Roche Virtuoso Používateľská príručka

Ki-67 (30-9) Image Analysis Software User Manual, v. 5.1

Ki-67 (30-9) Handbuch zur Bildanalyse-Software-Anwendung, v. 5.1

Manuel d’utilisation de l’application logicielle d’analyse d’images du Ki-67

(30-9), v. 5.1

Manual de usuario de la aplicación del software de análisis de imagen Ki-67

(30-9), v. 5.1

Manuale dell’utente del software per l’analisi delle immagini -

Ki-67 (30-9), v. 5.1

Uživatelská příručka softwarové aplikace pro analýzu obrazu

Ki-67 (30-9), v. 5.1

Brugermanual for Softwareapplikation til Ki-67 (30-9)-billedanalyse, v. 5.1

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Ki-67 (30-9), v. 5.1

Ki-67 (30-9) képelemző szoftveralkalmazás használati útmutató, v. 5.1

Gebruikershandleiding voor de Ki-67 (30-9) beeldanalyse-softwareapplicatie,

v. 5.1

Brukerhåndbok for Ki-67 (30-9) bildeanalyseprogram, v. 5.1

Podręcznik użytkownika oprogramowania użytkowego do analizy obrazu

Ki-67 (30-9), v. 5.1

Manual do utilizador para o software de aplicação para a análise de imagem

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Ventana Medical Systems, Inc. Part No.: 101407200 Rev. A

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Companion Algorithm Ki-67 (30-9) Image Analysis

Software User Manual, v. 5.1

Ventana Medical Systems, Inc. Part No.: 101407200 Rev. A

Companion Algorithm Ki-67 (30-9) Image Analysis Software User Manual

Copyright

Trademarks

BENCHMARK, CONFIRM, COMPANION ALGORITHM, VENTANA ISCAN HT, ISCAN, VIRTUOSO,

VENTANA, and the VENTANA logo are trademarks of Roche. All other trademarks are the property of their respective

owners.

Companion Algorithm Ki-67 (30-9) image analysis software is licensed for use between Ventana Medical Systems, Inc.

and a licensee, and only users authorized there under are permitted to access and use the software. Unauthorized use and

distribution may result in civil and criminal penalties.

Open Source and Commercial Software

Refer to the Virtuoso user guide for information on Open Source and Commercial Software programs.

Contact Information:

Ventana Medical Systems, Inc.

1910 E. Innovation Park Drive

Tucson, AZ 85755

USA

+1 520 887 2155

www.ventana.com

Ventana Medical Systems, Inc.

203 Ravendale Drive

Mountain View, CA 94043

USA

Roche Diagnostics GmbH

Sandhofer Strasse 116

D-68305 Mannheim

Germany

+49 621 7590

Part No.: 101407200 Rev. A i

Table of Contents

About the Companion Algorithm Ki-67 (30-9) image analysis software. . . . . . . . . .1

Topics Covered in this Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Who Should Read this Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Related Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Technical Support. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Connection Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Cyber Security . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

User Authorization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Securing Networks and Servers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Protecting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Chapter 1: Intended Use and Indications for Use . . . . . . . . . . . . . . . . . . . . . . . .5

Intended Use and Indications for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Summary and Explanation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Test Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Warnings and Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Pre-Analytical Variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Required Materials Not Provided. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Performance Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Assay Cutoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Chapter 2: Ki-67 Comparison and Reproducibility Studies . . . . . . . . . . . . . . . . . .9

Ki-67 Marker Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Staining Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Study Devices and Samples for the BenchMark XT Instrument and iScan Coreo Slide Scanner 9

Ki-67 Concordance with BenchMark XT Instrument and iScan Coreo Slide Scanner . . . . . . . 9

Intra-System and Inter-System Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Ki-67 Concordance with BenchMark ULTRA Instrument and iScan Coreo Slide Scanner. . . . 12

Ki-67 Concordance with BenchMark XT/ULTRA Instruments and VENTANA iScan HT Slide Scanner13

Ki-67 Concordance with BenchMark GX Instrument and iScan Coreo and iScan HT Slide Scanners14

Appendix A: Reagents (Antibody) Package Inserts. . . . . . . . . . . . . . . . . . . . . . .19

Reagents (Antibody) Package Inserts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

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Table of Contents Ki-67 (30-9) Image Analysis Software User Manual

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Part No.: 101407200 Rev. A 1

About the Companion Algorithm Ki-67 (30-9) image analysis

software

Welcome to the Companion Algorithm Ki-67 (30-9) image analysis software User Manual.

