Breast Cancer Genome Guided Therapy Study (BEAUTY)
Conditions: Invasive Breast Cancer
Sponsor: Mayo Clinic

  
MC1137 
Protocol Version Date: 23Aug2023 
Mayo Clinic Cancer Center 
MC1137 Breast Cancer Genome Guided Therapy Study (BEAUTY) 
 
 
Study Co-Chairs: 
 
 
 
 
 
 
Site Investigators:  
 
 
 
 
 
 
 
 
 
 
Study Co-investigators: 
 
 
 
  
  
 
  
 
  
 
 
  
 
 
 
Imaging Co-investigators: 
  
  
  
 
Statisticians: 
  
 
 
  
 
 
 
Systems Pharmacologist: 
 
 
√ Study contributor(s) not responsible for patient care 

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Document History 
Effective Date 
Activation 
March 5, 2012 
Addendum 1 
April 13, 2012 
Addendum 2 
August 15, 2012 
Addendum 3 
October 3, 2012 
Addendum 4 
February 11, 2013 
Addendum 5 
May 6, 2013 
Addendum 7 
October 30, 2013 
Addendum 8 
March 14, 2014 
Addendum 9 
June 12, 2014 
Addendum 10 
August 14, 2014 
Addendum 11 
October 17, 2014 
Addendum 12 
January 16, 2015 
Addendum 13 
June 2, 2015 
Addendum 14 
July 14, 2016 
Addendum 15 
February 23, 2018 
Addendum 16 
September 3, 2019 
Addendum 17 
November 10, 2020 
Addendum 18 
September 8, 2021 
Addendum 19 
October 19, 2023 
 
 
 
 
 

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Protocol Resources 
Questions: 
Contact Name: 
Patient eligibility*, test schedule, 
treatment delays/interruptions/adjustments, 
dose modifications, adverse events, 
forms completion and submission 
 
Forms completion and submission 
 
 
Research Study Nurse 
 
 
 
Protocol document, consent form, 
regulatory issues 
  
 
 
Non-paraffin biospecimens  
 
 
 
Pathology Coordinator 
 
 
   
CIM Genetics Counselor  
  
 
 
Adverse Events (AdEERS, MedWatch, 
Non-AER, AML/MDS) 
 
 
  
*No waivers of eligibility per NCI  
 
 
 

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Table of Contents 
Breast Cancer Genome Guided Therapy Study (BEAUTY) ........................................................................ 1 
Protocol Resources ........................................................................................................................................ 3 
Table of Contents .......................................................................................................................................... 4 
Schema .......................................................................................................................................................... 5 
1.0 
Background ................................................................................................................................... 6 
2.0 
Goals ........................................................................................................................................... 10 
3.0 
Patient Eligibility ........................................................................................................................ 12 
4.0 
Test Schedule .............................................................................................................................. 14 
5.0 
Stratification Factors ................................................................................................................... 17 
6.0  
Registration/Randomization Procedures ..................................................................................... 17 
7.0 
Recommended Treatment Plan ................................................................................................... 19 
8..0 
Dosage Modification Based on Adverse Events ......................................................................... 25 
9.0 
Ancillary Treatment/Supportive Care ......................................................................................... 25 
10.0 
Adverse Event (AE) Reporting and Monitoring ......................................................................... 25 
11.0 
Disease Evaluation ...................................................................................................................... 28 
12.0 
Molecular Breast Imaging (MCR Participants Only) ................................................................. 29 
13.0 
Follow–up Decision at Evaluation of Patient .............................................................................. 31 
14.0 
Body Fluid Biospecimens ........................................................................................................... 33 
15.0 
Information concerning research procedures .............................................................................. 43 
16.0 
Statistical Considerations and Methodology ............................................................................... 44 
17.0 
Pathology Considerations/Tissue Biospecimens ......................................................................... 50 
18.0 
Records and Data Collection Procedures .................................................................................... 60 
19.0 
Budget ......................................................................................................................................... 61 
20.0 
References ................................................................................................................................... 62 
Appendix I ECOG Performance Status ...................................................................................................... 64 
Appendix II Chemotherapy Guidelines ...................................................................................................... 65 
Appendix III RPPA Standard Antibody List .............................................................................................. 72 
Appendix IV Molecular Breast Imaging (MCR Participants Only) ........................................................... 74 
Appendix V Return of Test Results ............................................................................................................ 76 
Appendix VI Return of Results Gene List .................................................................................................. 89 
Appendix VII Digital Spatial Profiling of High Risk Hormone Receptor-Positive (HR+) Breast Cancer . 93 
Appendix VIII Gene Expression Analysis by the University of Bristol ..................................................... 97 
Appendix IX BCM/Mayo Clinic BEAUTY Collaboration Summary ........................................................ 99 
Appendix X SimBioSys Collaboration ..................................................................................................... 101 

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Schema 
 
Registration 
 
 
 
 
 
 
 
 
Her 2+ disease (regardless of ER level): 
Her 2- disease 
with ER ≥ 10%+: 
Her 2- disease with 
ER <10%: 
 
 
Paclitaxel + Trastuzumab then either AC 
or EC or FEC  
or 
Docetaxel/Pertuzumab/Trastuzumab 
then either AC or EC or FEC   
or 
Paclitaxel/Pertuzumab/Trastuzumab 
then either AC or EC or FEC  
 
 
 
 
Paclitaxel then 
AC or EC or FEC  
 
 
 
Paclitaxel then AC or 
EC or FEC  
or  
Paclitaxel/Carboplatin 
then AC or EC or FEC  
 
 
 
 
 
 
 
 
 
 
 
 
Discontinue neoadjuvant treatment due to: 
-   Tumor progression or toxicity 
       and refuse immediate surgery 
-   Refusal to continue study participation 
-   Intercurrent disease 
-   Administration of a non-protocol 
      anti-neoplastic therapy 
 
SURGERY 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
OFF STUDY 
Unable to 
achieve 
complete 
resection 
 
Disease 
resected 
 
 
 
 
 
 
 
Note:  Patients who have a hypersensitivity 
reaction may be switched to nab-paclitaxel 
 
OFF STUDY 
 
OBSERVATION 
 
 
 
 
 
 
 
 
 
 
EVENT 
MONITORING 
 

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1.0 
Background 
1.1 
Introduction 
Breast cancer is diagnosed in 195,000 women annually in the United States alone; 45,000 
women still die annually of this disease. Although many women now present with Stage I 
and II mammographically detected cancers and have excellent outcomes, 10–20% of 
newly diagnosed breast cancers present as locally advanced breast cancer (LABC) in 
which the risk of recurrence and death is significantly higher[1].  
 
Development of multi-agent adjuvant chemotherapy regimens has substantially improved 
the clinical outcomes for women with breast cancer [2]. The most effective adjuvant 
combination regimens include anthracyclines, such as doxorubicin or epirubicin 
(topoisomerase II inhibitors), the alkylating agent cyclophosphamide, and taxanes 
(currently docetaxel or paclitaxel), which are microtubule stabilizers. These different 
mechanisms of action were designed to provide maximal cell kill within the range of 
tolerable toxicity for each drug, and broad coverage in resistant cell lines within a 
heterogeneous tumor population, The current standard of care for women with locally 
advanced breast cancer is neoadjuvant therapy prior to surgical resection. 
 
While neoadjuvant chemotherapy does not lead to improved overall survival (compared 
to chemotherapy delivered after surgery), higher rates of breast conservation are achieved 
with neoadjuvant chemotherapy. Perhaps the most important neoadjuvant clinical trials 
performed to date were undertaken by the National Surgical Adjuvant Breast and Bowel 
Project (NSABP) and provide the largest randomized data to date comparing preoperative 
to standard adjuvant chemotherapy. The NSABP B18 trial randomized 1523 women to 
either preoperative or postoperative doxorubicin/cyclophosphamide for a total of four 
cycles [3]. Among those who were randomized to preoperative chemotherapy, 80% of 
patient had a reduction in breast tumor size; 49% had a clinical response (cCR: 36% and 
cPR: 13%); and 89% of the clinically node positive patients had a clinical nodal response.  
 
The NSABP-B27 trial was designed to determine the impact on breast cancer response 
rates and overall survival (OS) of the addition of docetaxel to preoperative doxorubicin 
and cyclophosphamide [4]. Women with operable breast cancer (n = 2411) were 
randomly assigned to receive preoperative doxorubicin and cyclophosphamide followed 
by surgery, doxorubicin and cyclophosphamide followed by docetaxel and then surgery, 
or doxorubicin and cyclophosphamide followed by surgery and then docetaxel. 
Tamoxifen was initiated for estrogen receptor (ER) positive tumors. Adding docetaxel to 
doxorubicin and cyclophosphamide did not significantly impact disease free [1] survival 
(DFS) or OS but was found to significantly reduced the incidence of local recurrences as 
first events (p = 0.0034). Thus, the primary benefit of neoadjuvant chemotherapy is to 
downstage tumors, thereby improving optimal surgical resection and increasing the 
probability of breast conservation [5].  
 
A meta-analysis was performed by the Early Breast Cancer Trialists Collaborative Group 
(EBCTCG) [6] that included eleven randomized clinical trials, performed between 1981–
1993 encompassing 4675 women, demonstrated that preoperative therapy was associated 
with 18% fewer mastectomies and no significant differences in breast cancer recurrence, 
breast cancer mortality, or death within 10 years of follow-up comparing preoperative 
regimens with postoperative regimens.   
 

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An additional important finding in these studies was that the achievement of a pathologic 
complete response (pCR) (i.e., elimination of invasive tumor in breast and axillary lymph 
nodes, as assessed by pathology at surgery) is a useful surrogate for prognosis in breast 
cancer patients overall, suggesting that chemotherapy sensitivity, in and of itself, is an 
independent predictor of DFS and OS. Pathologic complete response, which occurred in 
27% of patients in NSABP B-27 who received preoperative docetaxel, was a significant 
predictor of OS regardless of treatment (hazard ratio = 0.33; 95% CI, 0.23–0.47; p < 
0.0001). Pathologic nodal status after chemotherapy was a significant predictor of OS (p 
< 0.0001). The pCR rates of doxorubicin-containing regimens are in the range of 12%; 
combining doxorubicin and taxanes leads to pCR rates in the 25–27% range.  
Conversely, the prognosis is quite poor for patients with significant residual disease in the 
breast, lymph nodes or both [7-10] following neoadjuvant chemotherapy.  
 
Breast cancer is a heterogeneous disease, and there is a wealth of data demonstrating that 
the identification of pathways responsible for the malignant phenotype can lead to new 
drug development, leading to substantial gains in response and death. A growing 
understanding of the molecular, genetic, and biochemical changes that occur during the 
processes of carcinogenesis, progression, and metastasis has shifted drug development 
toward therapeutics that act on specific molecular targets responsible for the malignant 
phenotype. Recent advances in drug development have included drugs that target signal 
transduction (the HER receptor family, Ras, Raf, and MEK kinases); oncogenic proteins, 
such as BCR/ABL, cell-cycle regulating proteins, such as the cyclins, the cyclin-
dependent kinases, and inhibitors of cyclin-dependent kinases; and proteins involved in 
the tumor angiogenesis, such as endothelial growth factor receptors.  
 
While the most common drug target that drives the growth of breast cancer is the 
estrogen receptor, many women who present with locally advanced breast cancer have 
tumors that exhibit estrogen independent growth. An important pathway in breast cancer 
first described in the late 1980’s by Slamon et al [11], is the discovery of the human 
epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase. The HER2 protein 
(or ErbB2) is one of four structurally related receptor tyrosine kinases (HER1-4). The 
HER2 gene, is amplified in 20% of breast cancers and amplification leads to a substantial 
change in the malignant phenotype, including alterations in tumor size, lymph node 
status, angiogenesis, invasion, and metastasis. Blockade of the HER2 protein tyrosine 
kinase, either through the use of trastuzumab or small molecule inhibitors of the tyrosine 
kinase (lapatinib) has led to substantial reductions in recurrence, survival, and in the 
neoadjuvant setting substantial increases in pCR rates.   
Molecular phenotyping has now been incorporated into the classification of tumors 
undergoing neoadjuvant therapy.  These data have consistently demonstrated that 
response and pCR differs significantly by phenotype [12]. 
Patients with strongly ER positive tumors (those of the luminal A subtype) may have a 
low pCR rate, but subsequently have a favorable prognosis due to both the indolent 
natural history of the disease and the responsiveness of these tumors to anti-estrogen 
therapies. Conversely, many patients with triple negative breast cancer (i.e., 
ER/progesterone receptor (PgR)/HER2 negative by immunohistochemistry (IHC), or 
basal by molecular phenotyping) may have an excellent response to neoadjuvant 
chemotherapy, likely due to the high proliferative rate of these tumors. However, many of 
these patients subsequently relapse, and salvage strategies are lacking. 
 

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The optimal combination, sequencing, and schedule for neoadjuvant chemotherapy has 
not been established. Four cycles of doxorubicin/cyclophosphamide followed by 
paclitaxel is a common regimen for women with HER2/neu non-overexpressing LABC, 
and the addition of trastuzumab on a weekly schedule in combination with paclitaxel has 
been shown to be safe and effective for patients with HER2 positive disease [13]. 
However, several studies have examined variations to the standard regimen, with or 
without trastuzumab. The Eastern Cooperative Oncology Group (ECOG) 1193 trial, 
which compared doxorubicin followed by paclitaxel to the reverse sequence of paclitaxel 
followed by doxorubicin in the metastatic setting, demonstrated no difference in response 
rate [14]. Thus, clinical data to date suggests that the sequence of taxane and doxorubicin 
does not seem to impact efficacy, although randomized trials testing one sequence versus 
another have not been reported.    
 
Breast cancer is a genetically and clinically heterogeneous disease. While multiple 
commercial gene expression profiles are available and can identify patients at higher risk 
for recurrence, this technology has not necessarily led to new therapeutics for breast 
cancer. Clinical progress has been limited by a poor understanding of the genetic events 
which drive the malignant phenotype and by limited preclinical models to study the 
disease. The rapid advancement of next-generation sequencing technologies allows 
comprehensive characterization of genomic changes. This technology is particularly 
useful for analysis of the entire tumor genome for the identification of novel genetic 
alterations in genes and gene pathways associated with resistance to standard 
chemotherapy.   
 
Response to neoadjuvant chemotherapy is assessed by documenting whether residual 
invasive disease is present in the breast (complete pathologic response) as well as by 
residual cancer burden (RCB) classification (which takes into account amount invasive 
disease present in the breast and nodes).   
 
Post-treatment proliferation (Ki67), has been shown to be an important prognostic factor 
among women receiving neoadjuvant chemotherapy[27] and neoadjuvant endocrine 
therapy [28]. The persistence of elevated Ki67 after treatment is associated with poor 
outcome. Moreover, patients with residual disease whose Ki67 levels are low after 
treatment have similar outcomes to patients with a complete pathological response[27].  
 
Given the sequential nature of the regimens used in this study, we have the opportunity to 
interrogate the mechanisms of taxane resistance. For patients with elevated levels of Ki67  
(Ki67 ≥ 15%) prior to paclitaxel, genetic differences between the tumors  with elevated 
Ki67 levels after paclitaxel will be contrasted and compared to the tumors whose Ki67 
levels are not elevated after paclitaxel.   
 
Given that neoadjuvant chemotherapy followed by breast surgery is considered a 
standard for the initial treatment of locally advanced breast cancer, the neoadjuvant 
setting can be used for the development of new drugs in combination or following 
standard chemotherapy for women at high risk of breast cancer morbidity and mortality. 
Therefore, it is critical that the new drug development focus on the following: 
 
1)   The identification of patients at increased risk for treatment failure  
2)   Identification of novel mutations in genes and gene pathways that drive the 
malignant phenotype and those associated with resistance to standard therapy. 

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3)   The ability to establish human tissue xenografts derived from the primary tumor 
tissue to directly test whether drugs which target the mutations and structural 
variations in genes and gene pathways will alter the growth and survival of these 
cells. 
4)   A personalized approach to introduce new drugs to women with high risk tumors 
whose mutation pattern would be expected to confer response to those novel 
drugs.  
 
1.2 
Current state of genomics in breast cancer 
 
A core component in the development of personalized medicine is to identify specific 
gene mutations and their functional impact on signaling pathways involved in 
tumorigenesis. For example, the V600E mutation identified in BRAF through a 
sequencing effort of BRAF gene proved to be a unique drug target for BRAF inhibitors 
and the inhibitor is more efficacious in melanoma patients with this BRAF mutation. 
While first generation molecular signatures based on gene expression profiles are 
prognostic and appear to predict response to chemotherapy, the identification of genes 
and gene pathways that drive the malignant phenotype and are associated with resistance 
to standard therapy is a key step toward the realization of new therapies for breast cancer. 
High throughput assays including next generation sequencing, gene expression, and CpG 
methylation enable researchers to perform integrated analysis to identify molecular 
signatures associated with response to standard chemotherapy. Our group has extensive 
experience with these types of analyses. Under the NIH-Mayo Pharmacogenomics 
Research Network (PGRN), we have generated an in vitro model system consisting of 
300 human lymphoblastoid cell lines obtained from 3 different ethnic groups. For each 
cell line, we have dense genomic information including 1.3 million SNPs, 54,000 gene 
expression probe sets, 47,000 CpG methylation sites and over 280 micro RNAs, plus over 
20 different drug cytotoxicity phenotypes. With this information, we are able to perform 
integrated analysis of SNPs, expression, microRNA and CpG methylation with our drug 
cytotoxicity phenotypes to identify signatures associated with drug sensitivity. We also 
have experience with GWAS (genome wide association studies) and functional studies of 
GWAS signals using the lymphoblastoid cell lines in multiple disease settings including 
breast cancer. 
 
In women with primary breast cancer who will be treated with neoadjuvant chemotherapy 
we will obtain tumor tissue prior to neoadjuvant treatment, post paclitaxel +/-
trastuzumab, at surgery from those cases with residual tumor and at disease recurrence. 
These tissues will undergo whole exome sequencing and RNA seq of transcript levels 
and CpG methylation sites. Information gathered from these series of samples will enable 
us to identify signatures for Ki67 response, complete pathological response phenotype 
and the combination of both; identify mutations in genes of pathways that have known 
targets for therapy to individualize the patients with appropriate therapies, and identify 
additional novel mechanisms underlying different response phenotypes. Also, these 
tumor samples will be used to generate xenografts for future drug screening studies. 
Blood will be collected prior to the start of neoadjuvant therapy for isolation of germline 
DNA that will be compared to tumor DNA and will be used to assess the effect of 
polymorphisms on treatment response.   
 
1.3 
Molecular Breast Imaging (MCR Participants Only) 
 

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At Mayo Rochester we have developed Molecular Breast Imaging (MBI). This is a 
functional imaging tool that uses a small amount of injected radiotracer and a dual-head 
small field of view gamma camera system. Previous work has demonstrated a sensitivity 
of ~90% for the detection of lesions in the range 6-10 mm in diameter [15, 16] and shown 
that it can detect additional disease not seen on mammography.  
 
We will examine tumor grade and cell proliferation index from the pre-treatment biopsy 
specimen and correlate these with the uptake in breast tumors of the radiopharmaceutical, 
99mTc-sestamibi, prior to treatment. The images will be interpreted by breast imaging 
radiologists, and the type and distribution of radiotracer uptake will be documented 
according to the MBI lexicon [15].  
 
99mTc-sestamibi MBI exams will be performed at 3 time points; the first after 
registration on the study (prior to any chemotherapy), the second after completion of 
taxane chemotherapy (prior to starting AC or FEC), and the third after completion of all 
neoadjuvant chemotherapy (prior to surgery). Changes in the pattern of tracer uptake will 
be assessed in terms of its association with pathologic tumor response and changes in 
Ki67 levels  after NAC. 
 
1.4 
Summary of Background 
 
In summary, patients with breast cancer who do not achieve a pathologic complete 
response (pCR) have a higher rate of recurrence and death. Current standard 
chemotherapy with taxanes (with or without trastuzumab for HER2+ disease) followed 
by anthracyclines achieves pCR rates of 30-50%. Additionally, elevated Ki67 after 
chemotherapy has also been shown to be associated with poor clinical outcome. Drug 
development in oncology is now focused on the identification of abnormalities in genes 
and gene pathways and the development of drugs designed to target those pathways. 
However, the identification of novel somatic changes that are "druggable" must be 
functionally validated before clinical utility can be evaluated. This requires human breast 
cancer tissue (xenografts) in order to perform functional studies Our long range goal is to 
individualize patient care by sequencing individual patient tumors, identifying mutations 
and alterations in tumors associated with the malignant phenotype, and using this 
information to personalize breast cancer therapy. 
 
 
2.0 
Goals 
2.1 
Primary Objectives 
2.11a 
To ascertain the germline and somatic changes within gene and gene pathways 
that occur before and after completion of paclitaxel  
and examine whether these changes differ between tumors that respond to 
paclitaxel and those that do not respond to paclitaxel 
2.11b To ascertain the germline and somatic changes within gene and gene pathways 
that occur before and after completion of NAC and examine whether these 
changes differ between tumors that respond to treatment and those that do not 
respond to treatment 

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2.12 
To determine the frequency of known tumor mutations for which current drug 
therapies already exist (e.g. BRAF, C-KIT, EGFR mutation, KRAS, PTEN, 
PI3K). 
2.13 
Using breast cancer tissue obtained prior to chemotherapy in all patients and 
following the completion of chemotherapy (in patients with residual disease), to 
develop tumor xenograft cell lines for mechanistic and functional studies to 
determine whether mutations identified are associated with the malignant 
phenotype and response to associated drugs which target the gene and/or 
pathways.   
2.2 
Secondary Objectives  
2.21 
Assess the association between changes in 99mTc-sestamibi uptake and response  
to treatment following neoadjuvant chemotherapy (MCR participants only). 
2.22 
To determine the association between T cell sequence and to treatment following 
neoadjuvant chemotherapy  
2.23  
To determine the ability of pre-treatment homologous recombination deficiency 
(HRD) scores to discriminate between those patients who will have a pCR after 
taxane and anthracycline-based neo-adjuvant chemotherapy regardless of tumor 
subtype and within tumor subtypes. 
2.23a 
To determine if the pretreatment HRD score is associated with   
radiographic or Ki67 response to taxane therapy. 
2.23b To determine if the pretreatment or post taxane HRD score is associated 
with MRI response after AC based therapy in patients with residual 
tumor on MRI after taxane therapy.   
2.23c 
To determine if the pretreatment HRD score is associated with response 
to neoadjuvant chemotherapy as determined by pCR. 
2.24 
To examine the changes in HRD scores during the course of neoadjuvant 
chemotherapy and their association with surgical outcome. 
2.25  
To determine the association between baseline ER beta expression with 
  pCR after taxane and anthracycline-based neo-adjuvant chemotherapy in 
  patients with triple negative or ER alpha (1-10%), HER2 negative breast   
  cancer. 
2.25a 
To determine if pretreatment ER beta expression is   associated with 
radiographic or Ki67 response to taxane therapy in patients with triple 
negative or ER alpha (1-10%), HER2 negative breast cancer. 
2.25b To determine if pretreatment ER beta expression is associated with MRI 
response after AC based therapy in patients with residual tumor on MRI 
after taxane therapy in in patients with triple negative or ER alpha (1-
10%), HER2 negative breast cancer. 
2.25c 
To determine if pretreatment ER beta expression is associated with PDX 
“take rate” as well as “growth rate” in patients with triple negative or ER 
alpha (1-10%), HER2 negative breast cancer. 
2.26 
An exploratory analysis of mutation status of additional genes known to be 
involved in DNA-damage response.  Genes include AKT1, ATM, ATR, BAP1, 
BARD1, BRIP1, CDK12, CHEK1, CHEK2, CTNNB1, ERCC4, FAM175A, 

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FANCA, FANCD2, FANCE, FANCI, FANCL, KRAS, MLH1, MRE11A, 
MSH2, MSH6, MUTYH, NBN, PALB2, PIK3CA, PPP2R2A, PTEN, RAD50, 
RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54B, RAD54L, RPA1, 
TP53, TP53BP1, XRCC2, and XRCC3 
 
3.0 
Patient Eligibility 
3.1 
Inclusion Criteria  
3.11 
Age ≥18 years. 
3.12 
Histological confirmation of invasive breast cancer.  
3.13 
Confirmation of breast cancer lesion ≥1.5 cm in size by any clinical (physical 
examination measurement) or radiographic criteria (mammogram, ultrasound or 
MRI) in the ipsilateral breast. 
Note: Benign breast disease, LCIS or DCIS in the contralateral breast is 
allowed. Contralateral invasive breast cancer is allowed if disease is of 
clinically lower stage and the higher stage lesion will be the study lesion 
for all biopsies and tissue samples.  
Note:  Disease in axilla only is not eligible. 
Note: Patients that have a contraindication or inability to have an MRI may still 
be enrolled on study and not participate in the MRI at any of the study 
specific time points.  
Note:  For patients with bilateral disease the higher clinical stage disease will be 
the study lesion that will undergo study biopsies and tissue samples from 
surgery and the contralateral lesion will NOT undergo research biopsies 
and tissue samples. 
3.14 
Men or women who are to begin neoadjuvant chemotherapy for treatment of 
Stage I-III Her 2 negative, ER10% or greater breast cancer with paclitaxel 
followed by either the combination of 5-fluorouracil, epirubicin and 
cyclophosphamide (FEC) or the combination of epirubicin and 
cyclophosphamide (EC), or the combination of doxorubicin and 
cyclophosphamide (AC).  
OR 
Men or women who are to begin neoadjuvant chemotherapy for treatment of 
Stage I-III Her 2 negative, ER<10% breast cancer with paclitaxel with or without 
carboplatin followed by either the combination of 5-fluorouracil, epirubicin and 
cyclophosphamide (FEC) or the combination of epirubicin and 
cyclophosphamide (EC) or the combination of doxorubicin and 
cyclophosphamide (AC).  
OR 
Men or women who are to begin neoadjuvant chemotherapy for treatment of 
Stage I-III Her 2 positive breast cancer with one of the following regimens:  
1)  Paclitaxel/trastuzumab followed by either the combination of 5-fluorouracil, 
epirubicin and cyclophosphamide (FEC) or the combination of epirubicin 
and cyclophosphamide (EC) or the combination of doxorubicin and 
cyclophosphamide (AC). 

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2) Docetaxel/trastuzumab/pertuzumab followed by either the combination of 5-
fluorouracil, epirubicin and cyclophosphamide (FEC) or the combination of 
epirubicin and cyclophosphamide (EC) or the combination of doxorubicin 
and cyclophosphamide (AC). 
3) Paclitaxel/trastuzumab/pertuzumab followed by either the combination of 5-
fluorouracil, epirubicin and cyclophosphamide (FEC) or the combination of 
epirubicin and cyclophosphamide (EC) or the combination of doxorubicin 
and cyclophosphamide (AC). 
Trastuzumab and/or pertuzumab will be given concurrently with the taxane 
portion. Pertuzumab should not be given with the anthracycline portion of 
chemotherapy. Trastuzumab can be given concurrently with FEC (but not AC at 
the discretion of the local medical oncologist (see section 7.0).  
Note: Her2 positive disease is defined to be: HER2 score of 3+ by IHC or 
HER2 gene amplification by FISH. 
3.15 
Provide informed written consent. 
3.16 
Willing to return to Mayo Clinic in Rochester, MN, Mayo Clinic in Arizona, or 
Mayo Clinic in Florida for imaging correlative, surgery, and follow-up.  
3.17 
Willing to provide blood samples for correlative research purposes (see Sections 
6.2 and 14.1). 
3.18 
Willing to provide tissue samples for correlative research purposes (see 6.2 and 
17.1). 
3.19 
ECOG Performance Status ≤2. 
 
