Improving Diagnostic US for Reduction of Benign Breast Biopsies Using US-guided Optical Tomography
Phase: NA
Conditions: Breast Biopsy
Sponsor: Washington University School of Medicine

 
 
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Improving diagnostic US for reduction of benign breast biopsies using US-guided Optical 
Tomography 
 
Washington University School of Medicine 
Department of Radiology 
660 S. Euclid Ave., CB 8138 
St. Louis, MO 63110 
 
 
HRPO #: 201707042 
Protocol Version Date: 6/30/2021 
 
 
Principal Investigator: 
 
Debbie Bennett, MD 
 
 
 
 
 
debbie.bennett@mir.wustl.edu 
 
 
Sub-Investigators 
 
 
Modality 
Ian Hageman, M.D.  PHD                  Pathology 
Quing Zhu, Ph.D. 
 
 
Biomedical Engineering 
JingQin Luo                                        Biostatistics      
Kimberly Wiele, MD  
 
Radiology 
Steven Poplack, MD  
 
Radiology                                                                                                      
Jaimee Mannix, MD  
 
Radiology 
 
ClinicalTrials.gov#: NCT03842358 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent 
necessary to obtain informed consent, may not be disclosed to another party unless law or 
regulations require such disclosure.  Persons to whom the information is disclosed must be 
informed that the information is confidential and may not be further disclosed by them 
 
Protocol Revision History 
 
Initial Approval Version, 5/26/2017 
Version 2, 1/24/2018 
Version 3, 8/15/2018 
Version 4, 3/6/2019 
Version 5, 9/5/2019 
Version 6, 11/8/2019 
Version 7, 3/25/2020 
Version 8, 5/13/2020 
Version 9, 11/4/2020 
Version 9.1 6/22/2021 
Version 9.2 6/30/2021 
 

 
 
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Improving diagnostic US for reduction of benign breast biopsies using US-guided Optical 
Tomography  
Protocol Revision History 
 
Initial Version   
 
 
5/26/2017 
 
Table of Contents 
 
1. 
STUDY OVERVIEW ............................................................................................................3 
1.1 Significance of Breast Cancer Diagnosis ............................................................................ 3 
1.2 Optical Tomography Using Near Infrared Light Guided or Assisted With Ultrasound 
(NIR/US) ........................................................................................................................................ 3 
2 
OBJECTIVES ......................................................................................................................5 
3 
ELIGIBILITY CRITERIA .......................................................................................................6 
3.1 Inclusion of Women and Minorities ...................................................................................... 7 
4 
REGISTRATION PROCEDURES ........................................................................................7 
4.1 Confirmation of Patient Eligibility.......................................................................................... 7 
4.2 Patient Registration in the Siteman Cancer Center OnCore Database ............................ 7 
4.3 Assignment of UPN ............................................................................................................... 7 
5 
STUDY PROCEDURES .......................................................................................................7 
6 
US and OPTICAL IMAGING ..............................................................................................10 
7 
DATA COLLECTION AND ANALYSIS ...............................................................................10 
7.1 Data Analysis and Validation .............................................................................................. 12 
8 
ADVERSE EVENT MONITORING AND REPORTING..................................................... 144 
8.1 Definitions .......................................................................................................................... 144 
8.2 Reporting to the Human Research Protection Office (HRPO) ...................................... 166 
8.3 Reporting to the Quality Assurance and Safety Monitoring Committee (QASMC) at 
Washington University .............................................................................................................. 166 
8.4 Time Frame for Reporting Required Events ................................................................... 166 
9 
DATA AND SAFETY MONITORING ................................................................................ 166 
10.   STATISTICAL CONSIDERATIONS 
10.1 Study Design......................................................................... Error! Bookmark not defined. 
10.2 Study Hypotheses and Endpoints ....................................... Error! Bookmark not defined. 
10.3 Sample Size Justifications .........................................................................................18 
10.4 Data Analysis .................................................................................................................... 188 
11. REFERENCES ..................................................................................................................20 
 
 

 
 