Topics Covered in this Chapter

• Who Should Read this Manual (page 1)

• Introduction (page 1)

• Related Document (page 1)

• Technical Support (page 1)

• Connection Requirements (page 1)

• Cyber Security (page 2)

Who Should Read this Manual

System Administrators should read this user manual and use it for reference while operating the VENTANA Virtuoso software.

Introduction

The Companion Algorithm KI-67 (30-9) image analysis software assists the pathologist in the semi-quantitative measurement of

Ki-67 in tissues stained with the Ventana Medical Systems, Inc. CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody

(CONFIRM anti-Ki-67 (30-9)). This application generates a Ki-67 score that can be reviewed and accepted by the pathologist, or if

necessary, overridden by the pathologist. The image analysis application is an assist to the pathologist in the scoring and

interpretation of (CONFIRM anti-Ki-67 (30-9) staining on breast cancer tissues.

The Virtuoso Ki-67 (30-9) Digital Read Application allows the pathologist to view (CONFIRM anti-Ki-67 (30-9) stained slides as

images on a computer monitor, similar to what can be viewed under a microscope. While reviewing the image, the pathologist may

change magnification and move freely about the image.

Related Document

For additional information on the Virtuoso software, see the following VENTANA document:

• Virtuoso Reference Guide (PL-000123-EN)

Technical Support

Connection Requirements

Refer to the Virtuoso Reference Guide information on connection requirements.

Ventana Medical Systems, Inc.

1910 E. Innovation Park Drive

Tucson, AZ 85755

USA

Tel: +1 520 887 2155; for Technical Support, press 1

2 Part No.: 101407200 Rev. A

About the Companion Algorithm Ki-67 (30-9) image analysis software Ki-67 (30-9) Image Analysis Software User Manual

Cyber Security

Any device that is connected to a network (internally or externally) has the potential to be compromised by unauthorized access or

viruses. As with most devices, the software is designed to run on a computer utilizing Microsoft Windows and virus protection

software which requires the validation and implementation of the appropriate patches.

Some of the potential cyber security hazards are:

• Malicious software that alters the device software (such as viruses)

• Unauthorized access to the system that could compromise data safety

• Security of data transmitted over the Internet

Cyber security involves protecting data by preventing, detecting, and responding to malicious cyber attacks. Cyber attacks could

involve computer viruses which can completely erase data or hackers who alter files or even use the device as a host to attack other

devices. As serious as these hazards are, steps can be taken to maximize cyber security.

User Authorization

All software users must login with a valid user name and password. The user name and password are securely transmitted in

encrypted form over the Internet or Intranet. Once a user has logged in, the user remains active in the application until the user

explicitly logs out, closes the browser, or because the application closes after a period of inactivity.

Securing Networks and Servers

Network security consists of the provisions made in the computer network infrastructure, policies adopted by the network

administrator to protect the network, and the network resources that prevent unauthorized access.

The following are critical steps for securing a network server:

• Physical security (servers and network infrastructure behind locked doors)

• Use of robust passwords

• System and data backups (at regular intervals)

• Data protection

• Terminating unused services

• Restricting access to used services

The following are critical steps and methodologies used to secure network and servers:

• Data protection

• Data backups (at regular intervals)

• Refusal of automatic updates from off-the-shelf software

• Antivirus software for computers and servers

Protecting Data

Establishment of a network firewall and protection of the network against viruses using anti-virus software are effective methods to

protect data. Virus definitions should be kept up to date and regular scans of computers for spyware should be performed using a

legitimate anti-spyware application. If viruses or spyware are found, remove them immediately.

Ki-67 (30-9) Image Analysis Software User Manual About the Companion Algorithm Ki-67 (30-9) image analysis software

Part No.: 101407200 Rev. A 3

Evaluate Your Software Settings

The default settings of most software enable all available functionality. However, hackers may be able to take advantage of this

functionality to access devices. It is especially important to check the settings for software that connects to the Internet (browsers,

email clients, etc.). Apply the highest level of security available that still provides needed functionality.

