Roche uPath IVD User manual

uPath HER2 (4B5) image analysis for

Breast Algorithm Guide

Table of Contents

Introduction 1

Algorithm Summary and Explanation 2

Intended Use 3

Intended Use of Product 3

Purpose of Algorithm Guide 3

Clinical Signiﬁcance 4

Test Principles 5

Limitations 6

Network Characteristics 7

Data Security 8

Workﬂow for Using the uPath HER2 (4B5) Algorithm 9

Pathologist Workﬂow 11

Staining Characteristics 18

VENTANA anti-HER2/neu (4B5) Antibody Evaluation 18

uPath HER2 (4B5) Algorithm Stain Evaluation 18

Performance Characteristics 30

Method Comparison 30

Pathologist Reproducibility Studies 31

Scanner Reproducibility Studies 32

Troubleshooting 33

References 37

Introduction

The Roche uPath enterprise software (uPath enterprise

software) with the uPath HER2 (4B5) image analysis for Breast

algorithm (uPath HER2 (4B5) algorithm) is a software system

designed to assist in the semi-quantitative assessment of human

epidermal growth factor receptor 2 (HER2) protein expression

in immunohistochemically stained histologic sections from

formalin-ﬁxed, parafﬁn-embedded (FFPE) normal and neoplastic

tissues.

The uPath enterprise software 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 uPath enterprise software, the

pathologist can view digital images at various magniﬁcations,

add annotations, make tissue section measurements, perform

image analysis, and generate reports.

Note: The uPath HER2 (4B5) image analysis for Breast algorithm

is an adjunctive computer-assisted methodology to aid in the

acquisition and measurement of images from microscope

glass slides of breast tissue specimens that have undergone

immunohistochemical staining to determine the presence of

HER2 protein. To assure the validity of image analysis scores,

it is the responsibility of the pathologist to verify agreement

and employ appropriate controls as speciﬁed in the VENTANA

anti-HER2/ne u (4B5) antibody method sheet (available at dialog.

roche.com).

1 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Algorithm Summary and Explanation

For image analysis applications, the pathologist uses the uPath

enterprise software to select and outline one or several regions

of interest (ROIs), and each ROI may be viewed at various

magniﬁcations and then analyzed by the uPath HER2 (4B5)

algorithm. A count of the total number of target tumor cells

is generated, and tumor cells are stratiﬁed by whether they

are stained or unstained. Stained cells are stratiﬁed by stain

intensity and completeness of membrane staining. The uPath

HER2 (4B5) algorithm combines percent stained (stained,

unstained), stain intensity (weak, medium, strong) and stain

completeness (unstained, partial, complete) to generate a HER2

score on a scale of 0 to 3+. The uPath HER2 (4B5) algorithm can

generate a score for a particular ROI, or an aggregate score for

all selected ROIs on that slide. The pathologist may accept the

score provided by the uPath HER2 (4B5) algorithm, or override

the score with a different score. The uPath HER2 (4B5) algorithm

makes no independent interpretations of the data and therefore

should only be used by a qualiﬁed pathologist in conjunction

with histological examination, relevant clinical information, and

proper controls. The uPath HER2 (4B5) algorithm is designed and

indicated as an aid to the pathologist for the assessment of HER2

protein expression in breast tissue specimens stained with the

VENTANA anti-HER2/neu (4B5) antibody.

2 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Intended Use of Product

The uPath HER2 (4B5) image analysis for Breast algorithm

is intended for use as an aid to the pathologist in the semi-

quantitative detection of HER2 protein in formalin-ﬁxed,

parafﬁn-embedded neoplastic breast tissue. When used with

the VENTANA anti-HER2/neu (4B5) Rabbit Monoclonal Primary

Antibody, (VENTANA anti-HER2/neu (4B5) antibody) it is

indicated for use as an aid in the assessment of breast cancer

patients for whom Herceptin® (trastuzumab), KADCYLA® (ado-

trastuzumab emtansine) or PERJETA® (pertuzumab) treatments

are being considered.

