Clinical trial NCT04129216
Conditions: Breast Cancer
Keywords: breast cancer genomic assay hormone therapy
Sponsor: Johns Hopkins University
Start date: 2019-02-20; Completion date: 2022-11-10
Enrollment: 30 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The investigators would like to study the genetic and molecular outcomes that results after a short term neoadjuvant hormonal therapy on patients with breast cancer.

Detailed description:
Breast cancer is among the most common malignancies in women in the United States. Over the years breast cancer management have dramatically developed from the extensive surgical approach toward the breast conservative approach. This was mainly due to the introduction of chemotherapy and hormonal therapy. Hormonal therapy in particular has been shown to improve the oncological outcomes of the breast cancer. However, while this is well documented in the clinical outcomes. Little is known in regards what happens on the genetic level. As such in this study the investigators would like to study the genetic and molecular outcomes that results after a short term neoadjuvant hormonal therapy on patients with breast cancer.

The hypothesis of this study is that short-term, preoperative hormonal treatment will induce genetic changes associated with reduced proliferation, including lower Ki67 expression, and changes in Estrogen Receptor (ER) and Progesterone Receptor (PR) expression. The data from such investigation will be very helpful in advancing the individualized care to women with breast cancer.

Arms: arm 1: for premenopausal patients arm 2: for postmenopausal patients arm 3: for postmenopausal patients
Interventions: intervention 1: Tamoxifen Citrate intervention 2: Letrozole intervention 3: Exemestane intervention 4: Blueprint intervention 5: Mammaprint
Intervention descriptions: intervention 1: 10mg administered daily. Patients take this drug for 2-6 weeks intervention 2: 2.5mg is administered daily. Patients take this drug for 2-6 weeks intervention 3: 25mg is administered daily. Patients take this drug for 2-6 weeks. intervention 4: studies the genomics of the tumor and tumor behavior intervention 5: studies the genomics of the tumor

Protocol documents:
Prot_SAP_000.pdf: 
 
 
JHMIRB eFormA  01 
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Date: 10/18/2020    NCT04129216 
 
 
THE EFFECTS OF SHORT-TERM 
PREOPERATIVE TREATMENT WITH 
HORMONAL THERAPY ON GENE PROFILES 
IN 
BREAST CANCER 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
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Date: 10/18/2020 
Principal Investigator: Mehran Habibi, MD 
Application Number: IRB00130428 
 
 
JHM IRB - eForm A – Protocol 
 
 
 
• Use the section headings to write the JHM IRB eForm A, inserting the appropriate material 
in each. If a section is not applicable, leave heading in and insert N/A. 
• When submitting JHM IRB eForm A (new or revised), enter the date submitted to the field at 
the top of JHM IRB eForm A. 
 
**************************************************************************************
************* 
 
 
1. 
Abstract 
• Provide no more than a one page research abstract briefly stating the problem, the 
research hypothesis, and the importance of the research. 
 
Breast cancer is among the most common malignancies in women in the United States. Over 
the years breast cancer management have dramatically developed from the extensive surgical 
approach toward the breast conservative approach. This was mainly due to the introduction of 
chemotherapy and hormonal therapy. Hormonal therapy in particular has been shown to 
improve the oncological outcomes of the breast cancer. However, while this is well 
documented in the clinical outcomes. Little is known in regards what happens on the genetic 
level. As such in this study we would like to study the genetic, molecular and functional 
outcomes that results after a short term neoadjuvant hormonal therapy on patients with breast 
cancer. 
The hypothesize of this study is that short-term, preoperative hormonal treatment will induce 
genetic changes associated with reduced proliferation, including lower Ki67 expression, and 
changes in ER and PR expression as well as reduced ER pathway activity. The data from such 
investigation will be very helpful in advancing the individualized care to women with breast 
cancer. 
 
 
             Hypothesis   
• We hypothesize that short-term, preoperative hormonal treatment will induce genetic changes 
associated with reduced proliferation, including lower Ki67 expression, and changes in ER and 
PR expression. as well as changes in ER pathway activity measures 
 
2. 
Objectives  
 
The purpose of this study is to evaluate the effects of short-term preoperative treatment with 
hormonal therapy (tamoxifen, letrozole, and exemestane, administered according to standard of 
care) on gene expression profiles in patients with breast cancer. As such: 
A. Primary objective is to determine whether there is a reduction in the expression of 
proliferation marker Ki67, measured by IHC as well as single-gene read out, following 
short-term preoperative treatment with hormonal therapy.  