3.2 
Exclusion Criteria  
3.21 
Receiving any investigational agent which would be considered as a treatment 
for the primary neoplasm. 
3.22 
Other active malignancy ≤ 5 years prior to registration.  EXCEPTIONS:  Non-
melanotic skin cancer or carcinoma-in-situ of the cervix.   
Note:   If there is a history or prior malignancy, they must not be receiving any 
other treatment for their cancer.  
3.23 
Patients who are not planning to receive neoadjuvant chemotherapy. 
3.24 
Biopsy proven Stage IV disease. 
3.25 
Patients who are pregnant or nursing. 
 

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4.0 
Test Schedule 
 
 
Active Monitoring Phase 
Tests and procedures 
≤60 days 
prior to 
registration 
≤28 days 
prior to 
registration 
After 
registration 
but prior to 
start of 
taxane 
After the 
completion of 
taxane prior to 
anthracycline 
based 
chemotherapy 
(Cycle 1) 
Suspicion of 
disease 
progression 
during neo-
adjuvant 
treatment 
After completion 
of anthracycline-
based 
chemotherapy 
but prior to 
surgery 
(Cycle 2) 
At the 
time of 
surgery 
(Cycle 3) 
At first 
recurrence or 
post treatment 
if primary 
surgery is not 
to be 
performed 
Annual 
Observation 
(Years 1-6 
following 
surgery) 
History and exam, Wt, 
PS* 
 
X 
 
X 
 
X 
 
X 
X 
Height 
 
X 
 
 
 
 
 
 
 
Adverse Event 
Assessment10 
 
 
X 
X 
 
X 
 
 
 
Pregnancy Test8,13 
 
X 
 
 
 
 
 
 
 
Mammogram of the side 
of the study lesion 
X2,4 
 
 
 
 
X 
 
 
 
Breast ultrasound of the 
study lesion  
X4 
 
 
 
 
X 
 
 
 
Axillary ultrasound 
ipsilateral to study lesion 
X4 
 
 
 
 
X16 
 
 
 
Research Breast 
Biopsy9,15,R 
 
 
X 
X11 
 
 
 
X1,14,15 
 
Tumor evaluation by 
Breast MRI4 
X3,4 
 
 
X3,R 
 
X3 
 
 
 
99mTc-sestamibi MBI13,R 
 
 
X 
X 
 
X 
 
 
 
Blood specimen6,12,R 
 
 
X 
X 
 
X 
 
X 
 

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Active Monitoring Phase 
Tests and procedures 
≤60 days 
prior to 
registration 
≤28 days 
prior to 
registration 
After 
registration 
but prior to 
start of 
taxane 
After the 
completion of 
taxane prior to 
anthracycline 
based 
chemotherapy 
(Cycle 1) 
Suspicion of 
disease 
progression 
during neo-
adjuvant 
treatment 
After completion 
of anthracycline-
based 
chemotherapy 
but prior to 
surgery 
(Cycle 2) 
At the 
time of 
surgery 
(Cycle 3) 
At first 
recurrence or 
post treatment 
if primary 
surgery is not 
to be 
performed 
Annual 
Observation 
(Years 1-6 
following 
surgery) 
Diagnostic, surgical, & 
recurrent tumor 
specimen15,R  
 
 
X1 
 
 
 
X17, 18 
X5,9 
 
Evaluation of disease 
status  
 
 
 
 
X 
 
 
 
X 
1. Copies of the pathology report are to be submitted along with the tissue sample.   
2. Unilateral mammogram (for unilateral breast cancer) on side of index cancer required ≤60 days prior to registration. Contralateral mammogram 
(if unilateral breast cancer) required to be done ≤3 months prior to registration.   
3. Unilateral or bilateral after completion of paclitaxel ± trastuzumab but prior to the start of AC or FEC chemotherapy. * Patients with known 
bilateral disease must have a bilateral MRI performed at the follow-up time points 
4. Imaging examinations performed at outside facilities prior to study registration will be accepted following review by Mayo Clinic radiologists. If 
deemed inadequate for interpretation or if further evaluation is needed, additional imaging will be performed at Mayo Clinic per standard 
practice. Outside examinations will not be accepted following enrollment into the study. 
Breast MRI from all 3 time points will undergo central review by two Mayo Clinic Radiologists. Submit MRI electronically via GEPACS. 
5. At the time of recurrence, patients will return to Mayo Clinic for a fresh tumor sample from the locally recurrent or metastatic site. For patients 
who develop recurrence and are unable to return Mayo, tissue specimen and a copy of the pathology report will be requested from the treating 
institution. 
6. For validation of biomarker studies. 
7. Disease status evaluation by mammogram and physical examination if breast conserving surgery done or physical examination if breast 
mastectomy performed. 
8. For women of childbearing potential only. Must be done ≤ 7 days prior to the initial sestamibi MBI. 
9. Performing a tissue biopsy using imaging guidance is standard of care. The sample will be divided and some will go to pathology to establish a 
diagnosis (standard of care) and the other part will go to research. If a biopsy has been performed at an outside institution, then a repeat biopsy is 
required (if feasible). 
10. Adverse event assessment for MBI only.  
11. If no residual abnormality on MRI and no target lesion is seen at time of ultrasound guided biopsy, then no biopsy will be taken.  
12. See section 14 for collection time and preparation of samples 

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13. MCR participants only. 
14. If recurrence occurs in the study breast 
15. See section 17 for collection time and preparation of samples. 
16.  Axillary ultrasound at this time point is required ONLY for cases that were node positive at diagnosis. 
Axillary ultrasound is not required after chemotherapy on cases that were clinically negative at initial presentation. 
17. Tissue from any residual disease at surgery and from the lymph nodes if residual disease is confined to the lymph nodes must be submitted (see 
Section 17.243). Surgery and Pathology reports from all sites must be submitted within 30 days of date of surgery. ER, PR, Her2 and Ki67 
should be performed clinically on the surgical specimen. In the event that ER, PR, Her2 and Ki67 testing is not performed clinically, banked 
tissue (up to 8 unstained tissue slides) from tumor registry will be accessed for ER, PR, Her2 and Ki67 testing and charged to the study.  
18. For patients with bilateral disease the higher clinical stage disease will be the study lesion that will undergo study biopsies and tissue samples 
from surgery and the contralateral lesion will NOT undergo research biopsies and tissue samples. 
R 
Research funded (see Section 19.0). 
* 
Other standard of care tests done per treating physician discretion.   
 
 

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5.0 
Stratification Factors 
None  
6.0  
Registration/Randomization Procedures 
6.1 
To register a patient, access the Mayo Clinic Cancer Center (MCCC) web page and enter 
the registration/randomization application. The registration/randomization application is 
available 24 hours a day, 7 days a week. Back up and/or system support contact 
information is available on the Web site. If unable to access the Web site, call the MCCC 
Registration Office at
 between the hours of 8 a.m. and 4:30 p.m. Central 
Time (Monday through Friday). 
 
The instructions for the registration/randomization application are available on the 
 and detail the process for 
completing and confirming patient registration. Prior to initiation of protocol treatment, 
this process must be completed in its entirety and a MCCC subject ID number must be 
available as noted in the instructions. It is the responsibility of the individual registering 
the patient to confirm the process has been successfully completed prior to release of the 
study agent. Patient registration via the registration/randomization application can be 
confirmed in any of the following ways: 
• 
Contact the MCCC Registration Office
. If the patient was fully 
registered, the MCCC Registration Office staff can access the information from the 
centralized database and confirm the registration. 
• 
Refer to “Instructions for Remote Registration” in section “Finding/Displaying 
Information about A Registered Subject.” 
 
6.2 
Correlative Research 
 
A mandatory correlative research component is part of this study, the patient will be 
automatically registered onto this component (see Sections 3.1, 14.1 and 17.1). 
 
6.3 
Documentation of IRB approval must be on file in the Registration Office before an 
investigator may register any patients. 
 
In addition to submitting initial IRB approval documents, ongoing IRB approval 
documentation must be on file (no less than annually) at the Registration Office (fax: 
507-284-0885). If the necessary documentation is not submitted in advance of attempting 
patient registration, the registration will not be accepted and the patient may not be 
enrolled in the protocol until the situation is resolved. 
 
When the study has been permanently closed to patient enrollment, submission of annual 
IRB approvals to the Registration Office is no longer necessary. 
 
6.4 
Prior to accepting the registration, registration application will verify the following: 
• 
IRB approval  
• 
Patient eligibility 
• 
Existence of a signed consent form 
• 
Existence of a signed authorization for use and disclosure of protected health 
information  
 

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6.5 
At the time of registration, the following will be recorded: 
• 
Patient has/has not given permission to store and use his/her sample(s) for future 
research of breast cancer. 
• 
Patient has/has not given permission to store and use his/her sample(s) for future 
research to learn, prevent, or treat other health problems. 
• 
Patient has/has not given permission for MCCC to give his/her sample(s) to 
researchers at other institutions. 
 
6.6 
Treatment cannot begin prior to registration and must begin ≤21 days after registration. 
 
6.7 
Pretreatment tests/procedures (see Section 4.0) must be completed within the guidelines 
specified on the test schedule. 
 
 

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7.0 
Recommended Treatment Plan  
7.1 
Neoadjuvant taxane phase recommended treatment schedule for either HER2 negative or 
HER2+ breast cancer.  
 
Note:  The following recommended treatments are considered standard regimens for the 
neoadjuvant treatment of breast cancer. Taxane-based therapy can consist of paclitaxel 
(for HER2 negative breast cancer) or either paclitaxel or docetaxel for HER2+ breast 
cancer. For HER2+ breast cancer, either trastuzumab alone or the combination of 
trastuzumab + pertuzumab can be utilized. For ER≤10%, HER2 negative breast cancer, 
carboplatin may be combined with paclitaxel. 
 
 
 
 
 
 
 
OR 
 
 
 
 
 
 
 
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Patients 
with 
HER2- 
negative, 
ER≥10% 
breast 
cancer 
paclitaxel 
N/A 
80 mg/m2 
IV 
Day 1 
Weeks 1-
12 
 
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Patients 
with 
HER2 – 
negative,  
ER≥10% 
or greater  
breast 
cancer 
paclitaxel 
N/A 
175 mg/m2 
IV 
Day 1 
Weeks 1, 
3, 5, 7 

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OR 
 
 
 
 
OR 
 
 
 
 
Reference: Sikov et al, CALGB 40603, SABCS December 2013  
 
 
 
 
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Patients 
with 
HER2- 
negative, 
ER<10%  
breast 
cancer 
paclitaxel 
N/A 
80 mg/m2 
IV 
Day 1 
Weeks 1-12 
 
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Patients 
with HER2 
– negative,   
ER<10%  
breast 
cancer 
paclitaxel 
N/A 
175 mg/m2 
IV 
Day 1 
Weeks 1, 3, 
5, 7 
 
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Patients 
with HER2 
– negative,   
ER<10%  
breast 
cancer 
paclitaxel 
N/A 
80 mg/m2 
IV 
Day 1 
Weeks 1-12 
carboplatin 
after 
paclitaxel 
administered 
AUC6 
IV 
Day 1 
Weeks 1, 4, 
7, 10 

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OR 
 
 
 
OR 
 
 
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Patients 
with 
Her2 
positive 
disease 
paclitaxel 
N/A 
80 mg/m2 
IV 
Day 1 
Weeks 1-12 
trastuzumab 
after paclitaxel 
administered 
1st dose – 
4 mg/kg 
IV 
Day 1 
Week 1 
Subsequent 
doses – 
2mg/kg 
Day 1 
Weeks 2-12 
 
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Patients 
with 
Her2 
positive 
disease 
paclitaxel 
N/A 
175 mg/m2 
IV 
Day 1 
Weeks 1, 3, 
5, 7 
trastuzumab 
after 
paclitaxel 
administered 
1st dose – 
4 mg/kg 
IV 
Day 1 
Week 1 
Subsequent 
doses – 2mg/kg 
Day 1 
Weeks 2-12 
 
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Patients 
with 
Her2 
positive 
disease 
Trastuzumab 
Loading 
Dose 
[HERCEP 
LOAD] 
N/A 
8 mg/kg/day  
IV 
Day 1 only  
 
Trastuzumab 
[HERCEP] 
N/A 
6 mg/kg/day  
IV 
Day 1 
Weeks 4, 7, 
10 
Pertuzumab 
(Perjeta) 
Loading 
Dose 
After 
Trastuzumab 
840 mg/day  
IV 
Day1. 
 
Pertuzumab 
(Perjeta) 
[PERTUZU
MAB] 
 
420 mg/day  
IV 
Day 1 
 Weeks 4, 7, 
10 
Paclitaxel 
N/A 
80 mg/m2 
IV 
Day 1 
Weeks 1-12 

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OR 
 
 
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Patients 
with 
Her2 
positive 
disease 
Trastuzumab 
Loading 
Dose 
[HERCEP 
LOAD] 
N/A 
8 mg/kg/day  
IV 
Day 1  
 
Trastuzumab 
[HERCEP] 
N/A 
6 mg/kg/day  
IV 
Day 1 
Weeks 4, 7, 
10 
Pertuzumab 
(Perjeta) 
Loading 
Dose 
 
After 
Trastuzumab 
840 mg/day  
IV 
Day 1 
 
Pertuzumab 
(Perjeta) 
[PERTUZU
MAB] 
 
420 mg/day  
IV 
Day 1 
Weeks 4, 7, 
10 
Docetaxel 
N/A 
75 mg/m2 
IV 
Day 1 
Weeks 1, 4, 
7, 10 
 
Any dose adjustments or use of growth factors will be at the discretion of the treating 
medical oncologist. 
Patients may receive their chemotherapy at Mayo Clinic or at a local institution. Treating 
medical oncologist will be asked to submit a summary of chemotherapy. 
For Mayo Clinic Rochester (MCR) patients only:  Patients must return to Mayo Clinic 
Rochester after completion of paclitaxel ± trastuzumab taxane based chemotherapy for a 
physical examination, MRI, and MBI.  Patients will also undergo an image guided biopsy 
of any residual tumor. 
For Mayo Clinic Arizona (MCA) and Mayo Clinic Florida (MCF) patients:  After 
completion of taxane based chemotherapy, patients must return to MCA or MCF for a 
physical examination and MRI. Patients will also undergo an image guided biopsy of any 
residual tumor.  
For patients who develop an allergy to paclitaxel, nab-paclitaxel may be substituted, 
using a dose of 100 mg/m2 once weekly, to complete a total of 12 weeks of taxane-based 
chemotherapy (total number of weeks for paclitaxel and nab-paclitaxel is 12 weeks). 
 
7.2 
Neoadjuvant AC or FEC or EC phase – recommended treatment schedule 
 
Note:  The following recommended treatments are considered standard regimens for the 
neoadjuvant treatment of breast cancer. Therefore, oncologists should use one of 
the following anthracycline containing regimens. Note, for patients with HER2+ 
breast cancer, trastuzumab (but not pertuzumab)may be combined with FEC as 
indicated below. Trastuzumab and pertuzumab should not be combined with 

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doxorubicin, as no safety data exist. The starting doses and schedule of each of 
these drugs is listed below and considered standard in clinical practice 
 
 
 
 
 
OR 
 
 
 
 
OR 
 
 
 
 
 
 
Agent 
Dose 
Route 
Rx Days 
ReRx 
All 
patients 
doxorubicin 
60 mg/m2 
IV infusion 
Day 1 
 
every 2 weeks x 4 with 
growth factor support 
or 
every 3 weeks x 4 (with 
our without growth factor 
support 
cyclophosphamide 
600 mg/m2 
IV infusion 
 
Agent 
Dose 
Route 
Rx Days 
ReRx 
All patients 
5 – fluorouracil 
500 mg/m2 
IV push 
Day 1 
 
Epirubicin 
75-90 mg/m2 
IV infusion 
every 3 
weeks x 4 
cyclophosphamide 
500 mg/m2 
IV infusion 
 
Agent 
Dose 
Route 
Rx Days 
ReRx 
All patients 
Epirubicin 
75-90 mg/m2 
IV infusion 
Day 1 
every 3 
weeks x 4 
cyclophosphamide 
500 mg/m2 
IV infusion 

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For Her2 positive patients: 
 
 
 
 
OR 
 
 
 
Agent 
Dose 
Route 
Rx Days 
ReRx 
Her 2 
positive 
5 – fluorouracil 
500 mg/m2 
IV push 
Day 1 
every 3 
weeks x 4 
Epirubicin 
75-90 mg/m2 
IV infusion  
Cyclophosphamide 500 mg/m2 
IV infusion 
Her 2 
positive** 
Trastuzumab 
2 mg/kg 
IV infusion 
Days 1, 8 
and 15 
Weekly for 
12 weeks 
 
** = OPTIONAL - given data from ACOSOG Z1041 there is ample safety data to combine 
trastuzumab along with the FEC regimen for patients with Her2 positive breast cancer. 
Trastuzumab should not be combined with AC. 
 
MCR patients only:  After completion of all chemotherapy and prior to surgical 
resection, patients will undergo a physical examination, venipuncture, unilateral 
diagnostic mammogram, breast and axillary ultrasound (if pre-treatment axillary 
ultrasound was abnormal), MRI and MBI to evaluate the extent of residual disease.  
 
MCF and MCA patients:  After completion of all chemotherapy and prior to surgical 
resection, patients will undergo a physical examination, venipuncture, unilateral 
diagnostic mammogram, breast and axillary ultrasound (if pre-treatment axillary 
ultrasound was abnormal) and MRI to evaluate the extent of residual disease.  
 
 
 
Agent 
Dose 
Route 
Rx Days 
ReRx 
Her 2 
positive 
doxorubicin 
60 mg/m2 
IV infusion 
Day 1 
every 2 weeks x 4 
with growth factor 
support 
or 
every 3 weeks x 4 (with 
our without growth 
factor support 
 
cyclophosphamide 
600 
mg/m2 
IV infusion 

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7.3 
Surgical Approach 
 
Surgery MUST be performed within 10 weeks of completion of neoadjuvant 
chemotherapy. 
 
Surgical resection of the primary tumor can be by either lumpectomy or mastectomy at 
the discretion of the treating surgeon and the patient’s preference. Resection of the 
primary tumor to negative margins is recommended. Management of the axillary lymph 
nodes is also per surgeon discretion. Resection of positive lymph nodes is recommended.  
 
7.4 
Pathology 
 
Surgical specimens should be evaluated for residual disease in the breast and in the 
lymph nodes and reported using the elements for calculation of Residual Cancer Burden 
(RCB). Tissue from any residual disease in excess of diagnostic tissue at time of surgical 
resection will be flash frozen and submitted for research purposes (see section 17.243). 
Residual tissue from the surgical bed should be collected for research purposes, even if 
no residual cancer is present. In the event that ER, PR, Her2 and Ki67 testing is not 
performed clinically, banked tissue from tumor registry (up to 8 unstained tissue slides) 
will be accessed for ER, PR, Her2 and Ki67 testing. 
   
7.41 
If patient has bilateral disease, residual disease/tissue from the study lesion (the 
lesion that was biopsied and for which patient derived xenografts were 
generated) will be submitted. Any residual contralateral disease (non-index 
lesion) will have one representative section paraffin embedded and stored (see 
section 17.243). MCA and MCF will ship the flash frozen specimen and the 
paraffin embedded section (if indicated) to MCR (see section 17.2).   
 
 
8..0 
Dosage Modification Based on Adverse Events  
Any dose adjustments or modifications will be at the discretion of the treating medical oncologist.   
9.0 
Ancillary Treatment/Supportive Care 
Use of growth factors will be at the discretion of the treating medical oncologist.   
10.0 
Adverse Event (AE) Reporting and Monitoring 
10.1 
Adverse Event Characteristics 
 
CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.0 will be utilized for AE reporting.  A copy of the CTCAE version 4.0 can be 
downloaded from the CTEP web site 
   
 
10.11 Adverse event monitoring and reporting is limited to the research aspects of this 
study that are associated with the 99mTc-sestamibi MBI. The patient will be 
visually monitored by the nuclear medicine technologist from the time of 
injection of the radionuclide until image acquisitions are completed. Tc-99m 
sestamibi is an approved drug with an excellent safety profile. It has been used 

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routinely on a daily basis in over 40 patients a day in the Department of 
Radiology at Mayo Clinic for the last 15 years with no recorded adverse effects. 
Any side effects that occur during the MBI will be reported by the MBI staff in a 
note to file and sent to the clinical research coordinator for submission to the IRB 
following Mayo Clinic IRB policy.  
 
10.111 First, identify and grade the severity of the event using a copy of the 
CTCAE v4.0. Next, determine if the adverse event is related to the 
medical treatment or procedure.  
 
10.112 Assessment of Attribution 
 
When assessing whether an adverse event is related to a research 
procedure, the following attribution categories are utilized: 
 
Definite – The adverse event is clearly related to the investigational 
agent(s). 
Probable – The adverse event is likely related to the investigational 
agent(s). 
Possible – The adverse event may be related to the investigational 
agent(s). 
Unlikely – The adverse event is doubtfully related to the investigational 
agent(s). 
Unrelated – The adverse event is clearly NOT related to the 
investigational agent(s) 
 
10.2 
Expedited Reporting Requirements for Adverse Events that Occur in a Non-IND/IDE 
trial within 30 Days of the Last Administration of a Commercial Imaging Agent 1, 2  
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312) 
NOTE:  Investigators MUST immediately report to the sponsor ANY Serious Adverse Events, whether or not they 
are considered related to the investigational agent(s)/intervention (21 CFR 312.64) 
 An adverse event is considered serious if it results in ANY of the following outcomes:   
1) 
Death 
2) 
A life-threatening adverse event  
3) 
An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization for ≥ 24 
hours  
4) 
A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions  
5) 
A congenital anomaly/birth defect.  
6) 
Important Medical Events (IME) that may not result in death, be life threatening, or require hospitalization 
may be considered serious when, based upon medical judgment, they may jeopardize the patient or 
subject and may require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. (FDA, 21 CFR 312.32; ICH E2A and ICH E6). 
 
ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported to the sponsor within 
the timeframes detailed in the table below. 
Hospitalization 
Grade 1 
Timeframes 
Grade 2 
Timeframes 
Grade 3 Timeframes 
Grade 4 & 5 
Timeframes 
Resulting in 
Hospitalization  
≥ 24 hrs 
7 Calendar Days 
24-Hour 3 
Calendar Days 
Not resulting in 
Hospitalization  
≥ 24 hrs 
Not required 
7 Calendar Days 

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Expedited AE reporting timelines are defined as: 
o “24-Hour; 3 Calendar Days” – The AE must initially be reported via MedWatch within 24 hours of 
learning of the AE, followed by a complete expedited report within 3 calendar days of the initial 24-
hour report. 
o “7 Calendar Days” – A complete expedited report on the AE must be submitted within 7 calendar days 
of learning of the AE. 
1Serious adverse events that occur more than 30 days after the last administration of investigational 
agent/intervention and have an attribution of possible, probable, or definite require reporting as follows:  
Expedited 24-hour notification followed by complete report within 3 calendar days for: 
• 
All Grade 4, and Grade 5 Aes 
Expedited 7 calendar day reports for: 
• Grade 2 adverse events resulting in hospitalization or prolongation of hospitalization  
• Grade 3 adverse events 
For studies using PET or SPECT IND agents, the AE reporting period is limited to 10 radioactive half-
lives, rounded UP to the nearest whole day, after the agent/intervention was last administered.  Footnote 
“1” above applies after this reporting period. 
Effective Date:  May 5, 2011 
 
Additional Instructions: 
1. An increased incidence of an expected adverse event (AE) is based on the patients treated for this 
study at their site. A list of known/expected Aes is reported in the package insert or the literature, 
including Aes resulting from a drug overdose. 
2. 
 
  
 
Mayo Clinic Cancer Center (MCCC) Institutions: Provide copies, along with the UPIRTSO cover 
sheet, by fax 
 to the MCCC Regulatory Affairs Unit (RAU) Risk Information 
Specialist who will determine and complete IRB reporting. The RAU will submit to the MCCC 
SAE Coordinator. 
  
 
10.3 
Adverse events to be graded after 99mTc-sestamibi MBI is performed (see Section 4.0) per 
Common Terminology Criteria for Adverse Events (CTCAE) CTEP Version 4.0 grading 
unless otherwise stated:  
10.31 Submit via appropriate MCCC Case Report Forms (i.e., paper or electronic, as 
applicable) the following Aes experienced by a patient: 
 
10.311 Grade 2 AEs deemed possibly, probably, or definitely related to 99mTc-
sestamibi MBI. 
 
10.312 Grade 3 and 4 AEs regardless of attribution to 99mTc-sestamibi MBI. 
 
10.313 Grade 5 AEs (Deaths) 
 
10.3131 
Any death within 30 days of completion of chemotherapy or 
surgery 
 
 
 

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11.0 
Disease Evaluation  
 
11.1 
Neoadjuvant chemotherapy phase 
 
11.11 Ki67 response for those patients whose pre-treatment baseline Ki 67 level ≥ 15%. 
 
Ki67 levels will be obtained from a breast biopsy samples taken prior to 
chemotherapy, at the completion of paclitaxel as well as at the completion of 
anthracycline therapy. 
A Ki67 response is defined as a Ki67 value < 15% in the invasive component of 
the biopsy specimen or a biopsy specimen composed of neurotic and/or 
vacuolated cells (evidence of treatment effect) rendering Ki67 level 
undeterminable. OR a breast biopsy sample that contains no invasive disease will 
be considered to achieve a Ki-67 response.   
 
For those patients where no area of disease is seen under ultrasound and, as such, 
the radiologist chooses not to biopsy the breast,  these patients will be considered 
to achieve a Ki-67 response if central review of the 12 week MRI indicates a 
radiographic response (see 11.12) 
 
11.12    Radiographic response (regardless of pretreatment Ki67 level) 
 
Breast imaging will be performed at the completion of paclitaxel. Radiographic 
response is defined as a 30% or more decrease in the longest diameter of the 
lesion from pre-treatment size. 
 
11.2 
Surgery 
 
11.21 Pathological response  
A pathologic complete response is defined as no histologic evidence of invasive 
tumor cells in the surgical breast specimen and sentinel and/or axillary lymph 
nodes.  
 
11.22 Residual Burden of Disease 
 
RCB score is based upon four parameters of the residual tumor pathology review: 
the size of the residual tumor bed, the proportion of the tumor bed that contains 
invasive cancer, the number of axillary lymph nodes containing disease, and the 
diameter of the largest metastasis in an axillary lymph node.  
 
Determination of Residual Cancer Burden (RCB) is described at   
 
 
11.23 Ki67 response for those patients whose pre-treatment baseline Ki67 level ≥ 15%. 
A Ki67 response is defined as a Ki67 value < 15% in the invasive component of 
the pre-surgical biopsy specimen or a pre-surgical biopsy specimen composed of 

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neurotic and/or vacuolated cells (evidence of treatment effect) rendering Ki67 
level undeterminable.   
 
11.24    Radiographic response  
 
Breast imaging will be performed at the completion of NAC. Radiographic 
response is defined as a 30% or more decrease in the longest diameter of the 
lesion from pre-treatment size. 
 
 
11.3 
Post Surgery 
 
11.31  Local recurrence 
 
Local recurrence is defined as histologic evidence of ductal carcinoma in situ or 
invasive breast cancer in the ipsilateral breast or ipsilateral chest wall.  
 
11.32  Regional recurrence 
 
Regional recurrence is defined as the cytologic or histologic evidence of disease 
in the ipsilateral internal mammary, ipsilateral supraclavicular, ipsilateral 
infraclavicular and/or ipsilateral axillary nodes or soft tissue of the ipsilateral 
axilla. 
 
