Automated Method for Breast Cancer Detection
Phase: NA
Conditions: Breast Cancer Diagnosis
Sponsor: Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins

 
 
 
J18138; IRB00172441; NCT03863522  
Automated Method for Breast Cancer Detection   
  
  
  
Principal Investigator:   
  
  
Susan Harvey, M.D   
  
Co-Investigators:  
  
  
  
Christopher Vandenbussche, M.D, Cytopathology  
Ashley Cimino-Mathews. M.D, Surgical Pathology  
Syed Ali, M.D., Cytopathology  
Leslie Cope, Ph.D., Biostatistician  
Saraswati Sukumar, Ph. D, Laboratory PI*  
Bradley Downs, Ph. D, Postdoctoral Fellow  
Mary Jo Fackler, Ph. D, Research Associate*  
Christopher Umbricht, MD, Ph.D-Laboratory Co-I  
Antonio Wolff, MD- Medical Oncologist* 
Kala Visvanathan, M.B.B.S., M.H.S.  
  
(*) denotes individuals who have a conflict of interest 
which has been properly documented by JHM-IRB.   
The non-conflict designee is Dr. Christopher Umbricht.    
  
Clinical Coordinators:   
  
  
Ar’Lena Smith, M.S., CCRP  asmit227@jhmi.edu    
  
  
  
  
  
  
Mary Kate Jones  mjone242@jhmi.edu   
  
  
Version:  
November 7, 2018  
  
  
  
  
  
 
  
  
  
  

   
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TABLE OF CONTENTS  
  
1.  BACKGROUND, HYPOTHESIS, RATIONALE AND OBJECTIVES ................................. 3 
1.1 BACKGROUND ................................................................................................................................ 3 
1.2  HYPOTHESIS .................................................................................................................................. 4 
1.3 RATIONALE AND PRELIMINARY DATA ............................................................................................ 4 
1.4 OBJECTIVES ................................................................................................................................... 6 
2.  SELECTION OF PATIENTS ................................................................................................... 6 
2.1 ELIGIBILITY CRITERIA .................................................................................................................... 6 
2.2 INELIGIBILITY CRITERIA ................................................................................................................. 7 
2.3 SUBJECT RECRUITMENT ................................................................................................................. 7 
3.  PATIENT REGISTRATION .................................................................................................... 7 
4.  STUDY CALENDAR ................................................................................................................ 7 
5.  STUDY PARAMETERS ........................................................................................................... 8 
5.1 FNA PROCEDURE: FOR SUSPICIOUS LESIONS .................................................................................. 8 
5.2 CYTOMORPHOLOGY ....................................................................................................................... 8 
5.3 QUANTITATIVE AND MULTIPLEXED METHYLATION........................................................................ 9 
5.4 STRAT4 SUBTYPING ASSAY ........................................................................................................... 9 
5.5 ADDITIONAL ANALYSES ................................................................................................................. 9 
5.6 STUDY DESIGN ............................................................................................................................... 9 
6.  STATISTICAL CONSIDERATIONS .................................................................................... 10 
6.1  PRIMARY OBJECTIVES ................................................................................................................. 10 
6.2 SECONDARY OBJECTIVES ............................................................................................................. 11 
7.  ADVERSE EVENT REPORTING ......................................................................................... 12 
8.  RECORDS TO BE KEPT ....................................................................................................... 12 
9.  PATHOLOGY REQUIREMENTS ........................................................................................ 13 
10.  DATA AND SAFETY MONITORING PLAN ..................................................................... 13 
REFERENCES ............................................................................................................................ 14 
 
  
  
  
  
  
  
  
  
  
  
  
  
  

   
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Automated Method for Breast Cancer Detection  
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1. Background, Hypothesis, Rationale and Objectives  
  
