Clinical Validation for Non-Inferiority of Primary Diagnosis by WSI Hamamatsu NanoZoomer S360MD Digital Slide Scanner System Compared to Conventional Determination by Light Microscope
Phase: NA
Conditions: Pathology
Sponsor: Hamamatsu Photonics K.K.

Hamamatsu NanoZoomer S360MD Digital Slide Scanner System 
HCT-P001 Clinical Protocol for WSI Primary Diagnosis Non-Inferiority Study 
 
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Clinical Validation Protocol for Test of Non-Inferiority of 
Primary Diagnosis by WSI using Hamamatsu NanoZoomer 
S360MD Digital Slide Scanner System Compared to 
Conventional Determination by Light Microscopy 
 
 
Sponsor:  
Hamamatsu Photonics K.K. 
812 Joko-cho 
Higashi-ku 
Hamamatsu City, 431-3196 
Japan 
 
 
Sponsor Representative: 
Mark Holody, PhD 
Mark Holody, LLC 
mark.holody@yahoo.com 
13 Abeto St., Irvine, CA, 92620 
 
 
NCT#: 03991468 
Date: 09-JUL-2020 
 
 

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PROTOCOL SIGNATURE PAGE 
 
Sponsor’s (Hamamatsu Photonics K.K.) Approval 
 
Signature ________________________________     Date ______________________ 
Name: Susumu Suzuki  
Title:   Manager, Regulatory Management Group, Medical Compliance, Systems Division 
 
 
 
Protocol Number:   HCT-P001 
 
Study Title: Clinical Validation Protocol for Test of Non-Inferiority of Primary Diagnosis by WSI using 
Hamamatsu NanoZoomer S360MD Digital Slide Scanner System Compared to Conventional 
Determination by Light Microscope.  
I have read this protocol.  
By signing this protocol, I agree to conduct the clinical study according to this protocol, the principles of 
the Declaration of Helsinki (2008), the standards of Good Clinical Practice (as defined by the 
International Conference on Harmonization E6), ISO 14155 and applicable regulatory requirements. I will 
ensure that study staff fully understand and follow the protocol. 
I understand that failure to comply with the requirements of the protocol may lead to termination of my 
participation as an investigator for this study.  
Changes to the protocol will only be implemented after written approval is received from Hamamatsu 
Photonics K.K. and the Institutional Review Board or Independent Ethics Committee (as appropriate), 
with the exception of medical emergencies. 
 
Investigator Name, Affiliation and Address_____________________________________ 
__________________________________________________________________________ 
 
Signature ________________________________     Date ______________________ 
 
 
 

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Table of Contents 
PROTOCOL SIGNATURE PAGE ..............................................................................................2 
LIST OF TABLES ...........................................................................................................................5 
LIST OF FIGURES .........................................................................................................................5 
ABBREVIATIONS AND TERMS .................................................................................................6 
1. 
PROTOCOL SUMMARY ........................................................................................................8 
1.1 
Synopsis ..........................................................................................................................8 
1.2 
Schema ..........................................................................................................................11 
2. 
INTRODUCTION ..................................................................................................................12 
2.1 
Background ...................................................................................................................12 
2.2 
Rationale .......................................................................................................................13 
2.3 
Risk/Benefit: .................................................................................................................13 
3. 
OBJECTIVES AND ENDPOINTS ........................................................................................14 
3.1 
Primary Objective: ........................................................................................................14 
3.2 
Secondary Objectives: ...................................................................................................14 
3.3 
Endpoints ......................................................................................................................14 
4. 
STUDY DESIGN ...................................................................................................................15 
4.1 
Overall Design ..............................................................................................................15 
4.2 
Scientific Rationale for Study Design ...........................................................................15 
4.3 
End of Study Definition ................................................................................................15 
5. 
ADJUDICATION COMMITTEE ..........................................................................................19 
6. 
INCLUSION AND EXCLUSION CRITERIA ......................................................................19 
6.1 
Selection of pathology practice and pathologists ..........................................................19 
6.2 
Selection of pathology practice and pathologists cases ................................................20 
6.2.1 
Inclusion criteria: .............................................................................................22 
6.2.2 
Exclusion criteria: ............................................................................................23 
6.3 
Withdrawal or Discontinuation .....................................................................................23 
6.3.1 
Slide Withdrawal .............................................................................................23 
6.3.2 
Case Withdrawal ..............................................................................................23 
6.3.3 
Pathologist Withdrawal ....................................................................................24 
7. 
DEVICE UNDER STUDY .....................................................................................................24 
7.1 
System Description .......................................................................................................24 
7.2 
General Principles of Operation ....................................................................................26 
8. 
STUDY ASSESSMENTS AND PROCEDURES ..................................................................27 
8.1 
Preparation of Facility ...................................................................................................27 
8.2 
Training on Study Procedures and WSI Operation .......................................................28 

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8.3 
Selection of Case Slides ................................................................................................29 
8.4 
Validation of case and slide selection ...........................................................................31 
8.5 
Storage of case slides ....................................................................................................31 
8.6 
Whole slide scanning operations ...................................................................................31 
8.7 
Reading a Case ..............................................................................................................32 
8.8 
Adjudication of Discordant Readings ...........................................................................33 
8.9 
Pilot Studies ..................................................................................................................34 
9. 
STATISTICAL CONSIDERATIONS ...................................................................................34 
9.1 
Statistical Hypotheses ...................................................................................................34 
9.2 
Sample Size Calculations ..............................................................................................35 
9.3 
Populations for Analyses...............................................................................................36 
9.4 
Statistical Analyses .......................................................................................................36 
9.4.1 
Case Characteristics and Disposition, and Reader Pathologists ......................36 
9.4.2 
Primary Analysis of Non-inferiority of WSI Relative to Glass .......................36 
9.4.3 
Secondary Endpoints Analyses ........................................................................37 
9.4.4 
Other Analyses .................................................................................................38 
10. DATA MANAGEMENT AND DATA QUALITY ...............................................................38 
10.1 Data Management .........................................................................................................38 
10.2 Data Quality ..................................................................................................................40 
10.3 Data Retention Procedures and Period ..........................................................................40 
10.4 Publication Policy .........................................................................................................41 
11. DEVIATIONS FROM THE CLINICAL INVESTIGATION PLAN ....................................41 
11.1 Protocol Deviations .......................................................................................................41 
11.2 Device Deficiencies ......................................................................................................41 
12. SAFETY AND SUBJECT CONFIDENTIALITY .................................................................42 
12.1 Safety ............................................................................................................................42 
12.1.1 Adverse Events ................................................................................................42 
12.2 Patient Confidentiality ..................................................................................................42 
12.3 IRB and Protocol Approval ...........................................................................................42 
13. COMPLIANCE AND GOOD CLINICAL PRACTICES (GCP) ..........................................42 
13.1 Financial Disclosures ....................................................................................................43 
13.2 Monitoring ....................................................................................................................43 
14. BIBLIOGRAPHY ...................................................................................................................44 
REVISION HISTORY...................................................................................................................46 
 
 
 

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LIST OF TABLES 
 
Table 1: Schedule of Assessments .................................................................................................12 
Table 2: FDA List of Cases to be tested for Primary Diagnosis Study .........................................20 
Table 3: Definition of Discordance ................................................................................................33 
Table 4: Sample Size for 4 Readers and 4 Sites ............................................................................35 
 
 
LIST OF FIGURES 
 
Figure 1.  Study Schema ................................................................................................................11 
Figure 2.  Study Process: Slide Selection to Diagnosis by Reading Pathologists .........................17 
Figure 3.  Study Process: Adjudication of Diagnoses ....................................................................18 
Figure 4.  Communication between Scanner and Display .............................................................25 
Figure 5.  Example block diagram of typical components found in current WSI systems ............27 
Figure 6.  Example of CRF (using truncated CAP checklist) ........................................................40 
 
 
 
 

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ABBREVIATIONS AND TERMS 
Abbreviation 
Term 
AAP 
All Available Population 
AE 
Adverse Event 
AP 
Adjudicator pathologist 
CAP 
College of American Pathologists 
CFR 
Code of Federal Regulations 
CI 
Confidence Interval 
CMOS 
Complementary Metal-Oxide Semiconductor 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
 
 
DPA 
Digital Pathology Association 
eCRF 
Electronic case report form 
EDC 
Electronic Data Capture 
EP 
Enrolling Pathologist 
FDA 
Food and Drug Administration 
FFPE 
Formalin-Fixed Paraffin Embedded 
GCP 
Good Clinical Practice 
GT 
Ground Truth  
H&E 
Hematoxylin and Eosin 
HER2 
Human Epidermal Growth Factor Receptor 2 
ID 
Identifier 
IRB 
Institutional Review Board 
IT 
Information Technology 
LED 
Light Emitting Diode 
LM 
Light Microscopy 
MMRM 
Mixed Model Repeated Measures 
NA 
Numerical Aperture 
PDF 
Portable Document Format 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PIPS 
Philips IntelliSite Pathology Solution 
RP 
Reader Pathologist 

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Abbreviation 
Term 
SAP 
Statistical Analysis Plan 
SC 
Study Coordinator 
ST 
Study Technician 
US 
United States 
USB 
Universal Serial Bus 
VEP 
Verifying Enrolling Pathologist 
WSI 
Whole Slide Imaging 
 
 
 

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1. 
PROTOCOL SUMMARY 
1.1 
Synopsis 
TITLE OF TRIAL 
NanoZoomer S360MD Digital Slide Scanner System Clinical Validation Protocol 
TRIAL SITE(S) 
Four (4) sites in the United States (US) and possibly Canada. 
1. Ohio State University Wexner Medical Center, PI (Dr. Anil Parwani) 
2. Cleveland Clinic Foundation PI (Dr. Bin Yang, Dr. Scott Fitzpatrick) 
3. Tricore Reference Labs, PI (Dr. Richard Feddersen) 
4. Washington University Medical Center, PI (Dr. Jon Ritter) 
Primary diagnosis of glass slides and whole slide imaging (WSI) images done by four (4) reading pathologists 
Case screening and enrollment to use 1 pathologist to enroll cases and 1 pathologist to confirm case enrollment 
per site. 
OBJECTIVES 
Primary Objective:   
1. To evaluate the safety and accuracy of the Hamamatsu WSI test method compared to that of the 
reference method (conventional light microscope [Glass]) under clinical use conditions.  
Secondary Objectives:   
1. Evaluate the accuracy of the Hamamatsu WSI test method compared to that of the reference method by 
site, and by reader within sites. 
2. Evaluate the accuracy of the Hamamatsu WSI test method compared to that of the reference method by 
site, and overall for diagnostic subtypes separately. 
3. Evaluate the accuracy of the Hamamatsu WSI test method compared to that of the reference method by 
organ within site and overall. 
4. Evaluate the accuracy of the Hamamatsu WSI test method compared to that of the reference method by 
procedure type, within site and overall. 
5. Evaluate the agreement of the Hamamatsu WSI relative to glass slides for each diagnostic category and 
overall for each pathologist (intra-site/intra-reader/inter-modality precision). 
6. Evaluate the pathologist-to-pathologist precision overall and within diagnostic category within each site 
and for each modality (inter-reader/intra-site/intra-modality precision). 
PRIMARY ENDPOINT 
• 
The primary endpoint is the indicator of major discordance in primary diagnosis between ground truth 
case diagnosis and case diagnosis by each modality, WSI and Glass. 
METHODOLOGY 
Cases will be selected in consecutive order by the enrolling pathologist starting with cases at least one year old 
and verified to match inclusion and exclusion criteria, and the case distribution for that site.  Upon confirmation 
of enrollment, the slides necessary to make the diagnosis will be selected and included in the set of slides 
available to the reading pathologists.  Additional slides for the case will be excluded.  The original primary 
diagnosis reported in the clinical file for each case will be the Ground Truth (GT) diagnosis. 
Each pathologist will read each case enrolled at their site by both conventional light microscope (Glass) and by 
WSI and record their primary diagnosis only, including the correct subtype of the lesion/neoplasm, and related 