11.33  Distant recurrence 
 
Distant recurrence is defined as the cytologic, histologic, and/or radiographic 
evidence of disease in the skin, subcutaneous tissue, lymph nodes (other than 
local or regional metastasis), lung, bone narrow, central nervous system or 
histologic and/or radiographic evidence of skeletal or liver metastasis.  
 
11.34  Second primary breast cancer  
 
Second primary breast cancer is defined as histologic evidence of ductal 
carcinoma in situ or invasive breast cancer in the contralateral breast or 
contralateral chest wall.  
 
11.35  Second primary cancer (non-breast) 
 
Any non-breast second primary cancer other than squamous or basal cell 
carcinoma of the skin, melanoma in situ, or carcinoma in situ of the cervix is to 
be reported and should be confirmed histologically whenever possible. 
 
11.36  Death 
 
Underlying cause of death is to be reported. 
 
 
12.0 
Molecular Breast Imaging (MCR Participants Only)  

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12.1 
Molecular Breast Imaging procedure 
All patients will undergo MBI with 99mTc-sestamibi prior to start of neoadjuvant 
chemotherapy, at time of switching from paclitaxel ± trastuzumab to AC or FEC 
chemotherapy and after completion of neoadjuvant chemotherapy prior to surgery.   
 
For each scan, all patients will undergo cranio-caudal (CC) and medio-lateral oblique 
(MLO) views of the breast with the suspected lesion using a dual-detector MBI system at 
5 minutes post-injection of 4-8 mCi 99mTc-sestamibi. A CC and MLO view of the 
contralateral breast will also be performed. 
 
12.2 
Evaluation of MBI 
 
A radiologist will review the MBI study at each time point. Each exam will be interpreted 
in a blinded fashion using the established MBI lexicon [15]. The background 
parenchymal radiotracer uptake, number of lesions and their location, size of each lesion 
in three dimensions, intensity of radiotracer uptake, and associated findings will be 
evaluated and recorded. Final assessment codes, modeled after the Breast Imaging 
Reporting and Data System (BI-RADS) will be assigned on a per-breast basis. The 
interpreting radiologist will then be un-blinded and will correlate MBI findings with any 
available mammogram/ultrasound/MRI results.   
 
Tumor uptake of 99mTc-sestamibi in each lesion will also be quantified using software 
developed in-house. Algorithms for calculation of tumor uptake from opposing views of 
the breast have been validated in phantom models and computer simulations [16]. This 
will allow calculation of true tumor / background ratio. 
 
12.3 
Aims of MBI studies 
 
We will evaluate if tumor size can be satisfactorily assessed by MBI, if tumor size from 
MBI corresponds to tumor size evaluation by conventional imaging procedures 
(mammography, ultrasound, and MRI), and tumor size found at surgery, and if there are 
histopathologic features or biomarkers that correlate with tumor uptake of 99mTc-
sestamibi.  
 
The aims of this study are to a) determine the sensitivity of 99mTc-sestamibi for the 
evaluation of tumor size and function, b) to determine whether early changes in 
radiotracer uptake predicts eventual pathologic response, and c) to determine the 
correlation between tumor uptake and tumor grade and proliferation.  
 
12.4 
Participation in other MBI studies 
 
If a patient meets the inclusion criteria for more than one MBI-related protocol, we will 
ask the patient if he/she is willing to provide authorization for use of their MBI images 
from this study in the other MBI-related protocols. A check box on the Patient Consent 
form is provided to document whether the patient has specifically agreed to this request.   
 
If a patient agrees to participate in any additional research studies involving an MBI scan, 
the results from this study may be used in those protocols in lieu of performing an 
additional MBI scan provided that the MBI scan from this study meets the inclusion 

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criteria of the other study. This will eliminate unnecessary duplication of the MBI 
procedure in these patients.  
 
 
13.0 
Follow–up Decision at Evaluation of Patient   
 
 
13.1 
Prior to the drawing of any pre-treatment research bloods or MBI imaging (if 
applicable) 
 
 
13.11 If a patient withdraws consent for any reason before any pre-treatment 
research bloods are drawn or MBI imaging is done, the patient will be considered 
a cancel. On-study material and the End of Active Treatment/Cancel Notification 
Form must be submitted. No further follow-up is to be done 
 
13.2 
Neoadjuvant chemotherapy phase 
 
13.21 Patients who discontinue neoadjuvant chemotherapy due to toxicity or 
progression and choose to go directly to surgery will continue on study per 
protocol.   
 
13.22 Patients who discontinue neoadjuvant chemotherapy due to: 
 
− 
Tumor progression and/or toxicity on the taxane that do not go to 
anthracycline 
− 
Tumor progression and/or toxicity on anthracycline that do not go  to 
immediate surgery 
− 
Refusal to continue study participation 
− 
Intercurrent illness or pregnancy 
− 
Administration of alternative anti-neoplastic therapy  
 
will go off study. All data up until and including the point of discontinuation 
must be submitted. No further follow-up is required. 
 
Future treatments are at the discretion of the patient’s treating physician. 
 
13.23 Patients who do not receive the recommended treatment sequence of taxane 
based chemotherapy followed by anthracycline based chemotherapy will go off 
study. All data up until and including the point of discontinuation must be 
submitted. No further follow-up is required.Future treatments are at the discretion 
of the patient’s treating physician. 
 
13.3 
Surgery phase  
 
13.31 Patients who are not considered surgical candidates or who refuse to undergo 
surgery will go off study. All data up until and including the point of 
determination the patient is not a surgical candidate or the patient refuses to 
undergo surgery must be submitted. No further follow-up is required. Future 
treatments are at the discretion of the patient’s treating physician. 
 

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13.4 
Post surgery phase 
 
13.41 Patients whose disease could not be completely resected will go off study after 
post–operative visit. All data up until and including the post-operative visit must 
be submitted. No further follow-up is required. Future treatments are at the 
discretion of the patient’s treating physician. 
 
13.42 Patients who refuse to continue in the observation phase will go directly to event 
monitoring until death or a maximum of 6 years post-surgery. 
 
13.5 
Any point in the study  
 
13.51   If a patient is found to be ineligible by the Data Center, that is, it is determined 
that at the time of registration, the patient did not satisfy each and every 
eligibility criteria for study entry. The patient will go off study and all data up 
until the point of confirmation of ineligibility must be submitted.   
 

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14.0 
Body Fluid Biospecimens 
14.1 
Summary Table of Research Blood and Body Fluid Specimens to be Collected for this Protocol 
 
Correlative 
Study 
(Section for 
more 
information) 
Indicate if 
specimen is 
Manda-tory 
or Optional 
Blood or 
Body Fluid 
being 
Collected; 
processed 
and 
submitted by 
participating 
site 
Type of 
Collec-tion 
Tube 
(color of 
tube top) 
Volume 
to 
collect 
per tube 
(# of 
tubes 
to be 
collected) 
After 
registratio
n but prior 
to start of 
taxane 
After 
completion of 
taxane but 
prior to the 
start of 
anthracy-cline 
based chemo-
therapy 
(cycle 1) 
After 
comple-
tion of  
anthracycl
ine based 
chemother
apy, but 
prior to 
surgery 
(cycle 2) 
At first 
recurrence 
or post 
treatment if 
primary 
surgery is 
not to be 
performed 
Pro-
cess 
at 
site? 
(Yes 
or 
No) 
Additional 
processing 
required at 
site after 
blood 
draw? 
Temper-
ature 
Conditions 
for Storage 
/Shipping 
Visit Alias – 
Lab Use Only 
 
 
 
 
V1B 
(Visit 1 
Blood) 
V2B 
(Visit 2 Blood) 
V3B 
(Visit 3 
Blood) 
V4B 
(Visit 4 
Blood) 
 
 
 
Pharmaco-
genetics 
Mandatory 
Blood 
EDTA 
(purple) 
10 mL 
 (1) 
X 
 
 
 
No 
Yes 
Refrigerated or 
Wet ice 
Circulating 
Tumor Cells 
Mandatory 
Blood 
EDTA 
(purple) 
10 ml  
(1) 
X 
X 
X 
X 
No 
No 
Ambient 
Circulating 
Tumor Cells 
Mandatory 
Blood 
CellSave 
Tube 
(purple and 
yellow 
striped) 
20 ml 
(2) 
X 
X 
X 
X 
No 
No 
Ambient 
Proteomics 
Mandatory 
Blood 
EDTA 
(purple) 
10 ml  
(1) 
X 
X 
X 
X 
No 
No 
Refrigerated or 
Wet ice 
Lymphocytes 
Mandatory 
Blood 
Na Heparin 
(green) 
10 mL  
(1) 
X 
 
 
 
No 
No 
Refrigerated or 
Wet ice 
    RNA 
Mandatory 
Blood 
Paxgene 
(2) 
2.5 ml 
X 
X 
X 
X 
No 
No 
Ambient 

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14.2 Blood/Blood Products 
 
14.21 Mayo Clinic Rochester will use Special Study Cards in place of kits. 
 
All MCR samples must be collected Monday-Friday. 
 
14.22 Kits will be used at Mayo Clinic in Arizona and Mayo Clinic in Florida for 
this study. 
 
All specimens must be collected and shipped Monday – Thursday ONLY. 
Sample processing must be completed by 3 pm on Thursday for overnight 
shipping to MN. 
 
14.23 Label specimen tube(s) with protocol number, study patient ID number, and time 
and date blood is drawn. 
 
14.24 Collect and process all blood/blood products according to the table in 14.1.  
 
14.241 Gently invert EDTA purple top and Na Heparin tubes 8 to 10 times and 
place on wet ice for transport to BAP Hilton CL-21. Gently invert the 
CellSave tubes 8 to 10 times (keep CellSave and 1 EDTA tubes ambient) 
and transport to BAP Hilton CL-21. 
 
14.242 The CellSave tubes and (1) EDTA 10 mL purple top tube will be 
forwarded to Mike Campion at Hilton 10-66 for processing. The 
CellSave tubes will be processed per manufacture’s protocol for capture 
and enumeration of CTCs. Briefly, a ferrofluid reagent conjugated with 
antibodies to the epithelial cell adhesion molecule (EpCAM) is used to 
immunomagnetically separate epithelial cells. These captured cells are 
then stained with DAPI, cytokeratin 8, 18,19, and CD45 to help 
distinguish true CTCs from leukocytes. Cells that are DAPI+/CD45-
/CK+ are considered circulating tumor cells.  The EDTA tube will be 
processed per manufacture’s protocol for capture of epithelial cells. 
Briefly, this protocol is the same as the above protocol, however there is 
no staining of the captured cells. Any captured cells can be processed for 
molecular testing as seen appropriate. In this study, the captured cells 
will be frozen for future testing. 
 
14.243 BAP will process specimens as follows: 
 
Record receipt of specimens.   
 
DNA will be isolated from 8 mLs EDTA whole blood. 2 runs of DNA 
will be done. DNA will be stored at 4°C until a box is full and then 
transferred to and stored in a −80°C freezer.   
 
Extract Lymphocytes from Na Heparin whole blood using EBV slow 
freeze. Lymphocytes will be stored until the end of the study or upon 
request.  
 

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EDTA plasma will be collected from one EDTA tube. Centrifuge at lab 
standards and aliquot 1 mL into 4 aliquots, transfer WBC’s into cryovial 
and freeze at -80° C. 
 
CTC’s, (2) CellSave 10 mL tubes, and (1) EDTA 10 mL purple top tube 
will be forwarded to Mike Campion at Hilton 10-66 for processing. 
 
RNA (2) 2.5 mL PAXgene tubes will be collected.  BAP will process 
RNA from (1) 2.5 mLPAXgene tube and will store the second 2.5mL 
PAXgene tube at -80°C. 
 
14.244 Blood samples for MCA and MCF will be shipped to MCR for 
processing. Samples need to arrive within 24 hours of collection. The 
CellSave tubes and (1) EDTA tube must be kept ambient will be shipped 
to BAP Rochester and BAP will send to 
. For CTC 
processing. Green tube and EDTA tube for lymphocytes and proteomics 
and pharmacogenomics will be shipped to BAP (refrigerated). 
 
PAXgene tubes will be shipped to BAP ambient temperature. 
 
14.3  
Collection and Processing  
 
14.31  Overview of pharmacogenomic study 
 
Specimens (breast tumor and blood) will be obtained at initial time point prior to 
any chemotherapy. Additional tumor specimens will be obtained after completion 
of paclitaxel ± trastuzumab chemotherapy (prior to AC or FEC chemotherapy) 
and again at the time of definitive surgical resection (after completion of all 
chemotherapy) for sequencing. Additionally tissue from biopsy of any local or 
distant recurrence will undergo sequencing. 
 
Blood will be used to isolate DNA for a series of high throughput assays, 
including genome wide SNP analysis using 2.5 M Illumina Omini chips and 
exome sequencing.  
 
Blood will be collected and processed for DNA and lymphocyte isolation and 
proteomics at the 
. DNA will be used for exome 
sequencing in the 
b. DNA will also be sent 
for obtaining genome-wide SNPs at the 
 lab.   
 
Lymphocytes will also be collected for future generation of lymphoblastoid cell 
lines that will be used in functional studies to assess the effect of germline 
polymorphisms on protein function and response to therapy. 
 
Plasma will be collected for future proteomics. 
 
Appendix V addresses the issue of reporting the results of germline DNA 
analyses to the patient. 
 
14.32 Overview of Circulating Tumor Cells 
 

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Specimens will be obtained prior to initiation of chemotherapy, after completion 
of paclitaxel ± trastuzumab chemotherapy (prior to AC or FEC chemotherapy) 
and again after completion of all chemotherapy and at time of any disease 
recurrence. Blood will be used to measure the number of circulating tumor cells 
(CTCs) and isolate the intact CTCs for further testing. DNA isolated from the 
CTCs will be used for genotyping and comparison with the primary tumor. 
 
14.33 Overview of RPPA 
 
After RNA processing and collection in BAP lab, an aliquot of RNA from 
baseline (prior to chemotherapy), after completion of paclitaxel ± trastuzumab 
prior to AC or FEC chemotherapy) and from the time of surgery and from time 
of recurrence will be shipped to the Harris Lab at the University of Minnesota for 
quantitative expression profiling of APOBEC3B mRNA levels. 
 
14.4 
Shipping and Handling 
 
14.41 Shipping Specimens 
Verify ALL sections of the Blood Specimen Submission Form (see Forms 
Packet), BAP Requisition Form (provided in kit) and specimen collection labels 
are completed and filled in correctly. 
 
Ship (1) EDTA and Na Heparin tubes with a properly prepared cold pack. 
CellSave and (1) EDTA tubes will be shipped ambient at the top of the 
refrigerated shipper. See kit instructions for specific details for cold pack 
preparation (i.e., frozen or refrigerated) and proper packing of blood and cold 
pack to avoid freezing of specimens. 
 
Ship specimens, via Priority Overnight service, Monday – Thursday ONLY, to 
BAP Receiving according to the kit instructions. Do not send samples on 
weekends or just prior to federal holidays. 
 
The BAP kits will include a smart shipper label (3x5 white barcoded label) 
affixed to the shipping boxes. The smart shipper label is a pre-addressed return 
label, which replaces the need for an airbill. Shipping costs will be covered by 
BEAUTY if the shipping box provided with the BAP kit is used for shipping 
specimens to BAP Freezer.  
 
BAP Freezer will receive the samples and immediately forward specimens to the 
 
 
14.42 Handling Specimens 
 
Samples will be stored in BAP. 
 
14.5 
Background and Methodology 
 
 
14.51 Genome wide SNP analysis 
 

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2.5 M Illumina Omni chips will be used to obtain SNPs across the entire genome. 
These SNPs will serve as a quality control for both exome sequencing performed 
with the same DNA samples and that with tumor DNA. It will also serve as a 
base for further imputation to obtain SNPs outside exons, i.e. regulatory regions. 
The genome wide SNPs and imputed SNPs will then be examined in terms of its 
association with response phenotype.  
 
Sequencing will be analyzed through the pipelines created in the Mayo 
bioinformatics Core. All the sequencing results and genome wide SNP results 
will be transferred to the statistical group for quality control and statistical 
analyses.  
 
14.52 Exome sequencing 
   
DNA isolated from blood will also be used to perform exome sequencing. The 
sequencing results will be compared with sequencing results of tumor DNA from 
the same patient. The comparison will allow us to identify tumor specific 
mutations in individual samples. 
 
14.53 Future whole genome sequencing 
 
If the price becomes affordable during the period of this project, we will apply 
whole genome sequencing instead of exome sequencing of DNA isolated from 
individual blood samples.   
 
14.54 Ki 67 methodology and scoring 
 
Immunohistochemical staining of the Ki67 antigen will be performed in 
formalin-fixed paraffin-embedded tissue sections using the MIB-1 clone on 
either a Leica Bond III or Ventana BenchMark-XT automated stainer. Image 
acquisition and analysis will be performed using the Dako ACIS® III or the 
NanoZoomer Digital Pathology (NDP) 2.0HT instrument (Hamamatsu 
Corporation) along with the Definiens Tissue Studio software package version 
3.6. The percentage of positive staining tumor nuclei are quantitated by software 
after tumor selection by the technician. Results of tumor staining and appropriate 
controls will be reviewed by a pathologist. 
 
14.55 Assessment of Clonality 
 has developed a 
methodology to adapt genomic DNA isolated from flow cytometrically purified 
nuclei for use with genomic analyses including array comparative genomic 
hybridization (aCGH) and next-generation sequencing (NGS). Using this 
methodology, efficient high-resolution genomic analysis of clonal populations 
from patient biopsies is possible.  
We plan to apply this approach to analyze distinct clonal tumor populations 
within clinically annotated BEAUTY breast cancer specimens in order to study 
their evolution under the selective pressure of therapy and to evaluate potential 
mechanisms of therapy resistance. In addition we will apply these same methods 
to study a set of tumor grafts derived from patients enrolled in BEAUTY. 
Our initial focus will be comparing tissue obtained at the time of surgery from 
those patients with significant residual disease to those with partial and those 

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with complete response. Using samples from patients with varying degrees of 
response to standard chemotherapy, we will perform the following: 
1) Utilize core biopsies obtained at baseline (prior to chemotherapy) and 12 
weeks (after completion of paclitaxel), as well as any residual tumor tissue 
collected at the time of surgery. These tissues will be assessed by the study 
BEAUTY pathologist for determination of tumor cellularity, using an H&E 
cut from a cryosection (for baseline and 12 weeks). For the surgical 
specimen, a determination of tumor cellularity has already been conducted, 
and therefore the best surgical specimen will be utilized. 
2) For those patients with established xenografts, we will additionally send 
tumor samples derived from the 2nd generation of tumor growth. If 
xenografts are available from both baseline and surgery, tissues from both 
time-points will be sent. 
3) Following confirmation of tumor cellularity, a portion of each of these 
specimens from these 3 time points and the corresponding xenografts will 
be shipped to
 
 
 
 
The remaining portion of the tumor will be retained here at Mayo Rochester 
for RNA seq. 
4) For the portion of the tumor sent to 
, separation of the 
aneuploid and diploid populations will be performed using DAPI-based DNA 
content measures. Following this, 
 will profile each population 
with aCGH. 
5) The diploid populations and any remaining tissue will be shipped to Mayo 
Rochester for further studies analyzing T cell receptors (collaboration with 
. The DNA from sorted tumor populations will be submitted 
to 
 lab here at Mayo Clinic for sequencing.   
 
14.56   T cell sequencing profile 
 
Infiltration of immune cells including tumor-infiltrating T lymphocytes (TILs) in 
breast cancer tissues, and higher level of infiltration is considered to be a 
predictor of better clinical outcome of cancer patients (1). For example, the 
increased level of TILs in cancer tissues has been suggested as an independent 
predictive marker of response to neoadjuvant chemotherapy in breast cancer (2). 
However, most of studies are based on quantification of total TILs in tumors 
without examination of their functional characteristics that confer anti-tumor 
immune effects. 
 
T cell receptor (TCR) is primarily a heterodimer of alpha (TCRA) and beta 
(TCRB) chains.  These two genes cause rearrangement between VJ (alpha) and 
VDJ (beta) segments and determine functional diversity to recognize epitope of 
target cells. With the progress in next generation sequencing technologies, it has 
become possible to characterize millions of receptor clones representing the TCR 
repertoire of T cells from ~10 ml of blood or from cancer tissues (3). The TCR 
sequencing would be an effective way to examine the antigen specific expansion 
of T-cell subclones and in turn can help to characterize functional TILs of 
interest in response to cancer treatment. 

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The presence or absence of poly-(or oligo-) cloncal T cell population that may be 
associated with response to neoadjuvant chemotherapy will be investigated. The 
TCRA and TCRB cDNA of TILs in tumor biopsies that are collected before and 
after chemotherapy will be sequenced. To obtain cDNA samples of T 
lymphocytes, total RNAs will be isolated from peripheral blood mononuclear 
cells using Trizol reagent (Life Technologies, Carlsbad, CA) and then reversely-
transcribed into cDNAs using random primers. The synthesized cDNA samples 
will be amplified by polymerase chain reaction (PCR) to amplify the TCRA and 
TCRB cDNA with specific primer sets. The obtained amplicons will be ligated to 
adaptors, and further amplified onto the proprietary Ion Sphere particles by 
emulsion PCR, and then sequenced on the Ion Torrent PGM sequencer (Life 
Technologies, Carlsbad, CA). These experiments will be conducted in the 
University of Chicago Medical Center (
  
 
In this study, we plan to apply the next generation sequencing technology to 
profile TCR repertoire of T lymphocytes that could be related with response to 
neoadjuvant chemotherapy in breast cancer. Our findings will provide new 
insights for the establishment of immune markers predictive to neoadjuvant 
chemotherapy or can be applied as the surrogate biomarkers of monitoring 
patient’s response during treatment. 
 
To compare TCR profiled between two groups, 30 patients total, will be 
necessary, aiming for 15 each of responders and non-responders to neoadjuvant 
chemotherapy. The separated plasma for the proteomic samples and stored the 
frozen WBC aliquots (in storage), from both from baseline and surgery will be 
sent to 
 for sequencing. 
 
Circulating T cells in blood will be examined as control TCR profiles of 
individual patients. To obtain cDNA samples of T lymphocytes, total RNAs will 
be isolated from peripheral blood mononuclear cells using Trizol reagent (Life 
Technologies, 
 and then reversely-transcribed into cDNAs using 
random primers. The synthesized cDNA samples will be amplified by 
polymerase chain reaction (PCR) to amplify the TCRA and TCRB cDNA with 
specific primer sets. The obtained amplicons will be ligated to adaptors, and 
further amplified onto the proprietary Ion Sphere particles by emulsion PCR, and 
then sequenced on the Ion Torrent PGM sequencer (
 
 These experiments will be conducted in the University of Chicago Medical 
Center 
): 
 
 
 
 
  
Sequencing results will be analyzed by bioinformatics collaborator, 
 
 
 
From the sequencing data, the entire TCR profile will be compared between (1) 
blood and cancer tissue before treatment (clonality of TIL could be judged), (2) 

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blood and cancer tissue after treatment (clonality of TIL could be judged), and 
(3) between two tissues before and after the treatment (enrichment of certain T 
cell population(s) in cancer tissue and their relation with drug response can be 
examined). In addition, if the enrichment of specific type of TCR sequence(s) is 
identified, the information may be applied as immune markers to predict 
response of neoadjuvant chemotherapy.  
 
DNA sequencing results will be abstracted onto a secure database which 
will be maintained by 
 lab. The whole system will be 
protected by password and a backup disk will be kept in a locked file 
cabinet. 
 
14.57 APOBEC3B 
 
The human DNA deaminase APOBEC3B is a newly discovered genetic marker 
for breast cancer, with expression studies demonstrating its over-expression in 
~60% of breast cancers (
 in process). APOBEC3B 
mutates the genomic DNA of breast cancer cells causing an overall increase in 
mutation frequency (
), in process). It is highly likely that 
this mutator activity contributes to the heterogeneity of breast cancer and in turn 
to negative outcomes (i.e. higher APOBEC3B = faster tumor evolution = more 
likely to evolve resistance to therapy and immune responses). Here, we will take 
an unbiased approach to testing this in the context of this protocol. Quantitative 
expression profiling of APOBEC3B mRNA levels will be performed in 
comparison to those of the endogenous housekeeping gene TBP [17]). 
 
14.58 Proteomic studies 
 
As part of ongoing research, we will collect plasma for proteomic studies, 
according to patient consent information. Samples will be frozen at −70ºC by 
BAP. 
 
RPPA 
) 
Reverse phase protein array (RPPA) is a high-throughput antibody-based 
technique developed for Functional Proteomics studies to evaluate protein 
activities in signaling networks. Protein extracted from tumor samples will be 
analyzed for multiple proteins important in drug response and resistance. A list of 
the proteins is included in Appendix III. 
 
14.59a Homologous recombination assay (
) 
 
Homologous recombination (HR) is a critical pathway important in the repair of 
double and single stranded DNA.  Both hereditary and somatic alterations in 
genes responsible for HR are implicated in predisposition to cancer, but 
additionally, genetic alterations in this pathway appear to be critically associated 
with response to chemotherapy drugs which  
 
target genes in the HR pathway.   For example, mutations in BRCA1 and BRCA2  
have been demonstrated to have potential therapeutic relevance in the setting of 
DNA damaging agents and cells deficient in BRCA1 or BRCA2 are sensitive to 
PARP inhibitors.   

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Myriad developed a DNA-based HR score (HRD) based on genome-wide LOH 
analysis of ovarian tumors combined with comprehensive profiling of BRCA1 
and BRCA2 defects.  Recently, Telli et al assessed the  ability of the HRD score 
to predict pathologic response to neoadjuvant platinum-based therapy in early-
stage triple-negative and BRCA1/2 mutation-associated breast cancer and 
demonstrated that germline BRCA1/2 mutation status was known in all patients 
and 13/55 tumors were from carriers of deleterious BRCA1 (n = 9), BRCA2 (n = 
3) or BRCA1&2 (n = 1) mutations. There was a statistical association between 
response to treatment and HRD score (p= 0.004).  
 
Currently, a standard treatment employed in BEAUTY for high risk locally 
advanced breast cancer is taxane and  anthracycline-based therapy. However, 
there are no data regarding the role of HRD and either taxane response or 
response to AC therapy. Therefore, a key goal of these translational studies will 
be to assess the response to individual chemotherapy regimens according to HRD 
score. 
 
 
 
 
 
 
14.59b Analysis of estrogen receptor beta (ERβ) expression 
 
ERβ is a member of the nuclear receptor superfamily of proteins that functions as 
a ligand mediated transcription factor and is classically thought of as a tumor 
suppressor in multiple cell and animal model systems.  ERβ is known to be 
highly expressed in normal breast epithelial cells and tends to decrease in 
expression levels during progression from atypical hyperplasia to DCIS to breast 
cancer.  However, substantial levels of ERβ protein are detectable in about 30-
40% of all sub-types of breast cancer.  For these reasons, ERβ is being 
considered as a novel therapeutic target. 
 
Recent studies have also demonstrated that ERβ expression can sensitize breast 
cancer cells to the anti-cancer effects of multiple chemotherapy drugs.  Ongoing 
studies from the Hawse laboratory have also shown that ERβ can suppress TGFβ 
expression in triple negative breast cancer cells, a pathway that is known to lead 
to the development of chemotherapy resistance.  For these reasons, it is of 
interest to determine if ERβ positivity is associated with xenograft take rates 
and/or response to chemotherapy in triple negative breast cancer patients.   
 