Backup and Recovery

In order to develop a successful backup and recovery plan, comprehension of data accessibility needs and the potential impact of

data loss is essential. Automatic backup procedures need to be adopted using a data backup utility.

4 Part No.: 101407200 Rev. A

About the Companion Algorithm Ki-67 (30-9) image analysis software Ki-67 (30-9) Image Analysis Software User Manual

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Part No.: 101407200 Rev. A 5

Chapter 1: Intended Use and Indications for Use

This chapter shows comparison and reproducibility studies for the Ki-67 marker.

Intended Use and Indications for Use

The Virtuoso system provides automated digital slide creation, management, analysis, and viewing. It is intended for in vitro

diagnostic use as an aid to the pathologist in the display, detection, counting, review and classification of tissues and cells of clinical

interest based on particular morphology, color, intensity, size, pattern and shape.

The IHC Ki-67 (30-9) Digital Read and Image Analysis applications are intended for use as an aid to the pathologist in the detection

and quantitative measurement of Ki-67 protein in formalin-fixed, paraffin-embedded normal and neoplastic tissue. Ki67 results are

indicated for use in assessing the proliferative activity of normal and neoplastic breast tissue. When used with Ventana Medical

Systems, Inc. CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody Assay, it is indicated for use as an aid in the

assessment of Ki-67 status in breast cancer patients (but is not the sole basis for treatment).

Note: The IHC Ki-67 (30-9) Digital Read and Image Analysis applications are adjunctive computer-assisted methodologies for the

qualified pathologist in the acquisition and measurement of images from microscope glass slides of breast cancer specimens

stained for the presence of Ki-67 protein. The pathologist should verify agreement with the Image Analysis software

application score. The accuracy of the test results depends on the quality of the immunohistochemical staining. It is the

responsibility of a qualified pathologist to employ appropriate morphological studies and controls as specified in the

instructions for the CONFIRM anti- Ki-67 (30-9) Rabbit Monoclonal Primary Antibody assay used to assure the validity of the

Virtuoso System for IHC Ki-67 Digital Read and Image Analysis scores. The actual correlation of CONFIRM anti-Ki-67 (30-9)

Rabbit Monoclonal Primary Antibody to clinical outcome has not been established.

Summary and Explanation

The Virtuoso system Ki-67 (30-9) is an instrument and software system designed to assist the qualified pathologist in the consistent

quantitative assessment of protein expression in immunohistochemically (IHC) stained histologic sections from formalin-fixed,

paraffin-embedded (FFPE) normal and neoplastic tissues. The Virtuoso system can be used for review of digitized images of

histologic sections with image analysis algorithms (Companion Algorithm image analysis applications), or without Companion

Algorithm image analysis algorithms (Virtuoso Digital Read applications).

Digital Read applications present images on the computer screen in the same manner as one would see with a manual microscope,

inclusive of the pathologist's ability to select any areas of interest and the option of various magnification levels. For the Companion

Algorithm image analysis applications, the pathologist may use the system software to select and outline one or several field of views,

(FOVs), and each FOV may be viewed at various magnifications and then analyzed by the software; a count of the total number of

target cells and the number interpreted by the algorithm as positive and negative is generated. The pathologist can accept the score

provided by the algorithm, or may override the score with a pathologist score. The system requires competent human intervention

at all steps in the analysis process, and the software makes no independent interpretations of the data.

The Virtuoso system consists of a slide scanner, computer, monitor, keyboard, mouse, Companion Algorithm image analysis

algorithms, and software with a user interface. Virtuoso is an end-to-end digital pathology software solution that allows pathology

laboratories to acquire, manage, view, analyze, share, and report on digital images of pathology specimens. Using the Virtuoso

software, the pathologist can view digital images at various magnifications (as previously stated), add annotations, make

measurements, perform image analysis, and generate reports.

For in vitro diagnostic (IVD) use.

6 Part No.: 101407200 Rev. A

Chapter 1: Intended Use and Indications for Use Ki-67 (30-9) Image Analysis Software User Manual

Test Principles

The Companion Algorithm Ki-67 (30-9) image analysis software employs image analysis techniques to obtain Ki-67 scoring.