1.1  
Background  
  
1.1.1. The Problem   
  
In underdeveloped and developing countries of the world, breast cancer is the most 
frequently diagnosed cancer and the leading cause of cancer death. in 2008, 691,000 new cases were 
detected and 269, 000 women succumbed to the disease [1]. Breast cancer incidence is rising in 
underdeveloped world because of longer life expectancies, decreased burden of infectious diseases, 
and changes in reproductive risk factors [2, 3]. patients with breast cancer in the underdeveloped 
world experience longer diagnostic delays than patients in developed countries, leading to later-stage 
presentations [4].  However, optimal early detection strategies are not well-characterized in settings 
where population-based mammography screening is not yet available, primary care services are 
limited, and pathology and treatment services are available only at regional hospitals [5]. In many of 
these settings, health care systems to address breast problems and efficiently refer patients with 
symptoms concerning for cancer are in their infancy [6]. currently, there are delays for up to 10 
months in certain areas due to the lack of easy access to a clinical pathology laboratory or a 
pathologist [1, 7].  
  
In remote areas with highly overworked pathologists, a new, inexpensive, accurate and rapid 
test for use in screening clinics to detect malignancies would greatly assist in prioritizing those 
patients for quick and detailed evaluation at the regional hospital [8, 9]. Currently, there are no tests 
for accurate and quick determination of an abnormal lesion in the breast as benign or malignant in 
the underdeveloped world.  We have shown that DNA methylation that occurs specifically in breast 
cancer can serve a powerful marker for early detection of breast cancer in body fluids such as nipple 
aspiration, ductal lavage and core biopsy.   
  
 
1.1.2  DNA methylation   
  
DNA methylation is a molecular modification of DNA that is tightly associated with loss of 
gene expression [10]. The Sukumar lab has performed extensive work to derive methylated gene 
marker panels [11-17]which are specific to invasive ductal and lobular cancers. However, little work 
has been spent studying the methylation status of mammographically suspicious, biopsy proven 
benign lesions, and to select sensitive and specific markers that distinguish between malignant and 
benign lesions. Euhus and group have performed the most extensive analysis [18, 19] of this 
question. In one report [20] they studied DNA methylation of cyclin D2, APC, HIN1, RASSF1A, 

   
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and RAR-β in 290 benign and malignant breast epithelial cell samples obtained from palpable 
lesions by fine-needle aspiration (FNA) biopsy from 164 women. They concluded that tumor 
suppressor gene methylation increases in benign breast epithelium of high risk women. But could not 
identify any marker that clearly distinguished between benign and malignant lesions [20]. Markers 
that accurately distinguish between benign and malignant breast cancer are not available, and needed 
to be developed.   
  
  
  
1.1.3  DNA Methylation Marker Selection and a method for detecting methylated 
genes  
  
Towards the goal of developing markers that are specific to malignant breast cancer, 
Sukumar and co-workers identified methylation markers that are frequently methylated in breast 
cancer by performing large-scale methylation microarrays containing probes for 27K and 450K 
methylated regions (Illumina) in the genome. They also developed a sensitive and specific 
Quantitative Multiplex Methylation-Specific PCR (QM-MSP) technique [11, 15-17]. This method 
involves sodium bisulfite treatment of DNA, followed by a two-step reaction: multiplex PCR that 
amplifies both the methylated (M) and unmethylated (U) alleles of up to 14 genes in one reaction, 
followed by Q-PCR using primers and dual fluorophore labeled probes that amplify both the M and 
U alleles for each gene in a single well (22). The major advantages of this technique are: a wide 
dynamic range, its ability to detect a few M alleles in the midst of a vast excess of U alleles, and 
feasibility in small epithelial samples such as NAF, core biopsy and rFNA.   
  
1.2   
Hypothesis  
  
Many underdeveloped areas of the world lack both the equipment and trained personnel 
required by standard diagnostic methods like mammography. We hypothesized that well-chosen 
methylation markers, incorporated into an inexpensive, automated molecular test that could be 
applied in conjunction with ultrasound at the point of care, even in low resource areas could achieve 
sufficient accuracy to prioritize patients with suspected malignancy for quick and detailed evaluation 
at the regional hospital.    
  