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parameters (e.g., tumor subtype, tumor grade, margin status, lymph node involvement), if appropriate.  The order 
of reading by Glass or WSI will be randomly determined; and, readings by the two modalities will include a 
minimum of 4 weeks washout period.  
Upon completion of a reading at a site, two independent adjudicating pathologists will compare the primary 
diagnosis obtained using WSIs and using Glass to the GT diagnosis to determine if there is any discrepancy.  Any 
mismatch will be further judged as major or minor based on pre-specified case rules cited in the literature to 
categorize a “discordance” into pre-designated categories and to decide if the discordance would have led to a 
change in patient treatment (i.e., major) or none at all (i.e., minor).  If the two adjudicating pathologists do not 
agree on a “major” discordance, a 3rd independent pathologist shall review the case to determine majority 
agreement or if a consensus meeting of all three adjudicators is required. 
NUMBER OF CASES 
A total of 2000 cases will provide at least 90% power to demonstrate non-inferiority of WSI to Glass with a non-
inferiority margin of δ = 4% and type I error of 5%, if in fact WSI is non-inferior to Glass. 
MAIN CRITERIA FOR INCLUSION/EXCLUSION 
Inclusion criteria:  
Cases are eligible to be included in the study only if all of the following criteria apply: 
• 
Cases originating from and that were diagnosed at the local sites  
• 
Cases are available in the site’s archive 
• 
Cases are at least 1 year old since accessioning 
• 
Cases are selected because their primary diagnosis is consistent with the assigned target categories listed 
in Table 2 
• 
Cases have a set of slides representative of the primary diagnosis for which it has been selected 
Slide Selection for a given case must meet the following criteria: 
• 
Slide obtained by surgical pathology and prepared from formalin-fixed paraffin embedded (FFPE) 
human tissue 
• 
Slides must be stained with hematoxylin and eosin (H&E) and accompanying special stains 
(histochemical and/or immunohistochemical). 
• 
All special stain slides (histochemical and/or immunochemical) where the slide and stain is used for 
diagnosis, not prognosis 
• 
All chosen slides must demonstrate and be representative of the primary diagnosis; 1 H&E slide 
selection may suffice for biopsy cases 
• 
For resection cases, a minimum of 5 H&E slides must be selected, which represent the primary 
diagnosis. If represented with less than 5 H&E slides, additional H&E slides (primary, secondary, or 
benign slides) from same case may be used to fulfill minimum number 
• 
Slide is intact, has correct size/thickness, good edges, undamaged coverslip, without pen markings that 
can’t be removed, no air bubbles, tidy labels, and fulfills the quality checks per the general clinical 
practice 
Exclusion criteria:  
Cases are excluded from the study if any of the following criteria apply: 
• 
Case does not have relevant slides or if case information necessary for the study is missing 
• 
Case is still active (less than 1 year old) at the local site 
• 
Cases for which the control slides for immunohistochemistry and special stains are not available 

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• 
Two cases from same individual 
• 
Gross only cases that have no slides 
• 
Cases that are frozen section, cytology or hematology or immunofluorescence specimens only 
• 
Cases where the only available set of slides have evidence only of secondary or no diagnoses and not the 
primary diagnosis for which the case is being screened. 
Slides for a given case will be excluded if they meet the following exclusion criteria 
• 
Glass slide that is broken, has abnormal size/thickness, beveled edges, poor coverslip (cracks, waviness, 
scratches), is sticky, has many pen markings or dirt, contains air bubbles and overhanging labels that 
can’t be corrected, and if stain is severely faded. 
DEVICE DESCRIPTION 
Hamamatsu S360MD NanoZoomer Slide Scanner Digital Pathology System (NanoZoomer) is an automated 
digital slide creation, viewing, and management system. 
The Hamamatsu NanoZoomer S360MD Digital Slide Scanner creates diagnostic quality digital images of glass 
slides containing FFPE tissue.  Each image typically contains billions of image pixels, creating a digital image of 
the tissue on the original glass slide.  The NanoZoomer captures digital images of entire slides for duplication, 
annotation, storage, retrieval, image sharing and viewing to permit the pathologist to make a primary diagnosis 
without needing to view those glass slides through a light microscope. 
STATISTICAL METHODS 
All Available Population (AAP) is defined as all cases for which at least one out of four readers provides an 
evaluable outcome for either the WSI or Glass modality and will be used for the primary analysis.   
For the primary objective of demonstrating the WSI major discordance rate to be non-inferior to the Glass major 
discordance rate, a Mixed Model Repeated Measures (MMRM) logistic regression will be conducted.  The 
dependent variable will be the major discordance status (yes/no) and the independent variables will include 
modality (WSI or Glass) as a fixed effect and site, reader, and case as random effects.  From this model a two-
sided 95% confidence interval (CI) for the difference in major discordance rate (πWSI – πGlass) will be 
constructed.  If the upper bound of the 95% CI is less than the non-inferiority margin of 4%, then WSI would be 
considered non-inferior to Glass. 
 
 
 

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1.2 
Schema 
          
 
Figure 1.  Study Schema 
 
 
 
Case Selection 
& Validation
Glass
WSI
WSI
Glass
Adjudication
Randomization 

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Table 1: Schedule of Assessments 
 
Screening 
Reading 
Adjudication 
 
EP 
VEP 
SC 
ST 
RP-1 
RP-2 
RP-3 
RP-4 
Adj-1 
Adj-2 
Adj-31 
Enroll slide 
X 
 
 
 
 
 
 
 
 
 
 
Confirm eligibility 
 
X 
 
 
 
 
 
 
 
 
 
Enter GT 
Information 
X 
 
X 
 
 
 
 
 
 
 
 
Create WSI Images 
 
 
 
X 
 
 
 
 
 
 
 
Read slides2 
 
 
 
 
 
 
 
 
 
 
 
   WSI 
 
 
 
 
X 
X 
X 
X 
 
 
 
   Glass 
 
 
 
 
X 
X 
X 
X 
 
 
 
Discordance Status 
Adjudication3 
 
 
 
 
 
 
 
 
 
 
 
   WSI 
 
 
 
 
 
 
 
 
X 
X 
 
   Glass 
 
 
 
 
 
 
 
 
X 
X 
 
EP = Enrolling pathologist, VEP = verifying enrolling pathologist, SC = study coordinator, ST = study technician,  
RP = reader pathologist, Adj = adjudicator. 
1. When a major discordance differs between the first two adjudicators, a third adjudicator will review to break the tie. When all 
three results differ, a meeting will be held to reach consensus.  
2. Slides are read in a random order, specified separately for each pathologist at a site. 
3. Reader diagnosis concordance/discordance with the Ground Truth diagnosis will be adjudicated upon completion of a read. 
 
2. 
INTRODUCTION 
2.1 
Background 
Whole Slide Imaging (WSI) has emerged as an alternative way to view pathology slides for primary 
diagnosis instead of the conventional method of viewing slides of tissue under a traditional light 
microscope. There is growing adoption of the use of WSI for primary diagnosis in many countries (1-4).  
WSI is particularly advantageous for remote consultation over vast geographical regions where 
pathologists are not available, and for easier archiving of images of slide material that may have longer 
shelf life in digital form.  In the United States, WSI is increasingly used for teaching, archiving, 
consultation, and research.  Furthermore, the College of American Pathologists (CAP) has published 
recommendations to pathologists who wish to validate WSI in their clinical practices (5).  
However, quite recently, in a de novo authorization letter (6) and device summary (7), the Food and Drug 
Administration (FDA) has announced the authorization of the Philips IntelliSite Pathology Solution 
(PIPS) for use of WSI for primary diagnosis, specifically permitting WSI for in-vitro diagnostic use as an 
aid to the pathologist to review and interpret digital images of surgical pathology slides prepared from 
formalin-fixed paraffin embedded (FFPE) tissue, but not intended for use with frozen section, cytology, 
or non-FFPE hematopathology specimens. 

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Thus, the authorization of the Philips device for primary diagnosis, PIPS system, serves as the predicate 
device for any future 510(k) submissions for WSI systems seeking clearance for intended use of WSI, 
and, further, the FDA has indicated that a clinical study to prove non-inferiority of WSI device is a 
“special control” required to acquire that clearance.  
Hamamatsu is developing a digital slide scanner system, the NanoZoomer S360MD Digital Slide Scanner 
System (NanoZoomer), for the same intended use as the PIPS system.  Thus, Hamamatsu will make the 
submission in the form of a 510(k) premarket notification with the PIPS device as the predicate device 
and will accordingly adhere to the special controls that are established.  Hamamatsu will follow similar 
study designs to test the NanoZoomer system, and intends to use the data from this non-significant risk 
study for the 510(k) submission to clear its NanoZoomer system for the same intended use. Additional 
study design input became available from FDA presubmission review of protocol (20-25) 
2.2 
Rationale 
In recent years, there has been an abundance of literature testing the concordance of primary diagnosis by 
WSI versus determination by standard review of light microscopy (LM) of glass slides (8-10).  Many 
variations of experimental design are discussed encompassing topics of comparison to ground truth, 
concordance comparison, time necessary for washout period, targets for assessment of non-inferiority, 
sample size, choice of organ systems, effects of pre-training, adjudication of major and minor 
discrepancy, etc. 
The CAP has published guidelines for pathology practices interested in using such technology for primary 
diagnosis (5).  This society tried to standardize the methodologies for pathologists to consider for 
validation of WSI for primary diagnosis, further touching on the same study factors for consideration. 
The FDA has been in close communication with leading pathologists in the field of digital pathology on 
the design of studies to support WSI for primary diagnosis, as well as the major vendors interested in 
pursuing that application, mainly through the Digital Pathology Association (DPA).  The FDA has 
published guidance for technical specification (11).  Yet, for clinical validation, the FDA has presented 
the Agency thinking on clinical validation protocol considerations in two PowerPoint presentations (12-
13), touching on the kinds of cases to be tested, as well as the number of sites, pathologists and cases to 
be tested. 
Quite recently, the FDA has announced the de novo authorization of the PIPS for the intended use of WSI 
for primary diagnosis in a clearance letter (6) and device summary (7), which included more information 
on the design, conduct, and acceptance criteria for the non-inferiority study used to acquire clearance.   
Hamamatsu is closely following the scientific study design and performance targets that were used by 
Philips. 
2.3 
Risk/Benefit: 
Since this is a retrospective examination of cases, there is no apparent risk or benefit to the patient whose 
case is being used for the study.   
Because this study design does not call for the re-assessment of the primary diagnosis of the retrospective 
case, we do not envision a situation in which the original diagnosis will be questioned and reassigned.  