Pre-defined parameters are used to obtain Ki-67 scores. The identification of the nucleus is carried out automatically by the image

analysis algorithms. The steps involved in the analysis algorithms are:

1. Enhancing the image. This process increases the contrast to make the image more suitable for analysis.

2. Identifying the epithelial area. The epithelial area is the region of the image where there is the possibility of epithelial cells

being present.

3. Identifying the nucleus.

4. Classifying the cells based on extent, intensity, and thickness of nuclear staining.

5. Computing the score.

Warnings and Precautions

It is important that glass slides with acceptable staining quality be used.

Pre-Analytical Variables

Tissue preparation and staining should follow the recommendations provided in the CONFIRM anti-Ki-67 (30-9) assay package

insert. For optimal image capture using the Virtuoso software, it is recommended that the tissue be free of folds and be placed on

the slide with a minimum of 2 mm boundary from the edge on all sides. The cover slip and slide label (if present) should not overhang

the edges of the slide. For further information on scanning, please refer to the appropriate iScan slide scanner reference guide.

Procedure

Refer to the Virtuoso Reference Guide.

It is recommended that at least three field of view be selected for analysis when using the Companion Algorithm Ki-67 (30-9)

software.

Required Materials Not Provided

The Virtuoso system for Ki-67 (30-9) requires use of CONFIRM anti-Ki-67 (30-9), and any additional material or supplies listed in

the Ventana package insert, to stain tissues prior to analysis.

The iScan Coreo slide scanner or the VENTANA iScan HT slide scanner is required for scanning of the slides.

Results

The Companion Algorithm for Ki-67 (30-9) produces images and a staining score. The pathologist views the image and the

instrument score, makes an assessment, and reports a score which may not be the same as the instrument score. Refer to Virtuoso

Reference Guide for an example of a report.

Limitations

The algorithms are designed to work for Ki-67 cell nuclei staining. The test results are only as good as the quality and accuracy of

the immunohistochemistry slide that is imaged, and the subsequent image that is analyzed. The pathologist must validate the

CONFIRM anti-Ki-67 (30-9) staining run by examination of the CONFIRM anti-Ki-67 (30-9) control images to verify that the

Ki-67 (30-9) Image Analysis Software User Manual Chapter 1: Intended Use and Indications for Use

Part No.: 101407200 Rev. A 7

expected results have been obtained before images from patient slides are analyzed. The pathologist must follow the manufacturer's

recommendations for CONFIRM anti-Ki-67 (30-9) including using all the positive and negative quality control materials for each

staining run. If the control slides are not acceptable, the patient tissues need to be re-stained with acceptable results. (See the Ventana

CONFIRM anti-Ki-67 (30-9) package insert for details about quality control recommendations.) The pathologist must follow the

Ventana recommendations for surveying the entire breast cancer specimen to assess any heterogeneity in the CONFIRM anti-Ki-67

(30-9) staining, the degree of background staining, cytoplasmic staining, edge effect, etc. as recommended in the Ventana

CONFIRM anti-Ki-67 (30-9) user manual (available at www.ventana.com). If the images captured have different staining (nuclear,

cytoplasm, etc.), incorrect results will be generated. Incorrect results will also be generated if the image quality cannot be analyzed.

The software algorithms determine whether the quality of an image can be analyzed, based on pre-defined parameters. Refer to the

Virtuoso Reference Guide for more information.

The Ki-67 (30-9) image analysis algorithm will reject nuclei that are elongated regardless of the overall shape of the cell. For this

reason, tumors containing large numbers of cells with elongated nuclei may need to be evaluated manually. In addition, performance

of the Virtuoso system with the following types of breast cancers has not been evaluated: carcinoma in situ, carcinosarcoma, comedo

carcinoma, cystosarcoma phylloides, medullary carcinoma of the breast, mucinous variants of breast cancer, and spindle cell

carcinoma.

Performance Characteristics

Performance of the staining agent is described in the Ventana package insert for the CONFIRM anti-Ki-67 (30-9) staining process.

See Comparison and Reproducibility Studies in Chapter 2 for a description of the performance of the software.