1.3  
Rationale and Preliminary Data   
  
Assaying hypermethylated genes in fine needle aspirates of suspicious breast lesions that 
have been detected by ultrasound could help rapidly identify malignant tumors, where the 
methylation marker test is predicted to be positive. On the other hand, the test will score negative for 
the methylation markers if lesion is benign since the lesion would contain low or no detectable levels 
of methylation (9),(6;7).  In the last two years, using QM-MSP, we analyzed training and test sets of 
primary tumors from USA, China and Africa and developed an optimal methylated gene marker 
panel whose performance is at, or exceeding 90% sensitivity and specificity.    
  

   
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1.3.1  The Cepheid cartridge  
  
CEPHEID is a diagnostics company in California that has developed cartridges for the 
molecular detection of a large number of infectious diseases bacteria and viruses, including Anthrax 
and Ebola, with FDA approval for a large number of them.  The cartridge is able to perform sample 
extraction, and PCR-based detection of the desired analyte- RNA, DNA or cDNA. The dye detection 
limit is less than 1 nM for most analytes.   
  
The GeneXpert® System is available in a one, two, four, or 16-module configuration. All use 
the same GeneXpert analysis module, and the same patented cartridge technology. The GeneXpert® 
System returns most test results in about an hour, including sample preparation. The systems deliver 
results even faster than many alternative technologies, such as EIA or Immunoassay. The cartridges 
are single use, and detect up to 6 genes in the same cartridge through use of multiple calibrated 
fluorphor dyes with distinct excitation ranges.  Thus, we will use them for detection of multiple 
methylated genes from the same section of the core biopsy or FNA collection from the breast.   
  
 
  
1.3.2 Optimization of the Cepheid cartridge for breast cancer detection  
  
Next we optimized the conditions for analysis to achieve the lower limits of detection of 
methylated genes in the CEPHEID GeneXpert® cartridge. Further, we completed analysis of fine 
needle aspirates and touch preparations of xenografts of human breast cancer cells and of fine needle 
aspirates performed in surgically resected primary breast cancers very successfully in the cartridge. 
The cartridge is loaded with sodium bisulfite treated DNA, and performs the steps of QM-MSP 
within 3 hours, in contrast to the one week’s meticulous work by an expert technician.  
  
1.3.3 Determination of receptor status  
  
In addition to determining the methylation status of the tumor, sections or tissue lysate of the 
same lesion can be used in a separate cartridge on the GeneXpert® subtyping device to determine 
estrogen receptor (ER), progesterone receptor (PR), and HER2 status of the tissue which are key 
pathologic features responsible for determining treatment options.  Thus, the assay can be 
successfully employed in underserved areas of the world in the future. This assay is completed in 1.5 
hr.  We tested FNAs of xenografts and successfully determined the status of the four markers in this 
cartridge for several breast cancer cell lines.   
  
Figure 1:    
Left: GeneXpert cartridge.    
Right: A two cartridge module- GX11-2-L  
of the GeneExpert Diagnostic System with  
laptop is shown. An 8- and 16-module  
machine is currently installed in the  
Sukumar Lab.  
2.5 ” 

   
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Summary: Identifying the optimal set of 10 methylated gene markers suitable for use in the US, 
China and Africa has been completed.  Preliminary analysis of genes in the cartridge to determine 
their performance on FNAs was successful.  With our help, CEPHEID has designed two cartridges 
to analyze a total of 10 genes by a quantitative multiplexed methylation specific PCR that uses actin 
as the internal housekeeping gene control.  Preliminary analysis of FNA in the tumor subtyping has 
been partially successful.  While the assay works with high level of accuracy using FFPE sections, 
conditions for optimal detection of the subtyping markers in FNA needs further work.  
  
1.4  
Objectives  
  
1.4.1 Primary objectives:   
1) To determine if DNA methylation profile of 10 genes in the automated GeneXpert Cancer 
Detection cartridge correlates with diagnosis based on the gold standard of histopathology of 
the core biopsy or resected sample.  
2) To determine if DNA methylation profile of 10 genes as determined in the automated 
GeneXpert Cancer Detection cartridge correlates with FNA cytology.  
  