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For that reason, and because the cases are retrospective and at least 1 year old, we do not see any need to 
notify the patient as protocol does not call for any change in diagnosis.  
In the future, the device itself offers significant potential benefits, particularly for remote viewing of case 
material in geographies where pathologists are sparsely present. 
3. 
OBJECTIVES AND ENDPOINTS 
3.1 
Primary Objective:   
1. To evaluate the safety and accuracy of the Hamamatsu WSI test method compared to that of the 
reference method (conventional light microscope [Glass]) under clinical use conditions.  
3.2 
Secondary Objectives:   
1. Evaluate the accuracy of the Hamamatsu WSI test method compared to that of the reference 
method by site, and by reader within sites. 
2. Evaluate the accuracy of the Hamamatsu WSI test method compared to that of the reference 
method by diagnostic category within site and overall. 
3. Evaluate the accuracy of the Hamamatsu WSI test method compared to that of the reference 
method by organ within site and overall. 
4. Evaluate the accuracy of the Hamamatsu WSI test method compared to that of the reference 
method by procedure type within site and overall. 
5. Evaluate the agreement of the Hamamatsu WSI relative to glass for each diagnostic category and 
overall for each pathologist (intra-site/intra-reader/inter-modality precision). 
6. Evaluate the pathologist-to-pathologist precision overall and within diagnostic category within 
each site and for each modality (inter-reader/intra-site/intra-modality precision). 
3.3 
Endpoints 
The primary endpoint is the indicator of major discordance between ground truth case primary diagnosis 
and case diagnosis by each modality, WSI and Glass, separately. 
The secondary endpoints are: 
• 
The difference in major discordance error rates of both WSI and optical LM (Glass) modalities 
compared to the original sign-out primary diagnosis rendered at the site, by site and by reader 
within each site 
• 
The difference in major discordance error rates of both WSI and optical LM (Glass) modalities 
compared to the original sign-out primary diagnosis rendered at the site, by site and overall for 
each diagnostic category  
• 
The difference in major discordance error rates of both whole slide imaging (WSI) and optical 
LM (Glass) modalities compared to the original sign-out primary diagnosis rendered at the site, 
by site and overall for each procedure type 
• 
The difference in major discordance error rates of both WSI and optical LM (Glass) modalities 
compared to the original sign-out primary diagnosis rendered site, by organ within site and 
overall 

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• 
The percentage agreement of WSI relative to glass for each diagnostic category and overall for 
each pathologist  
• 
The percentage agreement for each diagnostic category and overall between each pair of readers 
within a site for a given modality (WSI or Glass).  
4. 
STUDY DESIGN 
4.1 
Overall Design 
The study is a multi-site, randomized-read order, retrospective, paired-design evaluation of the 
Hamamatsu NanoZoomer S360MD Slide Scanner system consisting of a review of archived, de-identified 
and previously signed-out slides representing main organ systems within surgical pathology.  Cases will 
include retrospective hematoxylin and eosin (H&E) stained formalin fixed tissue, and special stains 
and/or immunohistochemical stains (most commonly used is the brown chromophore) from the pathology 
practice, but will not include frozen sections, or cytological and hematological cases. 
A total of 2000 cases consisting of multiple organ and tissue types will be enrolled.  Cases will be divided 
over four (4) sites.  At each site, four pathologists will read all the cases assigned to their site using both 
WSI and Glass modalities in a randomized order and with a washout period of at least four weeks 
between readings, resulting in a total of 8000 planned WSI reads and 8000 planned Glass reads.  After 
any completed reader diagnosis case report form (CRF) at a site has been collected and cleaned and the 
CRF is completed, two adjudicators will review the reader’s diagnosis against the original diagnosis 
(ground truth) to determine whether the new and original diagnoses were concordant, minor discordant, or 
major discordant.  A third adjudicator will be used, if disagreement occurs between the first two 
adjudicators on the classification of a “major” discrepancy. If all three adjudication results differ, a 
meeting will be held to reach consensus among the three adjudicators. Figure 2 and Figure 3 display the 
process for the selection and reading of slides and the adjudication process, respectively. 
4.2 
Scientific Rationale for Study Design 
The use of multiple organs and tissue types obtained from various procedures provides a variety of cases 
upon which to base generalizability of the results.  Furthermore, a spectrum of diagnoses is included in 
these cases providing confidence in the generalizability of the results. 
The randomized order of the two modalities attempts to remove the bias associated with reading the same 
slide twice.  And, requiring that the two modalities be read with at least a four-week washout further 
decreases the chance of recall bias. 
Additional source information on the study design comes from the Philips scientific abstract (14), Digital 
Pathology Association presentation webinar (15), and a Philips presentation at the May 2017 Pathology 
Informatics Summit in Pittsburgh, PA (16). 
4.3 
End of Study Definition 
Each case will be considered complete when all study evaluations and study required assessments have 
been performed in accordance with the protocol.   

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It is anticipated that sites will have three to five months to screen and enroll slides, about two to three 
months to scan slides, and at least five to ten months to complete all reads. Adjudication will occur as 
soon as any one reading diagnosis is completed. Thus, adjudication will be occurring concurrently with 
reading of cases, and will end soon after all readings are completed.   

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Figure 2.  Study Process: Slide Selection to Diagnosis by Reading Pathologists 
 
 
 
SLIDE SELECTION
Site
Case
Path
Diag
Site 1
500 
(1-500)
1
EP
2
Glass
VEP
3
WSI
4
Site 2
500 
(501-1000)
5
EP
6
Glass
VEP
7
WSI
8
Site 3
500 
(1001-1500)
9
EP
10
Glass
VEP
11
WSI
12
Site 4
500 
(1501-2000)
13
EP
14
Glass
VEP
15
WSI
16

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Figure 3.  Study Process: Adjudication of Diagnoses 
ADJUDICATION
Diag
Round 1
Round 2
Round 3
Agree w/Adj 2
Stop
Glass
Adjudicator 1
Agree
Stop
Disagree
Adjudicator 3
WSI
Adjudicator 2
Disagree
Consensus 
Meeting
Decision
Agree w/Adj. 1
Stop

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The completion of the study at the site is defined as the date of the last reading of the last pathologist from 
the last participating site in the study.  
The end of the study is after the adjudicators have completed their review of the pathologists’ diagnoses 
and classified each reading as concordant or discordant; and, if discordant, as major or minor and the 
adjudication data has been entered into the clinical database, and the locked database has been transferred 
to the statisticians. 
5. 
ADJUDICATION COMMITTEE 
A panel of expert pathologists will convene to establish a charter with clear pre-established rules of 
adjudication for each type of case listed in Table 2.  Each case category from FDA list will have an 
adequate description of what constitutes a match, a minor discordance and a major discordance according 
to the definitions of major and minor discordance provided in Table 3. These pre-adjudication rules shall 
also specify that all deferrals shall be considered as minor or major discordances per application of 
adjudication rules for deferrals. A panel of adjudicators independent of the site shall be selected and 
trained on these rules. 
The committee to establish the pre-adjudication rules shall classify discordant results according to the 4 
harm categories (18) as shown in Table 3 and judge if the discordance in question would have led to a 
change in patient care or treatment plan.  Any discordance not leading to a change in treatment will be 
considered as a “minor” discordance and considered a match.  Any discordancy leading to a change in 
treatment plan for the patient would be judged as a “major” discordance, and therefore considered to not 
be a match.  
6. 
INCLUSION AND EXCLUSION CRITERIA 
6.1 
Selection of pathology practice and pathologists 
Four sites shall be selected such that: 
• 
There shall be as many as 3 academic and at least 1 community and/or private practice pathology 
sites, 
• 
The laboratory is accredited by the CAP 
• 
Each site will be assigned cases consistent with the types of cases read at that site and from the 
cases listed in Table 2.The four sites together shall cover the entire range of cases required in the 
protocol and are: 
• 
Ohio State University Wexner Medical Center, Columbus, OH 
• 
Cleveland Clinic Foundation, Cleveland, OH 
• 
Washington University Medical Center, St Louis, MO 
• 
TriCore Reference Laboratories, Albuquerque, NM 
• 
The practice has 4 board certified pathologists to serve as “reader” pathologist to conduct the 
study for primary diagnosis. The pathologists shall be both specialists and generalists, with 
acceptable proficiency testing and site performance metrics (e.g. case review rate by tumor board, 
amended reports) not outside the practice’s norm. 

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• 
The practices shall dedicate at least 1 enrolling pathologist (EP) to conduct screening and 
enrolling cases per inclusion and exclusion criteria, and at least 1 validation enrolling pathologist 
(VEP) to confirm case enrollment of EP  
• 
Each site shall have at least one dedicated scanning technician properly trained by the sponsor in 
WSI scanning operations per the sponsor’s operator’s manual. 
• 
Site can and will conduct the study as approved by the institutional review board (IRB) and in 
accordance with Good Clinical Practice (GCP) and Human Subjects Protection requirements 
6.2 
Selection of pathology practice and pathologists cases 
The FDA has provided a table of specific diagnoses, which are to be considered when selecting cases for 
inclusion in the primary diagnosis study, including organ system, and sample size for particular subtypes 
and procedures.  These are listed in Table 2 below.   
 
Table 2: FDA List of Cases to be tested for Primary Diagnosis Study 
(TOTAL 2000 CASES FOR THIS EXAMPLE) 
CNB = Core Needle Biopsy;      TUR = Transurethral Resection;     LEEP = Loop Electrosurgical Excision Procedure;  Dysp = Dysplasia; ECC = 
Endocervical Curettage; R/O ≡ Rule Out; 
ORGAN 
# OF 
CASES 
  
SUBTYPES (procedures) 
BREAST 
300 
  
50 
Benign/Atypical CNB 
  
 
50 
Benign/Atypical Lumpectomy 
  
50 
In-Situ Carcinoma CNB 
  
50 
In-Situ Carcinoma Lumpectomy 
  
50 
Invasive Carcinoma CNB 
  
50 
Invasive Carcinoma Lumpectomy 
PROSTATE 
300 
  
120 
Benign Core Bx 
  
 
30 
Benign Resection 
  
 
120 
Adenocarcinoma Bx 
  
  
  
30 
Adenocarcinoma Resection 
LUNG/BRONCHUS/Larynx/oral 
cavity/Nasopharynx 
100 
  
25 
Benign/Inflammatory Bx Only 
  
25 
Dysplasia Bx Only 
  
30 
Carcinoma Bx 
  
  
  
20 
Carcinoma Resection 
COLORECTAL 
150 
  
50 
Benign/Inflammatory Bx 
  
50 
Adenomas Including Severe Dysp Bx 
  
40 
Adenocarcinoma Endoscopic Bx 
  
  
  
10 
Adenocarcinoma Resection 
GE Junction 
100 
  
50 
R/O Barrett’s/Dysplasia Bx 
  
  
  
50 
Non-Neoplastic/Inflammatory Bx 

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Table 2: FDA List of Cases to be tested for Primary Diagnosis Study 
(TOTAL 2000 CASES FOR THIS EXAMPLE) 
CNB = Core Needle Biopsy;      TUR = Transurethral Resection;     LEEP = Loop Electrosurgical Excision Procedure;  Dysp = Dysplasia; ECC = 
Endocervical Curettage; R/O ≡ Rule Out; 
ORGAN 
# OF 
CASES 
  
