Clinical Evaluation of Opto-Acoustic Image Quality With the Gen 2 Imagio System
Phase: NA
Conditions: Breast Cancer Stage
Sponsor: Seno Medical Instruments Inc.

Seno Medical Clinical Study Protocol: GEN 2 Imaging-01 
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Seno Medical Instruments, Inc.  
Study Title: 
Clinical Evaluation of Opto-Acoustic Image Quality with 
the Gen 2 Imagio System 
Protocol Number: 
GEN 2 Imaging-01 
Study Device: 
Gen 2 Imagio® Breast Imaging System 
Sponsor:  
Seno Medical Instruments, Inc. 
Sponsor 
Contact/Representative: 
Shaan Schaeffer 
Vice President, 
Clinical Operations 
Seno Medical Instruments, Inc. 
8023 Vantage Drive, Suite 1000 
San Antonio, TX 78230 
210-615-6501
Medical Monitor: 
A Thomas Stavros, MD 
Chief Medical Officer 
Seno Medical Instruments, Inc. 
8023 Vantage Drive, Suite 1000 
San Antonio, TX 78230 
210-615-6501
Principal Investigator: 
Pam Otto, MD 
Chairman,  
Department of Radiology 
Medical Arts & Research 
Center - Radiology 
8300 Floyd Curl Drive 
San Antonio, TX 78229 
Study Duration:  
The study duration for each subject will be a single visit 
unless imaging is to be done again.  
Date: 10 Nov 2021 
Protocol Version: 4.0 
Confidentiality Statement: 
This document contains information that is the property of Seno Medical Instruments Inc. 
and is therefore provided to you in confidence for review by you, your staff, an applicable 
Institutional Review Board and regulatory authorities. It is understood that this information 
will not be disclosed to others without written approval from Seno Medical Instruments Inc., 
except to the extent necessary to obtain informed consent from those persons to whom 
Imagio may be administered. 
APPENDIX 16.1

APPENDIX 16.1

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PROTOCOL SIGNATURE PAGE 
 
 
Clinical Investigation:  
Clinical Evaluation of Opto-Acoustic Image Quality with the Gen 2 
Imagio System 
 
Reference: 
GEN 2 Imaging-01 
 
 
 
Signature Statement 
I have reviewed this clinical investigation protocol describing the design and specific provisions 
of the clinical investigation. I agree with the content of this document. 
 
 
 
 
Shaan Schaeffer 
Vice President of Clinical Operations 
 
 
 
 
Date 
A. Thomas Stavros, M.D. 
Chief Medical Officer 
 
 
 
 
Date 
Ann Waterhouse, RAC 
Vice President of Regulatory Affairs and Quality Assurance 
 
 
 
 
Date 
Pam Otto, M.D. 
Chairman of the Department of Radiology 
 
 
 
 
Date 
Elizabeth Kane, PhD 
Senior Biostatistician, Avania 
 
 
 
Date 
12Nov2021
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PROTOCOL SIGNATURE PAGE 
 
 
Clinical Investigation:  
Clinical Evaluation of Opto-Acoustic Image Quality with the Gen 2 
Imagio System 
 
Reference: 
GEN 2 Imaging-01 
 
 
 
Signature Statement 
I have reviewed this clinical investigation protocol describing the design and specific provisions 
of the clinical investigation. I agree with the content of this document. 
 
 
 
 
Shaan Schaeffer 
Vice President of Clinical Operations 
 
 
 
 
Date 
A. Thomas Stavros, M.D. 
Chief Medical Officer 
 
 
 
 
Date 
Ann Waterhouse, RAC 
Vice President of Regulatory Affairs and Quality Assurance 
 
 
 
 
Date 
Pam Otto, M.D. 
Chairman of the Department of Radiology 
 
 
 
 
Date 
Elizabeth Kane, PhD 
Senior Biostatistician, Avania 
 
 
 
Date 
10Nov2021
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INVESTIGATOR SIGNATURE PAGE 
 
 
Clinical Investigation:  Clinical Evaluation of Opto-Acoustic Image Quality with the Gen 2 
Imagio System 
 
 
Reference: 
GEN 2 Imaging-01 
 
 
 
Investigator’s Statement 
I agree to conduct this clinical investigation in accordance with the design and specific provisions 
of this clinical investigation plan; modifications to the clinical investigation are only acceptable 
with a mutually agreed upon clinical investigation plan amendment as approved by the Sponsor 
and involved IRB. 
I agree to await IRB approval of the clinical investigation plan and informed consent form before 
initiating the clinical investigation, to obtain consent from subjects prior to their enrollment, to 
collect and record data as required by the clinical investigation plan and associated case report 
forms, and to maintain documents related to the clinical investigation for the period of time 
required. 
 
Confidential 
This document contains confidential information belonging to Seno Medical Instruments, Inc. 
Except as may be otherwise agreed to in writing, by accepting or reviewing these materials, I 
agree to hold such information in confidence and not to disclose it to others (except where 
required by applicable law), nor use it for unauthorized purposes. 
 
 
 
 
 
Investigator name (print) 
 
 
 
 
 
Signature 
Date 
 
 
 
 
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SYNOPSIS 
Clinical investigation plan 
Title 
Clinical Evaluation of Opto-Acoustic Image Quality with the Gen 2 
Imagio System 
Short title  
GEN 2 Imaging-01 
Investigational device  
Name 
GEN 2 Imagio® Breast Imaging System 
Indication for Use 
The Imagio Breast Imaging System is indicated for use by a 
trained and qualified healthcare provider for evaluation of 
palpable and non-palpable breast abnormalities in adult subjects 
who are referred for a diagnostic imaging breast work-up, 
following clinical presentation or either screening or diagnostic 
mammography or other imaging examinations. The ultrasound 
mode should be initially used in a targeted fashion, to assess any 
focal area(s) of clinical or imaging concerns. In ultrasound (US) 
mode, the device can be used to assign a BI-RADS (Breast 
Imaging-Reporting and Data System) category to either breast 
tissue or a mass that is causing clinical or imaging concerns. 
Masses that are classified as BI-RADS categories 3 through 5 
can then be assessed using the Opto-Acoustic (OA) mode. In the 
OA mode, the Imagio Breast Imaging System provides 
information about the central nidus, boundary and peripheral 
zones, based on vascularity and blood oxygen saturation of the 
imaged tissues, to assist in the diagnosis of the benign or 
malignant mass(es) of interest. For ultrasound BI-RADS 3-5 
masses, using the OA features of the mass allows for improved 
classification of the mass of interest as compared to ultrasound 
alone. The OA mode is not indicated for ultrasound BI-RADS 1 
and 2 findings. The Imagio Breast Imaging System includes an 
artificial intelligence (AI) based software function to assist the 
users in assessing the BI-RADS Classifications. This device is 
not intended to be used as a replacement for mammographic 
screening or for definitive pathologic diagnosis. 
Sponsor 
Name 
Seno Medical Instruments, Inc. 
8023 Vantage Drive, Suite 1000 
San Antonio, TX 78230 
United States of America 
Contact details 
Shaan Schaeffer 
Vice President of Clinical Operations 
+1 210 615 6501 
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Investigation centers  
Number of centers 
One (1) 
Location of centers  
USA 
Clinical investigation design 
Objectives 
1. Obtain ultrasound only probe images and duplex probe 
OA/US (both gray scale and OA) probe images. This 
includes doppler and elastography imaging with the 
gray scale only ultrasound probe and ultrasound 
mode of the duplex OA/US probe.  
2. Provide breast and lymph node pathology results (except 
BI-RADS 1, 2 and 3 (as applicable)) 
Endpoints 
- 
Imagio (IUS+OA) vs Pathology Result assessment  
- 
Adverse Event Assessment 
Intervention 
Each subject will have the breast and, as applicable, the axillary 
lymph nodes imaged with the Gen 2 Imagio system. 
Duration of study visit 
One visit of up to 60 minutes unless Imagio imaging needs to be 
done again  
Total duration 
6 months 
Clinical investigation population 
Sample size 
A maximum of 38 healthy subjects ≥18 years old will be enrolled in 
the study. The 38 subjects will include the following:  
- 
4 BI-RADS 1  
- 
4 BI-RADS 2  
- 
8 subjects with BI-RADS 3 
- 
8 subjects with BI-RADS 4a 
- 
4 subjects with BI-RADS 4b 
- 
4 subjects with BI-RADS 4c 
- 
6 subjects with BI-RADS 5  
Inclusion criteria 
Subjects must meet the following criteria: 
- 
Signed and dated informed consent, prior to participating 
- 
At least 18 years of age 
- 
Have been referred for a breast US because of a breast 
lesion/mass finding via a palpable lump or per standard of care 
imaging 
- 
Willing and able to comply with protocol required scans 
Exclusion criteria 
Subjects who meet any of the following criteria will be excluded: 
- 
Pregnant or lactating 
- 
Has a condition or breast impediment at a location where the 
duplex probe will be in contact with the subject, which could 
interfere with the intended field of view 
- 
Photo-toxicity associated with currently taking, or having taken, 
photosensitizing agents within the previous 72 hours 
- 
Currently undergoing phototherapy 
- 
History of photosensitive disease or undergoing treatment for a 
photosensitive disease and is experiencing photosensitivity 
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- 
Previous adverse reaction to medical laser procedures 
- 
Prior benign excisional breast biopsy on breast of interest 
within the past 18 months 
- 
Current mastitis 
- 
-Prisoners 
Statistical analysis  
Analysis sets 
Full Analysis Set (FAS) Population 
The FAS population will be defined as all consented subjects who 
completed screening/enrollment and had a successful Imagio 
imaging procedure. The FAS population will be used as the 
secondary analysis population for the study endpoints.  
 