1.4.2 Secondary objectives:  
  
1) To compare the results of gene methylation based prediction of the FNAs as malignant or 
benign by the GeneXpert Cancer Detection cartridge to our known laboratory assay, 
quantitative multiplex-methylation specific PCR (QM-MSP).   
2) To compare QM-MSP results of FNA with QM-MSP results of section of the biopsy or 
resected tissue.  
3) To determine if the expression of ER/PR/Her2 and Ki67 in FNAs of suspicious, methylation 
positive breast lesions correlates with expression of ER/PR/Her2 and Ki67 in FFPE sections 
of tumors.  
  
  
2. Selection of Patients  
  
2.1  
Eligibility Criteria   
For this study, we require a total of 165 cases (women with invasive cancer) and 165 controls 
(women with benign breast disease), 18 years or older who have been recommended for ultrasound 
guided core needle biopsy of a suspicious breast lesion. Based on estimates that 40-50% of 
suspicious breast lesions will be malignant, we expect to enroll between 382 and 447 total subjects to 
provide the necessary cases and controls.  Accordingly, after enrolling 382 subjects we will calculate 
rates of malignant and benign disease, and estimate total enrollment requirements, and continue 
enrollment as needed. See statistical considerations for additional details.  
  

   
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Automated Method for Breast Cancer Detection  
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2.2  
Ineligibility Criteria  
Women may be excluded for any condition that in the opinion of the investigator may not 
make it safe to take part (e.g. comorbidity where stopping a concomitant medication is not in the best 
interest of the patient).  
  
2.3  
Subject Recruitment  
Subjects will be recruited through the Johns Hopkins Imaging Center site at Green Spring. 
Women will be offered participation if they are recommended to have a breast core needle biopsy of 
a suspicious lesion.   
  
  
3.  Patient Registration   
  
• 
Patients will undergo an informed consent process and subject registration number will 
be assigned.  
• 
Registered patients will be tracked by patient log.  This log will be submitted to the CRO 
on a periodic basis (quarterly, at minimum) until study closure and will include:  
o Subject name (or initials) o Registration number  o Date of registration  
o Date of birth  
o Race  
  
  
4. Study Calendar  
  
  
Baseline  
Day of 
Procedure  
Follow-Up 1(1)  
Follow-Up 2(2)  
Informed Consent  
X  
  
  
  
Eligibility Confirmed  
X  
  
  
  
Patient registration  
X  
  
  
  
PROCEDURES:  
  
  
  
  
- Fine Needle Aspiration (FNA)  
  
X  
  
  
- Core needle biopsy of suspicious lesion  
  
X  
  
  
OTHER ASSESSMENTS:  
  
  
  
  
Adverse Events call  
  
  
X  
X  
  
1: 1-2 days after procedure  
2: 10-14 days after procedure  
  
  

   
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5. Study Parameters  
  
5.1  
FNA Procedure: For Suspicious lesions  
  
Women ages 18 or older who have been recommended for ultrasound guided core biopsy for 
a suspicious breast lesion at the Green Spring Johns Hopkins Imaging site will be recruited into the 
study.  
  
The FNA will be performed on one ultrasound visible lesion per patient, using the method 
described by Fabian et. al. [21, 22] which is standard at Johns Hopkins and used the by the breast 
imagers. When recommended for the core biopsy, participants are asked to abstain from aspirin and 
other medications/supplements related to blood clotting and platelet function outlined in the 
eligibility criteria for a week prior to the procedure. For the procedure, 1% lidocaine local anesthesia 
is delivered at the skin (1 to 5 cc) followed by deeper infiltration (about 5 to 10 cc) of 1% lidocaine 
with or without 1:100,000 epinephrine at the site of each lesion; this will also be used for the core 
needle biopsy, which will immediately follow the FNA procedure. When more than one eligible 
lesion is visible in the same patient, one random lesion will be selected.   
  