Assay Cutoff

Clinical cutoffs used for the assessment of Ki-67 varies between laboratories. The performance of the Virtuoso system with

CONFIRM anti-Ki-67 (30-9) was evaluated at these commonly used clinical cutoffs: 0-0.99% was considered a negative test result

and 1-10% and >10% were considered positive test results. In some studies, the performance was evaluated at another commonly

used clinical cutoff: 0-10% was considered a negative test result and >10% were considered positive test results.

8 Part No.: 101407200 Rev. A

Chapter 1: Intended Use and Indications for Use Ki-67 (30-9) Image Analysis Software User Manual

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Part No.: 101407200 Rev. A 9

Chapter 2: Ki-67 Comparison and Reproducibility Studies

This chapter shows comparison and reproducibility studies for the Ki-67 marker.

Ki-67 Marker Studies

Staining Procedure

Refer to the CONFIRM anti-Ki-67 (30-9) package insert for the BenchMark XT, ULTRA and GX instruments, ultraView, and iView

detection.

Study Devices and Samples for the BenchMark XT Instrument and iScan Coreo Slide Scanner

The Ki-67 comparison and reproducibility studies for the Virtuoso Digital Read and Companion Algorithm image analysis software

consisted of 120 de-identified archived breast carcinoma sections immunohistochemically stained with CONFIRM anti-Ki-67

(30-9). Study test samples covered the ranges of 0-0.99%, 1-10% and >10%, and were interpreted at three different sites by three

different pathologists. All test slides were scanned at 20X magnification and all images were output in bif file format. For the image

analysis evaluation, a minimum of three fields of view were selected by each pathologist independently for each image.

Ki-67 Concordance with BenchMark XT Instrument and iScan Coreo Slide Scanner

The table below shows the concordance results for CONFIRM anti-Ki-67 (30-9) staining interpretation among three different sites:

1. Digital Read vs. Manual Method.

Table 2-1 Agreement - Digital Read vs. Manual (manual = true score).

Confusion Matrix

Digital

Site 1 Site 2 Site 3

(n = 120) (n = 118) (n = 114)

Neg Pos Neg Pos Neg Pos

Manual

Neg (0-0.99%) 7 11 18 1 21 2

Pos (1-10%, >10%) 0 102 9 90 11 80

% Agreement 91% 92% 89%

(95% CI) (84% - 95%) (85% - 95%) (81% - 93%)

10 Part No.: 101407200 Rev. A

Chapter 2: Ki-67 Comparison and Reproducibility Studies Ki-67 (30-9) Image Analysis Software User Manual

2. Image Analysis vs. Manual Method.

Intra-System and Inter-System Studies

The study was designed to demonstrate inter- and intra-Virtuoso system reproducibility for Virtuoso Digital Read and Companion

Algorithm image analysis applications. A designated subset of 40 cases that span the range of the Ki-67 scoring categories (0-0.99%,

negative, 1-10%, positive, and >10%, positive) were used.

1. Intra-pathologist/Inter-Day (pair-wise comparisons, Session1 vs. Session 2, Session1 vs. Session 3, Session 2 vs. Session 3).

Table 2-2 Agreement - Image Analysis vs. Manual (manual = true score).

Confusion Matrix

Image Analysis

Site 1 Site 2 Site 3

(n = 120) (n = 118) (n = 114)

Neg Pos Neg Pos Neg Pos

Manual

Neg (0-0.99%) 5 13 16 3 15 8

Pos (1-10%, >10%) 0 102 4 95 2 89

% Agreement 89% 94% 91%

(95% CI) (82% - 94%) (88% - 97%) (85% - 95%)

Table 2-3 Intra- Pathologist Digital Read.

Confusion Matrix

Intra-Pathologist Digital

Session 2 Session 3 Session 3

Neg Pos Neg Pos Neg Pos

2 38 1 37 1 37

Session 1

Neg 3 2 1 1 1

Pos 37 0 37 0 36

Session 2

Neg 2 10

Pos 38 037

% Agreement 98% 97% 100%

(95% CI) (87% - 100%) (87% - 100%) (91% - 100%)

Ki-67 (30-9) Image Analysis Software User Manual Chapter 2: Ki-67 Comparison and Reproducibility Studies

Part No.: 101407200 Rev. A 11

2. For Intra-Pathologist Image Analysis.

3. Inter-pathologist (pair-wise comparisons, Pathologist 1 vs. Pathologist 2, Pathologist 1 vs. Pathologist 3, Pathologist 2 vs.