Note: The uPath HER2 (4B5) image analysis for Breast algorithm

is an adjunctive computer-assisted methodology to aid in the

acquisition and measurement of images from microscope

glass slides of breast tissue specimens that have undergone

immunohistochemical staining to determine the presence of

HER2 protein. To assure the validity of image analysis scores, it

is the responsibility of the pathologist to verify agreement and

employ appropriate controls as speciﬁed in the VENTANA anti-

HER2/neu (4B5) antibody method sheet.

This algorithm is intended for in vitro diagnostic (IVD) use.

Intended Use

Purpose of Algorithm Guide

This uPath HER2 (4B5) algorithm guide (algorithm guide) is

intended to:

• Provide background information on the intended use of the

uPath HER2 (4B5) algorithm, test principles, and limitations.

• Deﬁne the necessary materials, IT, data security, and network

requirements.

• Show step-wise directions for running the uPath HER2 (4B5)

algorithm.

• Provide photographic images that illustrate how the uPath HER2

(4B5) algorithm should be used.

• Provide a tool to facilitate the use of the uPath HER2 (4B5)

algorithm on FFPE breast sections stained with the VENTANA

anti-HER2/ne u (4B5) antibody.

• Provide example images of challenging cases to provide

guidance on how to use the uPath HER2 (4B5) algorithm in their

evaluation.

• Show performance characteristics of the uPath HER2 (4B5)

algorithm.

3 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Clinical Signiﬁcance

Breast cancer is the most common carcinoma occurring in

women, and the second leading cause of cancer related death.

1,2

In North America, a woman’s chance of contracting breast

cancer is one in eight.

Early detection and appropriate treatment

therapies can signiﬁcantly affect overall survival.

3,4

Small tissue

samples may be easily used in routine immunohistochemistry

(IHC), making this technique, in combination with antibodies

that detect antigens important for carcinoma interpretation, an

effective tool for the pathologist in their diagnosis and prognosis

of disease. One important marker in breast cancer today is

the oncoprotein HER2.

5,6 ,7,8

The therapeutic drugs Herceptin

(trastuzumab), KADCYLA (ado-trastuzumab emtansine), and

PERJETA (pertuzumab), have been shown to beneﬁt some breast

carcinoma patients by arresting, and in some cases reversing

the growth of their cancer.

5,6,7,8,9

The drugs are humanized

monoclonal antibodies that bind to HER2 protein on cancer

cells.

5,8,9,10

In vitro diagnostics for the evaluation of HER2 status in breast

cancer patients are important to aid the clinician in determination

of HER2-targeted therapy.

5,6 ,7,8

The VENTANA anti-HER2/neu

(4B5) antibody is intended for use in the IHC-based detection of

HER2 protein expression in breast cancer. The uPath HER2 (4B5)

algorithm functions as an accessory to the VENTANA anti-HER2/

neu (4B5) antibody to aid in the assessment of breast cancer

patients for whom Herceptin (trastuzumab), KADCYLA (ado-

trastuzumab emtansine), or PERJETA (pertuzumab) treatment is

being considered.

4 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Test Principles

Criteria for intensity and pattern of cell membrane staining

for the uPath HER2 (4B5) algorithm’s score:

Staining Pattern Score HER2 Staining

Assessment

No membrane staining is observed 0 Negative

Faint, partial staining of the

membrane in any proportion of the

cancer cells

1+ Negative

Weak to moderate complete staining

of the membrane, >10% of cancer

cells

2+ Equivocal*

Intense, complete staining of the

membrane, >10% of cancer cells

3+ Positive

* Recommend reﬂex to ISH

The uPath enterprise software with the uPath HER2 (4B5)

algorithm employs image analysis techniques to obtain a HER2

score.

The uPath HER2 (4B5) algorithm uses pre-deﬁned parameters to

score images of tissue stained with the VENTANA anti-HER2/neu

(4B5) antibody.