SUBTYPES (procedures) 
Stomach 
100 
  
50 
Inflammatory Including R/O H. Pylori Bx 
  
 
40 
Polyps/ Neoplastic Bx 
  
  
  
10 
Polyps/ Neoplastic Resection 
SKIN 
175 
  
50 
Non-Neoplastic/Inflammatory Bx 
  
50 
Squamous/Basal Cell Neoplasms Bx 
  
  
  
75 
Melanocytic Lesions Bx 
LYMPH NODE 
100 
  
75 
For Presence/Absence Of Metastasis  
(no micrometastases smaller than 0.5 
mm) 
  
  
25 
Non-Neoplastic 
BLADDER 
100 
  
25 
Benign/Inflammatory/Non-Neo Bx 
  
25 
Dysplasia Bx 
  
25 
Noninvasive Carcinoma (TUR Or Bx) 
  
15 
Carcinoma TUR/Bx 
  
  
  
10 
Carcinoma Resection 
Gyn 
150 
  
40 
Endometrial Bx/Curetting 
  
 
10 
Hysterectomy for endometrial or cervical 
cancer 
  
 
25 
Cervix Bx/Curetting (Bx, ECC) 
  
25 
Cervix Bx/Curetting (Cone/LEEP) 
  
20 
Ovary Benign/Non-Neoplastic 
  
  
  
30 
Ovary Neoplastic 
LIVER/BD, NEO 
50 
  
40 
Core Bx 
  
  
  
10 
Wedge Bx or Resection 
Endocrine 
100 
 ALL 
COMERS 
50 
Pancreas 
  
30 
Thyroid  
  
10 
Parathyroid 
  
  
  
10 
Adrenal 
BRAIN/NEURO 
60 
  
10 
Non-Neoplastic 
  
 
25 
Neoplastic Bx 
  
  
  
25 
Neoplastic Resection 
KIDNEY, NEOPLASTIC 
50 
  
50 
All Comers (Consecutive Cases) 
Salivary gland 
50 
  
50 
  
Hernial/Peritoneal 
10 
  
10 
  

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Table 2: FDA List of Cases to be tested for Primary Diagnosis Study 
(TOTAL 2000 CASES FOR THIS EXAMPLE) 
CNB = Core Needle Biopsy;      TUR = Transurethral Resection;     LEEP = Loop Electrosurgical Excision Procedure;  Dysp = Dysplasia; ECC = 
Endocervical Curettage; R/O ≡ Rule Out; 
ORGAN 
# OF 
CASES 
  
SUBTYPES (procedures) 
Gallbladder 
10 
  
10 
  
Appendix 
10 
  
10 
  
Soft Tissue Tumors 
20 
  
20 
  
Anus/Perianal 
50 
  
50 
Bx 
Miscellaneous to reach 2000 
15 
  
15 
  
Sponsor should include a sufficient number of difficult and challenging diagnoses in the larger (>100) groups 
 
The total number of cases is to be 2000.  The total cases are to be subdivided among the four (4) 
independent clinical sites (academic, community hospital, and private laboratories.)  Each site will be 
assigned cases consistent with the types of cases read at that site and from the cases listed in Table 2.  The 
cases will be selected from actual cases diagnosed at that practice and taken from that site’s own database.  
However, it may be impractical for some sites to have sufficient cases of all types required under the 
protocol.  In such eventuality, the PIs will review any gaps in cases enrolled across the study and propose 
adjustments of allotted cases at certain sites where some unbalance with respect to all numbers of cases 
for certain sites may occur. However, each site will enroll at least 400 cases and no more than 600 cases. 
Ten extra cases at each site will be selected for self-familiarization testing after pathologist training. The 
selection of these ten cases will follow after the cases for the study have been collected. 
6.2.1 Inclusion criteria:  
Cases are eligible to be included in the study only if all of the following criteria apply: 
• 
Cases originating from and that were diagnosed at that local site  
• 
Cases are available in the site’s archive 
• 
Cases are at least 1 year old since accessioning 
• 
Cases are selected because their primary diagnosis is consistent with the assigned target 
categories listed in Table 2 
• 
Cases have a set of slides representative of the primary diagnosis for which it has been selected 
(see below for slide selection criteria). 
Slide selection for a given case must meet the following criteria: 
• 
Slide is obtained by surgical pathology and prepared from FFPE human tissue 
• 
Slides must be stained with H&E and accompanying special stains (histochemical and/or 
immunohistochemical) 
• 
All special stains slides (histochemical and/or immunohistochemical) where the slide and stain is 
used for diagnosis, not prognosis. 

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• 
A chosen slide must demonstrate and be representative of the primary diagnosis; 1 H&E slide 
selection may suffice for biopsy cases,  
• 
For resection cases, a minimum of 5 H&E slides must be selected, which represent the primary 
diagnosis. If represented with less than 5 H&E slides, additional H&E slides (primary, secondary, 
or benign slides) from same case may be used to fulfill minimum number 
• 
Slide is intact, has correct size/thickness, good edges, undamaged coverslip, without pen 
markings that can’t be removed, no air bubbles, tidy labels, and fulfills the quality checks per the 
general clinical practice 
6.2.2 Exclusion criteria:  
Cases are excluded from the study if any of the following criteria apply: 
• 
Case does not have relevant slides or if case information necessary for the study is missing 
• 
Case is still active (less than 1 year old) at the local site 
• 
Cases for which the control slides for immunohistochemistry and special stains are not available 
• 
Two cases from same individual 
• 
Gross-only cases that have no slides 
• 
Cases that are frozen section, cytology or hematology or immunofluorescence specimens only 
• 
Case where the only available set of slides have evidence only of secondary or no diagnoses and 
not the primary diagnosis for which the case is being screened. 
Slides for a given case will be excluded if they meet the following criterion: 
• 
Glass slide that is broken, has abnormal size/thickness, beveled edges, poor coverslip (cracks, 
waviness, scratches), is sticky, has many pen markings or dirt that cannot be removed, contains 
air bubbles and overhanging labels that can’t be corrected, and if stain is severely faded. 
6.3 
Withdrawal or Discontinuation  
6.3.1 Slide Withdrawal 
It is unlikely that slides will be withdrawn from the study after they satisfy inclusion and exclusion 
criteria.  However, if during the trial a slide somehow gets damaged or misplaced and cannot be used, or 
an inadvertent mix-up is discovered, this slide (or case) may be withdrawn and a protocol deviation noted.  
Such occurrence is expected to be very low. If necessary, another slide from the same case may need to 
be added or a new case may need to be enrolled in order to satisfy the overall number of cases examined 
for the trial.  As long as the new slide does not change the recorded ground truth primary diagnosis and 
synoptic information of subtype, grade, margin and lymph node involvement, then the RP will not be 
asked to re-read cases already read. These events will be documented. 
6.3.2 Case Withdrawal 
It is unlikely that cases will be withdrawn from the study after they satisfy inclusion and exclusion 
criteria.  However, if during the trial a case can no longer be used, or an inadvertent mix-up is discovered, 
this case may be withdrawn and a protocol deviation noted.  If necessary, a new case may need to be 
added to satisfy the overall number of cases examined for the trial. The substituted case shall be read by 

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all RPs for both modalities. These events will be documented.  Such occurrence is expected to be very 
low. 
6.3.3 Pathologist Withdrawal 
If a reading pathologist is unable to complete the study reads according to study schedules, a replacement 
pathologist may be selected by Hamamatsu.  In that situation there will be no re-reads of cases by the 
replacement pathologist (to be selected as similar as possible to the original pathologist), who will begin 
where the other pathologist left off, as long as the prior pathologist had completed reads for both 
modalities.  The primary analysis will use all readings by the original/previous pathologist, with the 
readings by the replacement pathologist only being used for cases that the original/previous reading 
pathologist did not read with both modalities. Such occurrence is expected to be very low, but there is 
some possibility of staff turnover at any location. 
7. 
DEVICE UNDER STUDY 
7.1 
System Description 
The NanoZoomer S360MD Digital Slide Scanner System shall include: 
1. The NanoZoomer unit to make whole slide digitized image scans of conventional histological 
glass slides of surgical tissue samples; 
2. The software (C13220-01MD, version 1.0.0) to operate the scanner to create and store WSI 
images, as well as to view, manipulate, and annotate the digital WSI image on the monitor; and 
3. The monitor for viewing the images. 
The subsystems of the NanoZoomer system are connected over an information technology (IT) network.  
The IT hardware/software that supports the application software are not part of the system, but may be 
located in a central server room separate from the workstation with the viewing software and display.  
The communication of data between scanner and display may be linked by a customer-provided wired 
network or a direct connected cable between the systems.  The display will be validated as part of the 
clinical study. 
Key Specifications are: 
Glass slide size 
26mm x 76mm 
Objective lens 
20x (NA 0.75) 
Scanning mode 
20x mode / 40x mode 
Scanning resolution / pixel 
20x mode (0.46micron) / 40x mode (0.23micron) 
Image acquisition 
Color CMOS sensor 
Light source 
LED 
Slide capacity 
360 slides 
NA = numerical aperture, CMOS = complementary metal-oxide semiconductor, LED = light emitting diode. 
 

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Figure 4.  Communication between Scanner and Display 
 
The Slide Scanner consists of the following blocks as shown above in Figure 4. 
• 
Slide Feeder: The slide feeder is manually loaded with glass slides and then transfers glass slides 
from the slide cassette to the macro scanning position using the mechanical hand. 
• 
Macro Scan: When the glass slide has moved to the macro image scanning position, the macro 
LED illuminates the slide and acquires the macro image of both specimen and barcode label.  The 
scanning area is automatically determined based on the macro image. 
• 
Micro Scan: After acquiring the macro image, the glass slide is moved to the micro image 
scanning position.  Then the micro LED illuminates the slide, and focusing points are 
automatically detected, and the scanning of micro images starts.  After the scanning has finished, 
the glass slide is moved back to the slide cassette. 
• 
Control Board: The control board controls both the micro and macro cameras together with the 
focusing lenses. 

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7.2 
General Principles of Operation 
The Hamamatsu NanoZoomer S360MD Digital Slide Scanner (Figure 5) creates diagnostic quality digital 
images of glass slides containing FFPE tissue (11).  Each image typically contains billions of image 
pixels, creating a digital image of the tissue on the original glass slide.  The NanoZoomer captures digital 
images of entire slides for duplication, annotation, storage, retrieval, image sharing, and viewing to permit 
the pathologist to make a primary diagnosis without needing to view those glass slides through a light 
microscope. 
The NanoZoomer can be loaded with as many as 360 glass slides with the mechanics of a slide feeder.  
The glass slide is transferred to the stage where the whole glass slide image is captured together with the 
barcode information present on each slide.  Then the glass slide is moved to the scanning position.  With 
the motorized stage, the 20x objective lens, the CMOS camera, and the LED, the NanoZoomer detects 
focusing points and scans the slide to acquire the digital slide image.  During the scanning, the image is 
automatically stored on a hard disc.  After scanning, the fully digitalized image can be observed with the 
viewing software. 
The computer controls the scanner via camera link and universal serial bus (USB) interface.  It also stores 
the images on a hard disk.  After the scanning has finished, the images can be displayed on the 
computer’s monitor. 
The system software does not have any software application that does automated image analysis to aid in 
computer-aided detection of diagnosis. 
 