 
 
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Secondary objectives are to determine whether short term treatment results in significant 
reductions in the Mammaprint risk score, or in ER or PR expression as measured by IHC and single-gene 
read out or a change in ER pathway activity.C.   Exploratory objectives:  
a. Identify differentially expressed transcripts after short-term pre-operative,    hormone 
treatment 
b. Identify transcripts for which gene expression is associate with measures of response, 
including changes in Ki67, ER and PR.  
c. Perform full-transcriptome set analysis to identify pathway level changes associated 
with treatment. 
d. Comparison of expression changes between two standard of care molecular  
diagnostics: 70-GS and 21-GA 
e. Perform OncoSignal pathway activity analysis on pre and post treatment samples to 
related pathway activity to patient response 
 
The participants of this study will be clinical breast cancer patients of the Principal Investigator 
(PI) or those referred by community physicians to the PI. The breast cancer patients will be 
evaluated by the PI or a study team member to assess if they meet inclusion/ exclusion criteria 
for this study. If the patient meets all the criteria, the consent process will take place. 
Participants will include 30 breast cancer patients. These patients will be recruited from Johns 
Hopkins University cancer treatment centers. 
This is a clinical trial with three treatment groups including tamoxifen, letrozole, and 
exemestane.Ten  pre-menopausal patients and men will be in their own group and will 
recievetamoxifen. Twenty post-menopausal patients will in a separate groupand will recieve 
letrozole or exemestane. Treatment selection will be at the discretion of the medical oncologist. 
Ten patients will be allocated to each treatment group, for a total of 30 patients 
Genomic data will be collected as a part of the standard of care 70 gene signature (70GS; trade 
name MammaPrint), which is read from the comprehensive-genome microarray at Agendia 
(Irvine, CA).   The 70GS will be performed on the baseline biopsy specimen as SOC, and 
experimentally on the surgical specimen collected after short-course endocrine treatment. We 
will then compare the genomic profiling between the two samples to measure changes in gene 
expression before and after treatment. Special care will be taken to verify with pathology that 
the baseline biopsy is large enough to run the commercially available 21 gene assay (21GA; 
trade name Oncotype) on, as this assay is also currently considered standard of care. For the 
OncoSignal test about 0.25mm3 FFPE tissue is needed from the same biopsy or surgical 
material. The tumor content should be at least 50%. If on a slide of 5 µm thickness about 25-50 
mm2 tissue is available, only 2 slides are required. 
 
 
3. 
Background   
 


 
 