Steps involved in image analysis:

• Detection of cells across the entire image.

• Classiﬁcation of cells as tumor cells or other cell types.

• Identiﬁcation of stained membrane and stratiﬁcation of

membrane into categories: partial or complete staining and

weak, medium, or strong intensity staining.

• Computation of the HER2 score by combining cell classiﬁcation,

stain intensity, and membrane classiﬁcation.

How HER2 image analysis algorithm identiﬁes tumor cells

and how the score is calculated:

• The uPath HER2 (4B5) algorithm identiﬁes tumor cells using

color, intensity, size and morphological features.

• Identiﬁed tumor cells are classiﬁed as stained using detected

membrane and pre-set thresholds.

• To calculate the HER2 score, the uPath HER2 (4B5) algorithm

uses the identiﬁed stained cells and stratiﬁes them by stain

intensity and completeness.

5 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

The uPath HER2 (4B5) algorithm is designed to work with the

VENTANA anti-HER2/neu (4B5) antibody. The test results are

only as good as the quality and accuracy of the IHC slide that is

imaged, and the subsequent image that is analyzed.

The pathologist must validate the VENTANA anti-HER2/neu

(4B5) antibody staining run by manual microscopic examination

of the HER2 control slides to verify that the expected results have

been obtained before images of slides are accessioned onto the

uPath enterprise software for analysis.

The manufacturer’s recommendations must be followed for

the VENTANA anti-HER2/neu (4B5) antibody including using

all positive and negative quality control materials for each

staining run. If the control slides are not acceptable with manual

microscopic examination, the tissues need to be re-stained with

acceptable results.

The pathologist must follow the recommendations for VENTANA

anti-HER2/ne u (4B5) antibody interpretation.

The uPath HER2 (4B5) algorithm is designed to be used

by a qualiﬁed pathologist in conjunction with histological

examination, relevant clinical information, and proper controls. It

is not designed to be a standalone tool, and requires competent

human intervention throughout the analysis process.

The uPath HER2 (4B5) algorithm may generate incorrect scores

if the captured images have abnormal staining (nuclear staining,

pigmentation, etc.).

The uPath HER2 (4B5) algorithm may reject tumor nuclei that

are elongated regardless of the overall shape of the cell. For this

reason, tumors containing large numbers of cells with elongated

tumor nuclei may need to be evaluated manually.

The uPath HER2 (4B5) algorithm has been trained, developed,

and validated on: invasive lobular, ductal, mucinous, medullary,

papillary, and micropapillary carcinomas as well as a few

metastatic lesions.

The uPath HER2 (4B5) algorithm has not been tested, or its

safety and effectiveness validated, when used with a personal

computer (PC) from home.

The uPath HER2 (4B5) algorithm identiﬁes DAB stained

membranes of any intensity and completeness. It uses a noise

threshold of 0.5% of all tumor cells to score a case as 1+. It

does not follow the method sheet guidelines of using a one cell

threshold for a score of 1+. This may result in some cases with

very few cells that have membrane staining being scored as 0

rather than 1+. This noise threshold was implemented to ensure

true negative cases are being called as 0.

The uPath HER2 (4B5) algorithm is designed to be sensitive to

the color brown (DAB staining) to ensure even faint membrane

staining is detected. For this reason, it can incorrectly score a

tissue sample containing brown artifacts such as pigmentation,

background staining in muscle ﬁbers or any other artifacts that

are stained brown. There are tools within the uPath enterprise

software that will allow the pathologist to exclude such areas.

These are discussed in more detail in the staining characteristics

section.

The uPath HER2 (4B5) algorithm is designed to be sensitive for

2+ cases. For this reason, the uPath HER2 (4B5) algorithm may

incorrectly score certain 1+ cases as 2+. Speciﬁcally, 1+ cases

with slightly less than 10% of tumor cells with complete staining

or cases with a high amount of tumor cell cytoplasmic staining

may be incorrectly scored as 2+. The pathologist may need to

override the score in these particular types of cases. Cytoplasmic

staining is discussed in more detail in the staining characteristics

section.