To determine ERβ protein levels in breast tumors, a 5 micron section will be 
stained with a highly specific and sensitive mouse monoclonal antibody 
(PPG5/10).  IHC stains will be performed on a Leica Bond III stainer using a 
1:75 dilution of this antibody purchased from Thermo Scientific.  IHC stained 
slides will be analyzed by a dedicated breast cancer pathologised and scored 
based on nuclear extent and intensity of staining.  A sum score will be calculated 
based on the estimated percentage of positive cells (less than 1% will be 
considered negative (0), 1%-25% (1), 26%-50% (2), 51%-75% (3) and 76%-

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100% (4)) and the intensity of staining (none (0), weak (1), moderate (2) or 
strong (3)).  Tumors will be grouped into ERβ negative/low (1-2), ERβ moderate 
(3-5) and ERβ high (6-7) using this sum score. 
 
14.59c Human Leukocyte Antigen (HLA) Typing 
Although the complex relationship between immune function and neoplastic cells 
is important in both carcinogenesis and cancer progression, at this time it is not 
fully understood.  On average, a malignant cell will accumulate 10 mutations that 
alter the antigenic profile of the tumor and can be presented by class I major 
histocompatibility complex (MHC) molecules to CD8+ T cells.  In addition, 
tumors often express proteins that aren’t normally expressed by that cell 
type.  Furthermore, tumor cells may undergo apoptosis, which can elicit an 
immune response, or can become senescent, resulting in the production of pro-
inflammatory cytokines.  Due to these changes, the host immune system can 
recognize and destroy cells that have diverged from normal development or are 
becoming malignant.   It has been suggested that tumors develop or progress in 
part as a result of loss of immunostimulatory antigens or suppression of the 
immune response. 
 
The Human Leukocyte Antigen (HLA) gene family is located on chromosome 6 
and encodes for a group of related proteins that are utilized by the immune 
system to distinguish self proteins from foreign/non-self proteins. HLA-A, HLA-
B, and HLA-C are class I genes and encode for proteins present on nearly all cells 
in humans, while HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQBI, HLA-DRA, 
and HLA-DRB1 are class II genes and encode for proteins that are present 
primarily on the surface of immune system cells.  Due to a high degree of 
sequence homology and the density of variable nucleotides, this region has been 
challenging to sequence.   Therefore, we will use a software package to assign 
HLA types using the previously generated exome sequencing data. In order to 
validate that the software is working appropriately in the BEAUTY population, 
we will compare the results generated by the software package with HLA-typing 
performed in the Tissue Typing Laboratory at Mayo Clinic in a subset of 
BEAUTY patients.  If  there is good concordance, the HLA types generated by 
the software will be used to test for an association with breast cancer subtypes as 
well as response to chemotherapy using the various response phenotypes 
generated in the BEAUTY study.   
 
14. 59d Evaluation for clonal hematopoiesis of indeterminate potential 
 
Clonal hematopoiesis of indeterminate potential (CHIP) is defined as somatic 
mutations in hematopoietic cells in an individual with normal hematologic 
indices and without an underlying hematologic disorder.  Whole exome 
sequencing in the normal population demonstrates that CHIP is an age-related 
phenomenon. CHIP is rarely identified in individuals younger than 40, but the 
incidence rises to more than 10% in persons above the age of 70.[18]  Mutated 
genes are involved in regulating DNA methylation, hydroxyl-methylation, 
chromatin remodeling, splicing, tumor suppression and signal transduction, with 
frequently mutated genes including DNMT3A, TET2, and ASXL1. In large 
population studies, the presence of these premalignant clones was associated with 
an increased risk of developing a future hematologic malignancy, coronary heart 

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disease, and all-cause mortality.[18, 19]  Further, in small retrospective studies of 
patients with solid organ or hematologic malignancy discovered that patients 
harboring CHIP at the time of primary malignancy diagnosis have an increased 
risk of developing a subsequent therapy-related myeloid malignancy (neither of 
these studies included patients with breast cancer).[20, 21]  We further 
hypothesize that breast cancer patients with these abnormalities in hematopoietic 
cells may additionally experience more neutropenia and thrombocytopenia 
during chemotherapy.  The correlative studies we propose aim to expand on the 
limited existing data on the incidence of CHIP in patients diagnosed with breast 
cancer, and explore the potential impact of CHIP on the development of 
cytopenias during treatment and subsequent therapy-related myeloid neoplasms.  
 
Whole exome sequencing results will be analyzed by the Beauty team 
bioinformaticians in collaboration with 
.  To determine the 
incidence of CHIP, we will evaluate 74 genes identified in the Supplemental 
Table S3 of the Jaiswal et al. 2017 study in the peripheral blood specimens 
collected prior to neoadjuvant chemotherapy.  We will utilize two variant 
detection programs, MuTect2 and VarDict to detect CHIP mutations.  Variants 
will be annotated using the Ensembl Variant Effect Predictor program.  Both 
callers have been reported to provide sensitivity in detecting low-allelic fraction 
variants while retaining respectable specificity in comparative evaluations.  In 
patients where somatic mutations consistent with CHIP are identified, we will 
evaluate pre-treatment hematologic indices, and if within references ranges, 
confirm a diagnosis of CHIP.  We will subsequently evaluate the entire cohort to 
correlate the presence of CHIP with delays or dose reductions in chemotherapy 
due to cytopenias, delayed count recovery after treatment, and developing 
therapy-related myeloid neoplasms.  
 
    
15.0 
Information concerning research procedures 
 
 
None 
 
 
 
 

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16.0 
Statistical Considerations and Methodology  
 
16.1 
Study Design and Specimen Collection 
 
A prospective cohort study is being conducted to collect biospecimens before during and 
after completion of neoadjuvant chemotherapy in participants with Stage I-III breast 
cancer to identify novel somatic changes within gene and gene pathways that are 
potentially “druggable.”  
 
A minimum of 200 and a maximum of 230 participants will enroll onto this study to 
ensure at least 200 eligible patients with sufficient pre-neoadjuvant treatment tissue and a 
post-initiation neoadjuvant treatment tissue is available for analysis. 
 
16.2 
Analysis Plans 
 
16.21  Pre-processing of genomic data  
 
16.211 Exome sequencing data 
Processing of the sequence reads will be done by mapping the short reads to the 
genome using Novoalign, followed by re-alignment and recalibration for greater 
efficiency in SNV calling. Gemline SNV calling for each sample will be 
completed using the union between GATK’s Unified Genotyper and SNVMix. 
Somatic mutations will be identified using the union of SomaticSniper, 
JointSNVMix2, MuTect, and GATK’ Somatic Indel detector – all of which 
simultaneously use the tumor and germline information to determine somatic 
status. We will then go back and use BEAGLE to refine and call missing 
genotypes using LD based imputation methods. All germline/somatic SNV and 
small insertions/deletions will be thoroughly annotated using BioR – which 
includes SIFT, PolyPhen2, and allele frequencies from numerous populations. 
Copy number variation will be assessed through PatternCNV – Mayo’s in-house 
solution to derive copy number data from exome capture studies. Finally, using 
data from the 2.5 Million variant Illumina Omni chip, we will estimate variants 
outside of the exons using the 1000 Genomes Project Data and state of the art 
imputation methods that utilize linkage disequilibrium. 
 
Variants will initially be prioritized based on whether they exist in genes relevant 
for breast cancer therapy (germline or somatic) or familial breast cancer risk 
(germline). These small lists of genes are reported in a user friendly HTML-
based report that allows rapid, easy, and simplified access to genomic variants 
that have the most potential for clinical implications. 
 
16.212 RNAseq data 
For paired-end RNA-Sequencing data analysis, we will use MAP-RSeq - a 
comprehensive computational workflow developed at Mayo Clinic to obtain a 
variety of genomic features from an RNA seq experiment 
(http://bioinformaticstools.mayo.edu/research/maprseq/). MAP-RSeq uses a 
variety of freely available bioinformatics tools along with in-house developed 
methods. The main goal of the MAP-RSeq workflow is to align, assess and 
deliver multiple genomic features. These include gene expression, novel 
transcripts, alternative splice sites, expressed SNVs, fusion transcripts, long non-

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coding RNAs, small insertions, small deletions, etc. Alignment of reads will be 
performed using Bowtie. Bowtie is a fast and memory efficient short sequence 
aligner. It aligns reads to the genome and maps them to a genome assembly. 
Unaligned reads from Bowtie are used by TopHat to map them to splice 
junctions. The aligned bam file from TopHat is used by HT-Seq software to 
calculate gene abundance per sample. Along with binary alignment (bam) and 
junctions bed file, TopHat also provides a list of expressed fusion transcripts 
using the TopHat-Fusion algorithm. 
 
At 
, we have also developed a novel computational method (ESNV-
Detect) to identify expressed single nucleotide variants from paired-end RNA-
Seq data. We use a combination of open access bioinformatics tools along with 
our in-house developed method to identify expressed single nucleotide variants 
(eSNVs) from RNA-Seq data confidently. ESNV-Detect uses consensus data 
from multiple aligners to call eSNVs. Features like number of reads at a 
nucleotide position, reference supporting reads, alternate allele reads, forward 
strand supporting reads, reverse strand supporting reads, base qualities, mapping 
qualities etc. are used to call an expressed SNV. A variety of sources such as 
1000 genome, dbSNP, and 5400 exome databases are used to annotate and 
identify known and novel variants from RNAseq datasets.  
 
Detailed QC reports, gene expression counts, fusion transcripts data and eSNVs 
will be obtained from MAP-RSeq and ESNV-Detect for each patient before and 
after therapy for further analysis. As a QC method, the concordance/discordance 
between the eSNVs detected from the RNAseq data will be compared with the 
exome sequencing data. 
 
16.213 Methylation data 
Methylation analysis will be completed from tumor taken before, during, and 
after neoadjuvant therapy using Illumina’s 450K methylation array to determine 
if any of the novel variants found from the sequencing of the tumor are contained 
or near known CpG sites (or islands) or if there is a change in CpG methylation 
pattern during and after  neoadjuvant therapy.  
 
16.22 Statistical/bioinformatic analyses of genomic data 
 
The primary objectives of this study are to ascertain the germline and somatic 
changes within gene and gene pathways that occur before and after completion of 
paclitaxel and examine whether these changes differ between tumors that respond 
to paclitaxel and those that do not respond to paclitaxel as well as to ascertain the 
germline and somatic changes within gene and gene pathways that occur before 
and after completion of all NAC and examine whether these changes differ 
between tumors that respond to treatment and those that do not respond to 
treatment.  These analyses will include a stratification by molecular subtype. We 
will take a discovery approach and will perform subset analyses throughout the 
course of this prospective study. Any interesting results will be further validated 
in subsequent cohort both within and outside of BEAUTY as well as through the 
use of laboratory functional studies as deemed relevant. 
 
The response phenotypes are defined in section 11.0 and include the following: 
Ki-67 response, residual cancer burden, radiographic response. The response 

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phenotypes will be assessed post paclitaxel and at surgery (following completion 
of all chemotherapy) 
 
Generalized linear models will be used to assess the association of the each 
somatic variant with the clinical endpoints described above. This will first be 
done using a univariate model and subsequently, we will adjust for known 
prognostic patient and tumor characteristics. Second, all somatic variants within a 
particular gene will be pooled into a single variable describing whether or not 
any somatic variant exists and we will evaluate the association between this 
pooled variable with the clinical endpoint under investigation. Third, rare variant 
collapsing methods [22, 23] will be used to determine the association of a 
“region” with the endpoint under investigation.  
 
Fourth, pathway association analyses will be completed in which the number of 
variants a tumor has in a given pathway will be associated with the endpoint 
under investigation, using analysis approaches similar to that described by Morris 
and Zeggini [24]. If necessary, population stratification can be employed using 
an eigen analysis of the genome-wide SNP variants measured on the germline 
DNA [25]. Lastly, if a variant is significant, we can perform a eQTL analysis 
using the RNAseq data to evaluate function. 
 
RNAseq data will be normalized using conditional quantile normalization (cqn). 
For each gene separately, a negative binomial model will be used to assess the 
association between the RNAseq gene counts and the clinical endpoints. The cqn 
parameters will be incorporated into the offset of the negative binomial models in 
order to normalize the data. Subsequently, we will redo analyzes adjusting for 
known prognostic variables. We will also evaluate if these differently expressed 
genes are in similar pathways as well as perform pathway analyses.  
 
The fusion genes detected from the RNAseq data will summarized descriptively 
across the entire cohort, across molecular subtypes, as well as across the 
endpoints described above. 
 
Methylation levels will be correlated with gene expression obtained by RNA seq 
and SNPs/mutations identified by sequencing. Generalized linear models will be 
used to examine the association between methylation status and each of the 
clinical endpoints.  
 
As an exploratory approach, the correlation between methylation levels, gene 
expression, SNPs/mutations and/or CNV will be evaluated using canonical 
correlation analysis. 
 
Adjustments for multiple testing of multiple variants will be made using both the 
simple (but conservative) Bonferroni adjustment along with permutation based 
adjustment [26]. False-discovery rates and q-values will be calculated [27].  
 
16.221 HRD deficiency  
 
An initial pilot of 5-10 samples will be sent to Myriad to assess quality of 
DNA in the specimens, without exchange of clinical data.   The HRD 
results obtained from these samples as well as  the HRD results obtained 

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from the remaining samples will be used in these analyses.  It is 
anticipated that the failure rate of the HRD assay will be 10%.  A sample 
size of approximately 120 patients is anticipated.    
 
Based on prior research by Myriad,  HR Deficiency is defined as either a 
high HRD score (≥42) or a BRCA1/BRCA2 mutation in the tumor.     
 
Extended Fisher’s exact test will be used to determine whether the 
proportion of patients with pretreatment HR deficiency differs among 
tumor subtypes (HER2- and ER+, HER2-and ER-, HER2+).   In 
addition, for patients who are not BRCA1/BRCA2 mutational carriers, 
whether pretreatment HR scores differs among molecular subtypes will 
be assessed using either Kruskual Wallis test or general linear F-test 
using the results of fitting a one factor ANOVA model and whether the 
proportion or patients with high HRD scores differ among among 
molecular subtypes will be assessed using chi-square test. 
 
Fisher’s exact test will be used to determine whether  
1. Radiographic or Ki67 response to taxane therapy rates 
differ with respect to pretreatment HR deficiency 
2. MRI response after anthracycline based chemotherapy in 
patients with residual tumor seen on MRI after taxane 
based therapy differs with respect to either pretreatment 
or post-taxane HR deficiency 
3. pCR to neoadjuvant therapy differs by pretreatment HR 
deficiency 
4. pCR to neoadjuvant therapy differs by post-taxane HR 
deficiency 
5. pCR to neoadjuvant therapy differs by post- 
anthracycline HR deficiency 
 
Logistic regression modeling will be used to assess the impact of 
pretreatment HR deficiency on determining the likelihood of pCR after 
other patient and disease characteristics known to impact pCR rates are 
accounted for in the model. To maintain model stability, 1 dependent 
parameter can be added to the model for every 15-20 patients included in 
the model. If feasible,  logistic regression modeling will also be used to 
assess the impact of pretreatment HR deficiency on determining the 
likelihood of pCR within molecular subgroups.  
 
Time series plots will be used to examine the changes in HRD scores 
throughout the course of neo-adjuvant treatment to visually assess 
differences between those who had a pCR and those who did not, for all 
patients as well as by tumor subtype and BRCA1/BRCA2 status. 
 
16.23 Molecular Breast Imaging 
MCR patients only:  Tumor uptake of 99mTc-sestamibi will be quantified using 
software developed in-house. Algorithms for calculation of tumor uptake from 
opposing views of the breast have been validated in phantom models and 
computer simulations [16]. This will allow calculation of true tumor / 
background ratio.  

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Spearman’s rank correlation coefficient will be used to assess (1) the strength of 
the association between the uptake in pre-treatment breast tumors to 99mTc-
sestamibi and tumor grade and cell proliferation index from the pre-treatment 
biopsy specimen. 
 
The percent change in uptake in breast tumors to 99mTc-sestamibi during 
neoadjuvant chemotherapy from pretreatment levels will be measured. 
Spearman’s rank correlation coefficients will be used to assess (1) the strength of 
the association between the percent change in uptake to 99mTc-sestamibi at 
completion of neoadjuvant chemotherapy and pathologic response and residual 
disease burden. 
 
16.3 
Sample Size justification: 
 
Table 1: Detectable effect sizes (odds ratio) for various analysis with N = 200 
 
 
Minor Allele Frequency or Variable Frequency 
for Region and Pathway Analyses 
Analysis 
Α 
5% 
10% 
20% 
Single Variant 
0.00001 
OR > 20 
8.9 
5.4 
Region/Gene 
0.0001 
19.3 
6.7 
4.4 
Pathway 
0.001 
10.1 
5.0 
3.6 
 
With a sample of 200 randomly ascertained subjects, we will be able to detect 80% of the 
variants in a region (assuming 20 variants in the region) with minor allele frequency of 
0.005 with probability 0.86[28]. 
 
The minimum detected odds ratio for pCR assuming that 40% of the 200 subjects 
enrolled will have a pCR is presented in Table 1 for a variety of minor allele 
frequencies/variable frequencies (5%, 10% and 20%) using a test of association with 
significant level α (0.00001, 0.0001 and 0.001). The significant levels used are to account 
for the testing of multiple hypotheses. Power was computed using the software package 
Quanto for a binary endpoint and assumed a dominant genetic model 
 
Accrual Targets 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
12 
0 
12 
Not Hispanic or Latino 
216 
2 
218 
Ethnic Category: Total of all subjects 
228 
2 
230 
Racial Category 
American Indian or Alaskan Native 
1 
0 
1 
Asian 
15 
0 
15 
Black or African American 
13 
0 
13 
Native Hawaiian or other Pacific Islander 
1 
0 
1 
White 
198 
2 
200 
Racial Category: Total of all subjects* 
228 
2 
230 
 
 

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Ethnic Categories 
Hispanic or Latino – a person of Cuban, Mexican, Puerto 
Rican, South or Central American, or other Spanish culture 
or origin, regardless of race. The term “Spanish origin” can 
also be used in addition to “Hispanic or Latino. 
Not Hispanic or Latino 
Racial Categories 
American Indian or Alaskan Native – a person having 
origins in any of the original peoples of North, Central, or 
South America, and who maintains tribal affiliations or 
community attachment. 
Asian – a person having origins in any of the original 
peoples of the Far East, Southeast Asia, or the Indian 
subcontinent including, for example, Cambodia, China, 
India, Japan, Korea, Malaysia, Pakistan, the Philippine 
Islands, Thailand, and Vietnam. (Note: Individuals from the 
Philippine Islands have been recorded as Pacific Islanders in 
previous data collection strategies.)  
Black or African American – a person having origins in any 
of the black racial groups of Africa. Terms such as “Haitian” 
or “Negro” can be used in addition to “Black or African 
American.” 
Native Hawaiian or other Pacific Islander – a person 
having origins in any of the original peoples of Hawaii, 
Guam, Samoa, or other Pacific Islands. 
White – a person having origins in any of the original 
peoples of Europe, the Middle East, or North Africa. 

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17.0 
Pathology Considerations/Tissue Biospecimens   
17.1  
Summary Table of Research Tissue Specimens to be Collected for this Protocol 
Correlative Study 
(Section for more 
information) 
Mandatory 
or 
Optional 
Type of 
Tissue to 
Collect 
After 
registration 
but prior to 
chemotherapy 
After the 
completion of 
taxane prior to 
anthracycline 
based 
chemotherapy 
At the 
time of 
surgery 
(cycle 3) 
At first 
recurrence or 
post treatment  if 
primary surgery 
is not to be 
performed 
Process 
at site? 
(Yes or 
No) 
Temperature 
Conditions 
for Storage 
/Shipping 
Visit Alias – Lab 
Use Only 
 
 
V1T (Visit 1 
Tissue) 
V2T (Visit 2 
Tissue) 
V3T (Visit 3 
Tissue) 
V4T (Visit 4 
Tissue) 
 
 
Tumor sample (for 
genotyping) 
Mandatory 
Breast 
tumor 
X 
X 
X+ 
X* 
Yes 
Frozen, Dry 
Ice 
Tumor sample (for 
establishing 
xenograft)++ 
Mandatory 
Breast 
tumor 
X1 
 
X1+ 
 
X1 
Yes, 
Place in 
PBS 
Refrigerated 
cold pack 
Tumor sample (for 
correlative studies) 
Mandatory 
Breast 
tumor 
X 
X 
X+ 
X* 
Yes 
Frozen, Dry 
Ice 
Diagnostic slides 
and representative 
tumor block) from 
original and/or 
recurrent  tissues 
(note, tumor cores 
are acceptable if an 
outside institution is 
unable to send 
blocks) 
Mandatory 
 
X 
 
X 
X 
 
 
* If patient develops disease recurrence – including local, regional, or distant disease site.  Biopsy of locally recurrent (local or regional) or metastatic lesion for those patients with disease 
recurrence. 
1  Tissue samples for xenografts must be placed  immediately into the 50 mL falcon conical tube containing 10 mL of PBS media in the procedure room. In MCR and MCF these are to be 
taken directly to the laboratory for xenografting. In MCA, place falcon tube containing tissue samples on wet ice until samples can be shipped refrigerate to Mayo Rochester.+ If 
preoperative imaging suggests complete response and complete response is confirmed by intra-operative frozen section then the lymph nodes should be evaluated for response as well.  If the 
lymph nodes from axillary dissection demonstrate significant residual disease then fresh tissue samples can be obtained from these nodes for the purpose of xenografting, genomic 
sequencing and/or storage.++ Please email Dr.  Liewei Wang’s laboratory  DL RST BEAUTY Xenograft Tissue Pickup Team, 
nd 
 
when you have a patient scheduled for biopsy and inform the team the date of biopsy. If biopsy is canceled for any reasons, please also send an email to inform 
 
 

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17.2 
Correlative Tissue Collection  
 
17.21 Xenograft tissue shipping boxes will be provided for this protocol. Shipping 
boxes can be ordered from the fax supply order form.   
 
Note: Tissue supplies to be ordered are: 
 
Falcon tubes: 50 mL Conical 332600 and the 15 mL conical is 332590 
PBS: GIBCO (catalogue number 10010-023) 
 
17.22 Fresh Tissue for xenograft 
 
Tumor for xenograft from MCA will be immediately stored in fresh PBS in a 
50 ml falcon tube and shipped overnight to:  
 
 
 
 
 
MCF will develop xenograft locally. Once the xenografts are established and 
frozen down, an aliquot of these cells will be shipped to Dr. Wang’s 
laboratory for future sequencing and drug studies. 
 
FFPE portions of the  xenografts will go from 
 and 
undergo pathology review by 
  
 
17.221 Core biopsies from breast tumor biopsy prior to initiation of 
chemotherapy (obtained in radiology) and breast tumor tissue from time 
of surgery (obtained in the operating room and frozen section laboratory) 
will be placed into a labeled specimen container with protocol number, 
study patient ID number, and time and date sample is obtained. In MCR 
these specimens will be delivered by study staff to 
 
From MCA the samples will be shipped overnight to 
 
 
to pick up the samples.  
Samples will be injected into mice at the Barrier area on 
 
 
 
17.23 Frozen Tissue for sequencing 
 
 
17.231   Tissues (Breast tumor) obtained at biopsy prior to chemotherapy, after 
completion of taxane prior to anthracycline based chemotherapy, and 
residual disease from time of surgery will be submitted. At time of 
surgery, tissue from frozen section lab after confirmation of residual 
viable disease will be submitted.  
 

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17.232   Tissue must be snap frozen in liquid nitrogen and remain frozen at 
temperatures between -60ºC and -80ºC until shipped and during 
shipping. 
 
17.233   Label specimen container with protocol number, patient ID number, and 
patient initials. Specimens must remain frozen at all times. 
 
17.24   Tissue for additional Correlative studies  
 
Tissue for correlative studies will be collected at 4 time points: prior to initiation 
of chemotherapy, after completion of taxane prior to anthracycline based 
chemotherapy, at surgery and at time of recurrence. At each time point, the 
biopsy will be performed following the completion of the MBI (if applicable) and 
conventional imaging studies (mammogram, ultrasound, and MRI) to minimize 
post-biopsy changes on imaging. 
 
17.241 Initial biopsy (prior to initiation of chemotherapy): Eight cores from a 14 
gauge core needle biopsy device will be obtained using an image-guided 
biopsy device. Tissue will be preferentially utilized for sequencing (3 
cores - frozen), establishing xenografts (3 cores – in PBS), FFPE in 15 
mL conical tube (1 core) and the remaining 1core will be fresh frozen for 
future studies. The FFPE specimen will be utilized for ER, PR, HER2, 
and Ki67. 
 
 
An aliquot of RNA derived from the RNA extraction will be shipped for 
quantitative expression profiling of APOBEC3B mRNA levels (
 
. 
 
An extract of protein from the frozen additional cores will be shipped to 
  
 
An aliquot of tumor DNA for HRD analysis will be shipped to 
 
 
 
17.242 2nd tumor biopsy (between taxane and AC or FEC chemotherapy): six 
cores from a 14 gauge core needle biopsy device will be obtained using 
an image-guided biopsy device. One core will be formalin fixed for 
FFPE for H&E and Ki67, ER/PR/HER2 and to quantitate and identify 
tumor cells. The additional cores (5) will be frozen in liquid nitrogen and 
sent to BAP for storage, one of which will be utilized for RPPA and two 
for DNA and RNA extraction. Cores from MCA and MCF will not be 
processed locally but will be shipped to MCR (5 cores shipped frozen, 1 
as FFPE). Upon arrival they will be handled as outlined above.  
 
An aliquot of RNA derived from the RNA extraction will be shipped for 
quantitative expression profiling of APOBEC3B mRNA levels 
 
 
 
An extract of protein from the frozen cores will be shipped to 
 
 
for RPPA  

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An aliquot of tumor DNA for HRD analysis will be shipped to 
 
 
 
17.243 At the time of surgery the surgical specimen will be processed through 
the frozen section laboratory. Any residual invasive disease will be tested 
for estrogen receptor (ER), progesterone receptor (PR), Her2, and Ki67 
per standard practice. If residual invasive disease is identified in the 
resected breast tissue any residual invasive tumor after tissue taken for 
clinical care will be collected and cut into three pieces. One piece will be 
flash frozen for DNA sequencing. A second piece will be flash frozen for 
RNA sequencing. The third piece will be placed in media for establishing 
xenografts. Any residual tissue from DNA/RNA specimens will be 
stored frozen in BAP for banking purposes.  
In the event that ER, PR, Her2 and Ki67 testing is not performed 
clinically, banked tissue from tumor registry (up to 8 unstained tissue 
slides) will be accessed for ER, PR, Her2 and Ki67 testing. 
 
Another piece will be flash frozen for future protein extraction for RPPA 
 
 – see Section 14).   
 
An aliquot of RNA derived from the RNA extraction will be shipped for 
quantitative expression profiling of APOBEC3B mRNA levels (
 
 
 
An aliquot of tumor DNA for HRD analysis will be shipped to Myriad 
Laboratories 
 
In cases where there is no residual invasive disease in the breast a sample 
of the residual tumor bed should be submitted flash frozen for storage. 
 
In cases where there is no residual invasive disease in the breast, but 
there is residual metastatic disease in the lymph nodes (sentinel node 
and/or axillary nodes), then tissue from the lymph nodes with disease 
will be flash frozen for DNA and RNA sequencing. 
 
In the case where there is multifocal disease remaining in the breast at 
the time of surgery a representative portion from each of the tumors will 
be submitted for processing.   
 
 
For patients with bilateral disease the tissue from the index side will be 
processed as outlined above and any residual disease from the 
contralateral side will be processed per usual pathology guidelines and a 
representative paraffin embedded block will be stored for research 
purposes for this study. 
 
