Roche uPath RUO User manual

Brand: Roche

Model: uPath RUO

Type: User manual

uPath HER2 (4B5) image analysis for

Breast Algorithm Guide (RUO)

Table of Contents

Introduction 1

Algorithm Summary and Explanation 2

Intended Use 3

Intended Use of product 3

Purpose of Algorithm Guide 3

Test Principles 4

Limitations 5

Data Security 6

Workﬂow for using the uPath HER2 (4B5) image analysis for Breast algorithm 7

Pathologist Workﬂow 8

HER2 (4B5) Image Analysis 12

Staining Characteristics 15

HER2 (4B5) Evaluation 15

Troubleshooting 25

Introduction

The Roche uPath RUO enterprise software (uPath RUO

enterprise software) with the uPath HER2 (4B5) image analysis

for Breast algorithm is a software system designed to assist

in the semi-quantitative assessment of protein expression in

immunohistochemically (IHC) stained histologic sections

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

neoplastic tissues.

The uPath RUO 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 RUO

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 tissue specimens IHC-stained for the presence of HER2

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

responsibility of the pathologist to verify agreement by employing

appropriate controls as speciﬁed in the PATHWAY anti-HER2/

neu (4B5) Rabbit Monoclonal Primary Antibody (PATHWAY HER2

(4B5)) method sheet (available at www.ventana.com).

For Research Use Only. Not for use in diagnostic procedures.

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

Algorithm Summary and Explanation

For image analysis applications, the pathologist may use the

uPath RUO 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) image analysis for Breast algorithm. A count of the total

number of target tumor cells and the number interpreted by the

uPath HER2 (4B5) image analysis for Breast algorithm as stained

or unstained is generated. Stained cells are stratiﬁed by stain

intensity and completeness of membrane staining.

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

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

selected ROIs on that slide. The pathologist can accept the score

provided by the uPath HER2 (4B5) image analysis for Breast

algorithm, or may override the score with a different score.

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

no independent interpretations of the data and therefore should

only be used by a qualiﬁed pathologist in conjunction with

histological examination and proper controls. It is designed as an

aid to the pathologist for the assessment of HER2 expression.

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

Intended Use of product

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

adjunctive computer-assisted methodology that supports the

detection and measurement of HER2 protein in formalin-ﬁxed,

parafﬁn-embedded normal and neoplastic breast tissue.

For Research Use Only. Not for use in diagnostic procedures.

Intended Use

Purpose of Algorithm Guide

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

Guide (Algorithm Guide) is intended to:

• Provide background information on the intended use of the

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

• Provide photographic images that illustrate how the uPath

HER2 (4B5) image analysis for Breast algorithm should be used.

• Provide pathologists with a tool to facilitate the use of the uPath

HER2 (4B5) image analysis for Breast algorithm on FFPE breast

sections stained with the PATHWAY HER2 (4B5) Assay.

• Provide example images of challenging cases to provide

guidance on how to use the uPath HER2 (4B5) image analysis

for Breast algorithm in their evaluation.

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

Test Principles

Criteria for intensity and pattern of cell membrane staining

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

algorithm score:

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

image analysis for Breast algorithm employs image analysis

techniques to obtain a HER2 score.

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

uses pre-deﬁned parameters to score images of tissue

stained with the PATHWAY HER2 (4B5) Assay.

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 according to the

PATHWAY HER2 (4B5) Assay method sheet.

How HER2 image analysis algorithm identiﬁes tumor

cells and how the score is calculated:

• The uPath HER2 (4B5) image analysis for Breast 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 that are in line with the

PATHWAY HER2 (4B5) Assay method sheet.

• To calculate the HER2 score the uPath HER2 (4B5) image

analysis for Breast algorithm stratiﬁes the identiﬁed, stained

cells by stain intensity and completeness according to the

PATHWAY HER2 (4B5) Assay method sheet.

Staining Pattern Score

No membrane staining is observed 0

Faint, partial staining of the membrane in

any proportion of the cancer cells

Weak complete staining of the membrane,

greater than 10% of cancer cells

Intense complete staining of the membrane,

greater than 10% of cancer cells

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

Table extracted from the Pathway HER2 (4B5) Assay method sheet.

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

designed to work for the PATHWAY HER2 (4B5) Assay. 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 PATHWAY HER2 (4B5) Assay

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 RUO enterprise software for analysis.

Use the manufacturer’s recommendations for the PATHWAY

HER2 (4B5) Assay including using all positive and negative

quality control materials for each staining run. If the control

slides are not acceptable with manual microscopic examination,

restain the tissue with acceptable results.

The pathologist must follow the recommendations for

PATHWAY HER2 (4B5) Assay interpretation.

Refer to the PATHWAY HER2 (4B5) Assay method sheet

(P/N 14427EN) and interpretation guide (P/N 1499100EN)

(available at www.ventana.com).

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

designed for use by a qualiