammographic and MRI-based evaluations carried out at UAMS as a part of the 
diagnostic process will be reviewed by study investigators.  Collected mammographic images 
would be used for measurement of volumetric mammographic breast density using Volpara 
software [6].  For each woman, standard views (CC and MLO views) will be reviewed. MRI images 
will be reviewed by Dr. Malak in order to measure benign parenchymal enhancement.  In the final 
dataset, mammographic and MRI-based measures will be stored with the participant’s study code.    
 
Withdrawal from the protocol 
Subjects may discontinue participation in the research at any time without penalty or loss of 
benefits to which the subject is otherwise entitled.  In the event that the participant is unable to 
attend study visits or tolerate study assessments, the Principal Investigator reserves the right to 
terminate the subject’s participation without the subject’s consent. Data from samples collected 
prior to withdrawal may be used in data analysis unless the subject provides a written request to 
the Principal Investigator requesting that data and/or any stored samples be destroyed. 
 
Reporting of protocol modifications, deviations or violations 
This study will be reviewed by the UAMS Institutional Review Board (IRB).  
Any modifications to the protocol will be submitted to the UAMS IRB for review and approval 
before any such changes are put into effect.  If modifications of the consent form are required, 
these will also be reviewed and approved by the IRB before the study proceeds.  In exceptional 
circumstances, involving major changes in the protocol, patients already entered into the study 
may be asked to sign a revised consent form (following IRB approval).  However, as this study 
does not involve patient treatment, and is no more than minimal risk, this last circumstance is not 
likely.  
Study activities will be reported to the UAMS IRB according to the guidelines for reporting outlined 
in IRB Policy 10.2. 
In addition, this study will undergo scientific review by the Cancer Institute’s Protocol Review and 
Monitoring Committee (PRMC). Approval by both the IRB and PRMC is required before the 
clinical trial can be activated. 
 
Disposition of data 
Data will be collected and recorded according to protocol requirements for all subjects enrolled to 
this trial. Data obtained during the study will be entered into the clinical trial management suite. 
Subjects will be registered in C3PR, a cancer Biomedical Informatics Grid (caBIG®, NCI) 
application of the clinical trial management suite. Data will be entered into OpenClinica through 
electronic web-based case report forms (CRFs). OpenClinica is a secure open source system for 
electronic data capture and clinical data management. Data from OpenClinica can be easily 
extracted for analysis throughout the study. 
Future research 
In the course of the present study, data is collected on many factors, including tumor 
characteristics, host characteristics, lifestyle factors, medical history, cancer therapies, 
gastrointestinal symptoms, comorbidities and clinical course for use in meeting stated study aims.  

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In addition to data, blood and fecal specimens are collected for analysis. For participants who 
agree to allow it, data collected in the course of the present study may be stored for future studies.   
Unused portions of blood and fecal samples may, with the participants’ permission, be securely 
stored for use in future research studies. Any future research would also be about factors that 
influence breast cancer risk and outcomes.  The informed consent has a separate section in which 
the participant is given the option to allow or decline use of their data and/or their biospecimens 
in future research.  If the subject declines to have data and/or biospecimens stored for future 
research, data and biospecimens will be used only for the research described in this protocol.  If 
the participant chooses, he or she can contact Dr. Fuhrman at any time with a written request to 
have his or her data be destroyed.   
In accordance with federal policy for NIH-funded investigators, the study shall send data to an 
NIH data repository. This data will be coded and will not contain any direct identifiers. The study 
is required to submit documentation that describes how the confidentiality of research subjects is 
protected, project protocols, and data variable descriptions. Access to the repository would be 
granted by an NIH-appointed Data Access Committee, which would review all applicant projects 
and put data-use agreements in place and only be available for the purposes of biomedical 
research.  
In addition, the informed consent has a section in which the participant is given the option to allow 
the research staff to contact them in the future regarding new clinical trials of which they would 
be eligible to participate. If the subject declines, they will not be contacted for future clinical trials. 
 
Benefits and Risks 
There will be no direct benefits to the study participants; however, knowledge gained from the 
study could potentially benefit patients in the future. 
Physical risks of blood withdrawal are limited to possible slight bruising at the site of needle 
puncture, the slight chance of fainting at the time of blood withdrawal, infection, bleeding and pain.   
There is a risk that the genetic information could be linked to the identity of the participant. This 
could result in genetic discrimination. Genetic discrimination occurs if people are treated unfairly 
because of differences in their genes that increase their chances of getting a certain disease. In 
the past, this could have resulted in the loss of health insurance or employment. Because of this, 
The Genetic Information Nondiscrimination Act of 2008, also referred to as GINA, was passed by 
Congress to protect Americans from such discrimination. The new law prevents discrimination 
from health insurers and employers. This act was signed into federal law on May 21, 2008, and 
went into effect May 2009. 
An unlikely risk to study participants is the potential for loss of confidentiality.  Measures to protect 
the confidentiality of study participants will be implemented as described in the Data Handling and 
Recordkeeping section below. 
Data Handling and Recordkeeping 
The Principal Investigator will carefully monitor study procedures to protect the safety of research 
subjects, the quality of the data and the integrity of the study.  All study subject material will be 
assigned a unique identifying code or number.  Data obtained during the study will be entered 
into C3PR and OpenClinica located in the UAMS clinical trial management suite. C3PR is a 
cancer Biomedical Informatics Grid (caBIG®, NCI) application that will keep a list of subjects 

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enrolled and specific information regarding their enrollment. Data will be entered into OpenClinica 
through electronic web-based case report forms (CRFs). OpenClinica is a secure open source 
system for electronic data capture and clinical data management. Data from OpenClinica can be 
easily extracted for analysis throughout the study. 
 