Per-protocol (PP) Population 
The PP population will be defined as all FAS subjects described 
above who meet the study eligibility criteria and have no other major 
protocol violations. The PP population will be used for primary 
analysis of the study endpoints. 
 
Safety Population 
The safety population will be defined as all subjects in whom the 
Imagio imaging procedure was attempted. This analysis population 
is the primary population for all safety analyses.  
Statistical design 
Summaries of each study endpoint and other 
clinical/demographic/safety data will be provided overall and by BI-
RADS of the breast masses to evaluate the consistency of the results 
across different type of breast masses with respect to BI-RADS. 
Additional analyses by clinical and/or demographic subgroups may 
be performed. Confidence intervals will be calculated at a 95% 
confidence level, unless otherwise specified. Given the nature of the 
study (i.e., early observational study), no formal statistical 
hypothesis testing is planned for this study.   
 
Baseline subject characteristics will be tabulated. Baseline subject 
characteristics will comprise the following: subject demographics 
(gender, age, race, ethnicity), height, weight, Fitzpatrick skin type,  
menopausal status, cup size, Standard of Care (SOC) imaging (i.e. 
mammography, Conventional Diagnostic Ultrasound (CDU), 
magnetic resonance imaging), Probability of Malignancy (POM) and 
BI-RADS, breast mass location and depth and size, lymph node 
location and size and depth, breast and lymph node medical/surgical 
history. For baseline subject characteristics that are continuous 
variables, the following descriptive statistics will be given: number 
of observations, mean, standard deviation, minimum, maximum, and 
median. For subject characteristics that are categorical variables, the 
frequency and percentage of subjects in each category will be 
provided. Summary tables will display demographic and baseline 
characteristics overall and by BI-RADS of the breast masses (3, 4a, 
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4b, 4c, 5).    
 
Adverse events, serious adverse events, and unanticipated serious 
adverse device effects will be summarized for the safety analysis 
population by presenting the number and percentage of subjects in 
each of these categories overall and by severity. 
 
 
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Table of Contents 
PROTOCOL SIGNATURE PAGE ...........................................................................................2 
INVESTIGATOR SIGNATURE PAGE ...................................................................................3 
SYNOPSIS .................................................................................................................................4 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ..........................................11 
REVISION HISTORY.............................................................................................................12 
 
INTRODUCTION ...........................................................................................................12 
1.1. 
Background and Significance ................................................................................12 
1.1.1. 
Imagio® Imaging System ............................................................................13 
1.1.2. 
SenoGram ....................................................................................................14 
1.1.3. 
Duplex OA/US Probe ..................................................................................15 
1.2. 
Indications for use ..................................................................................................15 
1.3. 
Prior Investigations ................................................................................................15 
1.3.1. 
Feasibility Study ..........................................................................................16 
1.3.2. 
System Verification Study ...........................................................................16 
1.3.3. 
PIONEER Study ..........................................................................................16 
1.3.4. 
Reader-02 Study...........................................................................................18 
1.3.5. 
MAESTRO Post-Marketing Study – Europe ...............................................18 
1.4. 
Study Motivation and Rationale ............................................................................19 
 
STUDY DESIGN AND OBJECTIVES ..........................................................................19 
 
SUBJECT POPULATION...............................................................................................19 
3.1. 
Inclusion/Exclusion Criteria ..................................................................................19 
3.2. 
Informed Consent...................................................................................................20 
3.3. 
Enrollment Failures ................................................................................................21 
 
STUDY PROCEDURE ...................................................................................................21 
4.1. 
Study Device ..........................................................................................................21 
4.2. 
Operator Credentials and Training .........................................................................21 
4.3. 
Subject Data ...........................................................................................................21 
4.4. 
Scan Procedure and Imagio Imaging .....................................................................22 
4.5. 
Post-imaging subject evaluation ............................................................................23 
4.6. 
Minimization of Bias .............................................................................................23 
 
SCHEDULE OF EVENTS ..............................................................................................24 
 
IMAGING DATA EVALUATIONS ..............................................................................24 
 
STATISTICAL CONSIDERATIONS.............................................................................25 
7.1. 
Statistical design and methods ...............................................................................25 
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7.1.1. 
General Statistical Considerations ...............................................................25 
7.1.2. 
Demographics, Medical History, and Other Baseline Characteristics .........25 
7.1.3. 
Analysis of Study Endpoints ........................................................................25 
7.1.4. 
Safety Analysis ............................................................................................26 
7.2. 
Sample size ............................................................................................................27 
7.3. 
Analysis populations ..............................................................................................27 
7.3.1. 
Full Analysis Set (FAS) Population .............................................................27 
7.3.2. 
Per-protocol (PP) Population .......................................................................27 
7.3.3. 
Safety Population .........................................................................................28 
7.4. 
Missing data ...........................................................................................................28 
7.5. 
Interim analysis ......................................................................................................28 
7.6. 
Statistical deviations ..............................................................................................28 
 
RISKS AND BENEFITS .................................................................................................28 
8.1. 
Potential Risks .......................................................................................................28 
8.2. 
Blinding..................................................................................................................29 
8.3. 
Anticipated Adverse Device Effects ......................................................................29 
8.4. 
Potential Benefits ...................................................................................................29 
8.5. 
Risk/Benefit Rationale ...........................................................................................30 
 
MEDICAL MONITOR ....................................................................................................30 
 ADVERSE EVENTS .......................................................................................................30 
10.1. Adverse Event Assessments ..................................................................................30 
10.1.1. 
Assessment of Severity ................................................................................31 
10.1.2. 
Evaluation of Severity..................................................................................32 
10.1.3. 
Evaluation of AE Relationship to Device ....................................................32 
10.1.4. 
Reporting of Adverse Events .......................................................................32 
10.1.5. 
Reporting Serious Device Adverse Event (SAE) and Unanticipated Serious 
Adverse Device Effect (USADE) Recording ..............................................33 
10.1.6. 
Reporting Adverse Event (AE) and Unanticipated Adverse Device Effect (UADE) 
Recording .....................................................................................................33 
10.2. Removal of Subjects from the Study .....................................................................33 
 PROTOCOL AND PROTOCOL AMENDMENT REVIEW AND     APPROVAL .....33 
11.1. IRB Committee ......................................................................................................33 
 MONITORING ................................................................................................................34 
 DEVICE STORAGE AND ACCOUNTABILITY .........................................................34 
 QUALITY ASSURANCE ...............................................................................................34 
 ETHICAL CONDUCT ....................................................................................................34 
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 PROTOCOL DEVIATIONS ...........................................................................................34 
16.1 
Corrective and Preventative Actions ...........................................................35 
 INVESTIGATOR DISQUALIFICATION CRITERIA ..................................................35 
 SUBJECT CONFIDENTIALITY ....................................................................................36 
 CASE REPORT FORMS AND STUDY RECORDS .....................................................36 
 DATA MANAGEMENT.................................................................................................36 
 RETENTION OF DATA .................................................................................................37 
 FINANCIAL DISCLOSURE ..........................................................................................37 
 PUBLICATIONS .............................................................................................................37 
 LITERATURE CITED ....................................................................................................37 
 
 
 
 
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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
AE 
Adverse Event 
BI-RADS 
Breast Imaging-Reporting and Data System 
CDU 
Conventional Diagnostic Ultrasound 
CRF 
Case Report Form 
CRO 
Clinical Research Organization 
eCRF 
Electronic Case Report Form 
EDC 
Electronic Data Capture 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
IRB 
Institutional Review Board 
IUS 
Imagio ultrasound using the duplex probe  
LOM 
Laser Optical Movies  
LOM/POM 
Likelihood/Probability of Malignancy 
MedDRA 
Medical Dictionary for Regulatory Activities 
NLR 
Negative Likelihood Ratio 
OA 
Opto-Acoustic 
OA/US 
Refers to the use of the Imagio system (i.e. both IUS and OA function 
combined + SenoGram) 
POM 
Probability of Malignancy 
QA 
Quality Assurance 
QAR 
Quality Assurance Radiologist 
SADE 
Serious Adverse Device Effect 
SAE 
Serious Adverse Event 
SOC 
Standard of Care 
UADE 
Unexpected Adverse Device Effect 
USADE 
Unanticipated Serious Adverse Device Effect 
 
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REVISION HISTORY  
Version  
Date 
Summary of Changes 
1.0 
5/12/2021 
Not applicable, first release.  
2.0 
7/14/2021 
Section 2 Study Design and Objectives – clarification to 
number 1:  This includes doppler and elastography imaging 
with gray scale only ultrasound probe and ultrasound mode of 
the OA/US Duplex probe 
Section 3 - Removal of inclusion criteria 5 
Section 4.3 Subject Data – Clarification to images required 
Section 4.4 Scan Procedure and Imagio Imaging – 
Clarification to number 3 – biopsy imaging required for up to 
3 subjects and timepoints for biopsy imaging clarified per 
probe 
Other Administrative Revisions 
  
3.0 
8/17/2021 
Remove exclusion for focal pain 
Administrative Revisions 
4.0 
11/10/2021 
Section 4.4 Remove the need for CDU Elastography and 
Doppler imaging prior to Imagio Elastography and Doppler 
Imaging.  Administrative Revisions 
 
 INTRODUCTION 
1.1. Background and Significance 
Breast cancer is the most common cause of cancer-related death for women [1]. According to the 
World Health Organization, the highest rate of breast cancer cases was observed in Europe in 2012 
[2]. Unfortunately, breast cancer remains difficult to definitively diagnose without performing a 
biopsy, despite the existence of multiple screening and diagnostic imaging methodologies.  
 