Pathologist 3).

4. Inter-Pathologist Image Analysis.

Table 2-4 Intra-Pathologist Image Analysis.

Confusion Matrix

Intra-Pathologist Image Analysis

Session2 Session 3 Session 3

Neg Pos Neg Pos Neg Pos

6 32 7 31 7 31

Session 1

Neg 8 5 3 7 1

Pos 30 1 29 0 30

Session 2

Neg 6 51

Pos 32 230

% Agreement 89% 97% 92%

5 (76% - 96%) (87% - 100%) (79% - 97%)

Table 2-5 Inter-Pathologist Digital Read.

Confusion Matrix

Inter-Pathologist Digital

Site 2 Site 3 Site 3

Neg Pos Neg Pos Neg Pos

27 91 34 82 34 82

Site 1

Neg 7 5 0 5 0

Pos 113 22 91 29 82

Site 2

Neg 27 22 4

Pos 91 12 78

% Agreement 81% 75% 86%

(95% CI) (73% - 87%) (66% - 82%) (79% - 91%)

Table 2-6 Inter-Pathologist Image Analysis.

Confusion Matrix

Inter-Pathologist Image Analysis

Site 2 Site 3 Site 3

Neg Pos Neg Pos Neg Pos

20 98 18 97 18 97

Site 1

Neg 5 3 0 3 0

Pos 115 17 98 15 97

Site 2

Neg 20 13 4

Pos 98 593

% Agreement 86% 87% 92%

(95% CI) (78% - 91%) (80% - 92%) (86% - 96%)

12 Part No.: 101407200 Rev. A

Chapter 2: Ki-67 Comparison and Reproducibility Studies Ki-67 (30-9) Image Analysis Software User Manual

Ki-67 Concordance with BenchMark ULTRA Instrument and iScan Coreo Slide Scanner

Study Devices and Samples for BenchMark ULTRA Instrument and iScan Coreo Slide Scanner

These Ki-67 (30-9) comparison studies for the Virtuoso Digital Read and Companion Algorithm Image Analysis software consisted

of 120 de-identified archived breast carcinoma sections immunohistochemically stained with CONFIRM anti-Ki-67 (30-9) Rabbit

Monoclonal Primary Antibody on the BenchMark ULTRA instrument. Study test samples covered the ranges of 0-10% (negative),

>10% (positive), and were interpreted at by one pathologist All test slides were scanned at 20X magnification and all images were

output in bif file format. Three fields of view were selected by the pathologist for each image when using the image analysis

application. 120 of 120 cases were able to be evaluated and were included in the analyses below.

The tables below shows the concordance results for CONFIRM anti-Ki-67 (30-9) assay stained on the BenchMark ULTRA

instrument.

Digital Read vs. Manual Microscopic Read

Image Analysis vs. Manual Microscopic Read

Table 2-7 Agreement (ULTRA Instrument) - Digital Read vs. Manual Microscopic Read

Manual Microscope Read

Digital Read

Positive Negative Total

Positive 52 12 64

Negative 0 56 56

Total 52 68 120

Positive Percent Agreement (PPA) n/N (%) (95% CI) 52/52 (100.0%) (93.1-100%)

Negative Percent Agreement (NPA) n/N (%) (95% CI) 56/68 (82.4%) (71.6-89.6%)

Overall Percent Agreement (OPA) n/N (%) (95% CI) 108/120 (90.0%) (83.3-94.2%)

Table 2-8 Agreement (ULTRA Instrument) - Image Analysis vs. Manual Microscopic Read

Manual Microscope Read

Image Analysis Read

Positive Negative Total

Positive 52 1 53

Negative 2 40 65

Total 77 41 118

Positive Percent Agreement (PPA) n/N (%) (95% CI) 52/77 (67.5%) (56.5-76.9%)

Negative Percent Agreement (NPA) n/N (%) (95% CI) 40/41 (97.6%) (87.4-99.6%)

Overall Percent Agreement (OPA) n/N (%) (95% CI) 92/118 (78.0%) (69.7-84.5%)