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1. STUDY OVERVIEW 
 
1.1. Significance of Breast Cancer Diagnosis  
 
Breast cancer is a heterogeneous disease with different histologic subtypes composed of different 
grades, growth rates, and metabolic activity levels resulting in a wide range of functional 
differences (1).  Additionally, benign breast disease encompasses a heterogeneous group of 
lesions that vary in vascular content, proliferative index, and metabolic activity, all of which may 
or may not be associated with future risk of breast cancer (2).   X-ray mammography is routinely 
used for breast cancer screening and diagnosis.   Ultrasound (US) of the breast is frequently used 
as a complement to mammography and breast MRI (3), and may aid in screening dense breasts 
(4-5).  Contrast enhanced mammography (CEM) is an FDA approved emerging technology 
indicated for lesion evaluation, determination of malignancy extent, neoadjuvant treatment 
response and as a substitute to MRI for high risk screening (6-9).  While the characteristics of 
malignant and benign breast lesions are well established by conventional imaging techniques (10-
12), the overlapping appearances of malignant and benign abnormalities result in hundreds of 
thousands of unnecessary biopsies, i.e. biopsies yielding benign results.  (13)  The positive 
predictive value of a screen detected abnormality recommended for biopsy is just over 25%, 
resulting in nearly 75% of unnecessary biopsy recommendations (14).  An optical tomography 
system that adds functional information like tumor related hemoglobin concentration may improve 
the accuracy of the breast US exam.  In particular, total Hemoglobin (tHb) values falling below a 
minimum value in the setting of a low or moderate suspicion abnormality (Breast Imaging and 
Database System, BI-RADS 4A or 4B assessment) may allow the diagnostic radiologist to 
downgrade the assessment to probably benign or benign (BI-RADS 3 or 2) and thereby reduce 
the rate of benign biopsies.  In a previous study when a minimum threshold value of tHb<50μm 
was used 45% of BI-RADs 4A and 4B abnormalities that subsequently were proven to be benign 
could have safely avoided biopsy [34].  At this point in time we have accrued and preliminarily 
analyzed results from 35 patients (In addition to the initial 20 patients, 15 patients from phase 2 
were also analyzed).  When using tHb<50μm as a minimum threshold 12 patients with BI-RADS 
4A or 4B abnormalities could be downgraded to a probably benign assessment and could have 
avoided biopsy, suggesting a 35% (12/35) reduction in benign biopsies. 
 
Furthermore, there is an additional clinical scenario that US-guided DOT may have a beneficial 
impact, in cases with discordant imaging pathology correlation in the setting of a high suspicion 
US abnormality. In current practice, if a high suspicion (BI-RADS 4C) lesion has a benign 
pathology result, it may be considered to have discordant imaging pathology correlation and is 
frequently recommended for repeat biopsy or surgical excision (15).  However, if the optical exam 
shows subthreshold tHb concentration, this may allow for less aggressive management, such as 
short term follow-up rather than additional tissue sampling. This is particularly applicable to a high 
suspicion irregular hypo-echoic mass, (which mimics malignancy on standard of care (SOC) US), 
with biopsy pathology of focal fibrosis.  Focal fibrosis typically has low tHb (35).   
 
 
1.2  Optical Tomography Using Near Infrared Light Guided or Assisted With Ultrasound 
(NIR/US) 
 
Diffuse optical tomography (DOT) and spectroscopy (DOS) have been explored for diagnosis of 
breast cancer and for predicting and monitoring neoadjuvant therapy responses of advanced or 
aggressive breast cancer (17-25, 34).  Utilizing DOT or DOS alone to perform breast cancer 

 
 
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diagnosis has been reported in many studies using different systems and optical wavelengths in 
the near infrared spectrum (17-25,34).   However, due to intensive light scattering in tissue, lesion 
localization and light quantification accuracy may not be fully realized (21-22).  New approaches 
taken by researchers in the field include ultrasound guided DOT (24-26, 28, 34), MRI-guided DOT 
or DOS (29-31), and x-ray guided DOT (32-33).  These approaches utilize a conventional imaging 
modality to guide the DOT or DOS for lesion localization and image reconstruction in order to 
improve the light quantification accuracy for more accurate diagnosis.   
 
Ultrasound-guided DOT, pioneered by our group, has demonstrated its potential role in 
differentiating malignant and benign breast abnormalities (24-25, 34), and in predicting and 
monitoring the neoadjuvant chemotherapy (NAC) response of breast cancer (26).  Our unique 
approach employs a commercial US transducer and NIR optical imaging sensors mounted on a 
hand-held US probe.  The co-registered US is used for lesion localization, and optical sensors 
are used for imaging tumor related vascularity.   
 
The technical aspects of the NIR optical imaging system have been described in detail [24].  
Briefly, NIR light is delivered from multiple source positions embedded within the probe with 
the central slot fitted with an ultrasound transducer and the reflected light (from normal and 
pathologic breast tissue)  collected by multiple detection fibers within the probe that are 
coupled to photomultiplier tubes (PMTs).  The transmitted NIR light is delivered to each 
transducer source position sequentially and reflected NIR light is detected in parallel from all 
PMT detectors.  The entire acquisition from all source detector pairs takes approximately 3 
to 4 seconds (figure 1).  Specific light wavelengths in the NIR spectrum are selectively 
absorbed by oxygenated (HbO2) and deoxygenated Hemoglobin (Hb) and allow for the 
quantification of the concentrations and distributions of tHb, Hb and HbO2. 
                