There are multiple reasons for the bias toward performing a biopsy beyond the psychological burden 
of a suspicious mass in the breast. Breast cancer is a heterogeneous disease with variability in 
genetic and hormonal influences, which contribute to its varied pathobiology [3]. Consequently, the 
clinical and radiographic manifestations and histopathology of breast cancer are variable. 
Furthermore, breast tissue is also heterogeneous, containing fat, glandular and fibrous tissue. The 
structure of the breast and the nature of breast cancer present a great challenge for imaging 
technologies to detect cancerous masses reliably and to differentiate them from benign masses.  
 
Seno Medical Instruments, Inc.’s imaging technology attempts to address this challenge by utilizing 
two very different real-time spatially co-registered and temporally interleaved modalities, opto-
acoustic (OA) and diagnostic ultrasound (US), thus providing co-registered and temporally fused 
functional and anatomical information. This combination has the potential to add diagnostic 
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information to help the radiologist evaluate breast masses in an efficient and cost-effective manner. 
Device Description 
1.1.1. Imagio® Imaging System  
The Imagio Breast Imaging System (Figure 1A) is a diagnostic functional and morphologic imaging 
system that combines optoacoustic (OA) imaging with conventional diagnostic ultrasound (US) 
imaging to aid in diagnostic evaluation of breast lesions. The Imagio Breast Imaging System 
acquires, processes, and displays co-registered OA and US images to provide structural and 
functional information about breast abnormalities in real-time. OA/US imaging technology 
combines the high contrast resolution of optical imaging with the high spatial resolution and deep 
penetration of ultrasound imaging.  
 
In OA imaging (also known as photoacoustic imaging in the literature), short nanosecond pulses of 
laser light are delivered into tissue. When that light is absorbed, for instance by hemoglobin 
molecules found in blood, those molecules undergo thermoelastic expansion and generate acoustic 
pressure signals that can be detected at the tissue surface using a handheld probe (Figure 1B & 1C). 
The probe has optical windows that deliver light to tissue and an array of piezoelectric transducers 
that detect acoustic signals. The received OA signal increases with both the absorption strength and 
concentration of light-absorbing molecules. Two strong absorbers in tissue are oxygenated and 
deoxygenated hemoglobin found in blood vessels. By performing OA imaging using two near-
infrared optical wavelengths (757 nm and 1064 nm) for which oxygenated and deoxygenated 
hemoglobin absorb light differently, the Imagio Breast Imaging System can estimate the relative 
oxygen saturation of hemoglobin in tissue. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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A 
B 
C 
D 
FIGURE 1. IMAGIO BREAST IMAGING SYSTEM (A); GRAYSCALE U/S PROBE (B); OA/US 
PROBE (C); EXAMPLE IMAGE AS 6-ON-1 DISPLAY (D) 
 
In addition to OA imaging, the Imagio Breast Imaging System provides conventional ultrasound 
imaging capabilities such as 2D B-mode (amplitude), color Doppler, power Doppler, and pulsed 
wave Doppler for blood/fluid flow analysis, user-adjustable image display settings, and the ability 
to measure and annotate images. The Imagio Breast Imaging System can provide overlaid OA/US 
images that co-register anatomical US B-mode images with functional OA mapped information in 
real-time. These images are presented as a 6-on-1 (six-up) display (Figure 1D). The grayscale US 
image provides morphologic information, whereas the OA/US images provide both morphologic 
and functional information. The morphologic information provided by OA/US includes: the 
presence or absence of detectable blood vessels as well as the number, size, shape, orientation, and 
polymorphism of blood vessels. The functional information provided by OA/US includes the 
relative amount of hemoglobin and relative oxygen saturation of hemoglobin. 
 
The Imagio Breast Imaging System was determined to be a Class 3B laser product for externally 
accessible emission from the handheld probe and a Class 4 laser product for internally accessible 
emission (during device maintenance/service). The Imagio’s laser subsystem is comprised of a laser 
head, laser controller, temperature controller, and footswitch. The laser head generates the pulsed 
laser emissions, directing them into an optical port which is coupled with a fiber optic cable 
connecting to the handheld probe. The laser controller provides real time control of laser emission 
timing and energy levels. The temperature controller regulates the laser head temperature to 
maintain optimal laser head operation by heating or cooling water that is then pumped through 
portions of the laser head at a constant flow rate. The foot switch must be engaged by the operator 
to allow laser light emission. Light is delivered from the fiberoptic cable through two optical 
windows in the face of the handheld probe to illuminate underlying tissue. During laser light 
emission, users, subjects and observers are required to wear provided laser safety eyewear meeting 
appropriate optical density specifications. The ultrasound imaging component of the device is based 
on conventional, currently existing ultrasound imaging principles. The ultrasound only probe is 
currently FDA approved as part of  510K K191007. 
1.1.2. SenoGram 
To assist the users in assessing Breast Imaging-Reporting and Data System (BI-RADS) 
classification, the Imagio Breast Imaging System is used in parallel with a SenoGram®. The 
SenoGram is a software tool, using machine learning, that helps the radiologist predict the 
probability of malignancy (POM) based on a set of reader-assigned feature scores and other relevant 
data. The purpose of the SenoGram is to help the users more accurately combine their various 
feature scores into a single overall score (POM). Unlike some of the more commonly used 
computer-aided diagnosis systems that use medical images as the input, SenoGram input comes 
from the users. The user enters 5 feature scores for internal ultrasound (IUS), 5 feature scores for 
OA and 4 other features: subject age, maximum diameter depth to posterior wall of mass, size of 
lesion, and mammographic BI-RADS density category (where available). IUS above refers to the 
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internal ultrasound functionality of the Imagio Breast Imaging System (i.e., without OA).When all 
data has been entered, the user interface graphically displays the SenoGram Likelihood/Probability 
of malignancy (LOM/POM) on a scale of 0 to 100. If the user assigns correct feature scores, the 
SenoGram will perform well; if the user assigns incorrect feature scores, the SenoGram will not 
perform well. The SenoGram classification model was trained using feature scores from the 
PIONEER Pivotal Readers, including feature scores from the expert reader assigned during the 
PIONEER Pivotal Study. 
1.1.3. Duplex OA/US Probe 
The handheld duplex OA/US probe (Figure 1C) comprises a linear 256 element transducer that can 
be used for conventional diagnostic ultrasound imaging.  In addition, the probe contains optics that 
are designed to transmit the system laser energy, which is uniformly distributed across two optical 
windows on the face of the probe. This delivers the light safely and uniformly into the tissue, 
creating OA signals that are then received by the ultrasound transducer in the probe to create co-
registered, temporally interleaved and fused functional OA and anatomic diagnostic ultrasound. The 
OA duplex probe footprint is no greater than 70 mm long by 40 mm wide, with the active ultrasound 
transducer size being 50 mm long by 4 mm wide. 
1.2. Indications for use 
The Imagio Breast Imaging System is indicated for use by a trained and qualified healthcare 
provider for evaluation of palpable and non-palpable breast abnormalities in adult subjects who are 
referred for a diagnostic imaging breast work-up, following clinical presentation or either screening 
or diagnostic mammography or other imaging examinations. The ultrasound mode should be 
initially used in a targeted fashion, to assess any focal area(s) of clinical or imaging concerns. In 
ultrasound mode, the device can be used to assign a BI-RADS category to either breast tissue or a 
mass that is causing clinical or imaging concerns. Masses that are classified as BI-RADS categories 
3 through 5 can then be assessed using the Opto-Acoustic (OA) mode. In the OA mode, the Imagio 
Breast Imaging System provides information about the central nidus, boundary and peripheral 
zones, based on vascularity and blood oxygen saturation of the imaged tissues, to assist in the 
diagnosis of the benign or malignant mass(es) of interest. For ultrasound BI-RADS 3-5 masses, 
using the OA features of the mass, allows for improved classification of the mass of interest as 
compared to ultrasound alone. The OA mode is not indicated for ultrasound BI-RADS 1 and 2 
findings. The Imagio Breast Imaging System includes an artificial intelligence (AI) based software 
function to assist the users in assessing the BI-RADS Classifications. This device is not intended to 
be used as a replacement for mammographic screening or for definitive pathologic diagnosis. 
1.3. Prior Investigations 
Early research proof of concept studies provided the company with supporting data to modify and 
enhance diagnostic performance of the Imagio. Optimization was achieved using the functional 
characteristics of real-time opto-acoustic imaging performed at 2 wavelengths of Nd:YAG 
(Neodymium-doped Yttrium Aluminium Garnet) and Alexandrite lasers. Imagio was subsequently 
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tested on phantoms simulating breast tissue. 
1.3.1. Feasibility Study 
A feasibility study was conducted using a total of 155 subjects to determine the ability of Imagio 
Gen 1 to detect benign and malignant features. The feasibility study provided data to further develop 
an algorithm to identify separate sets of features that characterize benign and malignant disease. The 
study included subjects with negative diagnostic ultrasound results and positive diagnostic 
ultrasound, including results of varying histopathology diagnoses, sizes, locations, depths, ages, 
ethnicities, and races. These subject-specific characteristics were intended to identify any unusual 
subpopulations. Exclusions for this study on the use of OA included pregnant subjects, males, 
subjects with obscured views, and subjects on medications that could activate dermatologic 
conditions (e.g. photosensitive skin conditions). 
 