After informed consent is obtained, the data will be collected, coded with a study ID number, and 
stored indefinitely.  
 
Data Analysis 
Analyses will be conducted according to study aims. As the aims are considered exploratory, P 
values < 0.05 will be considered statistically significant and no adjustments will be made for 
multiple comparisons. 
Aim 1. To assess the acceptability and feasibility of study procedures. 
We will describe numbers and characteristics of women who are 1) screened, 2) eligible, 3) 
consented, and 4) complete collections for each type of biospecimen. In a brief form which will 
accompany the fecal sample, we will ask enrolled women specifically about the acceptability 
of, and logistical difficulties with, the procedures for collection and packaging of the fecal 
specimen. 
Aim 2. To estimate proportions of patients with pathological complete response by host factors, 
breast cancer subtypes, tumor characteristics, reported gastrointestinal symptoms, and by 
categorical measures of gut microbial composition, diversity, and metabolic activity. 
We will estimate and compare rates of pCR by each factor using chi-squared tests for crude 
comparisons and multivariable logistic regression for adjusted comparisons. Factors of interest 
will include host factors such as 
age and menopausal status; and tumor characteristics 
including: intrinsic subtype assessed using clinically indicated measures (ER and PR status, 
Her2/neu status, and Ki67 positivity); standard clinical prognostic indicators (TNM stage and 
histologic grade), and subgroups defined using tertiles of all continuous gut microbial measures. 
Aim 3. To estimate the associations of pre-treatment gut microbial composition, diversity, and 
metabolic activity with anticancer immune responses, measured as relative abundance of 
immune infiltrates in pre- treatment FFPE breast tumor tissues, and as relative abundance of 
circulating immune cell subtypes (Th1, Th2, Th17, and Treg) in baseline and longitudinal blood 
samples. Bivariate and covariate-adjusted associations among measures will be evaluated 
using Spearman's rank test and General Linear Models, respectively. 
Aim 4.  To estimate associations of the measures of local and systemic immune responses with 
host factors such as age, BMI, and smoking status, and with measures of the breast 
microenvironment including mammographic density and benign parenchymal enhancement on 
breast MRI. Bivariate and covariate-adjusted associations among measures will be evaluated 
using Spearman's rank test and General Linear Models, respectively. 
 
Power and Sample Size: 
Aim 1: If we screen 60 potential participants, we should be able to estimate, with 95% 

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confidence, the true proportion of this patient population willing to participate in the study with 
precision of +/- 20%. 
Aim 2: If 40 participants are enrolled and complete study measures, and the expected pCR in 
the category with the greatest risk of mortality is assumed to be 20%, then we will have 47% 
power to detect a statistically significant trend in rates of pCR across tertiles of microbiome 
measures if the odds ratio for the highest tertile is 5.0. 
Aim 3 and 4: If we have 20 participants with both tumor infiltrate measures and measures of gut 
microbial composition, we will have adequate power (ß=80%) to detect relationships between 
the dependent variable (relative abundances of tumor infiltrating lymphocytes and circulating 
lymphocytes) and the independent variables gut microbial composition, diversity and metabolic 
activity; host factors, and measures of the tumor microenvironment) at a two-sided α=0.05 
significance level, when the true change in the dependent variables is 0.67 standard deviations 
per one standard deviation change in the independent variables.  
 
Ethical Considerations 
This study will be conducted in accordance with all applicable government regulations 
and University of Arkansas for Medical Sciences research policies and procedures.  This protocol 
and any amendments will be submitted and approved by the UAMS Institutional Review Board 
(IRB) to conduct the study. In addition, all study personnel must have completed human subject 
protection training.   
The formal consent of each subject, using the IRB-approved consent form, will be obtained before 
that subject is submitted to any study procedure.  All subjects for this study will be provided a 
consent form describing this study so they can make an informed decision about their participation 
in this study.  The person obtaining consent will thoroughly explain each element of the document 
and outline the risks and benefits, alternate treatment(s), and requirements of the study. 
The consent process will take place in a quiet and private room, and women may take as much 
time as needed to make a decision about their participation.  The privacy of all subjects will be 
maintained and questions regarding participation will be answered.  No coercion or undue 
influence will be used in the consent process.  This consent form must be signed by the subject 
and the individual obtaining the consent.  A copy of the signed consent will be given to the 
participant, and the informed consent process will be documented in each subject’s medical 
record. 
Dissemination of Data 
Results of this study may be used for presentations, posters, or publications. The publications will 
not contain any identifiable information that could be linked to a participant. 
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