17.244 If  patient develops disease recurrence – including local, regional, or 
distant disease site; a biopsy will be obtained (see section 17.3 below) 
and tissue will be distributed as follows: 
 

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Clinical sample for diagnostics (if not previously obtained) will be 
collected per routine clinical care for confirmation of disease recurrence. 
 
Additional samples for correlative sciences will be divided into 3 
specimens and prioritized as follows: 
 
1) DNA and RNA sequencing (fresh frozen) 
 
a)    An extract of protein will be shipped to
 
  
b)   A portion of tumor specimen will be converted to RNA and 
sent to the
 for 
quantitative expression profiling of APOBEC3B mRNA 
levels in comparison to those of the endogenous 
housekeeping gene TBP [17]. 
c.   If adequate tissue is available, a sample for HRD analysis 
will be sent to Myriad Laboratories . 
2) If residual tissue – fresh tissue will be sent for establishment of 
xenograft. 
 
17.245 Tissue from biopsy of recurrent disease – tissue will be utilized  for 
sequencing, establishing xenografts. Any additional tissue will be 
paraffin embedded for future studies. 
 
An aliquot of RNA derived from the RNA extraction will be shipped for 
quantitative expression profiling of APOBEC3B mRNA levels 
 
 
 
17.246 For MCA and MCR 
 
1) Same collection information as above. One core will be placed in 
formalin and used for histologic processing to confirm the 
presence of tumor. The container will be shipped to 
 
and immediately forwarded to 
 with the tissue 
submission paperwork. 
 will accession the formalin 
specimen and e-mail the pathology coordinator at 
 
 The pathology coordinator will pick up the formalin 
tissue container and take it to 
 for embedding.
 
will notify the pathology coordinator to pick up the tissue and 
store the sample with the 
 
.  
 
17.25 Correlative Science – evaluation of tumor bed tissue from surgery 
 
FFPE tumor blocks obtained from residual tumor and tumor bed samples 
collected at the time of surgery and banked in the 
 
all 3 sites will be obtained and 3 slides each of 5 microns thickness will be cut – 
1 will be stained for H&E and 2 unstained slides. The unstained slides will be 
stained for SNAIL IHC. 
 

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SNAIL can only be detected by IHC using FFPE tissue therefore stored samples 
from tissue registry will be used, rather than frozen tissue previously collected on 
this protocol. 
 
 
17.3 
Diagnostic slides from original tissue 
ALL original diagnostic slides and a  representative tissue block(s) containing invasive 
tumor from time of initial breast biopsy (note tissue cores are acceptable if the referring 
institution is unable to send blocks) will be obtained from the outside referring institution. 
Specimens should be clearly labeled and forwarded as soon as possible after surgery 
OR <30 days after registration for central review. ALL original diagnostic slides used to 
make the diagnosis of Breast cancer should be clearly labeled and forwarded < 30 days 
of registration for QA audit according to shipping instructions below. 
 
 
 
This Central Review is for research purposes only and not to verify diagnosis. 
Central pathology review will not be placed into the patient’s clinical record.  In the 
unlikely event a significant change is noted between the local pathology findings and the 
central pathology review the central pathologist will communicate the variances with the 
treating physician.    
 
17.31  Diagnostic slides from surgery 
 
Diagnostic slides of the tumor bed and any residual disease (DCIS or invasive) 
and of the lymph nodes from time of surgery will be obtained from Mayo Clinic 
AZ, Mayo Clinic FL and Mayo Clinic Rochester locations. Specimens should be 
clearly labeled and forwarded as soon as possible after surgery for central review.  
 
Additionally unstained slides from any residual invasive disease (breast or lymph 
node if no invasive disease in the breast) should be submitted from Mayo Clinic 
AZ and Mayo Clinic FL for tumor biomarker staining (ER, PR, Her2 and Ki67) 
as indicated below. 
  
ALL diagnostic slides and 10 unstained slides (charged and cut at 5 microns) for 
tumor biomarkers should be clearly labeled and forwarded to: 
 
 
 
This Central Review is for research purposes only (RCB, the primary endpoint) 
and not to verify diagnosis.  
 
 
 

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17.4 
Recurrent disease tissue 
 
17.41  Guidelines for tissue acquisition  
 
17.411 Image guided biopsies are recommended where feasible. The amount of 
tissue collected will follow the guidelines listed below. If a patient has 
more than one site of disease, only one site needs to be biopsied. 
 
17.412 Skin/chest wall:  An incisional/excisional biopsy is preferred.   If punch 
biopsy is performed, a minimum of 3 punch biopsies (≥ 5mm diameter). 
 
17.413 Lymph node: A goal of 3-6 core biopsy specimens obtained 
using an 18-gauge needle or larger. Smaller or less samples will be 
obtained at the discretion of the physician performing the biopsy. 
 
17.414 Liver: A goal of 2-3 core biopsy specimens obtained using an 18-gauge 
needle. 
 
17.415 Lung: Because of the risk of pneumothorax associated with core needle 
biopsies of lung nodules, core biopsies are not required.  If the tissue is to 
be obtained for clinical care; an additional 1-2 core biopsies are to be 
submitted for research purposes. 
 
17.416 Bone: Because the yield of malignant tissue from bone biopsies tends to 
be relatively low, if a patient has another accessible site of disease (i.e., 
skin, lymph node, liver), that site should be biopsied preferentially. If 
bone is the only biopsy-accessible site, then a goal of 3-6 core biopsy 
specimens will be obtained using an 18-gauge needle. 
 
17.5 
Background and Methodology 
 
17.51 Overview 
 
A series of high throughput assays including exome sequencing, RNA seq and 
CpG methylation will be applied using tumor samples from a series well 
annotated samples obtained at the base line (biopsy), after initial chemotherapy 
and during the surgery (after finishing neoadjuvant  chemotherapy). To control 
for possible batch effects, the exome sequencing of the tumor and germline DNA 
prior to treatment will be completed at same time. In addition, mRNA will be 
extracted from the tumor for RNA-seq at the same time as sequencing of the 
DNA. 
 
Integrated analysis will then be performed to identify signatures that are 
associated with complete pathological response. With those non responders, 
sequencing analysis will be used to identify mutations in genes within the known 
signaling pathways that have available drug targets, such as PI3K, AKT, EGFR, 
mTOR, etc. These analyses will allow future individualized the therapy with 
selective targeted agents. Additional integrated analyses will be also performed 
with data obtained with exome sequencing, RNA seq and CpG methylation. The 
integrated analysis will help identify additional novel mutations and novel 
mechanisms underlying the different response phenotypes, all of which could 

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also help identify novel drug targets. Finally, samples obtained during the biopsy 
will be immediately injected into scid mice to generate human tissue xenografts. 
These tissues will be passaged in mice (see IACUC protocol A49111) and will be 
used to evaluate new drug therapies based on tumor sequencing data.   
 
17.52 Exome sequencing of tumor DNA 
 
Summary:--tumor DNA will be isolated from specimens obtained at three 
different time points, prior to the start of neoadjuvant treatment, at surgery and at 
recurrence. DNA will be processed and plated at Mayo BAP lab and then send to 
sequencing lab for exome sequencing using the Agilent SureSelect Human All 
Exon kit 50Mb and the Illumina HiSeq 2000. Typically, over 80% of the target 
region is covered at a depth of greater than 30-fold, allowing for high confidence 
identification of sequence variants across the known protein coding region of the 
genome.  
 
Detailed Protocol--Paired-end indexed libraries are prepared following the 
manufacturer’s protocol (Agilent). Briefly, 3 ug of target DNA in 120 ul TE 
buffer is fragmented using the Covaris E210 sonicator. The settings of duty cycle 
10%, intensity 5, cycles 200, time 360 seconds generated double-stranded DNA 
fragments with blunt or sticky ends with a fragment size mode of between 150-
200bp. The ends were repaired and phosphorylated using Klenow, T4 
polymerase, and T4 polynucleotide kinase, after which an “A” base is added to 
the 3’ ends of double-stranded DNA using Klenow exo- (3’ to 5’ exo minus). 
Paired end Index DNA adaptors (Agilent) with a single “T” base overhang at the 
3’ end are ligated and the resulting constructs are purified using AMPure SPRI 
beads from Agencourt. The adapter-modified DNA fragments are enriched by 4 
cycles of PCR using InPE 1.0 forward and SureSelect Pre-Capture Indexing 
reverse (Agilent) primers. The concentration and size distribution of the libraries 
is determined on an Agilent Bioanalyzer DNA 1000 chip. 
 
Whole exon capture is carried out using the protocol for Agilent’s SureSelect 
Human All Exon kit 50MB. 500 ng of the prepped library is incubated with 
whole exon biotinylated RNA capture baits supplied in the kit for 24 hours at 65 
°C. The captured DNA:RNA hybrids are recovered using Dynabeads MyOne 
Streptavidin T1 from Dynal. The DNA is eluted from the beads and purified 
using Ampure XP beads from Agencourt. The purified capture products are then 
amplified using the SureSelect Post-Capture Indexing forward and Index PCR 
reverse primers (Agilent) for 12 cycles. Libraries are validated and quantified on 
the Agilent Bioanalyzer. 
 
Libraries are loaded onto paired end flow cells at concentrations of 4-5 pM to 
generate cluster densities of 300,000-500,000/mm2 following Illumina’s standard 
protocol using the Illumina cBot and HiSeq Paired end cluster kit version 1. 
 
The flow cells are sequenced as 101 X 2 paired end reads on an Illumina HiSeq 
2000 using TruSeq SBS sequencing kit version 1 and HiSeq data collection 
version 1.1.37.0 software. Base-calling is performed using Illumina’s RTA 
version 1.7.45.0. 
 
17.53 Sequence analysis of tumor RNA 

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Summary:  RNA will be isolated from the tumor samples obtained at three 
different time points, prior to the start of neoadjuvant treatment, at surgery, and at 
recurrence. RNA will be first made into cDNA library and then subjected to 
paired-end 51 bp sequencing using Illumina HiSeq 2000 at a coverage of about 
50 million reads per sample. 
 
Detailed Protocol--Libraries for mRNA-seq are prepared using the TruSeq RNA 
sample prep kit v1 following the manufacturer’s protocol (Illumina). PolyA 
containing mRNA is purified from total RNA using oligo-dT attached magnetic 
beads. The purified mRNA is fragmented using divalent cations at 95°C for 8 
minutes, eluted from the beads and primed for first strand cDNA synthesis in a 
single step. The RNA fragments are copied into first strand cDNA using 
SuperScript (Invitrogen) reverse transcriptase and random primers. Second strand 
cDNA synthesis is performed using DNA polymerase I and RNase H. The 
double-stranded cDNA is purified using a single Ampure XP bead (Agencourt) 
clean-up step. The cDNA ends are repaired and phosphorylated using Klenow, 
T4 polymerase, and T4 polynucleotide kinase followed by a single Ampure XP 
bead clean-up. The blunt-ended cDNAs are modified to include a single 3’ 
adenylate (A) residue using Klenow exo- (3’ to 5’ exo minus). Paired end DNA 
adaptors (Illumina) with a single “T” base overhang at the 3’ end are 
immediately ligated to the ‘A tailed’ cDNA population. The resulting constructs 
are purified by 2 consecutive Ampure XP bead clean-up steps. The adapter-
modified DNA fragments are enriched by 12 cycles of PCR using primers PE 1.0 
and PE 2.0 (Illumina). The concentration and size distribution of the libraries is 
determined on an Agilent Bioanalyzer DNA 1000 chip.  Twelve unique indexes 
are incorporated at the adaptor ligation phase if required for multiplex sample 
loading on the flow cells.  
Libraries are loaded onto paired end flow cells at concentrations of 6 pM to 
generate cluster densities of 400,000/mm2 following Illumina’s standard protocol 
using the Illumina cBot and cBot Paired end cluster kit version 1. 
 
The flow cells are sequenced as 51 X 2 paired end reads on an Illumina HiSeq 
2000 using TruSeq SBS sequencing kit version 1 and HCS version 1.1.37.0 data 
collection software. Base-calling is performed using Illumina’s RTA version 
1.7.45.0. 
 
17.54 RNA quantitative expression profiling of APOBEC3B 
 
Summary:  RNA will be isolate from the tumor samples obtained at three 
different time points, prior to the start of neoadjuvant treatment, at surgery, and at 
recurrence. RNA will be used for quantitative expression profiling of 
APOBEC3B mRNA levels in comparison to those of the endogenous 
housekeeping gene TBP [17]. 
 
Rationale:  The human DNA deaminase APOBEC3B is a newly discovered 
genetic marker for breast cancer, with expression studies demonstrating its over-
expression in ~60% of breast cancers (
 
APOBEC3B mutates the genomic DNA of breast cancer cells causing an overall 
increase in mutation frequency 
). It is highly 
likely that this mutator activity contributes to the heterogeneity of breast cancer 

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and in turn to negative outcomes (i.e. higher APOBEC3B = faster tumor 
evolution = more likely to evolve resistance to therapy and immune responses). 
   
17.55 CpG methylation analysis of tumor DNA 
 
Understand epigenetic regulation in tumor and their effect on gene expression as 
well as response phenotypes. DNA isolated from tumor tissues obtained at three 
different time points will be used to perform CpG methylation assays using the 
Illumina 450K methylation chips that contain over 47,000 methylation sites.  
 
17.56 Xenograft mice model  
 
To generate individual tumor line for drug screening, drug cytotoxicity assay, 
specimen obtained during the initial biopsy will be immediately injected into scid 
mice for the creation of human tissue xenografts (See IACUC A49111). The 
xenograft tumor samples will be used to determine the functional implications 
tumor mutations identified from exome sequencing and for future drug screening 
and cytotoxicity assay.  These lines will be also useful for future research 
purposes, such as testing novel compounds and different regimens. All the tumor 
tissues from xenografts will be stored on 
  
 
17.57 Stem Call Analysis 
 
The cancer stem cell (CSC) hypothesis suggests that many cancers, including 
breast cancer, harbor a subpopulation of malignant cells with stem cell properties 
that may drive metastasis and resist chemotherapy, resulting in resistance and 
relapse.  Two populations of CSCs have been identified: a mesenchymal-like 
population at the leading edge that stains CD24-/CD44+/ALDH- and a centrally-
located epithelial-like population that stains ALDH+.  Other groups have 
suggested that when tumors are treated with chemotherapy, often the CSCs are 
resistant.  Thus, although the tumor size and cellularity may decrease, when the 
tumor is not eradicated the CSCs may be enriched in the remaining population—
an interesting hypothesis that has not yet been tested to our knowledge.  
Interestingly, it has also previously been demonstrated that breast tumors 
containing increasing quantities of CSCs are capable of forming xenografts and 
that tumors expressing both CD24-/CD44+ and ALDH+ populations have the 
greatest tumor-initiating capacity. 
 
CSCs will be analyzed to test the hypothesis that cancer stem cells are enriched 
in residual tumor and that they are related to response to therapy.  
Immunohistochemical stains (CD24, CD44, ALDH, and potentially other related 
CSC markers) using commercially available antibodies will be performed on 
human breast tumor tissue, as well as patient-derived xenografts.  The quantity of 
cells that stain for each marker will be scored (% of tumor cells that stain 
positive) and the distribution of the positive-staining cells will also be noted 
(leading edge of tumor vs. center vs. uniformly distributed throughout).  This 
CSC data will be correlated with other clinical, imaging, and genomic 
information to better understand the role of CSCs in breast cancer. 
 
 
 
 

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18.0 
Records and Data Collection Procedures   
Initial Material(s)  
CRF 
Active-Monitoring Phase 
(Compliance with Test Schedule Section 4.0)  
On-Study Form 
≤2 weeks after registration 
Research Blood Submission Form 
Breast Biopsy Form 
MBI Related Adverse Event Form  
End of Active Treatment/Cancel Notification 
Form 
Research Tissue Submission Form 
≤30 days after registration 
 
Test Schedule Material(s)  
CRF 
Active-Monitoring Phase 
(Compliance with Test Schedule Section 4.0) 
After the 
completion of 
taxane prior 
to 
anthracycline 
based 
chemotherapy 
(Cycle 1) 
After 
completion of 
anthracycline 
based 
chemotherapy, 
but prior to 
surgery 
(Cycle 2) 
After 
completion 
of surgery 
Observation 
q 12 
months 
after 
surgery 
until 
recurrence 
At first 
recurrence 
or at 
completion 
of 
treatment if 
surgery is 
not to be 
performed 
Evaluation/Treatment 
Form 
X 
X 
 
 
 
Active Monitoring 
Measurement Form 
X 
X 
 
 
 
Suspicion of 
Progression on 
Neoadjuvant 
Chemotherapy 
X 
X 
 
 
 
MBI Related Adverse 
Event Form  
X 
X 
 
 
 
Surgery Reporting 
Form  
 
 
X 
 
 
Op & Path Reports 
 
 
X 
 
 
Tumor Marker on 
surgical tissues (clinical 
report) 
 
 
X 
 
 
Residual Cancer 
Burden Worksheet 
 
 
X 
 
 
Evaluation/Observation 
Form 
 
 
 
X 
 
Research Blood 
Submission Form 
X (see 
Section 14.0) 
X (see Section 
14.0) 
 
 
X (see 
Section 
14.0) 

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CRF 
Active-Monitoring Phase 
(Compliance with Test Schedule Section 4.0) 
After the 
completion of 
taxane prior 
to 
anthracycline 
based 
chemotherapy 
(Cycle 1) 
After 
completion of 
anthracycline 
based 
chemotherapy, 
but prior to 
surgery 
(Cycle 2) 
After 
completion 
of surgery 
Observation 
q 12 
months 
after 
surgery 
until 
recurrence 
At first 
recurrence 
or at 
completion 
of 
treatment if 
surgery is 
not to be 
performed 
Research Tissue 
Submission Form 
X (see 
Section 17.0) 
 
X (see 
Section 
17.0) 
 
X (see 
Section 
17.0) 
Breast Biopsy Form 
X (see 
Section 4.0) 
 
 
 
X (see 
Section 
4.0) 
Start of Adjuvant 
Endocrine Therapy 
Form 
 
 
 
X 
 
Changes in or 
Discontinuation of 
Adjuvant Endocrine 
Therapy Form 
 
 
 
X 
 
End of Active 
Treatment/Cancel 
Notification Form 
 
 
X 
 
 
 
 
Follow-up Material(s)   
CRF 
q. 12 months 
after  
recurrence1, 2 
Death 
New Primary 
Event Monitoring Form 
X 
X 
At each 
occurrence 
1. If a patient is still alive 6 years after surgery, no further follow-up is required. 
2. Submit copy of documentation recurrence to the MCCC Operations Office, Attention: QAS for 
MC1137. 
 
19.0 
Budget 
19.1 
Costs charged to patient:  routine clinical care 
19.2 
Tests to be research funded: molecular breast imaging (MCR Participants only), research 
blood samples, research tumor biopsy, and MRI between paclitaxel ± trastuzumab and 
AC or FEC chemotherapy. 

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20.0 
References 
1. 
Valero, V.V., A.U. Buzdar, and G.N. Hortobagyi, Locally Advanced Breast Cancer. The 
oncologist, 1996. 1(1 & 2): p. 8-17. 
2. 
Berry, D.A., et al., Estrogen-receptor status and outcomes of modern chemotherapy for patients 
with node-positive breast cancer. JAMA : the journal of the American Medical Association, 
2006. 295(14): p. 1658-67. 
3. 
Fisher, B., et al., Effect of preoperative chemotherapy on local-regional disease in women with 
operable breast cancer: findings from National Surgical Adjuvant Breast and Bowel Project B-
18. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 
1997. 15(7): p. 2483-93. 
4. 
Bear, H.D., et al., Sequential preoperative or postoperative docetaxel added to preoperative 
doxorubicin plus cyclophosphamide for operable breast cancer:National Surgical Adjuvant 
Breast and Bowel Project Protocol B-27. Journal of clinical oncology : official journal of the 
American Society of Clinical Oncology, 2006. 24(13): p. 2019-27. 
5. 
Buchholz, T.A., et al., Statement of the science concerning locoregional treatments after 
preoperative chemotherapy for breast cancer: a National Cancer Institute conference. Journal of 
clinical oncology : official journal of the American Society of Clinical Oncology, 2008. 26(5): p. 
791-7. 
6. 
Mauri, D., N. Pavlidis, and J.P. Ioannidis, Neoadjuvant versus adjuvant systemic treatment in 
breast cancer: a meta-analysis. Journal of the National Cancer Institute, 2005. 97(3): p. 188-94. 
7. 
Kuerer, H.M., et al., Pathologic tumor response in the breast following neoadjuvant 
chemotherapy predicts axillary lymph node status. The cancer journal from Scientific American, 
1998. 4(4): p. 230-6. 
8. 
Kuerer, H.M., et al., Clinical course of breast cancer patients with complete pathologic primary 
tumor and axillary lymph node response to doxorubicin-based neoadjuvant chemotherapy. 
Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 
1999. 17(2): p. 460-9. 
9. 
Kuerer, H.M., et al., Incidence and impact of documented eradication of breast cancer axillary 
lymph node metastases before surgery in patients treated with neoadjuvant chemotherapy. Annals 
of surgery, 1999. 230(1): p. 72-8. 
10. 
Symmans, W.F., et al., Measurement of residual breast cancer burden to predict survival after 
neoadjuvant chemotherapy. Journal of clinical oncology : official journal of the American 
Society of Clinical Oncology, 2007. 25(28): p. 4414-22. 
11. 
Slamon, D.J., et al., Human breast cancer: correlation of relapse and survival with amplification 
of the HER-2/neu oncogene. Science, 1987. 235(4785): p. 177-82. 
12. 
Esserman, L.J., et al., Pathologic Complete Response Predicts Recurrence-Free Survival More 
Effectively by Cancer Subset: Results From the I-SPY 1 TRIAL--CALGB 150007/150012, ACRIN 
6657. Journal of clinical oncology : official journal of the American Society of Clinical 
Oncology, 2012. 30(26): p. 3242-9. 
13. 
Buzdar, A.U., et al., Significantly higher pathologic complete remission rate after neoadjuvant 
therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: results of a randomized trial 
in human epidermal growth factor receptor 2-positive operable breast cancer. Journal of clinical 
oncology : official journal of the American Society of Clinical Oncology, 2005. 23(16): p. 3676-
85. 
14. 
Sledge, G.W., et al., Phase III trial of doxorubicin, paclitaxel, and the combination of 
doxorubicin and paclitaxel as front-line chemotherapy for metastatic breast cancer: an 
intergroup trial (E1193). Journal of clinical oncology : official journal of the American Society 
of Clinical Oncology, 2003. 21(4): p. 588-92. 

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Protocol Version Date: 23Aug2023 
15. 
Conners, A.L., et al., Lexicon for standardized interpretation of gamma camera molecular breast 
imaging: observer agreement and diagnostic accuracy. European journal of nuclear medicine and 
molecular imaging, 2012. 39(6): p. 971-82. 
16. 
Hruskaa, C.B. and M.K. O'Connor, Quantification of lesion size, depth, and uptake using a dual-
head molecular breast imaging system. Medical physics, 2008. 35(4): p. 1365-76. 
17. 
Refsland, E.W., et al., Quantitative profiling of the full APOBEC3 mRNA repertoire in 
lymphocytes and tissues: implications for HIV-1 restriction. Nucleic acids research, 2010. 38(13): 
p. 4274-84. 
18. 
Jaiswal, S., et al., Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J 
Med, 2014. 371(26): p. 2488-98. 
19. 
Jaiswal, S., et al., Clonal Hematopoiesis and Risk of Atherosclerotic Cardiovascular Disease. N 
Engl J Med, 2017. 377(2): p. 111-121. 
20. 
Gillis, N.K., et al., Clonal haemopoiesis and therapy-related myeloid malignancies in elderly 
patients: a proof-of-concept, case-control study. Lancet Oncol, 2017. 18(1): p. 112-121. 
21. 
Takahashi, K., et al., Preleukaemic clonal haemopoiesis and risk of therapy-related myeloid 
neoplasms: a case-control study. Lancet Oncol, 2017. 18(1): p. 100-111. 
22. 
Asimit, J. and E. Zeggini, Rare variant association analysis methods for complex traits. Annual 
review of genetics, 2010. 44: p. 293-308. 
23. 
Bansal, V., et al., Statistical analysis strategies for association studies involving rare variants. 
Nature reviews. Genetics, 2010. 11(11): p. 773-85. 
24. 
Morris, A.P. and E. Zeggini, An evaluation of statistical approaches to rare variant analysis in 
genetic association studies. Genetic epidemiology, 2010. 34(2): p. 188-93. 
25. 
Price, A.L., et al., Principal components analysis corrects for stratification in genome-wide 
association studies. Nature genetics, 2006. 38(8): p. 904-9. 
26. 
Westfall, P.H. and S.S. Young, Resampling-based multiple testing: examples and methods for p-
value adjustment. 1993, New York: Wiley. 
27. 
Storey, J.D., A direct approach to false discovery rates. . J Royal Stat Soc, Series B, 2002. 64(3): 
p. 479-98. 
28. 
Li, B. and S.M. Leal, Discovery of rare variants via sequencing: implications for the design of 
complex trait association studies. PLoS genetics, 2009. 5(5): p. e1000481. 
 

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Appendix I ECOG Performance Status 
 
Grade 
 
 
0 
Fully active, able to carry on all pre-disease activities without restriction (Karnofsky 90-100). 
 
 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a 
light or sedentary nature, e.g., light housework, office work (Karnofsky 70-80). 
 
 
2 
Ambulatory and capable of all self-care, but unable to carry out any work activities.  Up and 
about more than 50 percent of waking hours 
 
 
(Karnofsky 50-60). 
 
 
3 
Capable of only limited self-care, confined to bed or chair 50 percent or more of waking 
hours (Karnofsky 30-40). 
 
 
4 
Completely disabled.  Cannot carry on any self-care.  Totally confined to bed or chair 
(Karnofsky 10-20). 
 
 
5 
Dead 
 

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Appendix II Chemotherapy Guidelines 
Weekly Paclitaxel in the Neoadjuvant Treatment of Breast Cancer 
Cycles:  3   Emetogenic Potential:  2   Time:  3 Hours 
DRUG 
DOSE 
ROUTE 
DAYS 
COMMENTS 
Dexamethasone 
[DXM] 
20 mg/dose 
In 100 ml 0.9% NaCL IV infusion over 15 minutes 
immediately before Paclitaxel 
1, 8, 15, 22 
 
Famotidine 
[PEPCID] 
20 mg/dose 
In 50 mL 0.9% NaCL IV infusion over 15 minutes 
immediately before Paclitaxel 
1, 8, 15, 22 
 
Diphenhydramine 
[BEN] 
50 mg/dose 
In 100 mL 0.9% NaCl IV infusion over 15 minutes 
immediately before Paclitaxel. 
1, 8, 15, 22 
 
Paclitaxel 
[TAXOL] 
80 
mg/m2/week 
In 250 mL 0.9% NaCL IV infusion over 1 hour (use 
non-pvc container and tubing) 
1, 8, 15, 22 
Dose rounding to the 
nearest 5 mg. 
Retreatment:  Every 28 days for 3 cycles (12 doses of Paclitaxel) 
Reference:  Sparano JA, Wang M et al. Weekly Paclitaxel in the Adjuvant Treatment of Breast Cancer. The New England Journal of Medicine 
2008;358:1663-1671. 
 