The feasibility study demonstrated internal and external features representing tumor morphology 
[4-6].  The reader assigned POM was significantly higher for malignant masses than for benign 
masses [7]. The specificity was 8% higher for Imagio than for conventional diagnostic ultrasound 
(CDU) (24% for OA vs 16% for CDU) among those with benign disease, there was >40% relative 
reduction in the numbers with POM >2% in support of biopsy sparing [8]. Among subjects with 
benign disease, 54% of BI-RADS 4a subjects and 23% of BI-RADS 4b subjects had an Imagio 
POM <2% in support of downgrading [9].  There were no adverse events reported. 
1.3.2. System Verification Study 
A subsequent System Verification Study was performed, enrolling 44 subjects. The study was 
conducted at South Texas Radiology Imaging Center to evaluate the dual-wavelength performance 
of the Imagio Gen 1 Breast Imaging System in classifying suspicious breast masses for final tuning 
of the OA colorization algorithm prior to embarking upon the pilot phase of the PIONEER Study. 
The objectives of the System Verification Study were met, and further conclusions could be drawn. 
There were no reported adverse events (AEs). 
1.3.3. PIONEER Study 
A prospective multi-center trial, the PIONEER Study, was performed with  the Imagio Gen 1 
enrolling 2105 subjects enrolled across 16 US clinical sites [10]. Subjects were enrolled upon 
confirmation of having masses classified as BI-RADS 3-5 by CDU evaluation. Images were 
collected at the clinical sites and sent to an independent Imaging Core Lab, which processed and 
checked image quality prior to images being processed and read by seven independent readers. 
 
Of the 1,972 subjects in the Safety Population, which included all subjects exposed to an OA scan 
(including masses that were a quality assurance [QA] review fail), the overall mean age was 49 years 
(45 years for subjects with benign lesions and 58 years for subjects with malignant (cancer) lesions). 
The majority of the population was white (n = 1,558; 79%). Seventy-two subjects (<4%) were noted 
to have more than one mass. 
 
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The study had one primary effectiveness endpoint and five secondary effectiveness endpoints, all 
of which were achieved with statistical significance after accounting for multiplicity testing. 
 
Analyses were conducted on the intent to diagnose the population which consisted of any subject 
included in the Safety Population with at least one mass that passed QA review and had a valid 
biopsy (as applicable for BI-RADS 3, 4 and 5). Results were as follows: 
 
• Primary effectiveness endpoint (OA specificity) 
o An overall 14.9% absolute specificity advantage for OA/US vs IUS (p <0.0001) 
• Secondary effectiveness endpoint (OA/US sensitivity) 
o OA/US sensitivity was demonstrated to be non-inferior to IUS (p <0.0001), relative 
to a pre-defined 5% non-inferiority margin 
• Secondary effectiveness endpoint (OA/US specificity) 
o OA/US specificity was 43.0% which rejected the pre-planned 40% null hypothesis, 
supporting the OA/US secondary sensitivity hypothesis (lower bound of 99%, 
confidence bound >40%) 
• Secondary effectiveness endpoint (OA/US sensitivity) 
o OA/US sensitivity was 96.0% which rejected the pre-planned 90% null hypothesis, 
supporting the OA/US secondary sensitivity hypothesis (lower bound of 99%, 
confidence bound >94%) 
• Secondary effectiveness endpoint (downgrading of BI-RADS in benign masses) 
o OA/US reads downgraded 48.6% of IUS BI-RADS 3 benign masses to BI-RADS 2; 
a 25% alternative hypothesis was tested relative to no advantage (null hypothesis) 
o OA/US reads upgraded 21.3% of IUS BI-RADS 3 benign masses to 4a+ 
o OA/US reads downgraded 29.1% (1,578/5,418) of IUS BI-RADS 4a+ benign masses 
to a BI-RADS 2 or 3 
o Overall, in benign masses, OA/US downgraded 34.5% of BI-RADS (BI-RADS 4a+ 
to ≤3, or 3 to 2), in contrast to 6.0% being upgraded, for a net downgrade percentage 
of 28.5% 
• Secondary effectiveness endpoint (upgrading of BI-RADS in malignant masses) 
o OA/US reads resulted in upgrading 47.0% of malignant masses relative to IUS; a 
10% alternative hypothesis was to be tested relative to no advantage (null 
hypothesis). OA reads resulted in downgrading 27.3% of these reads.  
o Thus, for malignant masses, the net gain (using the same denominator) was 19.7% 
(47.0% - 27.3%). 
 
There were no serious adverse device effects (SADEs), unexpected adverse device effects 
(UADEs), or deaths reported during the Study. Ten subjects (0.5%) reported 11 AEs that were 
considered related to the OA procedure.1 None were serious. All were mild in severity. 
 
1 7 events of paraesthesia reported in 7 subjects (0.4%); and 1 event of burns second degree, procedural pain, 
erythema and skin warm reported in individual subjects (0.1% each). 
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1.3.4. Reader-02 Study  
The Reader-02 Pivotal Study met its primary endpoint and demonstrated that Imagio Breast 
Imaging Gen 1 System has better specificity than IUS at a fixed sensitivity of 98%. Specificity at a 
fixed sensitivity of 98% was analyzed using multi-reader multi-case (MRMC) analysis 
(Obuchowski-Rockette Dorfman-Berbaum-Metz (ORDBM) MRMC 2.51). Mean (average over all 
readers) fixed specificity (fSp) was found to be higher with statistical significance (two-sided 
p=0.027) for IUS+OA (47.2%, 95% CI=[35.9%,58.5%]) compared to IUS alone (38.2%, 95% CI= 
[24.9%, 51.6%]), with a difference in fSp of 9.0% (95%CI=[1.0%, 17.0%]). Thus, IUS+OA 
achieved the primary endpoint in the Reader-02 Pivotal Study. The decrease (i.e., improvement with 
IUS+OA compared to IUS alone) in  negative likelihood ratio (NLR) could not be established with 
statistical significance because the observed relative NLR (the ratio in NLR for IUS+OA and IUS) 
was 0.896 with a 95% CI= (0.693, 1.11) which included 1, indicating that no evidence of a 
difference in NLR was found. Based on descriptive statistics that do not control type I error and that 
cannot be generalized outside this particular study, the observed mean positive likelihood ratio 
(PLR) was 1.959 (95% CI: 1.870, 2.051) for IUS+OA (only as a descriptive result this suggests 
that averaging over all 15 readers in the study a positive test read (POM>2%) was observed about 
2 times more often among cases with cancer, compared to those without cancer), and the mean PLR 
was reported as 1.548 (95% CI: 1.498, 1.597) for IUS alone (only as a descriptive result this 
suggests that averaging over all 15 readers in the study a positive test read (POM>2%) was observed 
about 1.5 times more often among cases with cancer, compared to those without cancer). 
The descriptive observed relative PLR was 1.281 (95% CI: 1.231, 1.298). 
 
Partial area under the curve (pAUC) was lower in the hierarchical test order than NLR, which failed 
to achieve significance. Consequently, pAUC results are not part of the claim structure and are 
reported as descriptive statistics. The mean unscaled pAUC was 0.0244 (95% CI: 0.0230, 0.0258) 
for IUS+OA and 0.0205 (95% CI: 0.0191, 0.219) for IUS alone, with a difference of 0.0039. All 
readers had a larger point estimate of pAUC for IUS+OA than for IUS alone. 
 
Of the 480 subjects who contributed masses for reading in the Reader-02 study, 16 (3.3%) 
experienced at least one AE during the study, of which 6 (1.3%) occurred on the date of the Imagio 
procedure. Three subjects (0.6%) had at least one Imagio procedure-related AE. There were no AEs 
that were graded as severe; 14 (2.9%) were considered mild and two (0.4%) were graded 
moderate. All 
Imagio 
procedure-related 
AEs 
were 
considered 
mild. 
Two 
subjects 
(0.4%) experienced a serious adverse event (SAE), neither of which was Imagio procedure-related; 
one experienced rupture of a saline breast implant, requiring revision surgery, and one was 
diagnosed with Stage I non-small cell lung cancer and underwent resection of the affected lobe in 
the right lung. There were no SADEs or UADEs. 
1.3.5. MAESTRO Post-Marketing Study – Europe 
Another prospective multi-center study included results of 209 subjects with the Imagio Gen 1 
enrolling 215 breast masses classified as BI-RADS 4a or 4b by CDU [11]. Subjects were enrolled 
at 5 sites between 2015 and 2016. Masses that were classified as BI-RADS 4a or 4b by CDU were 
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entered into the MAESTRO study, and all were biopsied. The absolute OA/US specificity was 41.1% 
and the improvement in specificity with OA/US was also 41.1% because, by definition, the CDU 
findings in any benign mass classified as BI-RADS 4a or 4b would be falsely positive and 
specificity for CDU will be 0%. The OA/US sensitivity in MAESTRO was 95.5%. The NLR was 
0.11. There was one moderate adverse event, a hematoma post biopsy, that was unlikely related to 
the OA/US procedure. 
1.4. Study Motivation and Rationale 
This study performs the first clinical evaluation of the Gen 2 Imagio System in a clinical setting to 
assess image quality with both the IUS ultrasound only probe and the OA/US (duplex probe). This 
study assesses palpable and non-palpable breast abnormalities in adult subjects who are referred for 
a diagnostic imaging breast work-up, following clinical presentation, or screening mammography, 
or diagnostic mammography, or other imaging examinations. 
 STUDY DESIGN AND OBJECTIVES  
This clinical investigation is designed to evaluate the images produced by the Gen 2 Imagio System 
in a clinical setting.  
 