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Figure 5.  Example block diagram of typical components found in current WSI systems 
 
8. 
STUDY ASSESSMENTS AND PROCEDURES 
8.1 
Preparation of Facility 
The sponsor shall install a complete Hamamatsu NanoZoomer System at each of the 4 sites.  The system 
shall include the NanoZoomer Slide Scanner System, monitor, product software, and Operator’s Manual.  
In addition to the components of the Hamamatsu NanoZoomer system, additional off-the-shelf 
components shall be used with the system, such as a designated computer, input device, and perhaps a 
sponsor’s server or the site intranet for data storage.  

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The sponsor’s product specialists shall ensure the devices are operational and that all sites have all proper 
records and any relevant certificates of manufacture.  Likewise, the sponsor shall ensure any required 
maintenance is planned and conducted for the instruments prior to the start and during the course of study, 
as required.  Sponsor will track the serial number of each NanoZoomer installed at each site. The Sponsor 
will retain at the site any shipping records for each device. Device accountability logs will be used to 
account for equipment at site. The Sponsor will maintain accountability of each device.  All study records 
will be kept in a secure place at the site by the site coordinator. 
The site IT department shall ensure there is proper and secure IT services for data storage of scanned 
images, recording of medical information, and retrieval of images for viewing, and for proper and secure 
use of electronic data capture (EDC) systems for data collection. 
The WSI monitor viewing station cockpit and input device shall be standardized across all sites. Viewing 
images must be done at this defined monitor viewing station as the monitor is a required part of the 
NanoZoomer system as specified for the product and study. 
The pathologist shall use the light microscope conditions normally used for reading of glass slides at the 
site. 
8.2 
Training on Study Procedures and WSI Operation  
The sponsor, or its designated contract research organizations (CROs) or clinical professionals, shall 
conduct training according to pre-established and documented training modules, which may include: 
• 
Selection of sites and pathologists to participate in study 
• 
Shipment, installation, maintenance, and service of equipment by Hamamatsu personnel 
• 
Screening and enrolling of cases, slides, and establishment of ground truth for non-inferiority 
study, recording of case notes to be made available to reading pathologist, training slides, and 
selection of self-familiarization slides 
• 
Coding procedures for cases, slides, ground truth, and medical information selected for the 
enrolled case. 
• 
Coding procedures for the reader pathologist chosen to participate in the study 
• 
Storing, securing, and de-identifying Protected Health Information (PHI) for cases selected for 
study 
• 
Use of WSI equipment for scanning and image storage, coordination with IT systems, and 
viewing workstation setup and operation 
• 
Method to present case information available to pathologist  
• 
Method to conduct of self-familiarization process 
• 
CRO Installation, operation, and maintenance of EDC systems at site, validation and password 
protected access 
• 
CRO Entering, storing, and securing data in electronic case report form (eCRF) 
• 
CRO Randomization procedures for reading slides 
• 
CRO Adjudication charter and process to adjudicate reader pathologists’ diagnoses 
• 
Documentation and reporting on device deficiencies and protocol deviations 

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• 
CRO Procedures for data management to manage the data integrity (data rejection, audit trail, 
verification, validation, and locking database) 
• 
CRO GCP requirements training 
• 
CRO GCP monitoring procedures 
Training on Adjudication Process: 
• 
Rules for Adjudication of “Major” and “Minor” discrepancies,  
• 
CRO Operation of Adjudication Software    
Each training event shall be recorded in a training log as to what training was done, who was trained, who 
did the training, where the training was performed, and the date of training.  The training records shall be 
stored in a secure place by the study coordinator (SC). 
Training shall include training of the: 
• 
Entire site study staff (PI, sub-PIs, study coordinators, enrollment and validation enrollment 
pathologists, reader pathologists, WSI technicians, IT personnel) on study protocol, study 
processes, essentials of GCP, and human subjects training or applicable to those individuals. 
• 
Study coordinators on proper storage of equipment, essential study GCP documents and records, 
eCRF and data records management. 
• 
Technicians on operation of NanoZoomer scanner equipment, how to scan, review, check, 
annotate, and store scanned image, and training on what and how to fill out eCRF for scanning 
operation. 
• 
Information Technology (IT) staff, if relevant, responsible for proper storage and retrieval of 
scanned images. 
• 
EP on selection and validation of cases and slides for study, recording of ground truth, proper 
selection of slides from cases that demonstrate primary diagnosis, and collection of medical notes 
available to reader pathologist.  
• 
VEP for review and confirmation of the cases, slides, supplemental information, and ground truth 
diagnosis selected by the EP. 
• 
Reader pathologist (RP) on the protocol, study processes, instructions how to operate and view 
WSI slides for primary diagnosis, and how to fill out the eCRF for each case.  
• 
Adjudication pathologists on procedures for adjudicating a) differences between EP and VEP 
enrollment case selection, and b) diagnosis of WSI and LM readings if identical to ground truth 
or discrepant and if any discrepancy is major or minor in respect to patient treatment. 
Once trained, the reader pathologists shall be allowed to self-familiarize themselves in a mock primary 
diagnosis by WSI on a small set of training cases in order to become familiar with equipment operation.  
The pathologist will be allowed to conduct this trial diagnosis on 10 case slides chosen and selected per 
the same enrollment procedures for the cases for the study.  Once the pathologist feels comfortable with 
the self-familiarization process, he or she may begin the study. 
8.3 
Selection of Case Slides  
The site coordinator and/or EP shall search through the site’s database to locate and withdraw from the 
archive the assigned number of consecutive cases starting with archival cases at least 1 year old since 
accessioning and moving consecutively back in time.  They will choose cases categorized from FDA in 

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Table 2. The EP will screen and enroll cases per the inclusion and exclusion criteria, to record the ground 
truth primary diagnosis, to record the FDA category information (i.e., organ, procedure, subtype, and 
category), record the basic patient case note(s) that the reader pathologist will be presented with, to select 
the slides indicative of primary diagnosis and to collect the supplemental diagnostic information and 
synoptic checklist information, where available in site pathology report, and relevant to classification, 
subtype, grade, margin, status, etc.  The EP may consult with other pathologists within the site practice 
who may be subject matter experts in certain case categories, as long as those pathologists are not reader 
pathologists or VEPs.  This supplemental information will be in accordance with truncated CAP cancer 
protocols (17) developed to highlight the key synoptic information to take into account later when reader 
pathologist makes study diagnosis. 
GT diagnosis is the primary diagnosis based on the original sign-out (reported) primary diagnosis of cases 
that are enrolled in the study.  The ground truth will not be reestablished, and should not include 
secondary diagnoses.  Only a review of the final sign-out report (and not a review of the glass slides) will 
be used to record this information.  The original sign-out primary diagnosis (reported clinically), as well 
as any supplemental diagnostic information, will be captured verbatim. If site uses synoptic checklists in 
their pathology report, that information, too, will be recorded. 
From those cases, the EP will choose the H&E slide or set of H&E slides in the case that are consistent 
with the primary diagnosis.  The following rules will be followed when selecting slides: 
• 
For biopsy cases, the EP will select at least the H&E slides representative of the primary 
diagnosis. In most biopsy cases, this can be done with just 1 H&E slide, but the EP may choose 
more if needed to define the diagnosis. To minimize selection bias, the EP shall select the first 
H&E slide that encompasses that primary diagnosis.  
• 
For resection cases, the EP shall select the set of slides that are necessary to indicate the primary 
diagnosis, inclusive of the key CAP checklist abridged metrics of cancer type, grade, margins and 
if metastasized to lymph nodes.  
o If there are multiple levels, the EP may select only the level of the same block slide that is 
germane to the primary diagnosis.  
o To minimize selection bias, the EP shall choose the first available H&E slide that describes 
that condition(s), rather than the best slide.  
o A minimum of 5 H&E slides will be selected. If the set of slides selected to define the 
primary diagnosis is < 5, then the EP shall continue to pick H&E slides until a minimum of 5 
is achieved, and thereby the EP may pick further primary diagnosis slides, secondary 
diagnoses slides or slides irrelevant to any recorded diagnosis, whichever comes first. 
In addition, for any case where there are extra slides with special and/or immunochemical stains, the EP 
shall enroll all those, as long as these extra stains are used for making/confirming the diagnosis and not 
for prognosis or to guide treatment (e.g. human epidermal growth factor receptor 2 [HER2] stain in breast 
cancer). 
For enrolled slides, the EP shall remove all markings from the coverslip. If not possible, the slide shall be 
excluded, and another slide must be chosen. 
A priori defined slide and case data rejection criteria have been developed per the defined inclusion and 
exclusion criteria in section 6.  Some case data may be spurious or artifactual and may need to be 
disregarded, or slides may be damaged, aged, or lost, or there may be corrupted digital files, etc. 

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For every case enrolled, the EP shall make a determination if this case can be considered to be a “difficult 
and challenging” case. The EP will select “difficult and challenging” when one or more of the following 
apply: 
• 
It’s professional opinion of EP that case is “difficult and challenging” 
• 
Case used special stains or immunohistochemical stains to aid in diagnosis 
• 
Case used molecular tests to aid in diagnosis 
• 
Case used tumor boards to review case to aid in diagnosis 
• 
Other reason, and brief narrative to elaborate. 
The EP shall capture this information for every one of the 500 enrolled cases for that site. 
8.4 
Validation of case and slide selection  
Upon completion of the screening and enrollment by the EP, then one validation enrollment pathologist 
(VEP) shall review and confirm the screening and enrollment selection of the EP.  If the VEP disagrees 
with the case or slide selection of the EP, then both the EP and VEP shall meet for consensus decision.  If 
agreement cannot be reached, then that case shall be disqualified. 
All enrolled material and information shall be provided to the site coordinator for secure storage and to be 
kept blinded from reading pathologists. 
8.5 
Storage of case slides  
The site coordinator shall:  
a. Collect and store the cases, slides and medical notes, clinical history of the case, and final 
pathology report in a secure location,  
b. Place proper de-identification labels (e.g., hide names, numbers, barcodes) on slides with a code 
known only to the site coordinator, and confirm those labels will not interfere with the scanning 
process nor affect the integrity of the tissue on the slide. The printer and label operations shall be 
validated before the study to prove the study labels do not interfere with the scanning process and 
are robust for the duration of the study, when the study labels can finally be removed and the 
material returned to the archive. 
c. Remove all patient identifiers and assign coded subject ID to protect patient privacy, and  
d. Ensure the traceability code-defining patient ID with study assigned code shall be kept only at the 
site, stored in a secure location, not shared outside the practice, and kept blinded to reading 
pathologists. 
8.6 
Whole slide scanning operations  
The SC shall deliver the slides to be scanned to the scanning technician. 
The scanning technician shall receive the batch of de-identified case slides to be scanned from the site 
coordinator.  Then the technician shall: 