 
 
Weekly Paclitaxel/Carboplatin in the Neoadjuvant Treatment of Breast Cancer 
Cycles:  4   Emetogenic Potential:  3   Time:  5 Hours 
DRUG 
DOSE 
ROUTE 
DAYS 
COMMENTS 
Dexamethasone 
[DXM] 
20 mg 
in 100 ml NS IV infusion over 15 minutes 
immediately before Taxol 
1,8,15, 
 
Famotidine 
[PEPCID] 
20 mg 
IV infusion over 15 minutes immediately before 
Taxol 
1,8,15, 
 
Diphenhydramine 
[BEN] 
50 mg 
IV infusion over 15 minutes immediately before 
Taxol 
1,8,15, 
 

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Paclitaxel 
[TAXOL] 
80 mg/m2 
IV infusion in 250 ml 0.9% NaCL (non-PVC container) 
over 1 hour 
1,8,15, 
Dose rounding to the 
nearest 5 mg. If 
Inpatient, remain with 
the patient for the first 
15 minutes of the 
infusion. Monitor vital 
signs prior to 
administration of 
paclitaxel, 15 and 30 
minutes after initiation 
of paclitaxel, then 
hourly and at the 
completion of 
paclitaxel. 
Carboplatin 
[CBDCA] 
AUC = 6 
in 250 ml 0.9% NaCl or D5W IV infusion over 30 
minutes 
1 
Dosed using Calvert 
Formula with Cockroft 
& Gault Equation, actual 
body weight and 
maximum CrCL of 125 
mL/min. Creatinine 
clearance should be 
estimated using a 
minimum serum 
creatinine value of 0.7 
mg/dL. Dose rounding 
to the nearest 10 mg. 
RETREATMENT: Every 3 weeks  
 
 
 
 

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Herceptin + Weekly Paclitaxel 
Emetogenic Potential:  1     Time:  3 Hours   
DRUG 
DOSE 
ROUTE 
DAYS 
COMMENTS 
Dexamethasone 
[DXM] 
20 
mg/dose 
In 100 ml 0.9% NaCL IV infusion over 15 
minutes immediately before Paclitaxel 
1, 8, 15, 
22 
May decrease Dexamethasone dose if patient 
does not experience reactions to Taxol. 
Famotidine 
[PEPCID] 
20 
mg/dose 
In 50 mL 0.9% NaCL IV infusion over 15 
minutes immediately before Paclitaxel 
1, 8, 15, 
22 
 
Diphenhydramin
e [BEN] 
50 
mg/dose 
In 100 mL 0.9% NaCl IV infusion over 15 
minutes immediately before Paclitaxel. 
1, 8, 15, 
22 
 
Paclitaxel 
[TAXOL] 
60 – 90 
mg/m2 
In 250 mL 0.9% NaCL (non-pvc container and 
tubing) IV infusion over 1 hour 
1, 8, 15, 22 
Defaults to 80 mg/m2/dose 
Acetaminophen 
[APAP] 
650 mg 
PO prn fever 
prn 
 
Meperidine 
[DEM] 
25 mg/ 
dose 
25 mg IV push prn rigors, may repeat once if 
first dose ineffective 
prn 
 
Diphenhydramine 
[BEN] 
50 mg 
IV push prn fever & chills 
prn 
 
Trastuzumab 
[HERCEPLOAD] 
4 mg/kg 
loading 
In 250 ml NS IV infusion over 90 minutes 
1 
Day 1 at 2 mg/kg on subsequent cycles. 
Left Ventricular Function should be evaluated in 
all patients prior to and during treatment with 
Herceptin. 

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Trastuzumab 
[HERCEP] 
2 mg/kg 
In 250 mL NS IV infusion over 30 minutes 
8, 15, 22 
Infusion rate to remain at 90 minutes until at 
least one dose is well tolerated.  Then increase 
rate to 30 minutes. 
Retreatment:  Every 28 days x 3 cycles 
Reference:  N9831 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Reference: Datko F, D'Andrea G, Theodoulou M et al. Phase II study of pertuzumab, trastuzumab, and weekly paclitaxel in patients with metastatic HER2- 
overexpressing metastatic breast cancer. Cancer Research 2012;72(24 Supplement):P5-18-20 
 
 
 
Trastuzumab/Pertuzumab + Weekly Paclitaxel 
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Trastuzumab 
Loading Dose 
[HERCEP LOAD] 
N/A 
8 mg/kg/day  
IV 
 
Day 1 week 1 
 
Trastuzumab 
[HERCEP] 
N/A 
6 mg/kg/day  
IV 
day 1 
 
Weeks 4, 7, 10 
Pertuzumab 
(Perjeta) Loading 
Dose 
 
After Trastuzumab 
840 mg/day  
IV 
day 1 
week 1 
 
Pertuzumab 
(Perjeta) 
[PERTUZUMAB] 
 
420 mg/day  
IV 
Day 1 
Weeks 4, 7, 10 
 
 
Paclitaxel 
 
N/A 
 
80 mg/m2  
IV 
Day 1 
Weeks 1-12 

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Trastuzumab/Pertuzumab/Docetaxel  
Agent 
Time 
Dose 
Route 
Rx Days 
ReRx 
Trastuzumab Loading 
Dose 
[HERCEP LOAD] 
N/A 
8 mg/kg/day  
IV 
Day 1 Week 1 
 
Trastuzumab 
[HERCEP] 
N/A 
6 mg/kg/day  
IV 
Day 1 
Weeks 4, 7, 10 
Pertuzumab (Perjeta) 
Loading Dose 
After Trastuzumab 
840 mg/day  
IV 
Day 1 Week 1 
 
Pertuzumab (Perjeta) 
[PERTUZUMAB] 
 
420 mg/day  
IV 
Day 1 
Weeks 4, 7, 10 
Docetaxel 
N/A 
75 mg/m2  
IV 
Day 1 
Weeks 1, 4, 7, 10 
 
 
Reference: Gianni, L., T. Pienkowski, et al. (2012). "Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women with locally advanced, 
inflammatory, or early HER2-positive breast cancer (NeoSphere): a randomised multicentre, open-label, phase 2 trial." The lancet oncology 13(1): 25-
32. 
Schneeweiss, A., S. Chia, et al. (2013). "Pertuzumab plus trastuzumab in combination with standard neoadjuvant anthracycline-containing and 
anthracycline-free chemotherapy regimens in patients with HER2-positive early breast cancer: a randomized phase II cardiac safety study 
(TRYPHAENA)." Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 24(9): 2278-2284. 
 
 
 

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Combination ADR + CTX Followed by Paclitaxel 
Cycles:  4     Emetogenic Potential:  4     Time:  2 Hours 
DRUG 
DOSE 
ROUTE 
DAYS 
COMMENTS 
Doxorubicin [ADR] 
60 mg/m2 
IV push into free flowing IV of NS 
1 
Maximum cumulative ADR dose is 240 mg/m2. 
Cyclophosphamide [CTX] 
600 
mg/m2 
IV infusion in 250 mL NS over 30 
minutes 
1 
 
Aprepitant [EMEND125] 
125 
mg/day 
PO once pre-chemotherapy 
1 
Aprepitant 125 mg may be given on Gonda 10 or 
per Enterprise Orders prescription (Rx-EOP). 
Aprepitant [EMEND80] 
80 mg/day 
PO once daily Days 2 and 3 
2, 3 
Aprepitant 80 mg once daily Days 2 and 3 should 
be given to the patient via Enterprise Orders 
prescription (Rx-EOP). 
Filgrastim [GCSF] 
5 mcg/kg 
SQ 
3 through 
10 
Suggest rounding dose to nearest vial size (300 
mcg or 480 mcg).  May substitute Neulasta 
(pegfilgrastim). 
-- OR -- 
Pegfilgrastim 
(NEULASTA) 
6 mg 
SQ 
2 
May substitute Neulasta (pegfilgrastim) for GCSF.  
Begin at least 24 hours post chemo. 
Paclitaxel (Taxol) 
Cycles:  4     Emetogenic Potential:  2     Time:  4 Hours 
DRUG 
DOSE 
ROUTE 
DAYS 
COMMENTS 
Dexamethasone [DXM] 
20 mg 
In 100 mL NS IV infusion over 15 
minutes 
1 
 
Famotidine [PEPCID] 
20 mg 
IV infusion over 15 minutes 
immediately before Taxol. 
1 
 
Diphenhydramine [BEN] 
50 mg 
IV infusion over 15 minutes 
immediately before Taxol 
1 
 

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Paclitaxel [TAXOL] 
175 
mg/m2 
IV infusion in 500 mL 0.9% NaCL (non-
pvc container and tubing) over 3 hours 
1 
Inpatient monitoring with the patient for the first 
15 minutes of the infusion.  Monitor vital signs 
prior to administration of paclitaxel, 15 and 30 
minutes after initiation of paclitaxel, then hourly at 
the completion of paclitaxel. 
Filgrastim [GCSF] 
5 mcg/kg 
SQ 
3 through 
10 
Suggest rounding dose to nearest vial size (300 
mcg or 480 mcg).  May substitute Neulasta 
(pegfilgrastim). 
-- OR -- 
Pegfilgrastim 
(NEULASTA) 
6 mg 
SQ 
2 
May substitute Neulasta (pegfilgrastim) for GCSF.  
Begin at least 24 hours post chemo. 
Retreatment:  Every 2 weeks x 4 with growth factor support or every 3 weeks x 4 (with or without growth factor support) 
Reference:  C9741 
 
 
 
FEC (Fluorouracil + Epirubicin + Cyclophosphamide) 
Emetogenic Potential:  3     Time:  3 Hours 
DRUG 
DOSE 
ROUTE 
DAYS 
COMMENTS 
Epirubicin [EPIRUB] 
75 mg/m2/day 
IV push through a free-flowing IV line of 
0.9% NaCL 
1 
 
Fluorouracil [5FU] 
500 mg/m2 /day 
IV push 
1 
 
Cyclophosphamide [CTX] 
500 mg/m2/day 
In 250 mL 0.9% NaCL IV infusion over 30 
minutes 
1 
 
Retreatment:  Every 21 days 
Reference:   Buzdar, Ibrahim, Francis, et al. Significantly Higher Pathologic Complete Remission Rate After Neoadjuvant Therapy With Trastuzumab, 
Paclitaxel, and Epirubicin Chemotherapy: Results of a Randomized Trial in Human epidermal Growth Factor Receptor 2-Positive Operable Breast Cancer. 
JCO, Volume 23, Number 16, June 1, 2005. pp 3676-3685 
 
 

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Appendix III RPPA Standard Antibody List 
 

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Appendix IV Molecular Breast Imaging (MCR Participants Only) 
MBI Imaging Equipment 
Cadmium-Zinc-Telluride (CZT) detectors are essentially equivalent to conventional gamma cameras, and 
differ only in that the material used to detect radiation is a semiconductor material rather than a 
scintillating crystal such as Sodium Iodide. Mayo currently has 3 dual-detector MBI systems. One MBI 
system is FDA approved, and the other 2 devices are prototypes and do not have 510k FDA approval. 
While we will attempt to utilize the FDA approved device in this study, this is unit may not be available 
at the appropriate time and patients will be imaged on an available system to expedite their imaging study. 
The 3 devices are essentially equivalent in terms of their imaging capabilities and performance. The 2 
prototype detectors are mounted on modified mammographic gantries which were constructed in-house 
with assistance from the Department of Engineering, hence these 2 imaging systems are currently not 
FDA approved.  However, under the FDA’s Investigational Devices Exemptions Manual (HHH 
Publication FDA 96-4159), we believe that these 2 devices are exempt from the requirements of the IDE 
regulation, as they meet the requirements of a diagnostic device, i.e.,  
a) 
it is non-invasive and does not require an invasive sampling procedure that presents significant 
risk 
b) 
it does not by design or intention introduce energy into a subject  
c) 
it is not used as a diagnostic procedure without confirmation by another medically established 
diagnostic product or procedure. 
 
The Mayo-developed gantries comprise the support for mounting the detectors in a position for patient 
use, and controls to permit compression of the breast (15 lbs. force) between the two detectors. A safety 
stop was designed to limit the amount of compression that can be applied to the breast. These gantries 
have been safely used in over 2000 MBI patient studies to date. 
 
In terms of patient safety, the 2 prototype CZT detectors are electrically isolated and are essentially 
equivalent to conventional gamma cameras.  They utilize the same data acquisition/analysis computer that 
is used in some of our clinical gamma camera systems. Hence, we believe that use of these systems in this 
study is exempt from the requirements of the IDE regulations and only requires IRB approval. 
 
 
Radiation Exposure 
This protocol involves the administration of 4-8 mCi 99mTc-sestamibi.  The target organs are the upper 
and lower large intestine, which receive approximately 1.44 and 1.0 rads / 8 mCi respectively. The 
radiation dose to the breasts is 0.02 rads. Full details of the absorbed radiation dose to various organs 
from 99mTc-sestamibi are given below. 
 
The radiation dose from the combined injections of ~8 mCi 99mTc-sestamibi is significantly less than that 
from routine myocardial perfusion studies (60 mCi 99mTc-sestamibi) and the radiation dose to the breast is 
less than that from a mammogram.  
 
Radiation Dosimetry 
Absorbed radiation dose estimates for 99mTc-sestamibi, with an administered dose of 8 mCi and a 2 hour 
void *. 
 
Target Organ 
 
 
 
 
Dose (rads/8 mCi) 
Breasts  
 
 
 
 
 
0.05  
Stomach 
 
 
 
 
 
0.16 
Small Intestine  
 
 
 
 
0.80 

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Upper Large Intestine  
 
 
 
1.44 
Lower Large Intestine  
 
 
 
1.04 
Kidneys 
 
 
 
 
 
0.54 
Bladder Wall 
 
 
 
 
 
0.54 
Thyroid 
 
 
 
 
 
0.19 
Red Marrow 
 
 
 
 
 
0.14 
Ovaries  
 
 
 
 
 
0.40 
Testes  
 
 
 
 
 
0.08 
Whole Body 
 
 
 
 
 
0.14 
Cardiolite -  Kit for the Preparation of Technetium 99m Tc-Sestamibi for Injection, Bristol-Myers Squibb – 
Medical Imaging. T5-B001, May 2003. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Appendix V Return of Test Results 
1. BACKGROUND 
 
In MC1137 (the BEAUTY study), analysis of germline DNA is important for the identification of 
inherited mutations in cancer susceptibility genes, which can assist in patient counseling regarding 
treatment options. 
 
Approximately 5-10% of breast cancer is caused by germline mutations in cancer susceptibility genes.  A 
minimum of 200 and a maximum of 230 participants will enroll in BEAUTY.  Therefore, it is reasonable 
to expect that germline mutations will be present in approximately 10-23 BEAUTY participants.  Due to 
early-onset diagnosis and/or family history of cancer, we expect that many of these participants will have 
undergone clinical genetic testing previously as directed in their clinical care by the Mayo 
multidisciplinary team and will therefore already be aware of their genetic test results.  However, there is 
the potential that the research germline DNA analysis will identify mutations in clinically relevant genes 
that the patient has not been tested for and therefore is not aware of. Thus far, in the analysis of the first 
69 patients enrolled in MC1137, three BRCA1 and five BRCA2 gene mutations have been identified.  Six 
of these participants have undergone clinical testing previously and are thus aware of their results, while 
two patients have not yet had clinical genetic testing to our knowledge. 
 
We propose to offer the return of research germline DNA analysis results to MC1137 (BEAUTY) 
participants who are found to carry actionable germline mutations in cancer susceptibility genes and who 
are not already aware of their genetic test results through clinical testing.  Please refer to Table 1 for a 
complete list of genes.  The focus of our analysis will be on genes associated with hereditary breast 
cancer risk.  We will also analyze other known cancer-predisposition genes, but we expect that 
identifying mutations in these other genes will be exceedingly rare.  Mutations in all genes included in 
this proposal have associated medical management guidelines, such as National Comprehensive Cancer 
Network (NCCN) guidelines, and are thus actionable 
).  Mutations in genes that do not have 
medical management guidelines and are thus felt not to benefit the patient in terms of counseling will not 
be offered to be returned to the patient.  Mutations in genes causing childhood-onset conditions will not 
be returned, given that women enrolling in BEAUTY are all beyond the age when these disorders would 
have typically manifested.  Conditions with an autosomal recessive inheritance pattern will not be offered 
for return of results because if two mutations are detected, it is not possible to determine whether they 
occur in cis (on the same allele and thus not causative of the condition) or in trans (on opposite alleles and 
thus causative of the condition).  Clinical testing in a CLIA-certified laboratory is available in the United 
States for all conditions in order to confirm these research results.  Note, coverage of the exome 
sequencing may not be adequate to identify all disease associated mutations identified in Table 1.  Results 
will be returned only for those mutations identified with the current technology and coverage. 
 
Although there is no general consensus in the literature about whether individual research results should 
be returned to participants (1-3), ethical principles, studies of patient preferences, expert opinion in the 
literature, and national guidelines support the return of individual research results in certain situations.  
The ethical principles of beneficence and non-maleficence emphasize the need to act positively to 
maximize benefits and minimize harms for research participants.  Studies of participants’ attitudes toward 
return of research results indicate that the majority wish to receive study results (4, 5).  Many experts 
believe that individual research results could or should be returned under specific conditions, often 
including: the finding has important health implications with substantial risks, the finding is actionable, 
e.g. strategies are available to reduce expected morbidity or mortality, the test is analytically valid, and 
the participant has consented/opted to receive the results (1, 6-8).  Several national guidelines accept a 
similar stance that individual research results meeting certain criteria should be returned, including the 

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National Heart, Lung, and Blood Institute (NHLBI) and the National Cancer Institute Office of 
Biorepositories and Biospecimen Research (7, 11).  Furthermore, the majority of researchers accept that 
there is a fundamental “duty to rescue,” meaning that participants should be informed when an 
investigator discovers genetic information that clearly indicates a high probability of a serious condition 
for which an effective intervention is readily available (2, 3, 6, 9).   
 
All BEAUTY participants have been consented to indicate whether they would like to be contacted and 
given the opportunity to receive germline test results in the future.  Only those participants who checked 
“yes” will be contacted to offer return of research results if an actionable germline mutation is discovered. 
 
The Clinical Laboratory Improvement Amendments (CLIA) of 1988 regulates testing on humans for 
health purposes and requires laboratories to establish analytical validity.  Given that the germline testing 
in BEAUTY is not performed in a CLIA-certified laboratory, we will strongly recommend to all 
participants that their results be confirmed in a CLIA-certified laboratory before being used for clinical 
decision-making. 
 
2. METHODS 
 
2.1 ANALYTICAL METHOD 
 
Results from the germline DNA analysis will be analyzed by a genetic counselor on the 
BEAUTY team.  Germline variants in cancer-predisposition genes with existing medical 
management guidelines will be classified as benign, likely benign, variant of uncertain 
significance, likely deleterious, or deleterious using standard genetic variant software programs 
and databases including Alamut, Human Gene Mutation Database (HGMD), 1000 Genomes, 
Leiden Open Variation Database (LOVD), Breast Cancer Information Core (BIC), etc. 
 
2.2 RETURN OF RESULTS METHOD 
 
All women who previously opted-in to be contacted about germline test results, who are found to 
carry deleterious or likely deleterious germline mutations in a cancer-predisposition gene 
included in Table 1, and who have not had previous clinical testing that identified the germline 
mutation will be offered return of results.   
 
1. A BEAUTY study coordinator will contact each eligible participant by telephone to inform her 
that the blood genetic test results are now available, and offer the participant an appointment with 
the BEAUTY genetic counselor at no charge for further information about these results.  Please 
refer to the “Outline of Telephone Call for Study Coordinators” (section 1.2.3) for complete 
details regarding this phone call.   
The study coordinator will note that the participant does not need to make a decision about 
whether to accept or decline the appointment at this time, and offer to send a letter in the mail that 
summarizes the results disclosure procedure and includes information about genetic testing for 
hereditary cancer (see section 1.2.6, “Letter to Participants,” below).   
 
2. If the participant elects to proceed with an appointment with the genetic counselor, the participant 
will be given information about genetic testing for hereditary cancer, including risks, benefits, 
and limitations of test results, and an opportunity to ask questions (see section 2.4, “Outline of 
First Genetic Counselor Appointment,” below).   
 
a. In an effort to make sure that participants are fully informed and have adequate time to think 
through this information before making a decision about whether or not to learn the genetic 

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test results, participants will not be given their results during the first appointment.  Instead, it 
will be recommended that they consider their options and return for a second appointment 
during which they will learn their results.  They will be asked to call the study coordinator to 
schedule a second appointment if they choose to receive their research results.   
 
b. NOTE: the genetic counseling appointment can be performed in-person at Mayo Clinic or by 
telephone, depending on the participant’s preference.   
 
c. NOTE: the participant will not be charged for the genetic counseling appointment(s). 
 
d. Note: if the patient desires to receive the results immediately after the first appointment and 
this is appropriate based on the judgment of the genetic counselor, the second appointment 
can occur immediately after the first appointment. 
 
 
3. If the participant elects to receive their genetic test results, they will be given their research 
results at a second appointment (see section 2.5, “Outline of Second Genetic Counseling 
Appointment,” below).   
 
a. We will clearly inform them that the research results must be confirmed in a CLIA-certified 
laboratory before any clinical decision is made based on these results.   
 
i. 
NOTE: the genetic counselor will assist with coordinating the testing at a CLIA-certified 
laboratory. 
 
ii. NOTE: the cost of CLIA-certified testing will be submitted to the participant’s insurance 
company.  In the event that the cost of this testing is not covered or only partially covered 
by the insurance company, funds from the BEAUTY study are available to cover the 
remainder. 
 
b. A summary letter and additional gene-specific hereditary cancer information will be mailed 
to the participant.  A second copy will be included that the participant can share with their 
physician(s) if they chose to do so. 
 
c. A clinic note will be entered in the patient’s electronic medical records documenting the visit 
discussion, the research results, and whether the patient elected to proceed with confirmatory 
clinical testing. 
 
d. Should the participant have any questions or concerns about their clinical management based 
on their results, they will be referred back to their oncologist for clinical management after 
CLIA testing. 
 
4. If the participant chooses not to receive these research results: 
 
a. A letter will be sent summarizing the first appointment if the patient desires. 
 
b. A clinic note will be entered into the patient’s electronic medical records documenting the 
first appointment and that the patient declined to receive the research results.  The research 
results will not be entered into the patient’s records. 
 
2.3     OUTLINE OF TELEPHONE CALL FOR STUDY COORDINATORS 

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1. Introduce self/involvement in BEAUTY. 
2. Explain reason for phone call:  
a. 
You may recall filling out a consent form previously that asked if you were interested 
in learning more about your blood genetic test results.  You had checked “yes” that 
you would like the opportunity to receive these results.  We are contacting you as 
these research results are now available to give you the option to learn your results. 
3. Ask if the participant is still interested in receiving more information about her blood 
genetic test results and offer the participant an appointment with the genetic counselor (at 
no charge) for further information about these results. 
a. 
Are you still interested in receiving more information about your blood genetic test 
results?  Would you like to make an appointment with the genetic counselor (at no 
charge) for more information about your results? 
b. 
The purpose of the initial appointment with the genetic counselor is to talk about the 
type of genetic testing that was performed, and to talk through the risks, benefits, and 
limitations of learning the test results.   
c. 
This appointment can either be in-person at Mayo Clinic or over the telephone.   
d. 
The genetic counselor will not give you the research results during this first 
appointment.  If you decide after the first appointment with the genetic counselor that 
you want to receive your research results, you can call me [the study coordinator] 
back to set up a second appointment with the genetic counselor.  Two appointments 
are planned to make sure that you are fully informed and have adequate time to think 
through the risks of benefits of receiving genetic test results before you make a 
decision. During the second appointment with the genetic counselor, you will have an 
opportunity to ask any questions or discuss any concerns that you may have thought 
of, and you will then be given your results if you wish.  
4. The study coordinator will note that the participant does not need to make a decision about 
whether to accept or decline an appointment with the genetic counselor at this time, and will offer 
to send the participant a letter in the mail that summarizes the results disclosure procedure and 
includes information about genetic testing for hereditary cancer (see section 2.6, “Letter to 
Participants” below).   
5. The study coordinator will note that these preliminary research results will need to be verified in a 
certified clinical laboratory before they are used to make any decisions about the patient’s 
medical management.  If their insurance does not pay for this testing, funds from the BEAUTY 
study are available to cover the cost.  The participant will not be charged for the appointments 
with the genetic counselor.  
 
2.4 
OUTLINE OF FIRST APPOINTMENT 
 
1. Genetic counselor introduction and description of role in BEAUTY 
2. Ask participant’s understanding of why this appointment is taking place 
3. Discuss purpose of first appointment and plan for subsequent appointment if participant decides 
that they would like their results 
4. Description of testing that is performed in BEAUTY - analysis of cancer susceptibility genes 
5. Verify that participant has not already had genetic testing clinically/aware of their results 
6. Discuss participant’s personal/family history of cancer (formal pedigree not necessary) 
7. Education about genetic testing for hereditary cancer 
a. Sporadic vs. familial vs. hereditary cancer 
b. Background information about genetics 
c. Various genes that can cause hereditary cancer 
d. Cancer risks associated with gene mutations 

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e. Medical management recommendations 
f. Inheritance of gene mutations/ risks to relatives 
 
8. Risks, benefits, and limitations of test results 
a. 
Potential risks: 
i. 
Psychological impact 
1. Fear of developing a second cancer 
2. Having to make additional decisions about management 
3. Guilt of passing mutation on to children 
ii. Family 
1. Burden of sharing information with family 
2. Impact on family members/family relationships 
iii. Genetic discrimination 
1. Genetic Information Non-discrimination Act (GINA) 
2. Protects against health insurance and employment discrimination 
3. Does not cover life, long-term care, or disability insurance 
4. State laws 
b. 
Potential benefits: 
i. 
Possible explanation for cause of breast cancer diagnosis (if a breast cancer 
susceptibility gene is identified) 
ii. Opportunity to prevent/reduce risks for other cancers 
iii. Opportunity to share information with at-risk relatives 
iv. Potential for enrollment in clinical trials/ selected therapies  
c. 
Limitations: 
i.  Need to confirm research results in a CLIA-certified laboratory (will submit to insurance 
        company; if test is not covered or partially covered, funds are available to pay remaining 
        cost) 
9. No cost for genetic counseling 
10. Summary of research results will be in the medical records only if participant chooses to receive 
the results 
11. Participant questions 
12 Explain to participant that after thinking about this information, if they decide that they would 
like to receive their results, they can call the study coordinator to schedule a second appointment 
for results disclosure 
 
2.5 OUTLINE OF SECOND APPOINTMENT 
 
1. Answer any questions/concerns that participant has thought of since previous appointment 
2. Confirm that the participant would like to receive their research results 
a. 
If so, disclose results 
b. 
If not, offer opportunity for results disclosure in the future if they change their mind 
3. Discuss implication of results 
a. 
Cancer risks 
b. 
Medical management 
c. 
Inheritance/risks to relatives 
4. Recommend CLIA-certified laboratory testing 
a. 
Facilitate testing if patient wants to proceed. After results are received from CLIA-certified 
laboratory, contact participant by telephone to review results.   
5. Mail two copies of follow-up letter to participant (one for participant and one that they can give 
to their physician) 
 

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2.6 LETTER TO PARTICIPANTS 
 
Participant Name 
Address 
City, State Zip 
 
Dear Participant Name, 
 
Thank you for your continued participation in the BEAUTY study here at Mayo Clinic.  You may recall 
filling out a consent form previously that asked if you were interested in learning more about your blood 
genetic test results.  You had checked “yes” that you would like the opportunity to receive these results.  
We are contacting you as these research results are now available to give you the option to learn your 
results.  More information about genetic testing is included in the following pages.  
If you are interested in learning more about your blood genetic test results or if you have questions: 
Please call the study coordinator at 507-xxx-xxxx.  The study coordinator will set up an appointment for 
you to talk with the genetic counselor (
 
At the time of the initial appointment with the genetic counselor, you will be given more information 
about the genetic testing that was performed on the blood specimen that was collected at the time of your 
enrollment.   The risks, benefits, and limitations of learning your results will be discussed.  You will also 
have an opportunity to ask any questions you might have.  The genetic counselor will not give you the 
research results during this first appointment.   
If you decide after the first appointment with the genetic counselor that you want to receive your research 
results, you can call the study coordinator to set up a second appointment with the genetic counselor.  
Two appointments are planned to make sure that you are fully informed and have adequate time to think 
through this information before you make a decision about whether or not to learn the genetic test results.  
During the second appointment with the genetic counselor, you will have an opportunity to ask any 
questions or discuss any concerns that you may have thought of, and you will then be given your results if 
you wish.  
These preliminary research results will need to be verified in a certified clinical laboratory before they are 
used to make any decisions about your medical management.  If your insurance does not pay for this 
testing, funds from the BEAUTY study are available to cover the cost.   
These appointments can be in person at Mayo Clinic or over the telephone if you prefer, depending on 
what is most convenient for you. 
Results will not be available for up to 3 years from the date of this letter. 
If you are NOT interested in learning your blood genetic test results from this study, check the box below 
and return this letter in the enclosed envelope. 
    