The primary objectives of this study are: 
 
1. Obtain ultrasound only probe images and OA/US (both gray scale and OA) probe images. This 
includes doppler and elastography imaging with gray scale only ultrasound probe and 
ultrasound mode of the duplex OA/US probe.  
2. Provide breast and lymph node pathology results (except BI-RADS 1, 2 and 3 (as 
applicable)) 
 SUBJECT POPULATION 
A maximum of 38 healthy subjects ≥18 years old will be enrolled in the study. The 38 subjects will 
include the following via Mammo (BI-RADs 1 and 2) or CDU: 
  
• 4 Subjects with BI-RADS 1  
• 4 subjects with BI-RADS 2  
• 8 subjects with BI-RADS 3 
• 8 subjects with BI-RADS 4a 
• 4 subjects with BI-RADS 4b 
• 4 subjects with BI-RADS 4c 
• 6 subjects with BI-RADS 5  
3.1. Inclusion/Exclusion Criteria  
Inclusion Criteria: Subjects must meet all the following criteria to be included in the study: 
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1. 
Has a signed and dated informed consent, prior to initiation of any study-related 
activities. 
2. 
Is at least 18 years of age. 
3. 
Have been referred for a breast US because of a breast lesion/mass finding via a palpable 
lump or per standard of care imaging.  
4. 
Is willing and able to comply with protocol-required scans. 
 
Exclusion Criteria: Subjects who meet any of the following criteria will be excluded: 
 
1. 
Is pregnant or lactating. 
2. 
Has a condition or breast impediment (i.e. insect bites, poison ivy, rash, open wounds, 
chafing of the skin, scar, tattoos, moles, hematoma, nipple rings, etc.) which could 
interfere with the intended field of view. 
3. 
Is experiencing photo-toxicity associated with currently taking, or having taken, 
photosensitizing agents within the previous 72 hours such as some sulfur containing 
drugs, ampicillin, tetracycline. 
4. 
Is currently undergoing phototherapy.   
5. 
Has a history of any photosensitive disease (e.g., porphyria, lupus erythematosus) or 
undergoing treatment for a photosensitive disease and is experiencing photosensitivity. 
6. 
Has had an adverse reaction in the past to medical laser procedures, such as laser hair 
removal or laser tattoo removal. 
7. 
Has had prior benign excisional breast biopsy on breast of interest within the past 18 
months. 
8. 
Currently has mastitis. 
9. 
Prisoners 
These inclusion and exclusion criteria will be confirmed prior to the start of each scan procedure. 
3.2. Informed Consent  
Written informed consent will be obtained from every subject. Once consented, the subject will be 
considered enrolled in the study. Screen failure data will be collected. A subject is considered a 
screen failure post signing the informed consent. 
 
The informed consent interview will be performed, and each subject will be given personal time 
and the opportunity to ask questions. The time for decision making prior to giving consent or 
declining participation will include the time to slowly read and to discuss the consent form. The 
IUS (ultrasound only probe) and OA/US (duplex probe) imaging procedure will be performed after 
consent. The subject will receive a copy of the Informed Consent Form for their records. 
 
Upon enrollment, subjects receive a subject number that will be used on all documentation for the 
subject throughout the study. Subject numbers will be assigned in ascending order, and numbers 
will not be omitted or reused. The subject number is coupled with the site identification number for 
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unique identification of each subject.  
3.3. Enrollment Failures 
Subjects deemed enrollment failures according to the quality assurance review or failure to complete 
the biopsy evaluations for BI-RADS 3 (as applicable), BI-RADS 4 and 5 will be replaced during 
the active enrollment period of the study. The following subjects will be considered enrollment 
failures and will be followed until all AEs are resolved: 
 
• Subjects found to be ineligible after the scan is conducted 
• Subjects for whom the study device scans do not pass review (Imaging Core Lab Quality 
Assurance Radiologist (QAR) review) 
• BI-RADS  3 (as applicable) 4 and 5 subjects who have incomplete or unusable biopsy 
results   
 STUDY PROCEDURE 
4.1. Study Device 
The study will use the Gen 2 Imagio to generate grayscale ultrasound only (IUS )and OA/US images 
as applicable depending on the subjects mass or abnormality. The Gen 2 OA/US duplex probe and 
device is an investigational device in the US.  
The Imagio OA/US probe and device will not be used to make any subject 
clinical decisions during this study.  
4.2. Operator Credentials and Training 
The Imagio system will be operated only by a trained and qualified healthcare provider.  
The Subject, the Imagio Breast Imaging System User, and everyone else in the scan room must 
always wear Seno approved laser protective eyewear when the Imagio Breast Imaging System is 
powered on and the laser is enabled 
4.3. Subject Data 
Data to be collected: 
• 
Gender 
• 
Age 
• 
Race 
• 
Ethnicity 
• 
Fitzpatrick skin type  
• 
Breast and lymph node medical/surgical history 
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• 
Height 
• 
Weight 
• 
Inclusion/Exclusion criteria review 
• 
Breast Mass location and depth and size 
• 
Breast Mass feature Scores  
• 
Image Artifact Score   
• 
Lymph node location and size and depth  
• 
Lymph Node Scoring 
• 
Standard of Care (SOC) Imaging POM and BI-RADS 
• 
Imagio IUS POM and BI-RADS using ultrasound only probe and OA/US POM and 
BI-RADS post SenoGram for breast mass 
• 
IUS ultrasound only probe imaging,  duplex OA/US probe imaging (doppler and 
elastography images as applicable with duplex OA/US probe in ultrasound only 
mode), and SOC imaging for each breast study mass and study lymph node (as 
applicable) 
• 
Breast biopsy images for ultrasound only probe and duplex OA/US probe in ultrasound 
only mode and pre and post biopsy duplex OA/US probe in OA mode images as 
possible 
• 
Mass and lymph node pathology  and surgical report for BI-RADS 3 (as applicable), 
4 and 5 masses 
• 
Device Adverse Events/Effects 
• 
Reason for End of Study   
4.4. Scan Procedure and Imagio Imaging 
Each subject depending on BI-RADS score will have the breast and as applicable the axillary lymph 
nodes imaged with the ultrasound only probe and the Imagio OA/US duplex probe. The expected 
duration of a subject’s participation would be a total of one visit. The duration of the imaging session 
will be determined by the subject’s availability and may be up to approximately 60 minutes in 
duration. If there are technical difficulties, the scan procedure may extend beyond this timeframe. 
In addition, if there are technical issues with the saved images, the subject may be asked to return 
to repeat the scan procedure.   
Imagio imaging will be done per the Scan Protocol for each subject. 
 
1. Elasto and/or Doppler and scan with gray-scale ultrasound only probe as possible 
2. Elasto and/or Doppler and Scan with ultrasound portion of the duplex OA/US probe as 
possible. 
3. Biopsy imaging as possible (up to 3 subjects only) with the gray scale only probe and the 
duplex OA/US probe. 
 
a. Gray-scale ultrasound only probe and OA/US duplex probe in ultrasound only 
mode: 
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i. Pre biopsy.  
ii. Needle pre-fire and post fire 
iii. Post biopsy  
 
b. Duplex OA/US probe in OA mode 
 
i. Pre biopsy 
ii. Post biopsy 
 
 
4.5. Post-imaging subject evaluation 
Immediately after the scan procedure, the subjects will be questioned to assess for: 
 
• Any adverse device event/effect 
 
4.6. Minimization of Bias 
Potential for bias during this clinical investigation has been minimized by a well-controlled design, 
expected conduct under the terms of an approved study protocol, use of specific inclusion and 
exclusion criteria, careful definitions for study procedures and outcomes and prospectively defined 
methods of data analysis. No blinding/masking will be conducted. 
 