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a. Scan the slide at 40x magnification according to the standard Hamamatsu default scanning 
conditions and save the image in its proprietary native file format. 
b. Conduct an initial quality check of the scanned slide to confirm that all material on the slide was 
properly scanned. 
c. Assess if the image looks to be of sufficient quality to advance to the pathologist. 
d. Transfer the WSI image to the assigned secure drive with proper file nomenclature consistent 
with the patient de-identified code. 
If the technician deems that the scan quality is unsatisfactory, the technician may scan again, shall note 
that in the eCRF, review the slide quality of the re-scanned slide, and then store these images in the secure 
drive location.  If re-scanning does not result in a suitable WSI, the technician shall note that in the eCRF, 
and then bring this to the attention of the EP and VEP to decide if that case or slide should be replaced.  
Data or re-scans will be recorded. Up to 5 scans will be allowed. 
Once complete, all case and slide material shall be promptly returned to the coordinator for proper and 
secure storage. The WSI files shall be bundled into folders, each specific for each case. 
8.7 
Reading a Case 
At each site and after appropriate training, four pathologists participating at that site will read the same set 
of case slides, unique to each site.  Each pathologist shall read each case on a conventional light 
microscope (Reference Method; Glass) and on the Hamamatsu system (Test Method; WSI) in an order 
randomly determined.  
RPs will receive from the site coordinator the slides and/or WSI images to be read. The case study 
identification, patient age and gender, relevant supplemental clinical history, part and procedure 
information, and specimen gross description in the EDC system in addition to corresponding slides (either 
glass or WSI) will be available for RP viewing, but RP will never have access to the final sign-out 
diagnosis or GT information.  Pathologists may not request re-scans of any slide. No additional slides 
from the case can be requested.  Reading Pathologists will not be permitted to order additional re-cuts or 
stains outside of what was cut/stained during the original clinical review process (i.e., no additional slides 
will be created for the purpose of this study) and selected by the EP and VEP. 
All RPs will perform randomized (read-order) glass and WSI reads with a minimum 4-week washout 
period between modalities.  The order of reading will be randomly assigned such that each case will either 
be read first using glass slides and LM and then (following a 4-week wash-out period) via WSI or vice 
versa.  All digital reads will be executed using the Hamamatsu Pathology workstation.  Pathologists will 
access the Hamamatsu system via secure login/password.  For viewing by glass, the pathologist shall use 
the light microscope that they normally use at that site.  
When the RP reads the cases, all WSI and glass diagnoses, as well as supplemental information based on 
truncated CAP checklists developed for this study, will be entered into an eCRF using an EDC system.   
The RP will write a primary diagnosis in a narrative text in their words.  
Next, the reader pathologist shall fill in an abridged CAP checklist to capture supplemental diagnostic 
information specific to each of the organ malignancy types in the study, if applicable.  The abridged 
checklist will include only those items most relevant to the patient’s management, i.e., type, grade, 
margin and whether infiltration into lymph nodes.  The pathologist may use information from an abridged 

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CAP checklist to clarify and complete the diagnosis description that satisfies the primary diagnosis. The 
RP may use textbook reference materials to assist in determination of diagnosis. 
The reading pathologist will be trained and given instructions how to use the CAP checklist information 
when writing the text that shall describe the reading pathologist’s diagnosis.  
Possibility of Deferral:  Reader must do her or his best effort to determine a primary diagnosis. However 
if the reading pathologist feels strongly that he or she is not able to make a diagnosis, then the reader 
pathologist may record that in the case report form. The recording can be “deferral to Glass” in the case of 
WSI, or “deferral to specialist” in either case of Glass or WSI.  
RPs will not be able to review eCRFs for which a diagnosis is finalized (case signed-out). 
8.8 
Adjudication of Discordant Readings  
Once the RP eCRF is reviewed, queried, cleaned and completed, that reading will be sent to the 
adjudicators to adjudicate the endpoint. 
The adjudicator pathologist (AP) is part of an independent board of subject matter expert, board certified 
pathologists, who shall review and compare diagnoses on WSIs and on Glass to the GT, the original 
diagnosis that was rendered in the clinical practice.  The AP(s) shall not be presented with the slides or 
WSI images. The adjudicators shall be presented only with the ground truth primary diagnosis and 
synoptic information, the patient case notes available to the reading pathologist, and the reading 
pathologist primary diagnosis and supplemental CAP checklist records. 
When the adjudicator is presented with a case to adjudicate, the 1st and 2nd adjudicator will independently 
review the case.  If a disagreement involving a major discordance is present between the 2 adjudicators, 
such a case will go to the 3rd member to adjudicate.  Based on the determination of the 3rd member, the 
majority choice will be selected. If there is no majority from the rulings of the 3 adjudicators, then the 
adjudication will proceed to a consensus meeting for final decision. 
 
Table 3: Definition of Discordance 
Severity Definition 
Minor 
No Harm: 
a. 
Will not result in harm 
b. No change in prognosis or a change in prognosis that is unlikely to result in a 
change in treatment according to standard care. 
Minimal Harm [Grade 1] 
a. 
Further unnecessary noninvasive diagnosis test(s) performed [e.g., blood tests or 
non-invasive radiological examination]. 
b. Delay in diagnosis or therapy of < 6 months. 
c. 
Minor morbidity due to [otherwise] unnecessary further diagnostic effort(s) or 
therapy predicated on the presence of [unjustified] diagnosis. 
Major 
Moderate Harm [Grade 2] 

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Table 3: Definition of Discordance 
Severity Definition 
a. 
Further unnecessary invasive diagnostic test(s) [e.g., tissue biopsy, re-excision, 
angiogram, radionuclide study, or colonoscopy]. 
b. Delay in diagnosis or therapy of > 6 months. 
c. 
Major morbidity lasting < 6 months due to [otherwise] unnecessary further 
diagnostic effort(s) or therapy predicated on the presence of [unjustified] diagnosis. 
Severe Harm [Grade 3] 
a. 
Loss of life or limb, or other body part, or long-lasting morbidity [lasting > 6 
months.]. 
 
8.9 
Pilot Studies 
Before the study starts, the sponsor may choose to conduct one or more optional pre-study mock pilot 
trials of certain key procedures, specifically the screening and enrollment, reader pathologist diagnosis 
and adjudication procedure.  This pilot is an extension of the user acceptance testing and is to allow the 
sponsor to observe and optimize the combined operation and workflow of training, procedures and eCRF 
recordings to insure processes are operational as expected, and consistent amongst participants. 
The pilot will include participation of the actual pathologists and scanning technicians who are assigned 
in the study.  Each pilot may be a full or abbreviated test of the actual study procedure by those 
participants. Each pilot may use upwards of 5-10 de-identified cases from the archive.  And, so to not 
interfere with the cases selection process, these pilot cases are to be selected with accession dates less 
than 1 year from non-active cases. In order to insure pilots are being done correctly, the sponsor and 
consultants may review pilot data and compare against de-identified pathology reports and scans of de-
identified histochemical slides for that pilot case. 
9. 
STATISTICAL CONSIDERATIONS  
The statistical analysis plan (SAP) will be developed and finalized before database lock and will describe 
the set of data to be included in the analyses, and procedures for accounting for missing, unused, and 
spurious data.  The SAP may also include additional exploratory analyses not explicitly mentioned in the 
following sections.   
9.1 
Statistical Hypotheses 
The primary statistical objective of this study is to demonstrate that the safety and accuracy of the 
Hamamatsu system is non-inferior to the Diagnostic Reference Standard glass diagnosis in routine 
surgical pathology cases. 
Assuming a non-inferiority margin of 4%, consistent with the literature (8-10) and with the target to 
which the PIPS’ clearance was held, then the hypothesis to be tested can be written as:  
H0: πWSI - πGlass > 0.04  Versus  H1: πWSI - πGlass ≤ 0.04  

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where 𝜋𝜋𝑊𝑊𝑊𝑊𝑊𝑊 and 𝜋𝜋𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 are the major discordance rates for WSI and Glass respectively. 
9.2 
Sample Size Calculations 
Assuming E{𝑑𝑑𝑙𝑙𝑙𝑙} = ∆ and variance Var{𝑑𝑑𝑙𝑙𝑙𝑙 } = 𝜎𝜎2 where 𝑑𝑑𝑙𝑙𝑙𝑙 is equal to average over differences of error 
rates between WSI and Glass compared to GT diagnosis for case 𝑖𝑖 in site  𝑙𝑙, the following expression is 
applicable: 𝑛𝑛= ൫𝑍𝑍𝛼𝛼+ 𝑍𝑍𝛽𝛽൯
2(𝜎𝜎2)
(𝛿𝛿+ ∆)2
൘
 
=
൫𝑍𝑍𝛼𝛼+ 𝑍𝑍𝛽𝛽൯
2𝜋𝜋′𝑊𝑊𝑊𝑊𝑊𝑊(1 −𝜋𝜋′𝑊𝑊𝑊𝑊𝑊𝑊)𝜋𝜋′𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺(1 −𝜋𝜋′𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺){1 −𝜆𝜆+ (𝑟𝑟−1)(𝜉𝜉−𝜂𝜂)}
𝑟𝑟(𝛿𝛿+ ∆)2
 
For which 𝜋𝜋′𝑊𝑊𝑊𝑊𝑊𝑊 and 𝜋𝜋′𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺 are the probabilities that WSI and Glass methods agree (or minor 
discordance) with the GT diagnosis classification retrospectively; 𝜆𝜆 = Corr(𝑎𝑎𝑙𝑙𝑙𝑙𝑙𝑙1, 𝑎𝑎𝑙𝑙𝑙𝑙𝑙𝑙2) , the correlation 
between the classifications for the two modalities by the same reader; 𝜉𝜉  = Corr(𝑎𝑎𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙, 𝑎𝑎𝑙𝑙𝑙𝑙𝚥𝚥′̇ 𝑘𝑘), the 
correlation between two readers for a given modality; and, η = Corr(𝑎𝑎𝑙𝑙𝑙𝑙𝑙𝑙1, 𝑎𝑎𝑙𝑙𝑙𝑙𝚥𝚥′̇ 2), the correlation between 
two modalities and between two readers. 
Assuming power = (1 −β) = 0.90, α = 5% two-sided, a non-inferiority margin of 𝛿𝛿, a true difference 
between proportions of WSI and Glass equal to ∆ in favor of Glass, number of readers r = 4 and number 
of sites = 4, the required sample size is provided in Table 4 for different ranges of 𝜋𝜋′𝑊𝑊𝑊𝑊𝑊𝑊, ∆, λ, and (ξ–η). 
 