I do NOT want to receive my blood genetic test results from this study. 
 
Please be assured that regardless of your response, your involvement in the BEAUTY study and your care 
at Mayo Clinic will not be impacted in any way.  As always, we thank you for your participation in the 
BEAUTY study. 
 
Sincerely, 
 
 

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Genetic Testing for Hereditary Cancer 
 
Hereditary diseases are caused by gene mutations that are passed down through a family. A mutation is an 
alteration, or change, in a gene. Genetic tests can show whether you carry a gene mutation. Read this 
material to learn more about genetic testing.  The material outlines things to think about related to genetic 
testing.  It tells you what genetic test results may mean to you and your family.  
 
Causes of Cancer 
 
Around 40 percent of people in the general population are diagnosed with cancer.  Researchers do not 
know exactly what causes most cases of cancer.  It happens when cells accumulate genetic changes and 
begin to divide and grow faster than healthy cells do. The cells then build up and form a tumor.  
 
Most cancers are “sporadic”. This means they happen by chance.  Sporadic cancers are due mostly to 
aging, the environment, and lifestyle choices.   
 
Some cancers appear to be “familial,” meaning that there is more cancer in the family than would be 
expected by chance alone.  Genetic factors may play a role in familial cancers, but families share many 
things besides their genes.  For example, they may eat the same kinds of foods.  They may live in the 
same kind of environment.  They may have similar jobs.  These things in combination might cause 
several cases of cancer in a family. 
 
Only about 5 to 10 percent of breast cancers are “hereditary”, or inherited.  Certain gene changes, called 
mutations, that pass from parents to children can cause hereditary cancers.  If you have one of these 
mutations your risk for certain cancers is higher.  However, having a mutation does not guarantee that a 
person will develop cancer.  
 
 
 
 
 
 
 

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What are Genes and Mutations? 
 
Some background information about genes can help explain how you may inherit a higher risk for cancer.  
Inside of each one of your cells are your chromosomes.  Chromosomes are made of a long chain of a 
special chemical called DNA.  Your DNA provides instructions that tell your cells how to work.  DNA is 
divided into sections called genes.  Each gene determines a specific trait, such as your eye color or blood 
type. 
 
Mistakes or alterations in genes are called mutations. They are like words in a sentence that are spelled 
wrong or are in the wrong order. You can’t read the sentence correctly.  In the same way, mutations in 
genes can keep cells from doing their work correctly.  That can lead to genetic diseases or other problems. 
 
 
What Causes Hereditary Cancer? 
 
Hereditary cancer is caused by mutations in certain genes.  When these genes have no mutation and work 
normally, they stop tumor growth. But mutations in these genes increase the risk of certain cancers.   
  
If a family has a gene mutation, cancer often affects several family members in more than one generation.  
Family members who have hereditary cancer often get it when they are very young, such as under age 50.  
They may have more than one type of cancer.  They also may have rare cancers. 
 
Please remember that having an inherited gene mutation does not guarantee that an individual will get 
cancer.  Some people with gene mutations may live their entire life without developing cancer. 
 
 
How are Gene Mutations Inherited? 
 

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Inherited mutations can pass from a parent to a child.  You can inherit a gene mutation from either your 
mother or your father.  
 
Gene mutations cannot skip generations. If your parent has a gene mutation that you do not inherit, you 
cannot pass the mutation on to your children. 
 
Most gene mutations that cause hereditary cancer are inherited in an autosomal dominant pattern. This 
means that every child (both sons and daughters) of a parent who has a genetic mutation has one chance 
in two, or a 50-50 chance, to inherit the same mutation. 
 
 
 
Possible Test Results 
 
Positive test result  
A positive result means that a mutation was found in one of your genes.  This puts you at a higher risk to 
develop certain cancers.  Your health care provider will talk with you about options for managing your 
cancer risks.  
 
A positive test result also means that you could pass the mutation to your children.  Your siblings and 
parents may carry the mutation as well.  If your test result is positive, these family members may want to 
think about genetic testing.  
 
Negative test result  
 

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A negative test result means that the test did not find a gene mutation.  But the effect of this on your 
cancer risk is not always clear.  Your genetic specialist looks at the test result in light of your family 
history.  You may still be at increased risk of cancer if you have close family members with cancer. 
 
Genetic testing can find most mutations in genes. However, you could have a gene mutation that the test 
was not able to find.  
 
Deciding Whether to Receive Genetic Test Results 
 
There are a number of things to think about before receiving genetic test results.  
 
Benefits of genetic tests 
Genetic testing for cancer may: 
- Help show whether you are at higher risk for other cancers. 
- Help you make medical choices, such as what type of surgery to have or how often to have 
screening procedures in the future. 
- Explain the reason for your diagnosis of cancer, or for the cancer in your family. 
- Let other family members learn about their risks so they can decide whether to have testing 
too. 
 
Limitations of genetic tests 
Be sure to talk with your genetic specialist about what genetic testing can and cannot tell you. Genetic test 
results may not give you a clear answer about your cancer risk. The tests may help to find people or 
families with a high risk of cancer. But the tests cannot tell for sure who will or will not get cancer. 
 
Reactions to your test results 
• 
If your test result is positive:   
- Knowing your risk for cancer may make you feel in control and able to take charge of your 
health. Being able to inform and educate your family members about their possible cancer 
risks may give you a positive feeling. 
- However, knowing that you carry a gene mutation can be hard. You may feel anxious, sad, 
afraid or angry. You may think that you surely will get cancer. You may feel guilty about the 
chance that you may pass a gene mutation on to your children.  
- You may need to make some hard choices about the best cancer prevention method for you. 
Talking about your choices with a genetic specialist or other cancer specialist can help guide 
you. 
• 
If your test result is negative: 
- You may have a sense of relief to know that you do not have a gene mutation. If you don’t 
have an increased cancer risk, you may not need to have screenings more often or make 
decisions about measures to prevent cancer.  
- However, don’t let this relief lull you into a false sense of security. Even if your test result is 
negative, you can still get cancer. Women with other risk factors such as a family history of 
cancer or certain lifestyle choices may have an increased risk of cancer.  
- You may feel “survivor guilt” if other family members do carry a mutation and face a greater 
risk of cancer. In addition, because test results do not always give a clear answer, your health 
care provider may not be able to give you a definite conclusion about the meaning of your 
results. This can leave you feeling unsure about your cancer risk.  
 
Other things to think about  
• 
Confirming the preliminary research results:   

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- Your preliminary research results will need to be verified in a certified clinical laboratory 
before they are used to make any decisions about your medical management. 
• 
Financial issues:   
- Many insurance companies cover some or all of the cost of genetic testing.  If your insurance 
company does not cover the cost of your confirmatory genetic testing, funds from the 
BEAUTY study are available to help you. 
• 
Talking with your family:  
- Each person’s situation is different.  Some of your relatives may not want to know if a gene 
mutation is found within the family.  Think about whether you will share test results with 
your family members.  If you plan to share your results, think about how you will do this.  
Your health care provider or genetic specialist can help you find ways to share or ask for 
medical information within your family.  For example, he or she may write a letter about your 
test results that you can give to family members.  
• 
Concerns over health insurance discrimination:  
- There are federal and state laws to help keep your genetic information private.  The federal 
Genetic Information Nondiscrimination Act, or GINA, helps protect you against 
discrimination in health insurance and employment.  Laws to help protect you from 
discrimination in life insurance, long term care insurance and disability insurance vary from 
state to state.  
 
Follow-up Care 
 
If you have a positive test result, you may choose to take steps to lower your cancer risk.  What you 
choose to do depends on your age and medical history.  It depends on past treatments or surgeries you 
have had.  Your personal preferences also will affect your choices.  
 
Talk with your health care provider about your choices.  Your provider may suggest that you have 
screening exams more often.  Certain medicines may help lessen your risk for certain cancers.  To help 
prevent cancer, some women have surgery to remove healthy tissue, such as the ovaries.  Ask your health 
care provider to help you plan what is right for you.  
 
A Final Word 
 
Many things affect your decision about whether to receive genetic test results.  Take time to think about 
what is important to you.  Think about the benefits and limitations of genetic tests.  If you wish, talk with 
your family and close friends about your concerns.  Your health care provider and genetic specialist can 
give you more information to help you decide whether this is right for you. 
 
 
 
 

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2.7  EXAMPLE FOLLOW-UP LETTER TO PARTICIPANTS 
 
 
 
 
 
 
 
Participant Name 
Address 
City, State Zip 
 
 
Dear Participant Name, 
  
It was a pleasure talking with you about your choice to learn your research genetic test results.  As we discussed 
previously, your research results showed that you tested positive for a mutation in that causes         .  The specific 
mutation is in the ____ gene. Your result was confirmed by      , a CLIA-certified clinical laboratory.  The name 
of the specific mutation is:         .  A copy of your test result is included with this letter. 
 
The discovery of this mutation may influence your medical management and provides some potentially useful 
information for your at-risk relatives.  I encourage you to discuss these results with your doctor and with your family 
members.  I have provided a second copy of this letter for you, which you may share with your family or your 
physician if you would like.   
 
We hope this information is useful to you.  If you have questions when you receive this, please do not hesitate to 
contact us.  
 
As always, we want to express our appreciation for your continued participation in the BEAUTY study.   
 
 
Sincerely, 
 
 
 
  
 
 
 
 
 
 
 

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3. REFERENCES 
 
1. 
Levesque E, Joly Y, Simard J. Return of research results: general principles and international 
perspectives. The Journal of law, medicine & ethics : a journal of the American Society of Law, 
Medicine & Ethics 2011;39(4):583-92. 
 
2. 
Bredenoord AL, Kroes HY, Cuppen E, Parker M, van Delden JJ. Disclosure of individual genetic 
data to research participants: the debate reconsidered. Trends in genetics : TIG 2011;27(2):41-7. 
 
3. 
Bredenoord AL, Onland-Moret NC, Van Delden JJ. Feedback of individual genetic results to 
research participants: in favor of a qualified disclosure policy. Human mutation 2011;32(8):861-
7. 
 
4. 
Partridge AH, Burstein HJ, Gelman RS, Marcom PK, Winer EP. Do patients participating in 
clinical trials want to know study results? Journal of the National Cancer Institute 
2003;95(6):491-2. 
 
5. 
Shalowitz DI, Miller FG. Communicating the results of clinical research to participants: attitudes, 
practices, and future directions. PLoS medicine 2008;5(5):e91. 
 
6. 
Evans JP, Rothschild BB. Return of results: not that complicated? Genetics in medicine : official 
journal of the American College of Medical Genetics 2012;14(4):358-60. 
 
7. 
Fabsitz RR, McGuire A, Sharp RR, Puggal M, Beskow LM, Biesecker LG, et al. Ethical and 
practical guidelines for reporting genetic research results to study participants: updated guidelines 
from a National Heart, Lung, and Blood Institute working group. Circulation. Cardiovascular 
genetics 2010;3(6):574-80. 
 
8. 
Ravitsky V, Wilfond BS. Disclosing individual genetic results to research participants. The 
American journal of bioethics : AJOB 2006;6(6):8-17. 
 
9. 
Beskow LM, Burke W. Offering individual genetic research results: context matters. Science 
translational medicine 2010;2(38):38cm20. 
 
10. 
Kollek R, Petersen I. Disclosure of individual research results in clinico-genomic trials: 
challenges, classification and criteria for decision-making. Journal of medical ethics 
2011;37(5):271-5. 
 
11.  
Office of Biorepositories and Biospecimen Research (OBBR), National Cancer Institute.  
Workshop on Release of Research Results to Participants in Biospecimen Studies. Bethesda, 
Maryland; July 8-9, 2010. 
http://biospecimens.cancer.gov/global/pdfs/NCI_Return_Research_Results_Summary_Final-
508.pdf 
 
 
 
 

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Appendix VI Return of Results Gene List 
 
Gene name 
Syndrome/ 
associated 
cancers 
Listed in ACMG's 
recommendations 
for reporting 
incidental 
findings? 
Medical 
management 
guidelines? 
Inheritance 
Pattern 
Return to 
participants? 
APC 
Familial 
Adenomatous 
Polyposis 
Yes 
NCCN 
AD 
Yes 
BMPR1A 
Juvenile polyposis 
No 
NCCN 
AD 
Yes 
BRCA1 
Hereditary 
breast/ovarian 
cancer syndrome 
Yes 
NCCN 
AD 
Yes 
BRCA2 
Hereditary 
breast/ovarian 
cancer syndrome 
Yes 
NCCN 
AD 
Yes 
CDC73 
Familial primary 
hyperparathyroidi
sm/ 
Hyperparathyroidi
sm-jaw tumor 
syndrome/ 
Parathyroid 
adenoma with 
cystic changes/ 
Parathyroid 
carcinoma 
No 
GeneReviews 
AD 
Yes 
CDH1 
Hereditary 
Diffuse Gastric 
Cancer 
No 
Fitzgerald et al., J 
Med Genet 
2010;47:436-444 
AD 
Yes 
CDK4 
Hereditary 
Melanoma 
No 
Guidelines for 
CDKN2A but not 
CDK4; according to 
Puntervoll et al., J 
Med Genet 2013; 
50(4):264-270, 
phenotype cannot be 
distinguished. 
AD 
Yes 
CDKN2A 
Hereditary 
Melanoma 
No 
Eckerle Mize et al., 
in Cancer 
Syndromes: NCBI; 
2009 
AD 
Yes 
DKC1 
Dyskeratosis 
congenita 
No 
GeneReviews & 
Savage et al., 
Pediatr Blood 
Cancer 2009; 
53:520-523. 
XLR 
Yes 
EPCAM 
Lynch syndrome 
No 
NCCN for other 
Lynch syndrome 
associated-genes, 
not specifically for 
EPCAM 
AD 
Yes 

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Gene name 
Syndrome/ 
associated 
cancers 
Listed in ACMG's 
recommendations 
for reporting 
incidental 
findings? 
Medical 
management 
guidelines? 
Inheritance 
Pattern 
Return to 
participants? 
FH 
Hereditary 
Leiomyomatosis 
and Renal Cell 
Cancer 
No 
Provisional 
guidelines in 
GeneReviews; no 
consensus 
guidelines. 
AD 
Yes 
FLCN 
Birt-Hogg-Dube 
syndrome 
No 
GeneReviews 
AD 
Yes 
KIT 
Familial GIST 
No 
Provisional 
recommendations in 
Familial Cancer 
Suscetibility 
Syndromes 
handbook; no 
consensus 
guidelines. 
AD 
Yes 
MAX 
Hereditary 
Paraganglioma/Ph
eochromocytoma 
No 
GeneReviews; no 
consensus 
guidelines. 
AD 
Yes 
MEN1 
Multiple 
Endocrine 
Neoplasia type 1 
Yes 
GeneReviews & 
Thakker et al., J 
Clin Endocrin 
Metab 2012; 97(9): 
2990-3011. 
AD 
Yes 
MET 
Papillary renal 
cell carcinoma 
No 
Provisional 
recommendations in 
Familial Cancer 
Suscetibility 
Syndromes 
handbook; no 
consensus 
guidelines. 
AD 
Yes 
MLH1 
Lynch syndrome 
Yes 
NCCN 
AD 
Yes 
MSH2 
Lynch syndrome 
Yes 
NCCN 
AD 
Yes 
MSH6 
Lynch syndrome 
Yes 
NCCN 
AD 
Yes 
NF2 
Neurofibromatosi
s type II 
Yes 
GeneReviews & 
Evans et al., Br J 
Neurosurg 2005a; 
19: 5-12 
AD 
Yes 
PDGFRA 
Familial GIST 
No 
Provisional 
recommendations in 
Familial Cancer 
Suscetibility 
Syndromes 
handbook; no 
consensus 
guidelines. 
AD 
Yes 
PMS2 
Lynch syndrome 
Yes 
NCCN 
AD 
Yes 
PRKAR1A 
Carney complex 
No 
GeneReviews 
AD 
Yes 
PTCH1 
Gorlin syndrome 
No 
GeneReviews 
AD 
Yes 

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Gene name 
Syndrome/ 
associated 
cancers 
Listed in ACMG's 
recommendations 
for reporting 
incidental 
findings? 
Medical 
management 
guidelines? 
Inheritance 
Pattern 
Return to 
participants? 
PTEN 
Cowden 
syndrome 
Yes 
NCCN 
AD 
Yes 
RET 
MEN2 
Yes 
GeneReviews & 
American Thyroid 
Association 
Guidelines Task 
Force, Thyroid 
2009; 19(6): 565-
612. 
AD 
Yes 
SDHAF2 
Hereditary 
Paraganglioma/Ph
eochromocytoma 
Yes 
GeneReviews; no 
consensus 
guidelines. 
AD 
Yes 
SCG5 
Hereditary Mixed 
Polyposis 
Syndrome 
No 
Proposed in various 
papers; see Calva 
and Howe, Surg 
Clin North Am 
2008; 88(4):779. 
AD 
Yes 
SDHB 
Hereditary 
Paraganglioma/Ph
eochromocytoma 
Yes 
GeneReviews; no 
consensus 
guidelines. 
AD 
Yes 
SDHC 
Hereditary 
Paraganglioma/Ph
eochromocytoma 
Yes 
GeneReviews; no 
consensus 
guidelines. 
AD 
Yes 
SDHD 
Hereditary 
Paraganglioma/Ph
eochromocytoma 
Yes 
GeneReviews; no 
consensus 
guidelines. 
AD 
Yes 
SMAD4 
Juvenile polyposis 
No 
GeneReviews 
AD 
Yes 
STK11 
Peutz-Jeghers 
syndrome 
Yes 
NCCN 
AD 
Yes 
TERC 
Dyskeratosis 
congenita 
No 
GeneReviews & 
Savage et al., 
Pediatr Blood 
Cancer 2009; 
53:520-523. 
AD 
Yes 
TINF2 
Dyskeratosis 
congenita 
No 
GeneReviews & 
Savage et al., 
Pediatr Blood 
Cancer 2009; 
53:520-523. 
AD 
Yes 
TP53 
Li-Fraumeni 
syndrome 
Yes 
NCCN 
AD 
Yes 
TSC1 
Tuberous 
sclerosis 
Yes 
GeneReviews, de 
Vries et al., Eur 
Child Adolesc 
Psychiatry 2005; 
14(4): 183-190, & 
Roach et al., J Child 
Neurol 1999; 14(6): 
401-407. 
AD 
Yes 

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Gene name 
Syndrome/ 
associated 
cancers 
Listed in ACMG's 
recommendations 
for reporting 
incidental 
findings? 
Medical 
management 
guidelines? 
Inheritance 
Pattern 
Return to 
participants? 
TSC2 
Tuberous 
sclerosis 
Yes 
GeneReviews, de 
Vries et al., Eur 
Child Adolesc 
Psychiatry 2005; 
14(4): 183-190, & 
Roach et al., J Child 
Neurol 1999; 14(6): 
401-407. 
AD 
Yes 
VHL 
Von Hippel-
Lindau 
Yes 
GeneReviews & 
http://vhl.org/wordp
ress/library/Handbo
ok/handbook40.pdf 
AD 
Yes 
 
 
 
 

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Appendix VII Digital Spatial Profiling of High Risk Hormone Receptor-Positive (HR+) Breast 
Cancer 
Analytical Objective: In collaboration with the Mayo BEAUTY team, we will use the NanoString 
GeoMX Digital Spatial Profiling (DSP) platform to elucidate the immune architecture of high risk 
hormone-receptor positive HER2-negative (HR+) breast tumor samples from the BEAUTY study1. The 
initial experiments are designed to identify immune features of clinical high risk ER+/HER2 negative 
breast cancer in order to provide a new understanding of the immune landscape and biology of 
ER+/HER2- breast cancer both before and after chemotherapy.  The overarching goal is to compare the 
immune landscape for those that develop a local/regional recurrence with those tumors with no 
recurrence.  A secondary objective is to assess the immune landscape of tumors throughout the course of 
chemotherapy (baseline, after taxane, at surgery, and at the time of recurrence). 
Clinical Rationale: The use of immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment 
in the past decade. ICIs are believed to function primarily through inhibition of pre-existing host immune 
suppression. Consequently, an understanding of the baseline immune environment is critical for 
understanding and predicting the efficacy of immuno-therapy. Multiple clinical trials have demonstrated 
durable recurrence-free response with ICIs in “hot” or T-cell inflamed tumors such as melanoma or lung 
cancer. In contrast, the results of single agent ICIs in less immunogenic tumors such as breast cancer have 
been somewhat disappointing. This is particularly true in immunologically “cold” tumors like 
HR+/HER2-negative (i.e. luminal) breast cancer, that characteristically have lower amounts of tumor 
infiltrating lymphocytes (TILs)2. Clinical trials with a single agent ICI, anti-PD-L1 avelumab, showed 
that the objective response of single agent avelumab was only 2.8%3 in such tumors.  
There are several ongoing efforts to identify potential therapies to improve the efficacy of ICIs in luminal 
breast cancer. One such strategy is to combine conventional chemotherapy with ICIs. This strategy was 
evaluated in the recent I-SPY 2 trial in which patients with luminal breast cancer were randomized to 
receive standard neoadjuvant chemotherapy with or without pembrolizumab4. A 21% increase in 
complete pathological response (pCR) was observed when pembrolizumab was added to neoadjuvant 
chemotherapy (34% pCR with pembrolizumab plus chemotherapy vs. 13% with chemotherapy alone). 
However, the pCR rate in luminal breast cancer is still relatively low compared to more immunogenic 
tumors like triple negative breast cancer (TNBC), among which 60% of patients achieved pCR with 
pembrolizumab in combination with chemotherapy. 
Recent studies suggest that pre-existing host immune features may play a critical role in therapeutic 
response of less immunogenic HR+ breast tumors, and additional studies are needed to evaluate the 
immune landscape of these tumors within the context of response to both conventional and Immune 
therapies. Limited data exist on the immune architecture of luminal breast cancer, and the significance of 
pre-existing host immune features and outcome of patients with luminal breast cancer remains to be 
determined.  This is particularly true among patients with HR+ tumors with high risk of recurrence, 
generally defined as Grade 3 or Grade 2 with high proliferation. 
There are also data that suggest that the significance of the baseline immune architecture may be quite 
different in HR+ breast cancer, compared to more immunogenic breast cancer like TNBC. Higher levels 
of TILs are associated with improved outcome in TNBC or HER2+ breast cancer; whereas high TILs in 
luminal HER2-negative breast cancer is associated with relatively poorer overall survival2.. Such data 
reinforce the need for a more detailed understanding of the immune architecture of breast cancer 
subtypes. 
Quantification of TILs and analysis of the prognostic and/or predictive potential of TILs is a subject of 
great interest in breast cancer research at this time. However, the type, activity, and function of specific 
immune cells cannot be determined by simple pathological assessment of TILs. For example, one of the 
genes that is associated with poor outcome in luminal breast cancer is CD685 which is highly expressed in 

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both circulating and tissue macrophages. This observation illustrates the need to begin to break down the 
relationship between specific types of TILs and clinical outcome as a function of the immune 
environment that prevails in different breast cancer subtypes. Elucidation of the immune architecture in 
luminal breast cancer is critical in moving the field of immune-oncology forward in breast cancer. DSP 
provides a new tool that makes it possible to examine features associated with tumor cell-immune cell 
interaction at a level of detail that was heretofore unavailable. 
Samples: We will analyze tumor immune architecture as a function of clinical outcome following 
neoadjuvant therapy in a cohort of 52 high risk HR+/HER2- samples from the BEAUTY study.  All 
women received weekly paclitaxel for 12 weeks, followed by AC chemotherapy, followed by 
surgery.  Formalin-Fixed, paraffin embedded samples are available before treatment, after taxane, 
and after chemotherapy (residual disease).  Additionally, a subset have an FFPE sample available 
from a loco-regional or distant recurrence. 
For the primary aim, will assess in a co-primary endpoint of whether the immune landscape in the  
a) primary tumor biopsy (before chemotherapy) and b) surgical specimen (after chemotherapy) is 
associated with the risk of recurrence.  A secondary analysis will be to assess whether the immune 
landscape using the baseline tumor is associated with chemotherapy response (RCB 0/1 vs 2/3).  
Finally, we will assess whether the immune landscape changes over time comparing the pre-
treatment, b), after taxane c) surgical  and d) recurrence.   The latter will be an exploratory 
endpoint.   
GeoMX Digital Spatial Profiling:  DSP is a novel technology that allows simultaneous profiling of 
multiple proteins or RNAs while preserving the spatial information. The DSP technology is quite simple 
in concept and execution. FFPE sections (one 5-micron slide/sample) will be labeled with three 
fluorescent marker antibodies (pan-cytokeratin, anti-CD45, and anti-CD68) plus a DNA dye (SYTO13) to 
stain nuclei. These marker antibodies will be used to define regions of interest (ROIs). FFPE sections will 
also be incubated with the cocktail containing a panel of other “target” antibodies that are conjugated to 
synthetic oligonucleotides via a UV-cleavable cross linker. At present there are >40 well-validated target 
antibodies against immune function proteins (e.g. PD-L1, CD3, CD8, B2M, INO1, etc.), common tumor-
related proteins (ERBB2, Ki67, BCL2, etc) or housekeeping proteins for normalization. The list of 
rigorously validated target antibodies grows almost daily. The current list of validated protein targets is 
shown below. 
Figure 1A shows an ROI that has been segmented into tumor (pan-cytokeratin-green) and CD68-enriched 
(red) stroma. Stroma is segmented as SYTO13 positive/pan-cytokeratin negative.) Figure 1B shows a 
different ROI that has been segmented into tumor (green) and CD45-enriched (yellow) stroma. The 
individual segments within these ROIs are illuminated with UV light, releasing the oligonucleotides 
bound to the antibodies that were bound to their respective targets within each segment of each ROI. The 
oligonucleotides are “counted” using conventional nanoString technology, which provides a 5-log 
dynamic range for precise quantification of antibody binding to target proteins. Using this approach, we 
obtain an indirect measurement of the abundance of immune function proteins within the tumor in a 
spatially defined manner.  
 