            
Figure 1:  Hand-held US-guided Near Infrared system  
 
NIR System 
Combined 
NIR/US probe 

 
 
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       Examples of using NIR/US for breast cancer diagnosis are given below [34]. 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2. (a)  US image of a hypoechoic lobulated mass with internal echoes and septations located at 2 o’clock 
in the right breast of a 40-year-old woman.  US readings from the two readers were 4B and 4C.  (b)  The tHb 
map shows a diffused mass with a maximum value of 52.5
mol/L.  (c)  oxyHb map, and (d) deoxyHb map.  
Core biopsy revealed DCIS and ADH.    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.  (a) US image of a hypoechoic lobulated mass (arrow) in the right breast of a 72-year-old woman.  US 
readings from the two readers were 4B and 4C.   (b) tHb map showed an isolated mass, maximum 106.2
mol/L. (c) oxyHb map, and (d) deoxyHb map.   OxyHb map follows tHb map closely, and the deoxyHb 
distribution is quite diffused.  Core biopsy revealed DCIS, nuclear grade 2.  Pathology at surgery revealed 0.6 
cm DCIS with intraductal papilloma with atypia and microcalcification.   
 
2 
OBJECTIVES 
 
We have received 5-year NCI funding to support this study. 
 
The primary objective of this study is to assess the ability of US-guided DOT to reduce the rate of 
benign biopsy.  To achieve this objective we will simultaneously acquire co-registered US imaging 
data and US-guided DOT imaging data from a group of patients (~300) scheduled for US-guided 
breast biopsy.  We hypothesize that the additional optical information of tHb concentration and 
distribution will lead to a downgrading of the assessment of a subset of low to moderate suspicion 
µ
µ

 
 
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abnormalities based on conventional imaging (i.e. breast ultrasound with or without 
mammography) and thereby could lead to a reduction in benign breast biopsies (i.e. false 
positives). 
 
The secondary and complementary objective is to assess the impact on sensitivity of adjunctive 
optical data.  In particular we hypothesize that by defining a conservative tHb threshold below 
which malignancy is so rare that the addition of US-guided DOT will not significantly alter the 
sensitivity of the combined US-guided DOT & CI assessment, i.e. adjunctive US-guided DOT will 
not decrease true positives.  
 
A third objective is to assess the impact of adjunctive US-guided DOT data in the management 
of discordant pathology results.  In current SOC, the radiologist performing the needle biopsy 
routinely compares the imaging with the pathology results for concordance. Highly suspicious 
abnormalities (BI-RAD 4C, 5) that yield benign results are typically considered discordant and are 
recommended for repeat biopsy or surgical excision.  If the discordant abnormality has a tHb 
below the minimum threshold value, this might allow radiologists to forego a repeat biopsy or 
surgery.  For example, focal fibrosis may appear highly suspicious on US. Our initial data show 
that fibrotic lesions have low vascularity (28).  
   
Additionally as an exploratory objective, we will compare the results of US-guided DOT and in 
particular tHb concentration with contrast enhanced mammography (CEM) in the subset of 
patients who undergo CEM. 
 
3 
ELIGIBILITY CRITERIA 
 
Inclusion Criteria:  
• 
Female subjects ≥ 18 years old with ultrasound visible breast abnormalities (BI-RADS 
3*, 4A, 4B, 4C, and 5) referred for ultrasound-guided core needle biopsy or fine needle 
aspiration  
• 
Willing and able to provide informed consent 
 
(*note that while a BI-RADS 3 assessment is probably benign, a subset of patients with this 
assessment choose to undergo biopsy rather than follow up imaging).   
 
Exclusion Criteria: 
• 
Lesions located in the darkly pigmented nipple-areolar complex area 
• 
Subjects with breast implants 
• 
Abnormality in the mirror image location of the contralateral breast. 
• 
Additional abnormalities in the same region of the breast that would be included in US-
guided DOT imaging of the abnormality undergoing biopsy   
• 
Previous breast irradiation of the mirror image location of the contralateral breast 
• 
Lesions located at previous biopsy sites when biopsy occurred within the last six 
months. 
• 
Pregnancy 
• 
Superficial abnormalities located entirely within (i.e. less than) 5mm of the overlying skin 
 
 

 
 
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3.1 Inclusion of Women and Minorities 
 
Women and members of all races and ethnic groups are eligible for the study. 
 
 
4. REGISTRATION PROCEDURES 
 
The following steps must be taken in order to register patients to this study:  
 
1. Confirmation of patient eligibility  
2. Registration of patient in the Siteman Cancer Center OnCore database 
3. Assignment of unique patient number (UPN)  
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility by collecting the information listed below: 
 
1. Registering MD’s name 
2. Patient’s race, sex, and DOB 
3. Three letters (or two letters and a dash) for the patient’s initials 
4. Copy of signed consent form 
5. Completed eligibility checklist, signed and dated by a member of the study team 
6. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center OnCore Database 
 
All patients must be registered through the Siteman Cancer Center OnCore database. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  All data 
will be recorded with this identification number on the appropriate CRFs. 
 
5 STUDY PROCEDURES  
 
The study will ensue in two phases: phase 1 – training set, and phase 2 prospective trial. 
 
The first 20 patients undergoing US-DOT will comprise the training set to accomplish the aims 
of: 
1) assessing US-DOT data,  
2) assessing intra-observer variability in the assessment of US-DOT data 
3) assessing inter-observer variability in the assessment of US-DOT data. 
  
For phase 2, the prospective trial, up to 315 patients will be recruited with the expectation that 
approximately 5% will not successfully complete the study so that 300 patients will have valid 
US-DOT data for the ensuing reader study.   
 