Table 4: Sample Size for 4 Readers and 4 Sites 
𝝅𝝅′𝑾𝑾𝑾𝑾𝑾𝑾 
λ 
(ξ–η) 
𝐌𝐌𝐌𝐌𝐌𝐌 𝛔𝛔𝟐𝟐  
(𝛔𝛔𝟐𝟐for ∆= 𝟎𝟎) 
∆ = 𝝅𝝅′𝑾𝑾𝑾𝑾𝑾𝑾 – 𝝅𝝅′𝑮𝑮𝑮𝑮𝑮𝑮𝑮𝑮𝑮𝑮 
0.0 
-0.5% 
-1.0% 
-1.5% 
-2.0% 
0.6 
0.5 
0.150 
0.114000 
749 
976 
1326 
1904 
2968 
0.125 
0.105000 
690 
899 
1221 
1754 
2733 
0.100 
0.096000 
631 
822 
1116 
1604 
2499 
0.6 
0.150 
0.102000 
670 
874 
1186 
1704 
2655 
0.125 
0.093000 
611 
796 
1082 
1553 
2421 
0.100 
0.084000 
552 
719 
977 
1403 
2187 
0.7 
0.5 
0.150 
0.099750 
665 
852 
1154 
1653 
2568 
0.125 
0.091875 
604 
785 
1063 
1522 
2366 
0.100 
0.084000 
552 
718 
972 
1392 
2163 
0.6 
0.150 
0.089250 
587 
762 
1032 
1479 
2298 
0.125 
0.081375 
535 
695 
941 
1348 
2095 
0.100 
0.073500 
483 
628 
850 
1218 
1893 
0.8 
0.5 
0.150 
0.076000 
500 
646 
871 
1241 
1920 
0.125 
0.070000 
460 
595 
802 
1143 
1768 
0.100 
0.064000 
421 
544 
733 
1045 
1616 

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Table 4: Sample Size for 4 Readers and 4 Sites 
𝝅𝝅′𝑾𝑾𝑾𝑾𝑾𝑾 
λ 
(ξ–η) 
𝐌𝐌𝐌𝐌𝐌𝐌 𝛔𝛔𝟐𝟐  
(𝛔𝛔𝟐𝟐for ∆= 𝟎𝟎) 
∆ = 𝝅𝝅′𝑾𝑾𝑾𝑾𝑾𝑾 – 𝝅𝝅′𝑮𝑮𝑮𝑮𝑮𝑮𝑮𝑮𝑮𝑮 
0.0 
-0.5% 
-1.0% 
-1.5% 
-2.0% 
0.6 
0.150 
0.068000 
447 
578 
779 
1111 
1718 
0.125 
0.062000 
408 
527 
710 
1013 
1566 
0.100 
0.056000 
368 
476 
642 
915 
1414 
 
This table shows, a total of 2000 cases will provide more than 90% power to demonstrate non-inferiority 
of WSI to Glass with a non-inferiority margin of 4% as long as the proportion of agreements with Ground 
Truth is at least 60%, λ is at least 0.5, (ξ-η) is at most 0.15, and ∆ ≥ -1.5%.   
9.3 
Populations for Analyses 
All Available Population (AAP): AAP includes all cases for which at least one reader provides an 
evaluable outcome for either the WSI or Glass modality where an outcome is considered evaluable if the 
adjudication committee is able to classify the diagnosis as concordant, minor discordant, or major 
discordant with the Ground Truth diagnosis.  If a reading pathologist is unable to complete study reads, a 
replacement pathologist may be selected by Hamamatsu, the re-reads by the replacement pathologist are 
supplementary, and the primary analysis will use all readings by the original/previous pathologist, with 
the readings by the replacement pathologist only being used for cases that the original/previous Reading 
Pathologist did not read by both modalities. 
9.4 
Statistical Analyses 
This section is a summary of the planned statistical analyses of the primary and secondary endpoints 
described in detail in the study SAP. 
9.4.1 Case Characteristics and Disposition, and Reader Pathologists 
The characteristics of the cases selected at each site and overall will be descriptively summarized using 
numbers and percentages.  The characteristics of the cases will include, the case breakdown as specified 
in Table 2, including organ system, procedure type, overall classification. 
The number and percentage of deferrals will be summarized by reader, site, and overall for each modality.  
The reason for the deferral will also be provided.  
The characteristics of the reader pathologists at each site and overall will be descriptively summarized in 
regard to years in practice and specialty. 
9.4.2 Primary Analysis of Non-inferiority of WSI Relative to Glass 
The AAP will be used for the primary analysis.  To evaluate the major discordance rates between WSI 
and Glass, a mixed model repeated measures (MMRM) logistic regression will be performed.  The 
indicator of major discordance (yes/no) will be the dependent variable with modality (WSI or glass), as a 
fixed effect; and site, reader and case entered into the model as random effects; glass will be the reference 

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group. From this model, a two-sided 95% confidence interval (CI) for the difference in major discordance 
rate (𝜋𝜋𝑊𝑊𝑊𝑊𝑊𝑊 - 𝜋𝜋𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺𝐺) will be constructed.  If the upper bound of the 95% CI is less than the non-inferiority 
margin of 4%, then WSI will be considered non-inferior to Glass. 
A summary table will provide the number of deferrals and reasons for deferral (e.g., deferral to 
specialist, deferral to glass, etc.). Cases with diagnosis deferred for at least one modality (excluding 
missing observations) will be excluded from the primary analyses. 
The point estimates and the corresponding two-sided 95% confidence intervals of the major discordance 
rates will be provided separately for WSI and glass.  To support the clinical performance of NanoZoomer, 
the major discordance rate for WSI should not exceed 7%.   
 
Sensitivity Analysis 
Sensitivity analyses will be performed to evaluate the robustness of the results. All deferred plus missing 
data cases will be assumed as having major discordance with GT and included in the same MMRM 
logistic regression analysis as that of the primary analysis.  
 
Generalizability for Readers and Sites  
The WSI vs. glass difference in major discordance rate and associated 95% CI by reader will be 
constructed using the primary MMRM logistic regression model.  The 16 such confidence intervals for 
the 4 readers at the 4 sites will be descriptively displayed in a Forest Plot. The extent to which the 
respective confidence intervals in the Forest Plot are overlapping sheds light on their homogeneity, and 
reasonably homogeneous confidence intervals can support generalizability with respect to readers in the 
sense of comparability of estimated differences between WSI and Glass for the 4 readers at the 4 sites.  
Generalizability for sites will be addressed in a similar way to that previously discussed for readers within 
sites.  The 4 confidence intervals for the 4 sites can be descriptively displayed in a Forest Plot.  The extent 
to which the respective confidence intervals in the Forest Plot are overlapping sheds light on their 
homogeneity, and reasonably homogeneous confidence intervals can support generalizability with respect 
to sites in the sense of comparability of estimated differences between WSI and glass for the 4 sites.  
9.4.3 Secondary Endpoints Analyses 
The AAP will be used for all secondary endpoints analyses. Same as the primary analysis, missing 
observations and deferred outcomes will be excluded from these analyses. 
For the first 4 secondary endpoints, summary statistics and corresponding two-sided 95% confidence 
intervals for the major discordance rates and differences between the major discordance rates within each 
subgroup of interest (site, organ, diagnostic category, and procedure type) will be provided. 
To evaluate the Intra-reader/Inter-modality (within reader, between modality) precision, with glass as the 
comparator method, the percentage of overall agreement and 95% confidence intervals between the WSI 
and the Glass diagnostic categories will be computed for each reader at each site separately.   
To evaluate the Inter-reader/Intra-modality (between reader, within modality) precision, with glass as the 
comparator method, the percentage of agreement and 95% confidence intervals will be computed for each 
pair of readers at each site separately (6 pairwise comparisons per site).  

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9.4.4 Other Analyses 
The occurrence and rate of all technical issues during scanning and viewing of WSI images shall be 
tabulated. 
10. 
DATA MANAGEMENT AND DATA QUALITY 
10.1 
Data Management 
The study shall have a data management plan to define how the data is handled, managed, stored, secured, 
and how the processes for that are validated, and how the access is controlled. 
Case report forms (CRFs) and logs will be used to collect the data recorded in the course of the study, and 
shall be presented in an electronic data capture (EDC) system. 
An eCRF for selection and enrollment of cases is to be completed for each case by the EP and confirmed 
by the VEP.   
This information includes:  
• 
Site ID, organ, procedure and subtype,  
• 
Clinical history, age, gender, gross description notes 
• 
Inclusion & exclusion criteria,  
• 
Ground truth primary diagnosis, and diagnosis notes, and, where applicable, synoptic information 
of subtype, grade, margin and lymph node involvement 
• 
Number of slides for the entire case, number of slides selected for the study, and organ of the 
slide, and stain for that slide 
• 
Patient case notes. 
An eCRF or log for WSI scanning operation for creation of WSI images to be filled out for each case by 
the technician scanning the glass slides which shall include: 
• 
Clear instructions on what to do at what point along the way. 
• 
Field entries for:  
o Date and Time 
o Name of technician 
o Site Name/Number 
o Case # 
o Slide # if there is more than one slide for the case 
o Condition of scanned image, if not acceptable, and why 
 
If there is any gross out-of-focus, image aberration, color distortion, etc. 
 
If all material was scanned. 
 
If a rescan was required and why 
 
If final scan acceptable 
 
# of rescans 
An eCRF for Primary Diagnosis recording to be filled out by the pathologist reader, which shall include: 
• 
An eCRF for case and modality, with WSI-specific fields shown only for WSI cases 

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• 
Clear instructions on what to do at what point along the way 
• 
Field entries for:  
o Date and Time 
o Site name/number 
o Case # 
o Slide # if there is more than one slide for the case 
o Tissue and organ and stain used 
o Clinical history, age, gender, gross description notes 
o ID of pathologist conducting reading  
o Start time and date of reader’s review 
o Checkbox if reading by light microscope or WSI 
o Record of “Deferral” if to Glass, to specialist, or “Other” with free text field to describe. 
o Record of Primary Diagnosis:  
o Record all Abridged CAP checklist information as appropriate for resection cases, including 
primary diagnosis text narrative, as well as subtype, grade, margin, and if lymph node 
involved, etc. 
o End time and date of reader’s review 
An eCRF for the Adjudication Panel to be filled out by each Adjudication Pathologist, which shall 
include 
• 
Adjudication pathologist ID  
• 
Record of match, major or minor mismatch, and reason for decision, if needed 
• 
Record if adjudicator felt case should go to consensus meeting 
• 
Date of consensus meeting, consensus ID, final determination of discordance and rationale for the 
decision if needed. 
 
 
 

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• 
Note: The example shown is a case of a breast lumpectomy specimen with sentinel node 
excision, from which 3 slides were selected for the trial. 
 
• 
Reading Pathologist’s Name: Dr. X entered 
• 
Case Number:   
  Case #1 entered 
• 
Primary diagnosis:  
 
  Invasive breast carcinoma 
• 
Tumor histologic type:  
  Invasive ductal carcinoma (or just ductal) 
• 
Histologic grade:  
 
  Overall grade (1, 2 or 3) entered 
• 
Margin status:  
 
  Involved or uninvolved by invasive carcinoma entered 
• 
Lymph node involvement:  
  Positive or Negative for metastatic carcinoma entered 
Figure 6.  Example of CRF (using truncated CAP checklist) 
 
A CRO data manager shall create a pre-established validated database containing all the fields from the 
eCRF, which will be stored on a secure file server at the site or on a secure server of the CRO managing 
the EDC.  The database will be locked and stored in a secure place and transferred to the CRO study 
statistician for analysis.  All analyses will be conducted using data from that stored database. 
10.2 
Data Quality 
For each and every case reading, the CRO data manager will promptly and thoroughly review each eCRF 
to ensure all fields are complete, and query the coordinator to request a pathologist to correct and 
complete as quickly as possible any incomplete or illegible entries.  
Once the reader pathologist diagnosis is complete (whether WSI or glass), the data manager shall confirm 
completion of the case and advance the relevant data to the adjudicators.  
10.3 
Data Retention Procedures and Period 
The Sponsor and site should retain all data and study records in a controlled location for a minimum of 2 
years after completion of study or completion of FDA submission process. 