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We will use 3 fluorescent labeled antibodies to identify tumor cells (anti-pan-cytokeratin), leukocytes 
(anti-CD45), and macrophages (anti-CD68). Each ROI be “segmented” into two regions: a pan-
cytokeratin+ segment that includes tumor, plus a STYO13+ (nuclear stain)/cytokeratin- segment that 
includes tumor-adjacent stroma.  Twelve ROIs will be segmented for each sample: 6 ROIs that contain 
CD68-enriched stroma (Figure 1A), plus 6 ROIs that contain CD45-enriched stroma (Figure 1B). We will 
analyze 46 immune function proteins in tumor and stroma segments from each sample. Using this 
approach, we will test the hypothesis that macrophage (CD68)-associated vs. lymphocyte-associated 
(CD45+/CD68-) PD-L1 is prognostic for pCR in the neo-adjuvant setting. In addition, all other immune 
features will be tested for association with pCR. 
Figure 1: DSP images of segmented regions of interest. Two 300 micron ROIs were segmented. The 
segments include pan-cytokeratin-positive tumor cells (green), CD68-enriched stroma (Panel A, red), or 
CD45-enriched stroma (Panel B, yellow). 
 
Given the power of the technology, the uniqueness of the sample cohort, and the clinical significance of 
the question, we anticipate the results of these analyses will be of considerable clinical impact. The 
opportunity to use this powerful new technology to identify spatially-defined biomarkers is obvious. The 
wealth of genomic data that have been generated with these samples should inform subsequent studies 
and serve as preliminary data for future grant applications. We are also aware that some of these samples 
will be analyzed using the Fluidyne platform, and a cross-platform comparison would be of considerable 
interest, as well. 

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Appendix VII References: 
1 Goetz, M.P., et al., Tumor Sequencing and Patient-Derived Xenografts in the Neoadjuvant Treatment of 
Breast Cancer. Journal of the National Cancer Institute, 2017. 109(7). 
2 Denkert, C., et al., Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast 
cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy. Lancet Oncology, 2018 
Jan. 19(1): 40-50. 
3 Dirix, L.Y., et al., Avelumab, an anti-PD-L1 antibody, in patients with locally advanced or metastatic 
breast cancer: a phase 1b JAVELIN Solid Tumor study. Breast Cancer Research and Treatment, 2018 
Feb. 167(3): 671-686. 
4 Nanda et al. Proc American Society of Clinical Oncology 2017 
5 Waks, A.G., et al., The Immune Microenvironment in Hormone Receptor-Positive Breast Cancer 
Before and After Preoperative Chemotherapy. Clinical Cancer Research, 2019 Aug 1. 15(15): 4644-
4655. 
 
 

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Appendix VIII  Gene Expression Analysis by the University of Bristol 
Triple Negative Breast Cancer (TNBC) comprises around 20% of all breast cancer diagnoses, behaves 
more aggressively than other types of breast cancer and has targeted therapeutic options (Dent et al 2007). 
The majority of patients receive neoadjuvant chemotherapy in order to reduce tumour burden prior to 
their operation and this enables us to explore how tumours respond to chemotherapy drugs that are in use 
in routine clinical practice. In TNBC, there is a high-risk of early-relapse compared to other types of 
breast cancer, with most relapses having occurred by 3 years from diagnosis (Dent et al 2007 and Lietdke 
et al 2008). The relapse rate is significantly lower in those who obtain a complete pathological response to 
chemotherapy (Cortazar et al 2014 and Symanns et al 2017). 
 
In trying to understand why TNBC behaves so aggressively attention has turned to cancer stem cells 
(CSCs) (Thiagarajan P et al 2018). Previous work has identified that CSCs (Al Hajj et al 2003), or stem-
cell-like behaviour (Samanta et al 2014) could explain the aggressive characteristics of this type of breast 
cancer. TNBC has more CSCs as compared to other types of breast cancer (Dittmer et al 2018) and 
previous work has shown that CSCs increase in response to chemotherapy (Alamgeer et al 2014). In a 
previous study conducted at the Univsersity of Bristol, a set of 323 genes (whose expression was 
upregulated in purified murine normal mammary stem cells) was demonstrated to be highly prognostic of 
significantly shorter disease-free survival (DFS) in three human TNBC datasets (Soady et al 2015). This 
signature was particularly upregulated in the normal like breast molecular subtype as well as the BL1, M 
and MSL subtypes of TNBC but had prognostic ability for all subtypes of TNBC. Although this gene 
expression signature was highly prognostic of DFS in these retrospective analyses, chemotherapy 
response was not assessed. However, as is known, that response to chemotherapy is prognostic, a 
demonstration that the gene expression set (or a subset of these genes) could predict response to 
chemotherapy would provide a new tool for clinical management, enabling prediction of either de novo or 
acquired chemotherapy resistance and the possibility of changing treatment in patients as resistance 
emerges. 
For this analysis, the University of Bristol will use gene expression data in combination with pathological 
response to be used as a part of the discovery cohort for their gene expression signature. The University 
of Bristol will investigate whether the previously identified CSC-signature is associated with 
chemosensitivity in this cohort and, in addition, they will perform explorative analyses in which we will 
look for genes which expression is associated with treatment response. Genes associated with Epithelial 
to Mesenchymal Transition (EMT), a process associated with cancer stem-cell like behaviour, were 
associated with resistance to chemotherapy. The University of Bristol has approximately 1300 patients in 
their discovery cohort and 700 in two separate validation cohorts.  
 
Experimental approach: 
The statistical analysis for this gene expression work will be undertaken by statisticians based at the 
Cancer Research UK Integrated Cancer Epidemology Programme (CRUK-ICEP) at the University of 
Bristol as well as having input from Dr Anita Grigoriadis at King’s College London (KCL) who 
performed the statistical and bio-informatics analyses in the original stem cell gene signature study, and is 
leading in the cancer bioinformatics group at KCL 
 This will be based on the 
statistical plan as below. 
 
Details of Statistical Plan: 
Aim: The aim of our study is to predict neoadjuvant chemotherapy response in triple-negative breast 
cancer (TNBC) using gene expression.  
 
 
 

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Methods: 
Integration of gene expression datasets 
If the dataset arrays are all of the same version/type (for example Affymetrix arrays, Agilent arrays, RNA 
seq etc) then they will be processed concurrently. If the dataset arrays are different then they will be  
processed independently. Expression values will be summarized across whole genes independently. A list 
of common genes across all arrays will be built and the data will be converted to Z-scores independently. 
Afterwards, the data will be merged. There will likely be some batch effect so samples will be normalised 
using ComBat method and in downstream analyses, 'array type' will be included as a covariate. 
 
Molecular subtypes of TNBC 
Molecular subtypes of pre-processed and normalised raw data of TNBC will be determined by using the 
webtool 
This is based upon the paper by Lehmann et al 2011. The data 
will also be run through the updated and condensed 4-type 2016 classification. 
 
Classifier development and validation 
The overall TNBC dataset will be divided into training (dataset A will have 2/3 of total samples) and 
validation subset (dataset B will have 1/3 of total samples). Exact numbers will be pre-determined before 
analysis dependent on cohorts included in the final dataset. 
 
Linear models (univariate and multivariate (possible covariates will include age, T and N stage, grade as 
well as treatment received)) will be fit to dataset A and then an empirical Bayesian method will be used to 
compute the p-values corresponding to the t-statistics of differential expression. The genes will be ranked 
according to their most significant p-values, which will be adjusted for multiple testing by using 
Benjamini and Hochberg (BH) method. If any gene(s) are identified, the distribution of p value will be 
reviewed and used to empirically decide the cut-off to retain the genes for classification. 
 
The classifier will be validated on dataset B using 1) blind validation and 2) leave one out cross validation 
(LOOCV). In all the classification approaches, a non-parametric k-nearest neighbour (KNN) 
classification method will be applied for responders and non-responders to chemotherapy. 
 
TNBC is a heterogeneous disease. The above approach of classifier development and validation will be 
repeated for each of the molecular TNBC subtypes. Multi-class ROC analyses for all TNBC and each 
TNBC-subtype respectively will be performed to evaluation the classifier performance. The newly-
identified genes will be compared with a previously published panel to establish the degree of overlap 
(Soady et al 2015). 
 
There are pilot studies set up at the University of Bristol to examine whether they can capture this 
signature and look for dynamic changes throughout neoadjuvant chemo. 
 
 

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Appendix IX BCM/Mayo Clinic BEAUTY Collaboration Summary 
Project Description 
The 
 laboratories have a longstanding collaboration on two projects for which access to the 
BEAUTY quantitative MRI response and RNAseq data is requested. These two projects are: 
1. Development of PDX-based molecular signatures of differential response to taxane vs. platinum-based 
chemotherapeutics. 
2. Hypothesized requirement for the immune system for the growth of immunologically “hot” tumors, which 
have elevated expression of immune cell-associated gene expression. 
For project 1, BEAUTY data will be used for clinical validation of the taxane response signature. Based on our 
ability to predict response in other PDX datasets, and one clinical dataset for cisplatin/paclitaxel response, we expect 
that our signature will predict paclitaxel response near-quantitatively and significantly better than chance. 
 
For project 2, BEAUTY RNAseq data will be used to divide the clinical samples into “hot” and “cold” tumors using 
the
 lab’s computational cell type deconvolution algorithm. These classifiers will then be evaluated 
relative to the ability to grow PDX models from the clinical samples. Based on results from the BCM and HCI 
collections, we predict that the BEAUTY PDX will show similar results with only “cold” patient tumors yielding 
PDX models. 
 
Proposed Logistics and Activities 
During discussions in the meeting held August 5th, 2020, it was agreed that the BEAUTY trial RNAseq data would 
be made available to the 
 labs in a manner blinded to the quantitative MRI responses. 
However, we will need to know the ER/HER2 status for each sample as the PDX-based response predictor, and the 
“hot/cold” classification was developed exclusively using triple-negative models [That being said, we do have 
response data for four ER+ tumors so far, with additional models under study so we may be able to generate an ER+ 
chemotherapy response predictor at some point soon]. 
 
It is already known which clinical samples yielded PDX models. 
 
RNAseq FASTQ files will be transferred either via the Globus file transfer system or by other means. 
The 
 labs will process the RNAseq samples, conduct QC, and generate the Paclitaxel 
response predictions independently for triple-negative and ER+ clinical samples, and will determine “hot/cold” 
tumor classifications. 
 
Ranked and quantitative paclitaxel response predictions will be sent to the Mayo Clinic BEAUTY Executive 
Committee for evaluation relative to quantitative MRI responses. The MAE and RMSE will be determined for the 
predictions vs the know outcomes. The ROC curve will also be determined for patients that did and did not have 
pathological complete response (pCR) to chemotherapy.  
 
In addition, a set of genes (“multi-variate biomarker”) used in the predictive model will also be sent to the Mayo 
Clinic BEAUTY Executive Committee for building of a model by Mayo on a subset of triple-negative “cold” tumors 
identified to be such by Baylor based on the analysis of RNA-seq data. Mayo team will determine predictive power 
of the model using Leave-one-out or similar validation method. This predictive power of the multi-variate biomarker 
will be compared to the predictive power of models built on random sets of genes. It is anticipated as a positive 
result that the model built on the multi-variate biomarker will outperform almost all the models built on random sets 
of genes. A negative result of this experiment will be highly informative, as it would suggest that the multi-variate 
biomarker is not informative. 
 
Results of these analyses will be sent back to the 
 labs, and the BCM and Mayo Clinic 
groups will schedule a meeting to discuss. 
 
Assuming response prediction is successful, these results will be included in a manuscript with Mayo Clinic 
personnel participating as authors. 
 

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If our initial response prediction is not successful, we would like the opportunity to have one more attempt to refine 
the signature and re-evaluate relative to response. 
 
Depending on data availability for the TBCRC030 clinical trial completed at Dana Farber, the predictors may also 
be validated in this dataset for the publication. However, it may be advantageous to incorporate the paclitaxel 
(Mayo, RNAseq) and cisplatin (Dana Farber, Affy) data, as well as all available PDX data into a new set of 
predictors prior to use of TBCRC030 data. 
 
 

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Appendix X SimBioSys Collaboration 
 
Synopsis 
 
Data from the BEAUTY study will be shared with SimBioSys, Inc. for purpose of carrying out a 
validation of their TumorScope Algorithm for predicting response to NACT 
 
Objectives 
Primary Objective: To assess the accuracy of TumorScopeTM in predicting pathological complete 
response (pCR) among women with Triple Negative & HER2+ breast cancer who receive Neoadjuvant 
Chemotherapy (NACT) therapy. 
 
Hypothesis: TumorScopeTM can accurately predict whether a patient will achieve a pathological complete 
response (pCR) using the AJCC staging system’s definition of ypT0/Tis ypN0 as assessed by the local 
pathologist at the time of definitive surgery  
 
Secondary & Exploratory Objectives: 
Secondary & Exploratory objectives of the study are: 
 
1. To assess accuracy of TumorScopeTM in predicting pathological complete response(pCR) in women 
with HR+/HER2- Breast Cancer receiving NACT in combination with anti-Her2 therapy 
 
Hypothesis: TumorScopeTM can accurately predict whether a patient will achieve a pathological 
complete response (pCR) using the AJCC staging system’s definition of ypT0/Tis ypN0 as assessed 
by the local pathologist at the time of definitive surgery  
 
2. To compare accuracy of volumetric response of TumorScopeTM predictions to longitudinal imaging 
performed over the course of NACT  
 
Hypothesis: TumorScopeTM can accurately predict the reduction of tumor volume (measured in 
cubic centimeters) over the course of NACT 
 
3. To assess the relationship of pCR prediction from TumorScopeTM to Event Free Survival (EFS) 
 
Hypothesis: TumorScopeTM prediction of pCR is a prognostic marker for Early-Stage Breast 
Cancer as measured by Event Free Survival (EFS), independent of whether the pCR prediction is 
correct. 
 
Study Design 
A retrospective, independent, and blinded validation of the diagnostic accuracy of SimBioSys 
TumorScopeTM prediction of pathological complete response (pCR) will be undertaken. The predictions 

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will be performed using only information available at the time of diagnosis and compared to ground truth 
for actual patient outcomes. The study design is depicted in the figure below. 
The following variables will be shared with SimBioSys staff from the study database to perform 
predictions: 
 
• 
Demographic: Race: Caucasian/African American/Asian/Hispanic/Other 
• 
Age at start of NCT  
• 
Cancer T Staging: T1, T2, T3, T4 
• 
Cancer N Staging: N0, N1, N2, N3  
 
• 
Pathology Markers at time of Primary Diagnosis: 
• 
Estrogen Receptor Status  
• 
Progesterone Receptor  
• 
HER2+ Status: IHC (0 -3) or FISH Ratio 
• 
Tumor Grade: 1-3 
• 
Ki67: 0 – 100% (Optional, for analysis only) 
• 
Histological Type:  IDC/ILC/Mixed/Inflammatory 
 
1. Pre-Surgery Imaging 
• 
DCE MRI Series: Minimum of 3 timepoint 
• 
Initial Size of Tumor on MRI imaging 
 
2. NCT regimen 
• 
Regimen Given 
 
3. Dates for Volumetric Analysis (Optional): 
• 
Lag between diagnostic MRI and start of NCT 
• 
Lag between diagnostic MRI and any additional MRIs to determine which time points to 
compare volumes at 
• 
Lag between the start of NCT and surgery 
 
4. Data regarding the Pathology Specimens 
• 
weight of each breast pathology specimen for all specimens 
• 
dimensions (3 dimensions) of each breast and lymph node pathology specimen for all 
specimens 
 
Blinding & Unblinding Procedures 
This is a blinded study and any data indicative of potential response such assessment of pCR, Residual 
tumor size, post therapy imaging or any documented assessment of clinical response, recurrence of 
disease will not be utilized as an input or made available to SimBioSys staff at the onset of the study. Any 
subjects where data potentially indicative of response is accidently revealed will be excluded from the 
study and documented. 
 
The Mayo team will provide blinded data and follow procedures with absolute integrity. 
 
Submission of initial subject data will only include variables pre-identified as inputs into the model 
prediction. Upon delivery of initial data, SimBioSys will perform necessary validation and identify any 
subjects who should be excluded based on predefined criteria. Predictions of both primary, and secondary 
and exploratory endpoints will be performed and documented by SimBioSys staff. Simulation results will 

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be delivered to each site via email and confirmation of receipt will be required. The simulation prediction 
will be considered “locked” at this point and no changes to the results will be permitted. 
 
Principal investigators alongside collaborating biostatisticians at the study site will internally unblind the 
entire cohort to perform initial analysis. The site-specific analysis will be shared with SimBioSys staff for 
discussion. At this point, the analysis and results will be considered “locked”. 
 
Once study site specific analysis is finalized, data used for validation and exploratory analysis will be 
unblinded and sent to SimBioSys staff. This data includes: 
 
Output Variables 
These variables may be collected at any time after the initiation of the study but will be blinded to 
SimBioSys staff until final predictions are submitted. 
• 
pCR: Defined as ypT0/Tis & N0 
• 
Residual Size: Measurement of residual tumor post-surgery (centimeters) 
• 
Recurrence: Yes/No 
• 
Time to Recurrence: Weeks 
• 
Last Follow-up Date: Weeks 
 
Statistical Methods 
This study will be conducted in subjects who participated in Beauty Study (MC1137) at Mayo Clinic. 
Subjects will be selected based on inclusion criteria and availability of pre-treatment MRI. 
 
Statistical Evaluation (pCR) 
The investigational TumorScopeTM test device has two possible predicted pCR outcomes, yes (+) or no 
(−). Each patient will have actual clinical outcomes (i.e., ground truth) with regards to the pCR, yes (+) or 
no (−). Clinical sensitivity and specificity will be determined by comparing the investigational 
TumorScope TM predicted results to the ground truth at the subject level. 
 
The following 2x2 table is constructed to illustrate the co-primary endpoints: 
 
 
Ground Truth 
Total 
Yes (+) 
No (-) 
Investigational 
Device 
Yes (+) 
a 
b 
a+b 
No (-) 
c 
d 
c+d 
Total 
a+c 
b+d 
a+b+c+d 
 
Sensitivity is the proportion of subjects who are pCR responders in the ground truth (i.e., yes) are also 
predicted responders in the investigational test (i.e., yes); calculated as 100% x a/(a+c). 
 
Specificity is the proportion of subjects who are pCR non-responders in the ground truth (i.e., no) are also 
predicted non-responders in the investigational test (i.e., no); calculated as 100% x d/(b+d). 
 
Positive Predictive Value (PPV) is the proportion of subjects who are predicted pCR responders in the 
investigational test are also responders in the ground truth; calculated as 100 x a/(a+b). 
 
Negative Predictive Value (NPV) is the proportion of subjects who are predicted pCR non-responders in 
the investigational test are also non-responders in the ground truth; calculated as 100 x d/(c+d). 

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Diagnostic Accuracy is the proportion of correctly classified subjects among all patients; calculated as 
100 x (a+d)/(a+b+c+d). 
 
Exploratory Analysis 
Predicted Tumor Volume 
Using longitudinal MRIs, where available for each subject, segmented tumor volumes will be assessed by 
a practicing radiologist blinded to the prediction results. 
 
The segmented volumes will be compared against predicted volumes previously submitted from 
TumorScope to assess performance. Performance will be reported as a Pearson Correlation between 
predicted volume and actual volumes. In addition, median absolute error, median absolute deviation, 
mean error, and standard error will be reported on predicted response (%) vs actual response (%) 
calculated as: 
 
(Start Volume - End Volume)/Start Volume 
 
Other analysis will be performed at the discretion of SimBioSys and Principal Investigators. 
 
Event-Free Survival (EFS) 
The EFS time distribution will be estimated by the Kaplan-Meier method A point and interval estimate of 
the 3 year EFS rate and the 5 year EFS rate will be constructed from these estimates. A log rank test will 
be used to assess whether the EFS differs with respect to whether the patient had a pCR after NCT as well 
as assess whether EFS differs with respect to whether TumorScope predicted the patient would have a 
pCR or not.  
 
Target Subject Population 
All patients enrolled on MC1137 (Beauty) who have previously received standard of care NACT and 
meet inclusion/exclusion criteria will be included in this study. The Primary End Point population will 
only include TNBC and HER2+ patients as,  large, pooled analysis demonstrated strong association of 
pCR, when defined as no tumor in both breast and lymph nodes (ypT0 ypN0 or ypT0/Tis ypN0) 
following neoadjuvant therapy for breast cancer, with improved long-term benefit as measured by event-
free survival and overall survival [4]. 
 
Introduction 
 
Background 
Breast cancer remains the most common malignancy in women in the United States. About 1 in 8 U.S. 
women will develop invasive breast cancer over the course of her lifetime [1]. Neoadjuvant chemotherapy 
(NACT) is considered a standard of care for some women with locally advanced breast cancers [2, 3]. 
Recently, FDA recognized a complete response from NACT referred to as a pathological complete 
response as a surrogate marker for accelerated drug approval. Large metanalysis confirms that patients 
who achieve pCR after NACT have excellent event-free and distant disease-free survival especially in the 
Triple Negative and HER2+ subtypes of the disease [4]. pCR rates differ among cancer subtypes that are 
defined by the tumor’s ER, PR, and HER-2 receptor status. Specifically, pCR rates range from 10-15% in 
ER/PR+, HER2- cancers up to 70% in ERPR- HER2+ cancers [4]. Treatments are selected in clinical 
setting with the target to achieve pCR as this provides ability for physicians to prognosticate, escalate, or 
de-escalate therapy and develop treatment strategies post-surgery. Exploratory studies have shown factors 
that influence pCR rates include histology, morphologic features such as tumor size and enhancement 
pattern, and various imaging parameters [5-7]. Histological markers including higher Ki67 levels (greater 

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than 15% or greater than 35%, depending on the study) and greater percentages of tumor-infiltrating 
lymphocytes have been linked to higher pCR rates [10-12].  
 
Current prediction methods involve comparing images taken before vs. after NACT and take into account 
information from dynamic contrast enhanced MRI (DCE-MRI) and histologic features. Many of these 
methods require data gathered after NACT initiation [8, 9]. While early response to NACT may predict 
attainment of a pCR, patients who have started receiving treatment will have delayed opportunities to 
pursue, or may be precluded from, other therapeutic options such as de-escalation or clinical trials.  
 
Rationale for this Study 
Even though a strong standard of care exists for early-stage breast cancer, there is still significant 
variation in decision making. Varying opinions among clinicians result from lack of tools to standardize 
how data is utilized in the planning process. Patient expectations  are set using population-based statistics 
from clinical trials and references to literature, and the patient’s role in the decision-making process is 
informed only by standard medical reports (MRI/Pathology), their interaction with their healthcare 
providers, and online forums/engines.  
A reliable a priori prediction of pCR offers many benefits in clinical decision making oriented towards 
increased confidence of the decision for the clinician. Such a prediction would provide a patient-specific 
prognostication tool to supplement the commonly used clinic-pathological data points such as stage, 
grade and receptor status. However, none of the novel methods that have been recently developed have 
provided sufficient diagnostic accuracy to warrant use clinically. This justifies the urgent need for a 
comprehensive model, applicable to data obtained before NACT initiation, that can effectively 
differentiate between patients who are likely to achieve a pCR and those less likely to respond. Even 
though TNBC and HER2+ are chemosensitive compared to other breast cancer subtypes in the early-stage 
setting, residual disease rates post-NACT are still high and those with residual disease have an extremely 
poor outcome. Therefore, TNBC & HER2+ is a disease with high unmet medical need. 
 
Selection of study population 
Any patient previously indicated for Neoadjuvant Therapy in Early-Stage Breast Cancer who has 
completed therapy and whose surgical sample has been analyzed by a pathologist for attainment of pCR 
will be eligible for this study. The primary end-point analysis will be limited to Triple Negative and 
HER2+ subtypes. Exploratory analysis will be performed on all indicated patients. 
 
Inclusion Criteria 
For Inclusion in this study subjects must fulfill all the following criteria: 
1. A diagnostic Dynamic Contrast Enhanced MRI was obtained prior to therapy 
2. Patient is indicated for Neoadjuvant Therapy (HR+/HER2- subset only for secondary analysis) 
3. Patients who completed treatment & surgery at the study site between 1/1/2001 – 12/31/2020 
4. Treatment given was from the Standard of Care as defined by NCCN Guidelines listed below: 
 
For HER2- Patients: 
• 
Doxorubicin/Cyclophosphamide followed by Paclitaxel (AC-T) 
• 
Paclitaxel followed by Doxorubicin/Cyclophosphamide (T-AC) 
• 
Docetaxel/Cyclophosphamide (TC) 
• 
Docetaxel/Carboplatin (TCb) 
• 
Doxorubicin/Cyclophosphamide (AC) 
 
For HER2+ Patients: 
• 
Doxorubicin/Cyclophosphamide followed by Paclitaxel/Trastuzumab (AC-TH) 
• 
Doxorubicin/Cyclophosphamide followed by Paclitaxel/Trastuzumab/Pertuzumab (AC-THP) 

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• 
Paclitaxel/Trastuzumab (TH) 
• 
Paclitaxel/Trastuzumab/Pertuzumab (THP) 
• 
Docetaxel/Carboplatin/Trastuzumab (TCbH) 
• 
Docetaxel/Carboplatin/Trastuzumab/Pertuzumab (TCbHP) 
• 
Docetaxel/Cyclophosphamide/Trastuzumab (TCH) 
• 
Docetaxel/Cyclophosphamide/Trastuzumab/Pertuzumab (TCHP) 
 
Exclusion Criteria 
Any of the following are a criterion for exclusion from the study: 
1. Confirmed distant metastasis prior to therapy 
2. Patients with missing pathology or demographic input variables 
3. Patients with no DCE MRI prior to initiation of therapy 
4. Major MRI artifacts as defined by TumorScope including but not limited to poor fat suppression, 
significant coil flare, low signal to noise ratio, incomplete DICOM series and other non-correctable 
registration issues 
5. Use of experimental or novel therapy alongside standard of care 
6. Any patient with identifiable outcome information is shared prior to unblinding 
 
Data Sources 
 
Breast Magnetic Resonance Imaging (MRI) 
Any Breast MRI performed as part of routine clinical management are acceptable for use as an input for 
the study if they were dated prior to the first dose of treatment.  
Specifically, the T1-weighted DCE MRI images will be utilized both as inputs and validation (where 
available) for all subjects included in this study. Pre-Therapy (T0) Breast MRI images for all patients in 
the study will be sent to SimBioSys for quality review prior and then utilized for result predictions. 
 
Upon unblinding and analysis of results, up to 3 additional MRIs will be used for validation of secondary 
endpoints: 
• 
After the Neoadjuvant Treatment 1 Cycle 1 
• 
Before Initiation of Neoadjuvant Treatment 2 Cycle 1 (any drug in regimen is changed, where 
applicable, Inter-Regimen) 
• 
After completion of all Neoadjuvant Treatments but before Surgery (Pre-Surgery) 
 
Changes from the baseline in terms of volume (cc) will be initial segmented by SimBioSys staff and then 
assessed by the investigator for validation. 
 
Tumor Biopsy for Pathological Markers at Diagnosis 
Patient tumor characteristics from the original core needle biopsy as assessed by the local pathologist’s in 
the pathology report will be utilized for the subject inclusion, predictions, and result stratification.  
 
Definitive Surgery Assessment 
Assessment performed by the local pathologist after definitive surgery such as breast conservation 
surgery (BCS) or mastectomy with or without axillary lymph node dissection as part of the local standard 
of care approximately 10-35 weeks will be utilized as ground truth. If the assessment of any subjects is 
uncertain, specifically for the pCR outcome, they will be excluded from the analysis. 
 

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Invasive Disease-free Survival 
Invasive disease-free  survival is defined as the time from registration to any of the following events: 
progression of disease that precludes surgery, local or distant recurrence, second primary malignancy 
(breast or other cancers) or death due to any cause. 
 
 
Publications 
Mayo investigators will be included on any publications relating from this study or any future metanalysis 
performed using the corresponding results. 
 
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