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 SCHEDULE OF EVENTS 
Table 1 shows the schedule of events for this study 
 
Table 1. Schedule of events 
Evaluation 
Screening/ 
Enrollment/Baseline 
Imaging  
Visit1 
Day 0 
Breast and Lymph Node Biopsy Visit (if 
not done on same day as baseline) 
May be repeated if issue with initial 
biopsy  
 
*Up to 45 days from baseline  
Breast and 
Lymph Node 
(as applicable) 
Surgery 
 
 
  *Up to 60 
Days from 
baseline 
Informed Consent 
X 
 
 
Breast/Lymph Node Medical History 
X 
 
 
I/E Criteria 
X 
 
 
CDU (including elastography and 
doppler as applicable) and SOC 
Imaging  
X 
 
 
Imagio Imaging (including 
Elastography and Doppler as 
applicable) of breast and lymph 
node (as applicable). This includes 
biopsy imaging as applicable 
X2 
X 
  
SOC + Imagio Imaging uploaded to 
Core Lab 
X 
 
 
SOC Imaging, Imagio IUS and/or 
IUS+OA POM and BI-RADS Scoring 
X 
 
 
Pathology/Surgical Reports as 
applicable 
 
X 
X 
Adverse Event/Effect 
X 
 
 
End of Study 
X3 
X3 
X3 
1The Screening/Enrollment/ Imaging, and Biopsy Visits may take place on the same date as the discretion of the investigator 
2 BI-RADS 1 and 2 abnormalities will be scanned with the ultrasound only probe and possibly the duplex OA/US probe. BI-RADS 3-5 
masses will be scanned with both probes. 
3 Study will end at the final definitive diagnosis of the breast mass which could include confirmation via surgery report  
 IMAGING DATA EVALUATIONS 
All imaging results will be managed by an independent Imaging Core Lab, who will perform QA 
review of de-identified collected images and assembly of the designated image sets. A QA evaluator 
who is a QAR will perform an image/lesion pairing assessment once the core lab performs the QA 
of all demographic data, checks image completeness and quality, and resolves all discrepancies. 
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The QARs will check if lesions/masses scanned with Imagio at the site match the same lesion(s)/ 
mass(es) seen on all imaging modalities at the site as well as on the image guided biopsy.  
The Imaging Core Lab to be used in the study is: 
American College of Radiology (ACR)  
Center for Research and Innovation  
50 South 16th Street 
Suite 2800 
Philadelphia, PA 19102 
United States of America 
 STATISTICAL CONSIDERATIONS 
7.1. Statistical design and methods 
7.1.1. General Statistical Considerations 
Summaries of each study endpoint and other clinical/demographic/safety data will be provided 
overall and by BI-RADS of the breast masses to evaluate the consistency of the results across 
different type of breast masses with respect to BI-RADS. Additional analyses by clinical and/or 
demographic subgroups may be performed. Confidence intervals will be calculated at a 95% 
confidence level, unless otherwise specified. Given the nature of the study, no formal statistical 
hypothesis testing is planned for this study.  
7.1.2. Demographics, Medical History, and Other Baseline Characteristics 
For the full analysis set (FAS) population (defined in section 6.3), baseline subject characteristics 
will be tabulated. Baseline subject characteristics will comprise the following: subject 
demographics (gender, age, race, ethnicity), height, weight, Fitzpatrick skin type,  menopausal 
status, cup size, Standard of Care (SOC) imaging (i.e. mammography, CDU, and magnetic 
resonance imaging) POM and BI-RADS, breast mass location and depth and size, lymph node 
location and size and depth, breast and lymph node medical/surgical history.   
 
For baseline subject characteristics that are continuous variables, the following descriptive statistics 
will be given: number of observations, mean, standard deviation, minimum, maximum, and median. 
For subject characteristics that are categorical variables, the frequency and percentage of subjects 
in each category will be provided. Summary tables will display demographic and baseline 
characteristics overall and by BI-RADS of the breast masses (3, 4a, 4b, 4c, 5).    
7.1.3. Analysis of Study Endpoints 
Breast mass site pathology diagnosis (benign, malignant, high risk) will be summarized by 
presenting the frequency and percentage of breast masses in each BI-RADs category. A listing of 
the pathology results (benign specification, malignant specification, high risk specification) will be 
presented.  
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Breast mass feature scores 
Each of the following breast mass feature score data will be summarized by presenting the mean, 
standard deviation, number of observations, median, quartiles, minimum, and maximum: 
• For IUS:  
o External peripheral zone score (range 0-5) 
o External capsular/boundary zone score (range 0-6) 
o Internal shape score (range 0-5) 
o Internal sound transmission score (range 0-5) 
o Internal echotexture score (range 0-5) 
• For OA/US:  
o External peripheral zone radiating vessel score (range 0-5) 
o External capsular/boundary zone vessel score (range 0-6) 
o Internal vessel score (range 0-5) 
o Internal deoxygenated blush score (range 0-5) 
o Internal total hemoglobin score (range 0-5) 
 
3. Imagio/OA/US POM and BI-RADS for breast using SenoGram 
Investigator POM for breast using SenoGram will be summarized by presenting the mean, standard 
deviation, number of observations, median, quartiles, minimum, and maximum. Investigator BI-
RADS for breast using SenoGram will be summarized by presenting the frequency and percentage 
of breast masses in each BI-RADS category. SenoGram LOM for breast will also be summarized 
by presenting the mean, standard deviation, number of observations, median, quartiles, minimum, 
and maximum. 
 
Lymph node scoring 
Lymph node scoring (shape score, cortical thickness score, margin score, hilar compression-
displacement score, and size) will be summarized by presenting the frequency and percentage of 
lymph nodes in each category. The total score will be summarized by presenting the mean, standard 
deviation, number of observations, median, quartiles, minimum, and maximum. Imagio IUS 
classification of lymph node will be summarized by presenting the frequency and percentage of 
lymph nodes in each category.    
 
Lymph node site pathology (histopathology and cytopathology) results will be summarized by 
presenting the frequency and percentage of lymph nodes in each histopathology (positive for 
malignancy, negative for malignancy, indeterminate, extracapsular extension) and cytopathology 
(positive malignancy, negative for malignancy, indeterminate, other) category. A listing of 
pathology results will also be presented.     
7.1.4. Safety Analysis  
Adverse events (AE), serious adverse events (SAE), and unanticipated adverse device effects 
(UADE) will be summarized for the safety analysis population by presenting the number and 
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percentage of subjects in each of these categories overall and by severity and by the Medical 
Dictionary for Regulatory Authorities (MedDRA) System Organ Class / Preferred Term. A similar 
summary will also be provided for device-related and/or procedure-related AEs, SAEs, and UADEs. 
The number and percentage of subjects who withdrew from the study due to an AE will be presented 
overall and by System Organ Class and Preferred Term.  
 
A listing of all AEs will be provided that includes details such as AE term (System Organ Class and 
Preferred Term), days from procedure, seriousness, relatedness to procedure and device, severity, 
action taken, outcome.    
7.2. Sample size 
A maximum of 38 healthy subjects ≥18 years old will be enrolled in the study. The 38 subjects will 
include the following:  
 
• 4 subjects with BI-RADS 1- can be recruited from BI-RADS 0.  (Ex. Mammographic 
asymmetry caused by normal breast tissue, breast pain, etc.) 
• 4 subjects with BI-RADS 2- can be recruited from BI-RADS 0. (Ex. palpable lipoma, 
simple cyst, ductasia, angioma, etc.) 
• 8 subjects with BI-RADS 3 
• 8 subjects with BI-RADS 4a 
• 4 subjects with BI-RADS 4b 
• 4 subjects with BI-RADS 4c 
• 6 subjects with BI-RADS 5  
 
Given the nature of the study no formal hypothesis testing is planned, and formal power and sample 
size calculations were not performed. A sample size of 38 subjects is deemed adequate to provide 
a cross section of breast masses/lesions and lymph nodes.   
7.3. Analysis populations 
7.3.1. Full Analysis Set (FAS) Population 
The FAS population will be defined as all consented subjects who completed screening/enrollment 
and had a successful Imagio imaging procedure. The FAS population will be used as the secondary 
analysis population for the study endpoints.  
7.3.2. Per-protocol (PP) Population 
The PP population will be defined as all FAS subjects described above who meet the study 
eligibility criteria and have no other major protocol violations. The PP population will be used for 
primary analysis of the study endpoints.  
 
Within the PP population, summaries of each study endpoint will be provided overall and by 
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subgroups based on BI-RADS (3, 4a, 4b, 4c, 5) of the breast masses. 
7.3.3. Safety Population 
The safety population will be defined as all subjects in whom the Imagio imaging procedure was 
attempted. This analysis population is the primary population for all safety analyses.  
7.4. Missing data 
Given the nature of the study, analyses will be performed for observed (available) cases only. No 
imputation of missing data will be performed.   
7.5. Interim analysis 
Interim analyses are not planned for this study. 
7.6. Statistical deviations 
Any post-hoc, or unplanned, analyses not identified in the protocol will be clearly identified in the 
study report. 
 
 RISKS AND BENEFITS 
8.1. Potential Risks 
Subjects will be positioned in an identical manner to that of undergoing diagnostic ultrasound. The 
subject will be asked to wear protective eyewear once settled into position. The duplex probe and 
protective eyewear are designed to be wiped down with cleaner/disinfectant after every use.  
 
The Imagio laser classification falls under the Class IIIB laser designation. During OA imaging, the 
laser shutter is opened, and the laser delivers near infrared pulses to the subject’s target location 
through a fiber-optic beam delivery system built-into the duplex OA/US probe. Direct beam laser 
illumination from the OA laser can potentially cause damage to the unprotected eye. The risk to the 
eyes is mitigated using protective eyewear specifically designed for protection against laser 
illumination in the near infrared spectral range. This protective eyewear is required to be worn by 
operators, subjects, and any other personnel in the examination room. 
 
ANSI Z136.1-2014, American National Standard for Safe Use of Lasers, published by the Laser 
Institute of America defines the maximum permissible exposure safety threshold for skin exposure 
as 20 mJ/cm2 for the case of multiple near infrared laser wavelengths and low repetition rate 
nanosecond laser pulses. The repetition rate of the laser pulses is limited to assure that the laser-
induced pressure and heat dissipates from the illuminated volume during the time between two 
consecutive laser pulses. OA uses a pulse repetition rate of up to 20 pulses per second with laser 
fluence less than 20 mJ/cm2 at any point. 
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The Imagio Imaging System has been evaluated for acoustic output, biocompatibility, cleaning, and 
disinfection. The system has been found to conform with thermal, electrical, electromagnetic, and 
mechanical safety and has been found to conform to applicable medical device safety standards.   
 