NOTE: Buffered lidocaine may be used per investigator preference/institutional standard. Next, the  
FNA procedure itself is performed by sampling the lesion with a 22 to 25 gauge needle attached to a 
5 ml syringe with 2-3 passes through each lesion using ultrasound guidance thus confirming accurate 
targeting. Additional lidocaine may be given if needed for discomfort. The core needle biopsy using 
standard of care procedures will then occur.   
  
The standard post biopsy instructions, both verbal and written will be provided to all patients. 
An ice pack is applied to the breast(s) following the procedure, and the subjects are asked to wear a 
firm sports bra (alternatively, compression wraps or firm bandages may be used).   
  
In our present study, where 1 FNA procedure consists of 2 to 3 passes of the needle into the 
suspicious lesion, the mean pain rating is 1-1.5 (on a scale of 10); this is consistent with publications 
from the University of Kansas and the Johns Hopkins study [21, 22] .  We may also use a buffered 
1% lidocaine so as to minimize the burning sensation associated with unbuffered lidocaine.  With 
our present technique, we are experiencing an average epithelial cell yield of 10,000 cells, and about 
10% of samples contain less than 5,000 cells. DNA methylation studies can be performed with as 
low as 300 cells.  
  
5.2  
Cytomorphology   
  
  
A slide for cytomorphology, stained with Quik Dip (Mercedes Medical) will be prepared by 
the Sukumar Lab to be read by Dr. Vandenbussche. Results of the cytopathology review will not be 
given to subjects enrolled in the study; subjects will obtain results from the core needle biopsy 
recommended for definitive diagnosis.   
  

   
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Automated Method for Breast Cancer Detection  
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5.3  
Quantitative and Multiplexed Methylation   
  
DNA will be extracted and analyzed by the Cepheid’s Breast Cancer Detection cartridge and 
QM-MSP as previously described in the laboratory of Dr. Sukumar (10;11).  Both the cartridge and 
QM-MSP permit analysis of multiple methylated genes using the same aliquot of DNA with as few 
as 300 epithelial cells to provide a quantitative estimate of the level of methylation in each gene that 
exists in each sample.   
  
The cells will be smeared onto 4 uncharged slides.  The slides, in batches of 10, will be 
stained with Quik-Dip by the Sukumar lab and epithelial cells will be counted. The slides will be 
taken to the cytopathologist to determine their diagnosis (benign or malignant), the cytopathologist 
will create a report, deidentify the slide with a lab number and keep the lab personnel blinded to the 
diagnosis.    
  
Cells on the stained slide will be lysed using a custom lysis buffer.  An aliquot of the lysate 
will be used in the automated methylation assay. The results of the methylation assay will be sent to 
the cytopathologist to compare with standard histopathology based diagnosis report of the H and E 
stained section of the tumor.  
  
5.4  
STRAT4 subtyping assay   
  
A cell lysate from a second unstained slide will be analyzed in the automated STRAT4 
(ER/PR/HER2/Ki67) assay cartridge.   
  
STRAT4 results will be compared to standard estrogen receptor (ER), progesterone receptor 
(PR), human epidermal growth factor receptor 2 (HER2) and Ki67 (ER/PR/HER2/Ki-67) by 
IHC/FISH performed routinely be the pathlab for comparative accuracy of the markers.    
  
5.5  
Additional analyses  
  
In addition, FNA lysate or FFPE sections of the resected lesion or core biopsy will be 
accessed –   
  
a) To compare the predictions of the FNAs as malignant or benign and gene quantitation by 
the GeneXpert Cancer Detection cartridge to our known laboratory assay, quantitative 
multiplex-methylation specific PCR (QM-MSP).   
  
b) To perform the STRAT 4 assays in the cartridge using FFPE section of the core of the 
same malignant lesion to determine concordance between assays performed on FNAs 
versus resected or biopsied tissue  
  
5.6  
Study design  
  

   
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Because this study represents the first use of FNA samples in the GeneXpert cartridge, it will 
necessary to establish that: 1) a standard FNA sample yields enough cells to perform the assay, and 
2) lock down the decision rule for calling a sample malignant based on DNA methylation levels, 
before 3) obtaining an unbiased estimate of performance.  Accordingly, the study will be divided 
into pilot, training and test phases, respectively, to accomplish these 3 tasks.    
  