Limitations

6 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

7 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Network Characteristics

A 1Gbps network connection between the uPath enterprise software and the image management system (IMS) is recommended.

Table 1. Roche uPath image analysis server speciﬁcations for small labs.

Parameter Details

Processor CPU @ 3.6 GHz

Number of cores 4

RAM 6 x 8GB (48 GB)

Hyper-threading Disabled

Hard disk 240 GB SSD

Operating system Microsoft Windows Server 2016

Virtual Machine (VM) support Yes

VM additional information Performance will be different to the physical servers due to VM overhead.

Antivirus Symantec Endpoint Protection Version 12

Power 110V/220V, 800 Watts (2)

Ports 2 USB, HPE Eth 10/25Gb Adaptor

Uninterruptible power supply Recommended

Table 2. Roche uPath image analysis server speciﬁcations for medium to large labs.

Parameter Details

Processor CPU @ 3.6 GHz (2 processors)

Number of cores 8 per processor / 16 in total

RAM 12 x 8GB (96 GB)

Hyper-threading Disabled

Hard disk 240 GB SSD (2x)

Operating system Microsoft Windows Server 2016

Virtual Machine (VM) support Yes

VM additional information Performance will be different to the physical servers due to VM overhead.

Antivirus Symantec Endpoint Protection Version 12

Power 110V/220V, 800 Watts (2)

Ports 2 USB, HPE Eth 10/25Gb Adaptor

Uninterruptible power supply Recommended

Malicious software or unauthorized instrument access can result

in data loss or instrument unavailability.

To avoid infection by malicious software or unauthorized access

and misuse of the instrument, the following recommendations

are essential:

• Do not install or run any other software on the instrument.

• Make sure that other computers and services on the network

are properly secured and protected against malicious software

and unauthorized access. For example, the laboratory

information system (LIS), archiving share, backup share, or

service.

• Customers are responsible for the security of their local area

network, especially in protecting it against malicious software

and attacks. This protection might include measures, such as

a ﬁrewall, to separate the device from uncontrolled networks.

This protection might also include measures that ensure that the

connected network is free of malicious code.

• Restrict physical access to the instrument and all attached IT

infrastructure (computers, cables, network equipment, and so

on).

• Make sure that instrument backup and archive ﬁles are

protected from any unauthorized access and disaster. This list

includes the remote storage location, disaster discovery sites,

and the secure transfer of backup ﬁles.

• If possible, use a ﬁrewall to restrict network trafﬁc.

• USB ﬂash drives can be used for several kinds of backups and

restores. Wrong handling of a USB ﬂash drive may result in data

loss or malfunction of the instrument.

• Use only USB ﬂash drives that are tested and installed by your

local Roche service representative.

• At any one time only one USB device can be in use. Before

inserting a USB ﬂash drive, check that no other USB device is

inserted.

• Before removing a USB ﬂash drive, choose the Eject button in

Windows.

• The default operating system (OS) conﬁguration provided with

the server should not be altered as this has implications on the

hardened OS conﬁgurations.

• To prevent a virus from infecting the uPath enterprise software,

use the USB ﬂash drive exclusively on the instrument. Do not

store other data on this USB ﬂash drive.

Data Security

8 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Workﬂow for Using the uPath HER2 (4B5) Algorithm

Materials Provided

• uPath HER2 (4B5) algorithm

Materials Required but not Provided

• uPath enterprise software

• Breast tissue slides stained with the VENTANA anti-HER2/neu (4B5) antibody, using the ultraView Universal DAB Detection Kit,

stained on a BenchMark ULTRA instrument.