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Breast cancer is the most common malignancy and the second leading cause of cancer deaths 
among women [1]. As a heterogeneous and phenotypically diverse disease, breast cancer may 
necessitate divergent treatment strategies, among which cytotoxic chemotherapy remains an integral 
part of therapy at any given stage. However, cytotoxic therapy is not considered a “targeted 
therapy”, because it does not act on cellular pathways involved in cell growth and progression [1]. 
The real targeted therapy would be based on the identification of a molecular growth pathway and 
on the modification of its activity in order to block the growth and progression of cancer. With 
regard to breast cancer, the endocrine manipulation is considered as a targeted therapy [2]. The role 
of estrogen on tumor cell growth is well established in breast cancer. Identification of the estrogen 
receptors (ER) provided the first target for anti-estrogenic therapeutic agents [1-3]. Tamoxifen, the 
prototype SERM (Selective Estrogen Receptor Modulator), has been the gold standard treatment for 
early and advanced disease and its activity on ER is considered the first form of molecular targeted 
therapy [1, 4]. Aromatase inhibitors (e.g., anastrozole, letrozole, or exemestane) which directly 
inhibit the production of estrogen, have shown superiority over tamoxifen in adjuvant and 
metastatic setting [5-8], also they appear to fare better than tamoxifen in the neoadjuvant setting [8], 
where therapeutic agents are administered before the main treatment (e.g. in the preoperative 
phase).  Following the success of adjuvant endocrine treatments [9-11], more studies have been 
emerging with the use of endocrine agents in the neoadjuvant setting (PO24 trial [12], IMPACT 
trial [13] and PROACT trial [14]). The objective of endocrine therapy in the neoadjuvant setting is 
primarily to produce sufficient response in the tumor to allow mastectomy in inoperable tumors and 
potentially breast conservation in patients initially proposed to undergo mastectomy [8]. Treatment 
response to these “targeted therapies” differs vastly across different populations; even patients with 
the same stages of disease can have markedly different responses and overall outcome [10]. 
Therefore, an ideal therapy should be tailored based on individual characteristics so that each 
patient receives a treatment with an appropriate response and the least adverse effects. Genetic 
breakthroughs in the last few decades have provided us with a unique opportunity to address this 
challenge. The emergence of genomic techniques (e.g. complementary DNA microarrays) and their 
ability to simultaneously measure the expression of thousands of genes have led to the identification 
of biology-based prognostic profiles [15-18], several of which have been validated and are in 
clinical use. The development of prognostic profiles has been particularly useful in guiding 
decision-making about adjuvant therapy along with well-established clinical prognostic factors (e.g. 
patient age, comorbidity, tumor size, grade, and nodal status) [19]. These molecular prognostic 
profiles can augment but do not replace classic clinical factors. They offer the potential for use in 
clinical practice for prognostic stratification and treatment selection for patients with breast cancer, 
particularly if they are hormone receptor-positive.  Among the available prognostic profiles, the 
Amsterdam 70-gene prognostic profile (Mammaprint) was one of the first gene expression array-
based marketed for prognostic purposes [20]. The test was developed using a supervised DNA 
microarray analysis of gene expression arrays on frozen tissue from primary breast tumors. A 
mathematical model is used to calculate a score that stratifies patients as having a breast cancer with 
an associated poor-prognosis (High risk: 29% risk of metastasis after 10 years, without adjuvant 
treatment) or good-prognosis (Low risk: 10% risk of metastasis after 10 years without adjuvant 
treatment) [21]. Validation of the Mammaprint gene expression profile demonstrated that the profile 
is a powerful prognostic factor to predict outcome in lymph node-negative and lymph node-positive 
breast cancer. Recent reports, that the most important of them is MINDACT trial [22, 23], confirm 
this predictive value for Mammaprint signature. Although it was originally approved for use with 
unfixed, frozen tissue, it has now been adapted for use with formalin-fixed, paraffin-embedded 
tissue. Neoadjuvant endocrine therapy has been increasingly employed in clinical practice to 
improve the disease outcome [6-8], but little is known of the molecular effects of these agents. 
Particularly, the genetic impact of endocrine therapy directly on breast cancer remains largely 
unexplored. Most studies that examined the anti-estrogen treatment of ER positive breast cancer, 
have measured changes in clinical, pathological, proliferative or surgical outcomes [7, 25, 26] and 


 
 
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one study investigated the effect of neoadjuvant treatment on ER pathway activity (34; Inda et al; 
Mol Can Ther 2020 19 680-9) To our knowledge, there are few studies that have shown molecular 
effects as the early (10-14 days) or late (3-4 months) changes in gene profile after preoperative anti-
estrogen treatment of ER-positive breast cancer [ 26, 27]. Most reports of the transcriptional 
profiling of estrogen responses in breast cancer cell lines are results of in vitro [28-31] or animal 
models [32]. Thus, identification of in vivo responses needs to be more elaborated. On the other 
hand, these studies generally examined one type of neoadjuvent therapy, therefore a comparative 
study on this subject can be very useful. Preoperative endocrine therapy in neoadjuvant settings 
revealed to be ideal for this purpose because it allows the direct assessment of response to 
treatment, and tumor is readily available for multiple time point biopsies [7]. 
 
The FLEX Registry (NCT03053193) is a large-scale, population based, prospective registry. All 
patients with stage I to III breast cancer who receive MammaPrint testing on a primary breast tumor 
are eligible for entry into the FLEX Registry, which is intended to enable additional substudies at 
low incremental cost. This study will utilize FLEX’s adaptive study infrastructure to capture clinical 
data.  
 
 
 
4. 
Study Procedures 
 
a. Study design, including the sequence and timing of study procedures   
(distinguish research procedures from those that are part of routine care). 
 