  
Pilot: FNA specimens from the first 5 malignant and 5 benign tumors will be smeared onto 
uncharged slides, stained and the epithelial cells will be enumerated to ensure that the procedure 
results in collection of adequate number of cells (>300). If not, additional passes of the needle (up to 
5) will be instituted for the FNAs that follow.  The remaining cells, if any, will be stored as slides for 
future optimization of the assay, if needed.  
  
Training Set:  A sample size of 80 cases with invasive disease and 80 controls with benign disease 
is proposed.  With the projected incidence of methylated gene markers in benign disease tissue at 
higher than threshold values at 10%, we project a specificity of 80%.  If needed, we will test other 
tumor specific genes known to us to improve the panel such that a panel of up to 10 genes will 
function with >90% specificity.   
  
Test Set:  A sample size of 80 cases with invasive disease and 80 controls with benign disease is 
proposed.  . If sensitivity and specificity exceeding 90% is achieved in the training set, we will enroll 
patients for FNAs for the Test set.    
  
We propose to use DNA methylation as biomarkers in cells obtained by fine needle aspiration in 
a total of 330 women who have suspicious breast lesions recommended by ultrasound guided core 
needle biopsy for definitive diagnosis.  If needed, IRB approval will be requested for the addition of 
new patients to compensate for FNAs with sparse or no cells, so that projected numbers are fulfilled.  
   
  
6. Statistical Considerations  
   
The above study plan will recruit 5 women with benign disease and 5 women with malignant 
disease to undergo FNA prior to core biopsy in the Pilot phase.  80 evaluable subjects with benign 
disease and 80 with malignant disease will be recruited for FNA prior to core biopsy in Training 
phase.  An additional 80 women with benign disease and 80 women with malignant disease will be 
recruited for the Test phase if the criteria for the assay are met in the Training set. If not, the assay 
will be fine-tuned further in the lab with modifications to increase sensitivity and specificity.  
  
6.1   
Primary Objectives  
  
6.1.1  Pilot Phase  
  
The goal of the pilot study is to establish that standard FNA samples contain enough cells to 
complete the proposed assays.  The number of epithelial cells obtained from a typical FNA will be 

   
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modeled using a Poisson distribution, with parameters estimated from the first 10 subjects (5 
malignant, 5 benign).   
   
If the expected probability of obtaining the required 300 cells is less than 99%, additional 
passes of the needle will be taken to achieve this goal.  
  
 
6.1.2  Training Phase  
  
  
The first step in evaluating the ability of the GeneXpert cartridge to distinguish benign from 
malignant disease, is to establish a decision rule specific for FNA material in the cartridge. Only 
gold standard pathology on core biopsy samples is used as a standard for comparison during the 
training phase; results from FNA cytology will not be considered when determining the decision 
rule.  Clinical considerations lead us to target a sensitivity of at least 0.90.  Accordingly, we will fix 
sensitivity at 0.90, selecting the threshold on cumulative methylation to achieve that level, and 
reporting the conditional specificity along with confidence intervals.   
  
 
6.1.3  Test Phase  
  
  
Both primary objectives require the comparison of cartridge results to binary diagnoses made 
by the study pathologist, so statistical methods for the two objectives are identical. Cumulative 
methylation values will be calculated for each sample and disease status predicted according to the 
rule locked down in the training phase. Sensitivity and specificity will be reported, along with exact 
binomial confidence intervals.   
   