Potential risk of data disclosure will be addressed by proper data management procedures (e.g. 
immediate study number assignments, de-identification of data, and with the subject number kept 
in a place of physical security). 
 
Possibility of physical injury is primarily associated with violation of procedures of safe use of 
lasers. Subjects and all personnel will wear protective eyewear to minimize the risks to the eyes. 
The risk of skin injury is minimal with the use of safe levels of laser illumination.  
8.2. Blinding 
Subjects will not be blinded to best practice study procedures but will not have access to the Imagio 
OA/US evaluation result. 
 
The site investigators will have access to all subject data including the Imagio OA/US images and 
will assess and document the results of the OA/US study by completing the electronic data entry 
form (electronic case report form (eCRF)).   
 
The site investigators will have access to the background clinical information and to the subsequent 
histopathology report.     
8.3. Anticipated Adverse Device Effects 
List of potential risks and discomforts: 
 
• Tingling or warmth of the skin during the Imagio scan (0.4% or a 1 in 269 chance) that 
resolve at completion of the scan 
• Exposure to the acoustic output 
• Contamination due to insufficient cleaning 
• Phototoxicity  
8.4. Potential Benefits 
There are no direct benefits to subjects participating in this study. However, evaluating the opto-
acoustic image quality of the Gen 2 System in a clinical setting may contribute to improved 
diagnostic information. We hope that improved Imagio OA/US image quality will contribute to 
more accurate diagnosis of breast masses in future clinical use. 
 
The Gen 2 System will not be used to make any clinical decisions.  
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8.5. Risk/Benefit Rationale 
Seno Medical Instruments, Inc. believes that any potential risk presented by this investigation has 
been minimized and that adequate testing, safeguards, and safety monitoring have been incorporated 
into the design of the product and it’s accessories to further mitigate subject risk and exposure to 
potentially harmful laser light. 
 
Other risks associated with the device are based on nonclinical laboratory testing  and have been 
mitigated per ISO 14971. The device is a Class 3B laser product for external exposures (i.e., through 
the imaging probe) and Class 4 for internal exposures (i.e., during maintenance). Laser 
exposure is below known safety limits for skin and eye (when wearing appropriate 
laser safety eyewear).Therefore, this observational study, done to assess images is felt to be safe 
and aligned with the previously approved P200003 Imagio Breast Imaging System.   
 
 MEDICAL MONITOR 
The medical monitor is a physician who will provide safety oversight for the investigation. 
Responsibilities of the medical monitor include, but are not limited to: 
 
• Provide medical and scientific input to clinical data and subject medical safety data  
• Maintain ongoing assessment of the safety profile of the investigational device during the 
investigation 
• Provide medical surveillance and evaluation of Serious Adverse Events (SAEs) and 
unanticipated adverse device effects (UADEs). 
 
The medical monitor for this study is: 
A. Thomas Stavros, MD 
Chief Medical Officer 
Seno Medical Instruments, Inc. 
8023 Vantage Drive, Suite 1000 
San Antonio, TX 78230, USA 
Phone: 210-615-6501 
 ADVERSE EVENTS 
10.1. Adverse Event Assessments 
Criteria outlined in this section will be used to evaluate each subject during their participation in the 
study. 
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10.1.1. Assessment of Severity 
10.1.1.1. Adverse Event (AE) 
An AE is defined as any untoward medical occurrence in a subject. This means any clinically 
adverse sign, symptom, syndrome, or illness that occurs or worsens during the visit, regardless of 
causality, that is not otherwise being measured in the study. It includes any written, electronic, or 
oral communication with regard to a company sponsored clinical investigation that alleges 
deficiencies related to the investigational device resulting in an undesirable occurrence or untoward 
deviation in health away from the subject’s baseline. Only device related adverse events will be 
collected during this study.  
 
Each AE will be evaluated for its seriousness, severity, and association with the Imagio. Adverse 
events will be followed until resolution. 
10.1.1.2. Serious Adverse Event (SAE) 
The severity of AEs will be assessed according to the 21 CFR 812 SAE definition. A device-related 
SAE will be defined as an SAE in which the Imagio device relationship cannot be ruled out. An AE 
that meets any of the following criteria will be considered an SAE for protocol purposes: 
1. Led to death 
2. Led to a serious deterioration in the health of the subject that either: 
 
• Resulted in a life-threatening illness or injury 
• Resulted in permanent impairment of a body structure or a body function 
• Required in-subject hospitalization or prolongation of an existing hospitalization 
• Resulted in medical or surgical intervention to prevent permanent impairment of a body 
structure or a body function. 
 
Additionally, an AE that meets either of the following criteria will be considered reportable as a 
serious event to meet SAE regulatory reporting criteria: 
 
3.  Resulted in persistent or significant disability or incapacity; or  
4. Important AEs that are not immediately life-threatening or do not result in death or 
hospitalization but, based on the physician’s medical judgment, may jeopardize the subject, or 
may require intervention to prevent one of the other outcomes listed above. 
10.1.1.3. Unanticipated Adverse Device Effect (UADE) 
An UADE is “any unanticipated adverse effect on health or safety or any life threatening problem 
or death caused by, or associated with, a device, if that effect, problem, or death was not previously 
identified in nature, severity, or degree of incidence in the investigational plan or application, or 
any other unanticipated serious problem associated with a device that relates to the rights, safety, 
and welfare of subjects”. An observer in this study must report any UADE effect to the Institutional 
Review Board (IRB) within 24 hours of learning of the occurrence. Seno will then report the UADE 
effect, as required by the Food and Drug Administration (FDA) guidelines.  
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10.1.2. Evaluation of Severity 
The severity of AEs will be assessed according to the following definitions:  
 
Mild: The AE is noticeable to the subject but does not interfere with routine activity. The AE does 
not require any action in connection with the study. 
 
Moderate: The AE interferes with the subject’s routine activity but responds to symptomatic 
therapy or rest. The AE may require action concerning the study.  
 
Severe: The AE significantly limits the subject's ability to perform routine activities despite 
symptomatic therapy. The AE may require action concerning the study. 
 
Life-Threatening: The AE is of a nature that the subject is at immediate risk of death, even if not 
related to study activities. The AE may require action concerning the study.  
10.1.3. Evaluation of AE Relationship to Device 
The relationship between an AE and the study device (investigational device) will be determined 
by the Principal Investigator based on his or her clinical judgment and the following definitions: 
 
Table 2. Evaluation of AE Relationship to Device 
Likely related 
AEs with clear temporal and/or spatial relationship to Imagio 
device, with no clear alternative underlying cause. 
Probably related 
AEs with reasonable temporal and/or spatial relationship to 
Imagio device that cannot be readily explained by the subject’s 
clinical state or other agents/therapies. 
Possibly related 
AEs with fair temporal and/or spatial relationship to Imagio 
device, with no clear alternative underlying cause. 
Not related 
AEs that do NOT follow a temporal and/or spatial relationship 
to the Imagio device and can be readily explained by an 
alternative cause, such as an accidental injury or expected 
progression of an underlying or concomitant disease. 
10.1.4. Reporting of Adverse Events 
The Investigator will determine only device related adverse device effects. AEs will be reported on 
the AE CRF page, and each will be classified according to the aforementioned criteria. Adverse 
event reporting for each subject will begin upon the subject entering the Imagio scan room and 
continue through the Imagio procedure and subjects exit of the Imagio scan room. Any pre-existing 
conditions that are detected as part of the enrollment procedures must be reported in the medical 
history and not as an AE unless they worsen as a result of the Imagio procedure. Regulatory 
reporting standards will be followed for qualifying serious or unexpected events as per 21 CFR 812. 
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10.1.5. Reporting Serious Device Adverse Event (SAE) and Unanticipated Serious Adverse 
Device Effect (USADE) Recording 
The Investigator shall complete the SAE/USADE form. If the event has been classified as a 
SAE/USADE, details of medical history and concomitant medications (as applicable) will 
accompany the form.  The Investigator must report the SAEs and USADEs promptly within 24 
hours of the event occurring.  
 
All SAE and USADE data collection forms will be collected and placed within regulatory binder 
and submitted to the IRB as required per IRB guidelines.    
10.1.6. Reporting Adverse Event (AE) and Unanticipated Adverse Device Effect (UADE) 
Recording 
The same guidelines listed above under 9.1.4 apply to UADEs. Device related AEs will be recorded 
within the adverse event data form.  
10.2. Removal of Subjects from the Study 
A subject may be withdrawn from the study for any of the following reasons: 
 
• A serious or intolerable adverse event/effect or medical event occurs that in the 
investigator’s opinion would preclude subject from continuing with the study 
• Seno terminates the study 
• Subject enters the study in violation of the protocol, and it is subsequently discovered, and 
it is not in the subjects best interest to continue 
• Subject deviates from the protocol during the study and it is not in the best interest of the 
subject or sponsor for subject to continue 
• Pregnancy 
• Subject withdrawal of consent or subject requests to be withdrawn 
 PROTOCOL AND PROTOCOL AMENDMENT REVIEW AND     
APPROVAL 
Seno and the IRB will be required to approve the initial study protocol and applicable documents. 
Any amendment to the study protocol must be approved by the sponsor. A protocol amendment 
may not be implemented until after it has been approved by the IRB unless immediate 
implementation of the change is necessary for subject safety. In this case, the protocol change must 
be documented in an amendment.  
11.1. IRB Committee 
The protocol and the informed consent form must have the approval of a properly constituted IRB 
Committee responsible for approving clinical studies. The signed IRB Committee approval letter 
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must specify the date of protocol and informed consent form approval and identify the documents 
approved including the investigator's name, the protocol version, date, and title. Any subject 
materials or advertisements used to recruit subjects should also be reviewed and approved by the 
IRB Committee. 
 MONITORING 
Monitoring of the study will be performed in accordance with FDA guidelines and Good Clinical 
Practice.  
 