The breast cancer patients will be evaluated by the PI or a study team member to assess if they 
meet inclusion/ exclusion criteria for this study. If the patient meets all the criteria, the consent 
process will take place. This protocol is designed to be a companion substudy of FLEX 
Registry, which prospectively observes patients receiving MammaPrint as a part of their care, 
and data collection will occur within FLEX’s eCRF infrastructure. All patients consenting to 
screening for this study will be co-enrolled in FLEX, with appropriate dual informed consents 
obtained to participate in both studies. 
Participants will include 30 breast cancer patients. These patients will be recruited from Johns 
Hopkins University cancer treatment centers. 
This is a clinical trial with three treatment groups including tamoxifen, letrozole, and 
exemestane. Ten pre-menopausal patients and men will be in their own group and will receive 
tamoxifen. Twenty post-menopausal patients will in a separate group and will receive either 
letrozole or exemestane. Treatment selection will be at the discretion of the medical oncologist. 
Ten patients will be allocated to each treatment group, for a total of 30 patients.Full genome 
Mammaprint will be run on the biopsy specimen and the final surgical specimen. We will then 
compare the gene profiling between the two results. OncoSignal pathway analysis will make use 
of the same biopsy specimen for evaluating the ER pathway activity scores.  
The Oncosignal results will not be shared with patients(subjects), treating physicians; nor will 
be used for patients’ management; It only will be used for research purposes. 
In addition, some clinical data will be collected from the patient’s medical records, 
operative/pathology reports, and clinical notes.  This information will include information such 
as details of the cancer diagnosis, details of the surgery and further treatment of the disease.  


 
 
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Study design is illustrated in Figure 1.  
Figure 1. Study Scheme 
 
Figure 1. Study Scheme 
Assays and Tests used in the study: 
 
Mammaprint: This a strong diagnostic tool that will be used in the study as Breast Cancer Assay 
and will provide invaluable information regarding the genomics of the patient and their tissue sample. We 
will be running the test twice to ascertain the differences between the samples due to a short term hormonal 
therapy course.  
• OncoSignal: This is a test that measures biologically active pathway activities of 4 different 
pathways (ER, AR, PI3K and MAPK). In this study especially the ER-pathway activity is 


 
 
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relevant since ER staining positive tumors are selected with an intend for hormonal treatment. 
From previous studies it is known that, although tumors can be ER positive in staining, the 
pathway is not always equally active (34). High ER pathway activity on the primary biopsy 
leads to a better response to hormonal therapy as compared to samples with a low ER activity 
on the primary biopsy, although all are ER staining positive. Also the change in pathway 
activity is higher in patients that respond to hormonal therapy as compared to patients that do 
not, or suboptimally respond, to hormonal treatment. The test requires 0.25mm3 FFPE tissue 
with a tumor percentage of at least 50%. In principle the same biopsy material can be used as 
collected for mammaprint but the processing of the FFPE samples is different 
 
Study Drug Information(Dosage, Administration Frequency, Duration): 
 
Tamoxifen: 20mg is administered daily. Patients take this drug for 2-6 weeks. 
 
Letrozole: 2.5mg is administered daily. Patients take this drug for 2-6 weeks 
Exemestane: 25mg is administered daily. Patients take this drug for 2-6 weeks. 
 
b. Study duration and number of study visits required of research participants. 
 
We anticipate this study to be done in 2 years. Following consent, and prescription of hormonal 
therapy, patients will have only one study visit prior to surgery, to take a baseline core research 
biopsy. The surgical sample will be used for post-treatment analysis 
 
c. Blinding, including justification for blinding or not blinding the trial, if applicable. 
d.  
This is a single arm trial and so will not be blinded. 
 
 
 
e. Justification of why participants will not receive routine care or will have current therapy 
stopped. 
 
Participants will receive routine care. Normally, patients are scheduled for surgery after 
radiologic and other workups are completed. After surgery, they will receive hormonal therapy. 
In our study, hormonal therapy will commence after patients are recruited to the study and 
informed consent is signed, prior to surgery. Hormone therapy will continue after surgery.  
 
 
f. Justification for inclusion of a placebo or non-treatment group. 
 
There is no placebo or non-treatment group. 
 
g. Definition of treatment failure or participant removal criteria. 
 