Ki-67 (30-9) Image Analysis Software User Manual Chapter 2: Ki-67 Comparison and Reproducibility Studies

Part No.: 101407200 Rev. A 13

Ki-67 Concordance with BenchMark XT/ULTRA Instruments and VENTANA iScan HT Slide

Scanner

Study Devices and Samples for BenchMark XT/ULTRA Instruments and iScan HT Slide Scanner

The Ki-67 comparison and reproducibility studies for the Virtuoso Digital Read and Companion Algorithm image analysis software

consisted of 30 de-identified archived breast carcinoma sections immunohistochemically stained with CONFIRM anti-Ki-67 (30-9)

Rabbit Monoclonal Primary Antibody. Study test samples covered the ranges of 0-10% (negative), >10% (positive), and were

interpreted at by one pathologist. All test slides were scanned at 20X magnification and all images were output in bif file format.

Three fields of view were selected by the pathologist for each image when using the image analysis application.

Digital Read vs. Manual

Image Analysis vs. Manual

Table 2-9 Agreement - Digital Read vs. Manual Microscopic Read

Digital Read

Manual Microscope Read

Negative Positive Total

Negative 10 1 11

Positive 1 18 19

Total 11 19 30

Positive Percent Agreement (PPA) n/N (%) (95% CI) 18/19 (95%) (75-99%)

Negative Percent Agreement (NPA) n/N (%) (95% CI) 10/11 (91%) (62-98%)

Overall Percent Agreement (OPA) n/N (%) (95% CI) 28/30 (93%) (79-98%)

Table 2-10 Agreement - Image Analysis vs. Manual Microscopic Read

Image Analysis Read

Manual Microscope Read

Negative Positive Total

Negative 11 0 11

Positive 2 17 19

Total 13 17 30

Positive Percent Agreement (PPA) n/N (%) (95% CI) 17/19 (89%) (69-97%)

Negative Percent Agreement (NPA) n/N (%) (95% CI) 11/11 (100%) (74-100%)

Overall Percent Agreement (OPA) n/N (%) (95% CI) 28/30 (93%) (79-98%)

14 Part No.: 101407200 Rev. A

Chapter 2: Ki-67 Comparison and Reproducibility Studies Ki-67 (30-9) Image Analysis Software User Manual

Ki-67 Concordance with BenchMark GX Instrument and iScan Coreo and iScan HT Slide

Scanners

Study Devices and Samples for BenchMark GX Instrument and iScan Coreo and iScan HT Slide Scanners

The Ki-67 comparison and reproducibility studies for the Virtuoso Digital Read and Companion Algorithm image analysis software

consisted of 30 de-identified archived breast carcinoma sections immunohistochemically stained with CONFIRM anti-Ki-67 (30-9)

Rabbit Monoclonal Primary Antibody. Study test samples covered the ranges of 0-10% (negative), >10% (positive), and were

interpreted by two pathologists. All test slides were scanned at 20X magnification and all images were output in bif file format. For

the image analysis evaluation, a minimum of three fields of view were selected by each pathologist independently for each image.

The results were analyzed for each pathologist individually and also averaged across the two pathologists. The average overall

percent agreement (OPA) was determined as the weighted average for the two pathologists who independently scored the same

cases, where the weight of a pathologist's individual OPA rate was determined by the number of cases considered evaluable by that

pathologist.

Digital Read vs. Manual Microscopic Read, iScan Coreo Slide Scanner

The average OPA for Digital Read vs. Manual Microscopic Read for iScan Coreo slide scanner was 83.3%.

Table 2-11 Pathologist #1 Agreement - Digital Read vs. Manual, iScan Coreo Slide Scanner

Digital Read

Manual Microscope Read

Negative Positive Total

Negative 25 5 30

Positive 0 0 0

Total 25 5 30

Overall Percent Agreement (OPA) n/N (%) (95% CI) 83.3%

Table 2-12 Pathologist #2 Agreement - Digital Read vs. Manual Microscopic Read,

iScan Coreo Slide Scanner

Digital Read

Manual Microscope Read

Negative Positive Total

Negative 23 5 28

Positive 0 2 2

Total 23 7 30

Overall Percent Agreement (OPA) n/N (%) (95% CI) 83.3%

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