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10.4 
Publication Policy 
The results of this study may result in publication.  
The study will be registered on www.clinicaltrials.gov. 
11. 
DEVIATIONS FROM THE CLINICAL INVESTIGATION PLAN 
The site personnel shall document all protocol deviations and device deficiencies in logs to document the 
event and date, as well as clear explanations of the event.  Protocol deviations and device deficiencies 
shall be recorded in these logs by the scanning technician, study coordinator or enrolling and/or reader 
pathologists. Any protocol deviations noted by CRO during monitoring and data management will be 
directed to site study coordinator to enter into protocol deviation log. 
11.1 
Protocol Deviations  
A protocol deviation is defined as any change or alteration from the procedures stated in the clinical 
investigation plan, consent document, recruitment process, or study materials (e.g. questionnaires) that 
were originally approved by the IRB where the change or alteration itself is not IRB approved.  
All protocol deviations must be reported to Hamamatsu or their authorized representatives (CRO study 
monitors) through the protocol deviation log form. In addition, the Investigator is required to adhere to 
IRB of records’ procedures for reporting protocol deviations.  Deviations shall be documented in writing 
and maintained in the Investigator and clinical study management files.   
Per 21 CFR §812.140 (a) (4), Investigators are required to maintain accurate, complete and current 
records, including documentation showing the dates of, and reasons for, each deviation from the CIP.  
Failure to comply with the CIP may result in Investigator termination of participation [21 CFR §812.46 
(a)] in the current study. 
Repeated protocol deviations will lead to site retraining.  Continued deviation from the CIP may result in 
site or investigator suspension or disqualification and is at the discretion of the Sponsor.  Suspension 
and/or disqualification actions, including the rationale, will be documented. 
For protocol deviations, study processes, including washout, modality read order, and matching of slides 
with correct case information will be closely managed.  A deviation will be filed for any case part that has 
missing, damaged, or broken slides.  These cases will be reviewed for consistency with the protocol 
across all Reading Pathologists.  
11.2 
Device Deficiencies 
A device deficiency is when a device not working as designed. The site personnel shall document any 
device deficiencies by recording the event narrative and date.  All device deficiencies must be reported to 
Hamamatsu or their authorized representatives or CRO (study monitors) through an appropriate log. Any 
incident with the operation of the WSI device will be recorded and assessed for impact on scanning, file 
storage and file viewing operations. 
Sponsor may terminate study conduct due to poor product performance. 

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12. 
SAFETY AND SUBJECT CONFIDENTIALITY 
12.1 
Safety 
For the purposes of this study, the Hamamatsu NanoZoomer S360MD Digital Slide Scanner System is 
not considered a significant risk IVD device (21 CFR §812.3(m) and poses a low risk to the physical 
safety of study participants.  Findings from the current study will not be used to diagnose or impact 
treatment of subjects.  No invasive sampling techniques will be utilized as cases/slides are selected from 
patient archives.  
12.1.1 Adverse Events 
The study does not anticipate any adverse events (AEs) as this is a retrospective study without possibility 
to impact patient care.   
12.2 
Patient Confidentiality  
All reports and communications relating to Subjects in the study will identify Subjects by their Subject ID 
number only. The site coordinators will de-identify all cases and slides of patient identifiers, and replace 
the labels with a code for Subject ID for cases and slides being used in the study.  All other personnel of 
the study, with the exception of the study monitor of the sponsor, sponsor’s agent or CRO, will be blinded 
to the actual patient information.  The locked database that goes to the CRO statistician and to the sponsor 
shall be free of any patient information before transfer to sponsor and CRO statistician.  If there is a 
privacy breach, the sponsor will take reasonable steps to expunge such private information from its 
systems. 
12.3 
IRB and Protocol Approval  
The protocol must be submitted and approved by the site IRB of record before the start of the study.  The 
study will ask for a waiver of informed consent consistent with other reported studies since active slides 
will not be included.  The IRB will be informed that study results may lead to publication and 
presentation at scientific meetings.  There is no anticipated risk to patients with this study. The site will 
not start the study until the site IRB approval is available.   
Amendments to the CIP will be reviewed by and agreed upon by the Sponsor and the Principal 
Investigators prior to implementation.  Amendments will be approved by the sites’ IRBs as required by 
applicable IRB procedures.  All amendments will be tracked in the CIP revision history. 
Any additional requirements imposed by the IRB or FDA shall be followed. 
13. 
COMPLIANCE AND GOOD CLINICAL PRACTICES (GCP) 
The trial will be carried out in accordance with Good Clinical Practice (GCP) as required by US Code of 
Federal Regulations (CFR) applicable to clinical studies (45 C.F.R. Part 46, 21 C.F.R. Part 54, 21 C.F.R. 
Part 56, and/or 21 C.F.R. Part 812) and ICH E6.  
All key personnel (all individuals responsible for the design and conduct of this trial) will have completed 
Human Subjects Protection Training. 

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The sponsor, or sponsor-designated CROs or Clinical Professionals, shall conduct site monitoring, 
including site qualification, site initiation, site monitoring, and site closure, per ICH EG Good Clinical 
Practice (19) to ensure the study is undertaken according to GCP. 
13.1 
Financial Disclosures 
Investigators and sub-investigators will provide the sponsor with sufficient, accurate financial information 
as requested in accordance with 21 C.F.R. Part 54 to allow the sponsor to submit complete and accurate 
financial certification or disclosure statements to the appropriate regulatory authorities.  Investigators are 
responsible for providing information on financial interests during the course of the study and for 1 year 
after completion of the study. 
13.2 
Monitoring 
The sponsor, or sponsor representatives and/or CRO monitor, shall conduct site monitoring at site 
qualification, initiation, interim and close out periods, as needed, to ensure the integrity of the data 
collection and that the study is done per GCP. 
 
 

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14. 
BIBLIOGRAPHY 
1. 
Thorstenson S, Molin J, Lundström C. Implementation of large-scale routine diagnostics using 
whole slide imaging in Sweden: digital pathology experiences 2006–2013. J Pathol Inform. 2014;5-
14. 
2. 
Al-Janabi S, Huisman A, Nap M, Clarijs R, van Diest PJ. Whole slide images as a platform for 
initial diagnostics in histopathology in a medium-sized routine laboratory. J Clin Pathol. 
2012;65(12):1107–1111. 
3. 
Evans AJ, Chetty R, Clarke BA, et al. Primary frozen section diagnosis by robotic microscopy and 
virtual slide telepathology: the University Health Network experience. Hum Pathol. 
2009;40(8):1070–1081. 
4. 
Cheng, C.L. et. al.al. Enabling digital pathology in the diagnostic setting: navigating through the 
implementation journey in an academic medical centre.  J Clin Pathol. 2016;0:1–9. 
5. 
Pantanowitz L, Sinard JH, Henricks WH, et al. Validating whole slide imaging for diagnostic 
purposes in pathology: guideline from the College of American Pathologists Pathology and 
Laboratory Quality Center. Arch Pathol Lab Med. 2013;137(12):1710–1722 
6. 
FDA Clearance Letter for Philips Device for WSI, April 12, 2017 
7. 
FDA Device Summary, Evaluation of Automatic Class III Designation for Philips IntelliSite 
Pathology System (PIPS), April 13, 2017  DEN 160056 
8. 
Bauer, T.W. et. al., Validation of Whole Slide Imaging for Primary Diagnosis in Surgical 
Pathology.  Arch Pathol. Lab Med, 2013, April, Vol 137, 518-524 
9. 
Snead D.R. et. al. Validation of digital pathology imaging for primary histopathological diagnosis. 
Histopathology. 2016 Jun; 68(7):1063-72. 
10. Goacher E, Randell R, Williams B, Treanor D. The Diagnostic Concordance of Whole Slide 
Imaging and Light Microscopy: A Systematic Review. Arch Pathol Lab Med. 2016 Jul 11, 151-161 
11. Technical Performance Assessment of Digital Pathology Whole Slide Imaging Devices. Issued 
February 25, 2015.  Guidance for Industry and Food and Drug Administration Staff. 
http://www.fda.gov/ucm/groups/fdagov-public/@fdagov-meddev-
gen/documents/document/ucm435355.pdf 
12. Yun-Fu Hu, FDA PowerPoint Presentation: FDA Regulation of Digital Pathology Devices, 
13. Anderson, Nick, FDA PowerPoint Presentation: Whole Slide Imaging (WSI) Devices – FDA 
perspective, June 6, 2014 
14. Feldman M et. al., A Large Multicenter, Retrospective Non-Inferiority Study to Evaluate Diagnostic 
Concordance Between Optical vs. Digital Microscopic Diagnoses in 2000 Surgical Pathology 
Cases. Mod Pathol. 2017;30(S2):395A 
15. Abels, Ester, 2017 Digital Pathology Association Webinar Series, Session 2, “WSI and Primary 
Diagnosis: The Journey Begins; What FDA’s Approval of WSI Means for You and Digital 
Pathology.” May 11, 2017 
16. Feldman, Michael, Philips Presentation at Pathology Informatics Summit “Clinical Studies DeNovo, 
Philips IntelliSite Pathology System, May 23, 2017 
17. College of American Pathologist Cancer Protocols, 
http://www.cap.org/web/oracle/webcenter/portalapp/pagehierarchy/cancer_protocol_templates.jspx?
_adf.ctrl-state=12rukqd6ih_4&_afrLoop=16011323070214#! 
18. Wack et al, a multisite validation of whole slide imaging for primary diagnosis using standardized 
data collection and analysis, J Path Inform. 2016 7:49   
19. ICH Guidance for Industry, E6 Good Clinical Practice: Consolidated Guidance 
20. Holody, M, Presubmission for NanoZoomer S360 Digital Slide Scanner System, Q171418/A001 
21. Cui, C, Re: Written Feedback for Hamamatsu NanoZoomer S360 Digital Slide Scanner System 
22. Cui, C, Record of Meeting for NanoZoomer S360 Digital Slide System  

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23. Holody, M, Meeting Minutes Disagreement-Q171418/A001 – Pre-Submission Meeting Minutes for 
NanoZoomer S360 Digital Slide System 
24. Cui, C, Re: Q171418/A002 Meeting Minutes Disagreement Received, 2017 Dec 28, Email 
25. Cui, C, Re: Q171418/A002 Review Complete 2018 Jan 3, Email 
 
 

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REVISION HISTORY 
 
Rev 
Description of Change 
Page 
Number 
Date 
1.0 
First Release 
n/a 
September 10, 
2018 
2.0 
Clarification of study as non-significant risk study.  Clarification 
of minimum numbers of slides as H&E slides. Clarification that 
any replacement case must be read by all 4 RPs for both 
modalities. Clarification of use of both logs and CRFs for data 
collection. Clarification that any protocol deviations noted by 
CRA or Data Manager staff will be directed to site study 
coordinator to be entered into on-site protocol deviation log.  
Clarification that protocol deviations and device deficiencies to 
be recorded in logs. Clarification of adverse events for 
retrospective study. Clarification of slide or case replacement. 
Identification of 4 sites. Clarification of participants in 
Monitoring and function to confirm the integrity of the data. 
Clarification of adjudicator board. Clarification of data review 
for pilots. 
 
July 28, 2019 
3.0 
Modifications to the statistical analysis sections based on 
discussions with the Food and Drug Administration. 
 
October 16, 
2019 
4.0 
Modifications to be in accord with FDA instructions re: 
adjudication of deferrals, removal of period & sequence term for 
analysis, and removal of interaction terms for analysis. 
 
July 9, 2020