Monitoring will be performed by Seno or Seno’s representatives on a periodic basis. During 
monitoring sessions, data recorded and entered directly into the eCRF will be considered 
source. Any data recorded outside of the eCRF will be source and the eCRF will be verified against 
it for accuracy and completeness. The monitor should have direct access to source data for 
verification purposes. Such verification is essential to quality control.   
 
Delegation of study tasks will be the responsibility of the Principal Investigator and recorded on the 
Study Delegation Log.  
 DEVICE STORAGE AND ACCOUNTABILITY 
Seno will ship Imagio devices to the participating site. Seno personnel will unpack and install the 
study device as well as conduct and document operator training for study personnel. 
 
The Principal Investigator and designated site personnel will be responsible for ensuring that only 
authorized personnel have access to the study device for study purposes. Accountability of the 
devices to be used in the study will be maintained. 
 QUALITY ASSURANCE 
The sponsor, or the sponsor’s representative, may conduct internal audits. Audits may include, but 
are not limited to, device supply, presence of required documents, the informed consent process, 
and comparison of eCRFs with source documents. Source documents would include any records of 
the first-time data is recorded during the study beyond the data recorded directly into the eCRF plus 
the study images. The IRB may request access to source data during the conduct of the study.   
 ETHICAL CONDUCT 
This study will be conducted in accordance with the ethical principles outlined within FDA and 
Good Clinical Practice (GCP) regulations as well as local IRB requirements.   
 PROTOCOL DEVIATIONS 
Protocol deviations may occur in two ways: 
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• Deviations from the protocol, contrary to protocol specifications (i.e., deviations from the 
protocol procedures, eligibility, instructions for use, visit windows, etc.).  
 
• Deviations affecting the endpoint outcome not previously specified in the protocol (i.e., 
deviation that had not been previously considered in the protocol or eligibility criteria, but 
having a clear impact on the primary outcome measure) 
 
The investigator is not allowed to deviate from this protocol except as specified in FDA regulations. 
The Investigator should not implement any deviation from or changes to the protocol without 
approval by Seno and prior review and documented approval/favorable opinion from the IRB of a 
protocol amendment, except where necessary to eliminate immediate hazards to study subjects, or 
when the changes involve only logistical or administrative aspects of the study (e.g., change in 
monitors, change of telephone numbers). While every effort should be made to avoid protocol 
deviations, should a deviation be discovered, Seno must be informed immediately.  
 
Deviations will be documented in the eCRF. The Investigators will also adhere to procedures for 
reporting investigation deviations to their IRB committee in accordance with their specific reporting 
policies and procedures.  
 
Prior to final database lock, all protocol deviations will be reviewed and categorized as major (i.e., 
those that affect measurement or interpretation of the primary endpoint or related to safety) or minor 
(those not affecting the primary endpoint). The presence of major deviation may exclude subjects 
from the Intention to Diagnose (ITD) analysis population upon review by the Protocol Deviation 
Committee. 
16.1 Corrective and Preventative Actions 
Seno Medical and her representatives will evaluate protocol deviations during monitoring visits. 
Individual event corrective and preventive actions may be recommended at that time.  
 INVESTIGATOR DISQUALIFICATION CRITERIA 
Seno Medical reserves the right to terminate an investigator/investigational site for any of the 
following reasons: 
• Failure to secure subject informed consent including protection of personal data prior to 
enrollment. 
• Failure to report unanticipated adverse device effects within 24 hours of discovery (to 
Seno Medical) and to the IRB committee within its required reporting time after learning 
of the event. 
• Failure to report serious adverse device effects within 24 hours of discovery. 
• Repeated investigational plan deviations. 
• Repeated failure to appropriately complete case report forms. 
• Failure to enroll an adequate number of subjects. 
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• Loss of or unaccounted for investigational product inventory. 
 SUBJECT CONFIDENTIALITY 
Confidentiality will be maintained in accordance with FDA and IRB regulations. Subject names 
must not be revealed to the sponsor or sponsor’s representatives. Only the subject identifier 
(number) will be recorded in the eCRF and if the subject’s name appears on any other document, it 
must be redacted and replaced with the subject identifier before a copy of the document is supplied 
to the sponsor or sponsor’s representatives. Study findings stored on a computer will be stored in 
accordance with local data protection laws. In the event of inadvertent communication of such 
information, immediate steps to redact the information from all study files will be implemented, 
with appropriate documentation in the subject study file.  
 CASE REPORT FORMS AND STUDY RECORDS 
Data will be recorded using an electronic data capture (EDC) system, according to FDA regulations. 
The database is considered validated when the expected results are the same as the actual results, 
and the end users verify that the database performs according to the requirements. 
 
The data will include collecting images plus the Laser Optical Movie (LOM) to be sent to Seno 
Medical Instruments, Inc.  
 
The validation report, design procedures, testing results, source code, and test eCRF cases are filed 
in the central files. Database maintenance will be provided throughout the study, as well as user 
support and administration (access and site user rights set-up, removal, etc.). 
 
The Medical Dictionary for Regulatory Authorities (MedDRA) will be used to code adverse device 
effects.  
 
The investigator is responsible for maintaining adequate and accurate medical records from which 
information will be transferred into the study database.  
 DATA MANAGEMENT 
A detailed plan for the data management activities will be prepared. The structure of the database 
will be based on the eCRFs and validated according to the Data Validation Plan, prepared by the 
Clinical Research Organization (CRO). Discrepancies revealed by data validation will be delivered 
for resolution with Quality Control Reports. The clean database will be locked after all detected 
discrepancies have been solved and the database has been updated accordingly. Only authorized 
and well-documented updates are possible after the database lock. The data will be secured by daily 
back-ups to the CRO’s server (details are included in the Data Management Plan). 
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 RETENTION OF DATA 
All records must be retained by Seno or Seno’s representatives for a period of two years after the 
latter of the following two dates: the date the study is closed or terminated or the date of the last 
approval of a marketing application or when records are no longer needed to support a regulatory 
submission. All study records may be relevant to regulatory inspection.  
 
If the Seno Representative in charge of the study retires, relocates, or for other reasons withdraws 
from the responsibility of keeping the study records, custody must be transferred to a person who 
will accept the responsibility. 
 FINANCIAL DISCLOSURE 
Investigators will be asked to provide financial disclosure prior to authorization to begin the study 
as well as after the study is completed. Investigators will also be expected to share any situations 
which could introduce specific bias. 
 PUBLICATIONS 
The results of the study are the property of Seno Medical Instruments, Inc. All publications 
(manuscripts, abstracts, or other modes of presentation) must be submitted at a time determined by 
Seno Medical Instruments, Inc. and must be reviewed and approved in writing by Seno Medical 
Instruments, Inc., in advance of submission. Co-authorship with any Seno Medical Instruments, Inc. 
personnel will be discussed and mutually agreed upon before submission of a manuscript to a 
publisher. Seno Medical will prepare a final study report following the conclusion of the study. 
 
 LITERATURE CITED 
1. Torre LA, Islami F, Siegel RL, Ward EM, Jemal A. Global cancer in women: Burden and 
trends. Cancer Epidemiol Biomarkers Prev. 2017. 
2. Lakhani, S, Ellis. I, Schnitt, S, Tan, P, van de Vijver M. WHO Classification of Tumours of the 
Breast, Fourth Edition. In: IARC WHO Classification of Tumours, No 4. ; 2012. 
3. Russo J. The pathobiology of breast cancer. 2016. 
4. Kruger RA, Liu P. Photoacoustic ultrasound: Pulse production and detection in 0.5% Liposyn. 
Med Phys. 1994. 
5. Oraevsky AA, Esenaliev RO, Jacques SL, Tittel FK, and Medina D. “Breast Cancer 
Diagnostics by Laser Opto-Acoustic Tomography”, OSA Trends in Optics and Photonics on 
Advances in Optical Imaging and Photon Migration,” R.R. Alfano and J.G. Fujimoto, eds. 
(OSA, Washington, DC), 2, pp. 316-321, 1996.   
6. Oraevsky AA, Esenaliev RO, Jacques SL, and Tittel FK. “Laser Opto-Acoustic Tomography 
for medical diagnostics: principles”, Proc. SPIE 1996; 2676: 22-31. 
7. Richards-Kortum R, Sevick-Muraca E. Quantitative optical spectroscopy for tissue diagnosis. 
Annu Rev Phys Chem. 1996. 
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8. Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst. 
1990. 
9. Weidner N, Semple JP, Welch WR, Folkman J. Tumor Angiogenesis and Metastasis — 
Correlation in Invasive Breast Carcinoma. N Engl J Med. 1991. 
10. Wray S, Cope M, Delpy DT, Wyatt JS, Reynolds EOR. Characterization of the near infrared 
absorption spectra of cytochrome aa3 and haemoglobin for the non-invasive monitoring of 
cerebral oxygenation. BBA - Bioenerg. 1988. 
11. Roggan A, Friebel M, Dörschel K, Hahn A, Müller G. Optical Properties of Circulating 
Human Blood in the Wavelength Range 400–2500 nm. J Biomed Opt. 1999. 
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