The study flow chart that integrates US-guided DOT collection into the standard of care (SOC) 
clinical workflow is shown below (figure 3).  SOC clinical workflow includes review of patient 
information and diagnostic information, such as conventional imaging results with  BIRADs 

 
 
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assessments.  The study coordinator will approach eligible patients prior to biopsy to assess their 
willingness to participate in the study. Patients that are willing to participate will sign informed 
consent and undergo US-guided DOT imaging before biopsy. A subset of patients will also 
undergo CEM. 
 
Breast radiologist coinvestigators will perform (or supervise certified breast US technologists) 
in all NIR/US studies.  As noted above, they will not be blinded to any clinical data.  The 
function of the imaging specialist in US guided DOT imaging is to ensure that the correct 
anatomic location of the index lesion is imaged.   
 
The study radiologist that performs/supervises the US-guided DOT imaging will complete CI 
and US-guided DOT case report forms (CRFs) blinded to the pathology result of the ensuing 
biopsy.  In the event that the patient also undergoes CEM the study radiologist will complete 
CI and CEM CRFs blinded to the pathology result of the ensuing biopsy.  Details of CRF 
completion and related reader study are noted in section 7 – Data Collection and Analysis 
below. 
 
 

 
 
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Figure 3. Study work flow 
 
Training Set: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prospective Trial   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Patients referred for US guided biopsy  
Review of subject eligibility and imaging data 
including 
BIRADS assessment 
US/NIR Imaging Exam (& CEM in subset) 
Breast biopsy or FNA performed (Ground truth 
to determine disease status for this study.) 
Informed consent obtained for eligible patients 
Subset analysis: DOT - 
CEM 
20 patients with BI-RADS 3, 4 or 5 
Assessments who agree to participate in the 
study 
Reader training 
Variability analysis 
Reader Study 
Analysis of DOT 
impact on Diagnosis 

 
 
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6. 
US and OPTICAL IMAGING  
 
A hand-held hybrid probe, consisting of a commercially available US transducer located in the 
middle and near-infrared source and detector optical fibers distributed at the periphery (see Figure 
1), will be used for scans.  For each patient, co-registered digital US images from the commercial 
ultrasound unit and optical measurements from our NIR optical imaging device are acquired 
simultaneously at multiple locations including the lesion of interest region and the same region in 
the mirror-image contralateral breast.  The contralateral location is chosen as the reference 
site.  The difference between measurements obtained from the lesion and the reference site is 
used for optical imaging reconstruction.  Optical absorption distributions at four different 
wavelengths are reconstructed and lesion total hemoglobin concentration (tHb), and oxygenated 
and deoxygenated hemoglobin concentrations (HbO2 and Hb) are computed from absorption 
maps.  The total data acquisition time is about 10 minutes once the lesion is identified by US.  
Following each patient study, the near infrared images will be reconstructed by Dr. Zhu’ group.  
 
Safety and Setup for Imaging 
 
The US-guided NIR device has been used safely by Prof. Zhu’s group in the past 14 years for 
~500 patients [24,25,34].  There is no known risk associated with the use of the device. The light 
sources are low-power laser diodes with wavelengths in the NIR range and are fiberoptic coupled 
to the probe.  The laser light sources can only be activated by pushing a button on the touch 
screen laptop when the surface of the probe, i.e., the tips of the fibers, is in contact with the skin.  
 
7. 
DATA COLLECTION AND ANALYSIS 
 
Study specific data will be collected from subjects after informed consent is obtained.  The 
following data may be collected on the patient questionnaire: demographic information (name, 
DOB, Age, Race and Hispanic or Non-Hispanic) MRN#, imaging reports (e.g. mammography, 
US, PET, MRI, etc.), core biopsy pathology reports, surgical pathology reports if available, breast 
cancer risk information, etc. 
 
In some cases, the pathologist may review existing biopsy specimens with special staining to look 
for signs of increased blood supply and vessel density. This will not require additional slicing of 
standard of care biopsy specimens; thus, samples will not be labeled or stored for the research 
study. The results of these tests will not be placed in the patient medical record. The results of 
these tests may be given to the investigators to correlate to the results of the NIR/US procedures. 
 
Dr. Ian Hagemann, specialist in breast pathology, will assess pathological findings. 
 
 
 
The goal of this study is to assess the impact of adjunctive US-guided DOTon the accuracy 
of breast biopsy recommendations for abnormalities undergoing US-guided breast biopsy.  
In particular to assess the effect of combining US-guided DOT with CI to: 1.  reduce the rate 
of benign breast biopsy, i.e. decrease false positive biopsy recommendations, and 2. to 
maintain sensitivity, i.e. maintain true positive recommendations. 
 
Additional goalsof this study are to assess the role of US-guided DOT in managing discordant 
biopsy results and assess the correlation of optical imaging parameters with the presence 

 
 
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and intensity of contrast enhancement of the abnormality undergoing biopsy in patients who 
have undergone CEM. 
 