 
6.1.4  Subgroup Analysis   
  
This study will include women who were referred for mammography after presenting with 
symptomatic disease as well as asymptomatic women who were identified in the course of routine 
screening, and it is possible that the assay performs differently in these two populations.  
Accordingly, in addition to overall performance, we will evaluate and report performance in each of 
these populations separately.   
  
6.2  
Secondary Objectives  
  
Spearman rank correlation will be used to compare the DNA methylation levels as measured 
on the cartridge to measurements obtained by QM-MSP. Individual genes will be evaluated, as well 
as the cumulative methylation index.  Likewise, Spearman correlation will be used to compare levels 
of ER/PR/Her2 and Ki67 as measured by the study pathologist, with those obtained using the 
cartridge.   
  
6.2.1 Sample Size Considerations  
  

   
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Our sample size considerations are motivated by the need for precise estimates of 
performance, so that an apparently successful model can be carried forward with confidence. Our 
current methylated marker panel achieves both sensitivity and specificity higher than 90% for 
distinguishing between normal/benign and malignant disease when evaluated using QM-MSP, and 
so sample size requirements are calculated at that level.  At the proposed sample size, at a sensitivity 
and specificity of 90%, the estimation errors for the conditional specificity are controlled at +/-10%.  
   
In addition to 10 pilot patients, we will recruit 80 subjects in each group, in each of the two 
major phases (training and test).  This number was selected to control the precision of the confidence 
intervals on sensitivity and specificity. Specifically, with a sample size of 80 in each group, and 
sens/spec of 90%, the performance can be estimated to within 10% percentage points (lower, 95% 
confidence bound =80%), a level of confidence we feel is appropriate for this study.   
   
We estimate that 40%-50% of suspicious lesions will turn out to have malignant disease after 
path exam of the core biopsy, in which case the limiting factor in obtaining the necessary 330 lesions 
will be collecting 165 malignant cases.  Assuming the lower value of 40% malignancy, we expect to 
have to enroll 165/0.4=413 patients in order to obtain at least the required 165 cases. At the more 
extreme ends of the sampling distribution, there is a 10% chance that we can obtain the necessary 
cases in as few as 382 enrollments, and conversely a 10% chance of falling short even with 447 
enrollments .  Accordingly, after enrolling 382 subjects we will calculate rates of malignant and 
benign disease, and estimate total enrollment requirements, and continue enrollment as needed.  
  
  
7. Adverse Event Reporting  
  
  
Subjects will be contacted by phone 1-2 days post-FNA and core biopsy to assess adverse 
events.  There is some complexity in this reporting as a core needle biopsy will immediately follow 
the FNA. The cause, FNA vs core biopsy, of the AE will be inseparable; therefore, determining 
which process led to the AE will be impossible.  
   
  
Subjects will also be contacted by phone approximately 10-14 days post-FNA to assess 
adverse events.  Bruising that is still present after 14 days will be considered an AE.  If a subject 
reports bruising at 14 days, we will ask her to be seen by the breast imagers for assessment. A 
postFNA/biopsy hematoma that requires surgical evaluation will be considered a severe adverse 
event  
(SAE).  All adverse events will be recorded and reported to the Clinical Research Office as well as  
to the IRBs as required by guidelines for adverse event reporting.    
  
  
8. Records To Be Kept   
In addition to the hospital chart, a separate patient study folder will be kept which will include the 
patient's signed, dated informed consent document.  
  

   
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9. Pathology Requirements  
  
For laboratory/pathology samples:  
  
Laboratory samples will be collected in the clinic by Dr. Susan Harvey or their qualified 
investigators at Johns Hopkins Imaging at Green Spring.  Cytopathology will be assessed by Dr. 
Vandenbussche and a laboratory identifier will be provided. The Sukumar lab will measure 
methylation in all samples.   
  
10.  Data And Safety Monitoring Plan  
  
This trial will be conducted in accordance with the Data and Safety Monitoring Plan (DMSP) of the 
Johns Hopkins University’s Sidney Kimmel Comprehensive Cancer Center.   
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  

   
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