Treatment failure happens if the patient doesn’t comply with the hormonal therapy treatment 
plant. At such cases, the patient will be removed from the study. If any patient miss more than 
two doses of the medication they will be removed from the study. 


 
 
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Because the duration of the study is short, and the medications are well tolerated, we expect 
very  few non-compliant patients. We will add additional patients to the study if we lose any due 
to this clause. 
 
 
h. Description of what happens to participants receiving therapy when study ends or if a 
participant’s participation in the study ends prematurely. 
 
This is expected to be a rare event as patients are only followed for 2-6 weeks, until surgery, and 
the hormonal therapy administered in this study is well studied and carries minimal risk of long 
term effects or consequences. In such cases, patients will continue their standard of care plan as 
advices by their care providers.  
 
 
5. 
Inclusion/Exclusion Criteria  
 
A. Inclusion criteria: 
• Treatment-naïve, histologically confirmed invasive ductal breast cancer between stages 1 to 3. 
• Co-enrollment in the FLEX Registry 
• ER+ PR+ confirmed hormone receptor status measured by immunohistochemistry(IHC) 
• Patients should understand their condition and be able to give informed consent to participate 
 
 
B. Exclusion Criteria: 
• History of  hormonal therapy, chemotherapy, radiation therapy, or novel therapy to treat the 
current breast cancer.  
• Allergic reactions/hypersensitivity to tamoxifen, letrozole, or exemestane or any of their 
ingredients. 
• Any contraindication to hormonal therapy,  such as history of thromboembolic disease or 
uterine cancer. 
• Patients without invasive disease (stage 0) 
• Patients with metastatic breast cancer(stageIV) 
• Patients that are HER2+ by IHC/FISH. 
 
Drugs/ Substances/ Devices 
a. The rationale for choosing the drug and dose or for choosing the device to be used. 
 
The drugs in this study are FDA approved mediations and drugs of choice for hormone therapy 
according to NCCN (National Comprehensive Cancer Network) guidelines. We will use the 
standard recommended daily doses of the drugs. 
MammaPrint has 510k FDA clearance as a device and is a cost-effective vehicle for evaluating 
the genomic objectives of this study. The patient population considered for enrollment in this 
trial are candidates for receiving MammaPrint as a part of their standard of care. 
 
The rationale for using the Oncosignal in addition to Mammaprint is that Oncosignal shows 
differences in pre treatment ER pathway activity scores between patients, although all patients 
are ER staining positive this does not always relate to an active pathway. Mammaprint test can 


 
 
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show the gene level changes and oncosignal can show the whole pathway activity before and 
after treatment 
 
 
 
 
 
 
b. Justification and safety information if FDA approved drugs will be administered for non-
FDA approved indications or if doses or routes of administration or participant 
populations are changed. 
 
The drugs that will be used are FDA approved to be administered for an FDA approved 
indication. 
 
c. Justification and safety information if non-FDA approved drugs without an IND will be 
administered. 
 
No non-FDA approved drugs without an IND will be administered 
Study Statistics  
a. Primary outcome variable: The primary outcome variable is Ki67 expression, a measure of 
tumor proliferation 
a. Secondary outcome variables: Secondary outcomes include ER and PR expression and 
pathway activity patterns pre and post treatment  
b.  
c. Statistical plan including sample size justification and interim data analysis.           
 
Primary Objective: Change in Ki67 expression after treatment will be calculated by subtracting 
%Ki67 expression at time of surgery from the baseline level. We will then fit a linear model to 
determine whether short-term treatment significantly reduces expression, while adjusting for choice 
of treatment, ER and PR level, and other possible cofounders.   In a secondary analysis, we will 
evaluate individual treatment groups separately. 
 
Sample size justification:  Power calculations are based on the use of a one-sided, one-sample t-test 
to evaluate a decrease in Ki67. The proposed sample size of 30 subjects offers at least 90% power at 
an alpha of 0.05, to detect a decrease greater than 0.55s.d.  For the analysis of individual treatment, 
groups of 10 offer at least 90% power to detect decreases greater than 1s.d.  
       
Secondary objectives 
Determine whether short term treatment results in significant reductions in Mammaprint risk score, 
ER or PR expression as measured by IHC. 
 