Figure 4 – Reader Study Design 
 
 
To accomplish the aims of assessing the impact of adjunctive US-guided DOT on reducing benign 
biopsy recommendation (i.e. FPs), while maintaining sensitivity (i.e. TPs) we will conduct a reader 
study of the 300 subject trial.  Three of four study radiologists will serve as study readers for each 
case.  (To eliminate potential bias a study radiologist performing the biopsy will not serve as a 
reader for that case.)  As noted above the study radiologist who performs or supervises the US-
guided DOT exam will be one of the readers.  Two additional study radiologist readers will 
subsequently and independently review the available pre-existing patient information and CI data, 
(excluding the US-guided biopsy exam) and complete CI and DOT CRFs blinded to the pathology 
result of the abnormality that underwent biopsy.  The CRFs include a BI-RADS assessment. The 
reader will first complete the CI CRF blinded to the US-guided DOT result, then after reviewing 
the optical data (tHB concentration and distribution) the reader will complete the DOT CRF 
including the impact of US-guided DOT information on the BI-RADs assessment, i.e. no change, 
decreased or increased.    
   

 
 
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Similar to the CI plus DOT workflow, for cases that have CEM, a CI CRF will be completed first, 
followed by a CEM specific CRF including a BI-RADS assessment and the impact of CEM on the 
BIRADs assessment will be recorded.   As noted above, the study radiologist that supervises the 
US-guided DOT and CEM exams will serve as one of the study readers.  For the subsequent two 
readers we will alternate which adjunctive modality (i.e. CEM or US-DOT) is reviewed first and 
separate the subsequent review of the second adjunctive modality by a minimum of 4 weeks to 
prevent recollection bias).   
 
Figure 4 diagrams the core analysis.  Combined CI and US-guided DOT BI-RADS 2 and 3 
assessments will be considered test negative, and BI-RADs 4A, 4B, 4C, and 5 assessments will 
be considered test positive. For negative tests, the biopsy proven malignant group (Gc, see Fig. 
1(b)) represents false negative results, and the benign group Gd represents true negatives and 
reflects the potential reduction in benign biopsy recommendation if US-guided DOT were used as 
SOC.  Ga represent true positives and Gb represent false positives utilizing adjunctive  US-guided 
DOT.  A comparison of the Ga groups i.e, CI alone vs. CI & DOT will constitute the assessment 
of the effect of adjunctive US-guided DOT on sensitivity.  
 
A small minority of patients assessed as BIRADS 3-Probably Benign and recommended for short 
term follow up imaging on the basis of CI will elect to undergo biopsy.  We will separately track 
the effect of supplemental US-DOT on this small subset. 
 
7.1 
Data Analysis and Validation 
 
US-guided DOT database and Training:   
Part 1 - Using typical tHb levels and features (e.g. peripheral enhancement, shadowing) from 
biopsy proven benign and malignant cases obtained from earlier studies we will train the four 
study radiologists.  The goals of the training are to familiarize the study radiologists with US-
guided DOT imaging and typical hemoglobin distributions (tHb, oxyHb, deoxyHb) of different 
benign and malignant disease processes and to assess intra- and inter-observer variability.    
Data from the training study will not be used in the primary analysis.  
 
Part 2 - Approximately 20 patients will be enrolled for the training phase. Inter-reader agreement 
will be assessed based on Fleiss’s Kappa coefficient across the three radiologists and Cohen’s 
Kappa between two radiologists, each associated with 95% confidence interval. The 20 patients 
will provide 83.32% power for testing agreement between two readers using the Kappa statistics 
to detect a true Kappa value of ≥0.75 against an unacceptable Kappa value of ≤0.3, based on 1-
sided normal test at a 0.1 level. 
 
Intra-observer variability will also be assessed in the training phase.  Each radiologist will read 
each patient’s images twice (or even three times). After the first round of reading, the patients’ 
images will be stored and the radiologists will be asked to review the same patients’ images some 
time later, after the image orders have been perturbed and patient ID and previous evaluation 
results have been blinded. Integrating data from repeated evaluations from multiple radiologists 
on the 20 patients, generalize linear mixed effects model will be used to fit the reading results to 
characterize inter- and intra-reader variability based on an intra-class correlation coefficient (ICC). 
If a persistent large intra-reader variability is found (e.g., with ICC >0.3), we will perform a focused 
interview with the radiologists to identify possible factors yielding inconsistency in reading results. 
Considering the influential factors, we will revise the training phase by providing more structured 
instructions illustrated with imaging examples to clarify potential issues. We expect improvement 
with structured instruction, but if intra-reader variability persists, we will consider requiring 

 
 
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repeated evaluations from the same radiologist on the same patient in order to robustly evaluate 
the diagnosis of US-guided DOT. 
 
 
Up to 315 participants will be enrolled to yield 300 participants with valid US-DOT data to evaluate 
the primary and secondary objectives as below. 
 