• VENTANA DP 200 slide scanner

Warnings and Precautions

1. For in vitro diagnostic (IVD) use.

2. For professional use only.

3. CAUTION: In the United States, Federal law restricts this device to sale by or on the order of a physician. (Rx Only)

4. To report suspected serious incidents related to this device, contact the local Roche representative and the competent authority of

the Member State or Country in which the user is established.

Workﬂow

1. The breast tissue on a glass slide is stained with the VENTANA anti-HER2/neu (4B5) antibody using a BenchMark ULTRA instrument.

2. Image acquisition (whole slide scanning) is performed with the VENTANA DP 200 slide scanner at 20x magniﬁcation at one z-plane.

3. Once digital images are acquired, these images are transferred from the computer associated with the VENTANA DP 200 slide

scanner to the image management system (IMS) on a centralized server.

4. Following transfer to the server, a case will be created in the uPath enterprise software. Case creation can occur automatically

through communication with the laboratory information system (LIS) using identiﬁcation information (i.e., tissue type and primary

antibody) contained in the barcode label of the glass slide or entered manually into the uPath enterprise software. Refer to the uPath

enterprise software User Guide (PN 1018943EN).

5. If the uPath HER2 (4B5) algorithm is installed (must be installed on a separate server from the uPath enterprise software and the IMS)

and a 20x image is accessioned with the appropriate stain and tissue type, the uPath enterprise software then automatically triggers

whole slide analysis (WSA).

6. WSA automatically analyzes the entire scanned image.

7. Once WSA is complete, the pathologist is notiﬁed within the uPath enterprise software viewer with a blue bar that reads “analysis

successful”.

8. The pathologist may select speciﬁc ROIs to score using the ROI and Exclusion tools within the uPath enterprise software.

9. The pathologist reviews the image analysis results and either accepts the score or manually overrides it.

Staining

• Tissue preparation and staining should follow the recommendations provided in the VENTANA anti-HER2/neu (4B5) antibody method

sheet.

• All proper controls should be reviewed and slides should be re-stained if the staining does not meet the guidelines outlined in the

VENTANA anti-HER2/neu (4B5) antibody method sheet.

9 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

• The uPath HER2 (4B5) algorithm requires use of the VENTANA anti-HER2/neu (4B5) antibody, and any additional material or supplies

listed in the VENTANA anti-HER2/neu (4B5) antibody method sheet, to stain tissues prior to analysis.

• The VENTANA anti-HER2/neu (4B5) antibody detects HER2 protein in FFPE breast tissue stained with the ult raView Universal DAB

Detection Kit on a BenchMark ULTRA instrument.

Image Capture

A VENTANA DP 200 slide scanner is required for scanning the slides. Images are required to be scanned at 20x magniﬁcation. It

is recommended that the tissue be free of folds and ink. If large sections of the image are out of focus, it is recommended that the

slides be rescanned. For further information on scanning, please refer to the VENTANA DP 200 slide scanner IVD User Guide (PN

1017149EN).

General Navigation: uPath enterprise software

The uPath enterprise software is meant to be customizable to individual and site needs including but not limited to report

conﬁguration and user interface. This algorithm guide will focus on the tools necessary for using the uPath HER2 (4B5) algorithm only.

For further information on the uPath enterprise software, please refer to the uPath enterprise software User Guide.

10 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Pathologist Workﬂow

Opening a Case in uPath Enterprise Software

To access images of breast tissue stained with the VENTANA anti-HER2/neu (4B5) antibody, double-click on a case, or select a case

and press the viewer tab (Figure 1).

A screen with all images associated with a case will appear (Figure 2).

Figure 2

Figure 1

11 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Once a glass slide stained with the VENTANA anti-HER2/neu (4B5) antibody is scanned on a VENTANA DP 200 slide scanner at

20x, the image is imported into the uPath enterprise software and associated with a case. The uPath HER2 (4B5) algorithm will

automatically trigger WSA. Times to complete the WSA precomputing step depends on server speciﬁcations, image sizes, and the

number of images in the queue. When displayed, “waiting to start auto-analysis” speciﬁes the images are in queue and yet to be

analyzed and “analyzing” is used when WSA is being performed (Figures 3 & 4).