Change in risk score ER and PR expression will be evaluated as described above for Ki67, using 
linear regression to model the change in each variable while adjusting for ki67 in addition to the 
covariates described above. Expected power is the same as for the primary analysis. 
 
 
Exploratory analyses 
Identify differentially expressed transcripts after short-term pre-operative, hormone treatment 


 
 
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Identify transcripts for which gene expression is associate with measures of response, including 
changes in Ki67, ER and PR expression.  
We will fit empirical Bayes linear models [32] to identify genes for which expression is associated 
with response to treatment, controlling type I error at an FDR of <10%. Wilcoxon Rank Sum tests 
will be used to perform Gene set enrichment analysis on the results to identify pathways that are 
significantly altered after short-term treatment. [33] 
 
 Show differences in pre treatment ER pathway activity scores between patients, although all 
patients are ER staining positive this does not always relate to an active pathway. Show differences 
in ER pathway activity scores between pre and post treatment samples, indicating a measurable 
biological effect of the treatment. 
 
 
 
d. Early stopping rules.  
 
There are no early stopping rules.  
 
 
6. 
Risks 
 
a. Medical risks, listing all procedures, their major and minor risks and expected frequency. 
 
Important drug adverse effects like increased risk of thromboembolism would be considered a  
major risk. The other non-specific side effects may be considered as minor risk. Since the drugs 
are administered over a short period, it is highly unlikely to develop an adverse event that 
requires chronic use of the drug. 
 
The Oncosignal test has minimal risks: 
 
It is noninvasive, does not require an invasive sampling that presents significant risk, does not 
introduce energy into a subject and is not used as a diagnostic procedure without confirmation 
of the diagnosis by another medically established diagnostic product or test 
The Oncosignal results will not be shared with patients(subjects), treating physicians; nor will 
be used for patients’ management; It only will be used for research purposes. 
 
 
b. Steps taken to minimize the risks. 
 
Known adverse effects of tamoxifen and anastrozole will be recorded using a checklist every 
week by the research assistant. Medical monitoring will be made by both the principle 
investigator and the Institutional Data and Safety Monitoring Board. Patients are informed about 
all common and important adverse effects and they are advised to report as soon as they notice 
them. They are also advised to take some preventive measures for example for the prevention of 


 
 
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deep vein thrombosis. In any circumstances, all adverse effects are taken care of by the research 
team immediately and effectively. 
Participants are provided with the investigators contact information and a list of frequently 
asked questions for any concerns they might have. 
 
 
c. Plan for reporting unanticipated problems or study deviations. 
 
Any unanticipated problem or study deviation will be recorded and reported per IRB guidelines. 
 
d. Legal risks such as the risks that would be associated with breach of confidentiality. 
 
There is a minimal risk of losing confidential data. However, all measures advised by IRB to 
minimize this risk will be done. Best practices will be followed, including the assignment of a 
study ID and the de-identification of PHI from study data. 
 
 
e. Financial risks to the participants. 
 
N/A 
 
7. 
Benefits 
a. 
Description of the probable benefits for the participant and for society. 
 
You will not directly benefit from being in this study. If you take part in this study, you may help others in 
the future. 
8. 
Payment and Remuneration 
a. 
Detail compensation for participants including possible total compensation, proposed 
bonus, and any proposed reductions or penalties for not completing the protocol. 
 
Participants will not receive compensation. 
 
9. 
Costs 
a. 
Detail costs of study procedure(s) or drug (s) or substance(s) to participants and 
identify who will pay for them. 
 
Costs of the study will be paid for using the grant funding this study. 
 
 
References: 
 
1. Orlando L, Schiavone P, Fedele P, Calvani N, Nacci A, Rizzo P, et al. Molecularly targeted 
endocrine therapies for breast cancer. Cancer treatment reviews. 2010;36 Suppl 3:S67-71. Epub 
2010/12/07. 
2. Miller WR, Larionov A. Molecular effects of oestrogen deprivation in breast cancer. Molecular and 
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3. Osborne CK, Schiff R. Estrogen-receptor biology: continuing progress and therapeutic implications. 
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4. Coates AS, Keshaviah A, Thurlimann B, Mouridsen H, Mauriac L, Forbes JF, et al. Five years of 
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