       Aim 1.1 Assess the impact of US-guided DOT on potential reduction of benign biopsies 
The primary objective of this prospective study is to assess the impact of US-guided DOT on the 
potential reduction of benign biopsies, a.k.a, improve specificity. We hypothesize that the 
adjunctive use of US-guided DOT will reduce the rate of benign breast biopsy.  Using these 
criteria, the first endpoint, specificity = Gd/(Gb+Gd), will be calculated as the proportion of subjects 
with a negative assessment (BI-RADS 2 or 3) and  with no cancer at biopsy.  With the exception 
of a small minority of patients with BI-RADS 3 assessments who elect to undergo biopsy and who 
will be tracked separately, all subjects in this study are considered positive using the current SOC, 
because they have been recommended for biopsy.  Hence, 1) the specificity in this group for the 
SOC is 0% by definition; 2) the estimate of specificity for the DOT system represents the 
improvement in specificity; and 3) the estimate of specificity is also an indication of the percentage 
of benign biopsies that might be avoided if the NIR data were used by the radiologist in the biopsy 
decision making process. 
  
Aim1.2 Assess the impact of US-guided DOT as an adjunct to CI on maintaining high sensitivity 
To also ensure that the potential for false negatives is not a significant risk with the use of the US-
guided DOT, we will also assess the probability of potentially misclassifying a malignant 
abnormality. By definition, FN=Gc/(Gc+Ga), which is 1 minus sensitivity.  Our earlier study [Zhu 
reference] indicated that both radiologists reached 96.6% to 100% sensitivity with the tHb data 
included, and therefore had a 0-3.4% probability of missing a malignant lesion.  
  
All CRFs including CI, DOT, CEM and biopsy results will be entered by our study radiologists, or 
study coordinator or study pathologist into the OnCore data base to be exported to Dr. Luo for 
statistical analysis.  An ad hoc analysis of the CI, CEM and optical imaging characteristics and 
histopathologic features of false negative cases will be performed to identify suspicious features 
that always indicate a level of suspicion warranting biopsy. 
 
Aim1.3 Explore the potential benefit of managing post biopsy BI-RADS 4C and 5 lesions 
From a total of 300 patients who undergo the US-DOT exam, we expect to recruit a small subset 
of patients with BI-RADS 4C or 5 lesions whose biopsy results are benign.  We will correlate initial 
biopsy results, optical data and gold standard final pathology for this subset of patients. This pilot 
data will allow us to assess the potential benefit of US-guided DOT in managing discordant 
abnormalities, i.e. reducing additional invasive procedures of a subset of benign abnormalities.   
 
Exploratory Aim 1.4 Explore and compare US-guided DOT and CEM 
In this exploratory aim, we will evaluate the agreement (Kappa coefficient) of US-guided DOT and 
CEM, as both modalities measure alterations in lesion vascularity.  We will compare BI-RADS 
assessments of CI&CEM vs. CI&US-guided DOT as well as correlate imaging data such as 
intensity of enhancement in CEM and the total hemoglobin level of US-DOT.   
 
Alternative plan: If US-guided DOT is not comparable with CEM we will evaluate the capability 
of US-guided DOT in improving the accuracy of combined CI&CEM because CEM may become 
SOC in a significant subset of patients. 

 
 
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8. 
ADVERSE EVENT MONITORING AND REPORTING  
 
The entities providing oversight of safety and compliance with the protocol require reporting as 
outline below. 
 
The Washington University Human Research Protection Office (HRPO) requires that all events 
meeting the definition of unanticipated problem or serious noncompliance be reported as outlined 
in Section 8.2. 
 
There are minimal side effects associated with the NIR/US, therefore we will not be tracking AEs.  
All SAEs that occur within 24 hours of an NIR/US exam will be brought to the attention of the PI 
and a determination will be made if they are related or unrelated to study participation. Any SAE 
that is related to the NIR/US intervention, will be reported to the IRB.   
 
8.1 
Definitions 
 
8.1.1 Adverse Events (AEs) 
 
Definition: any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease.  
  
Attribution (Relatedness), Expectedness, and Seriousness: the definitions for the 
terms listed that should be used are those provided by the Department of Health 
and Human Services’ Office for Human Research Protections (OHRP). A copy of 
this 
guidance 
can 
be 
found 
on 
OHRP’s 
website 
at: 
http://www.hhs.gov/ohrp/policy/advevntguid.html.  
 
8.1.2 Serious Adverse Event (SAE) 
 
Definition:  any adverse drug experience occurring at any dose that results in any 
of the following outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed above 
 
 

 
 
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8.1.3 Unexpected Adverse Experience 
 
Definition: any adverse drug experience, the specificity or severity of which is not 
consistent with the current investigator brochure (or risk information, if an IB is not 
required or available). 
 
8.1.4 Life-Threatening Adverse Experience  
 
Definition: any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have 
caused death. 
 
8.1.5 Unanticipated Problems 
  
• 
unexpected (in terms of nature, severity, or frequency) given (a) the 
research procedures that are described in the protocol-related documents, 
such as the IRB-approved research protocol and informed consent 
document; and (b) the characteristics of the subject population being 
studied;  
• 
related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the 
incident, experience, or outcome may have been caused by the procedures 
involved in the research); and  
• 
suggests that the research places subjects or others at a greater risk of 
harm (including physical, psychological, economic, or social harm) than 
was previously known or recognized.  
 