Once the image is completely analyzed via WSA in the uPath enterprise software, “analysis successful” is displayed underneath the

slide image within the viewer (Figure 5). Images cannot be scored prior to successful WSA completion.

Figure 5

Figure 4

Figure 3

12 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Drawing Whole Tumor ROI(s): Selecting Tumor Area

Use the Freehand tool button within the ROI dropdown menu (Figure 6) to select the tumor area(s) on the IHC slide image to be

analyzed. Figure 7 illustrates an image that has a single ROI drawn. Additional ROIs can be drawn. Each area selected will cause an

ROI to appear in the Slide Panel (Figure 8).

Figure 7

Figure 8

Figure 6

13 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Drawing Whole Tumor ROI(s): Exclusion Area

When drawing the ROIs, the exclusion of certain areas may be required; speciﬁc areas to be avoided or omitted as well as examples

will be addressed within the staining characteristics section below. Use the Freehand exclusion tool within the Exclusion dropdown

menu (Figure 9) to exclude speciﬁc areas (Figure 10). If large areas of the image are blurry or out of focus, rescan the slide.

Excluded areas will not be analyzed by the uPath HER2 (4B5) algorithm, and the stained and unstained tumor cells within this area

will be excluded from the total analysis area. If the ROI has already been analyzed, and an exclusion is used, the ROI will need to be

reanalyzed and the overlay and score will be updated appropriately.

Drawing a high number of exclusions, especially complicated exclusions using the Freehand tool, can be time consuming and impact

workﬂow efﬁciency with only a marginal impact on the ﬁnal score. If a case requires a high number of exclusions, the pathologist

should

• Draw multiple ROIs and exclude portions of tissue they deem unscorable with minimal use of the Exclusion tool.

• Limit exclusions and manually override the score with another score.

Drawing Whole Tumor ROI(s): Deletion

If a selected whole tumor ROI is not optimal, it can be deleted. Select the whole tumor ROI by clicking on the center of the ROI on

the image and then clicking the Delete button within the Slide Panel (Figure 11) or within the slide image near the ROI (Figure 12). A

conﬁrmation window will appear. Select Conﬁrm to delete the selected ROI. Select Cancel to retain the ROI.

Figure 9

Figure 11

Figure 12

Figure 10

14 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

Once all whole tumor ROI(s) and/or exclusion areas have been drawn, the image is ready to be analyzed. Select the whole tumor ROI by

clicking on the center of the ROI to be analyzed or clicking on the ROI in the Slide Panel. For each ROI, click the Image Analysis button

either within the Slide Panel (Figure 13) or next to the ROI (Figure 14).

Once the HER2 analysis is complete, the result appears in the Slide Panel in two locations: under Slide Score and next to the ROIs

(Figure 15). The Slide Score is based on a summation of the HER 2 status across all selected ROI(s), which is the score that will appear

in the report.

You can also see more detailed information in the Slide Score ﬂip-out and the ROI Details ﬂip-out, by clicking the ﬂip-out icon (Figure

16). The Slide Score ﬂip-out will appear (Figure 17). Clicking the same ﬂip-out icon again will also hide this information.

Figure 15

Figure 17

Figure 16

Figure 13

Figure 14

15 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

HER2 Image Analysis: Color Overlay

Once the ROI(s) Analysis button has been pressed and the tissue analyzed, a color overlay will be displayed on the ROI. In the image

below (Figure 18), the red overlay represents cell membranes determined to be positively stained for HER2. When grabbing the image

(left-clicking with the mouse and moving the image) the overlay disappears (Figure 19). When the mouse button is no longer pressed,

the overlay reappears (Figure 18).

Figure 18

Figure 19

16 uPath HER2 (4B5) image analysis for Breast Algorithm Guide

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