8.1.6. Noncompliance 
  
Definition: failure to follow any applicable regulation or institutional policies that 
govern human subject research or failure to follow the determinations of the IRB. 
Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB.  
 
8.1.7 Serious Noncompliance  
 
Definition: noncompliance that materially increases risks, and results in 
substantial harm to subjects or others, or that materially compromises the rights or 
welfare of participants.  
 
8.1.8 Protocol Exceptions  
 
Definition: A planned deviation from the approved protocol that are under the 
research team’s control. Exceptions apply only to a single participant or a singular 
situation. Pre-approval of all protocol exceptions must be obtained prior to the 
event.  
 
 

 
 
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8.2 
Reporting to the Human Research Protection Office (HRPO)  
 
The PI is required to promptly notify the IRB of the following events:  
• 
Any unanticipated problems involving risks to participants or others which occur at 
WU, any BJH institution, or that impacts participants or the conduct of the study.  
• 
Noncompliance with federal regulations or the requirements or determinations of 
the IRB.  
• 
Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study.  
 
These events must be reported to the IRB within 10 working days of the occurrence of 
the event or notification to the PI of the event. The death of a research participant that 
qualifies as a reportable event should be reported within 1 working day of the occurrence 
of the event or notification to the PI of the event. 
 
8.3 
Reporting to the Quality Assurance and Safety Monitoring Committee (QASMC) at 
Washington University 
 
The PI is required to notify the QASMC of any unanticipated problem occurring at WU or 
any BJH or SLCH institution that has been reported to and acknowledged by HRPO as 
reportable.  (Unanticipated problems reported to HRPO and withdrawn during the review 
process need not be reported to QASMC.) 
 
QASMC must be notified within 10 days of receipt of IRB acknowledgment via email to a 
QASMC auditor. 
 
8.4 
Time Frame for Reporting Required Events 
 
As noted above minimal adverse events are associated with NIR/US.  If we are made 
aware of an AE/SAE then we will report this to the HRPO in the timeline noted in section 
8.2. 
 
9. 
DATA AND SAFETY MONITORING  
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the Washington 
University Quality Assurance and Safety Monitoring Committee (QASMC) semi-annually 
beginning six months after accrual has opened (if at least five patients have been enrolled) or one 
year after accrual has opened (if fewer than five patients have been enrolled at the six-month 
mark). This report will include: 
 
• 
HRPO protocol number, protocol title, Principal Investigator name, data coordinator name, 
regulatory coordinator name, and statistician  
• 
Date of initial HRPO approval, date of most recent consent HRPO approval/revision, date 
of HRPO expiration, date of most recent QA audit, study status, and phase of study  
• 
History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason  
• 
Study-wide target accrual and study-wide actual accrual  

 
 
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• 
Protocol activation date  
• 
Average rate of accrual observed in month 1, month 2, and subsequent months  
• 
Expected accrual end date  
• 
Objectives of protocol with supporting data and list the number of participants who have 
met each objective  
• 
Measures of efficacy  
• 
Early stopping rules with supporting data and list the number of participants who have met 
the early stopping rules  
• 
Summary of toxicities  
• 
Abstract submissions/publications  
• 
Summary of any recent literature that may affect the safety or ethics of the study  
 
The study principal investigator and Research Patient Coordinator will monitor any potential 
issues on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 
10. Statistical Considerations 
 
10. 1 Study design. 
 
The study consists of two phases. The training phase consists of 20 eligible participants as 
detailed in Section “US-guided DOT database and Training” to assess sources of variabilities. 
The formal phase is a prospective study to enroll up to 315 eligible participants who will undergo 
SOC biopsy and fall in the BIRAD categories of 3, 4A, 4B, 4C, or 5 to yield 300 with valid US-
DOT data.  
 
10. 2 
Study hypotheses and endpoints. 
 
Study Hypothesis 1 for aim 1.1:  The first hypothesis is that the measured tHb 
distribution and values can be potentially used as an adjunct for radiologists to reduce 
unnecessary biopsies in low risk patients with benign pathological results. The benign 
biopsy spare rate can be calculated as Gd/(Gb+Gd) in Fig.11, which is equivalent to  the 
specificity, the primary endpoint of Aim1.1.  We observed in previous studies that 
specificity could be improved by 45% on average if NIR data were used in the decision-
making process.  Note that the specificity in this group of biopsied patients under the 
SOC is 0% by definition.  In this study, we conservatively set the minimum acceptable 
specificity at 30%.  The null and alternative hypotheses, which demonstrate superiority 
when the NIR imaging data are used as an adjunct to the diagnostic assessment versus 
when the SOC diagnostic data are used alone, are given by 
                                             H0: Specificity ≤ 30% versus H1: Specificity > 30% 
This aim will be considered a success if the lower limit of the 95% confidence interval on 
specificity exceeds 30%. This hypothesis will be tested at the one-sided 0.025 level, rather than 
0.05, to account for the fact that two hypothesis tests (on specificity and sensitivity) are planned.  
This aim is considered a success if the lower limit of the 97.5% confidence interval on specificity 
exceeds 30%. 
Study Hypothesis 2 for aim 1.2:  The second hypothesis is that, while reducing unnecessary 
benign biopsies, the addition of adjunct information from US-DOT still maintains a high sensitivity 

 
 
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in capturing nearly all malignant tumors.  This can be evaluated based on sensitivity, which is 
calculated as Ga/(Gc+Ga), as shown in Fig.11. 
             Ho: sensitivity ≤ 90% versus H1: sensitivity>90% 
Based on previous study, we anticipate the sensitivity will be in the range of 96-100%.  This aim 
is considered a success if the lower limit of the 95% confidence interval for sensitivity is at least 
90%.  This hypothesis will be tested at the one-sided 0.025 level, rather than 0.05, to stringently 
account for the fact that two hypothesis tests (on specificity and sensitivity) are planned. This aim 
is considered a success if the lower limit of the 97.5% confidence interval for sensitivity is at least 
90%.   
 
10.3 
Sample size justifications. 
 
We expect ~300 eligible women who will undergo SOC biopsy with valid US-DOT data and fall 
in the BIRAD categories of 3, 4A, 4B, 4C, or 5, rendering ~210 pathologically benign biopsies 
and 90 malignant biopsies. For Study Hypothesis 1, prior study results indicate the specificity 
was estimated to be no less than 45%. A total of 83 benign biopsies are needed to achieve 
80.18% power to test Study Hypothesis 1 at a 2.5% level.  This calculation is based on a 1-sided 
exact binomial test by conservatively assuming the true specificity is 45%, while 210 benign 
biopsies achieve 99.4% power to test Hypothesis 1. We also calculated the sample size based 
on the more stringent requirement that the lower limit of the 1-sided 97.5% conservative Clopper-
Pearson binomial exact confidence interval (CI) (60) to the estimated specificity (of 45%) should 
exceed 30%. The 210 benign biopsies will allow us to estimate the 1-sided 97.5% exact CI to be 
between 37.7%~100%, thus satisfying this more stringent requirement.  For Study Hypothesis 
2, 90 malignant biopsies will achieve 89.33% power to conduct Study Hypothesis 2 at a 1-sided 
2.5% level, based on a 1-sided exact binomial test. With 90 malignant biopsies, the sensitivity 
can be estimated at 98% with a 1-sided 97.5% exact CI of 92.06%~100%, thus satisfying the 
stringent criterion requiring a lower limit of >90%.  
In calendar year 2016, over 7,000 women came to the Siteman Cancer Center at WashU for 
diagnostic evaluation.  Approximately 17% had suspicious or highly suggestive malignancy 
findings and were recommended for tissue sampling.  Over 700 women underwent US guided 
biopsy, which is more than 10 times larger than the potential study subjects each year. 
 
             
10.4 
Data Analysis 
 
All data will be analyzed as it is without imputations. Summary statistics will be used to summarize 
data including mean, median, standard deviation, inerquartile range for continuous data and count 
(percentage) for categorical data.  
 
All evaluable subjects will be analyzed. In addition to the specificity and sensitivity of US-
guided DOT results, PPV, NPV, and overall accuracy will be calculated.  The two study 
hypotheses will be tested by a binomial exact test and by constructing the one-sided 
97.5% Clopper-Pearson exact confidence intervals for specificity and sensitivity. The lower 
limit will determine the achievement of study goals.  We will explore the data further to 
refine the classification algorithm and the cut-off threshold. ROC analysis will evaluate the 
quantitatively measured tHb for pathological results of malignant biopsies. Besides tHb 
and its distribution, other parameters (such as oxyHb, deoxyHb, and distributions) will be 
either linearly incorporated with tHb to maximize the ultimate area under ROC or non-
linearly through a tree-based classification model (e.g., using the R package “rpart”) (62).    

 
 
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The area under the ROC will be calculated to gauge the model’s overall discriminative 
ability for biopsy results. 
For the exploratory study (discordant biopsy results), the imaging data (e.g., tHb, oxyHb etc) will 
be summarized as appropriate in the subset of participants whose BIRAD results yield highly 
suspicious abnormalities (BI-RAD 4C, 5) but pathology yield benign results. Two sample t-test or 
Wilcoxon rank sum test as appropriate will be applied to compare this subset versus those with a 
malignant biopsy.  
For the exploratory Study (CEM DOT correlation), we will compare BI-RADS assessments of 
CI&CEM vs. CI&US-guided DOT as well as correlate imaging data such as intensity of 
enhancement in CEM and the total hemoglobin level of US-DOT using Pearson or Spearman 
correlation coefficients.  We will evaluate concordance/disconcordance between US-guided DOT 
and CEM using agreement measure (Kappa coefficient) and will test the disconcordance using 
McNemar test.   
 
 

 
 
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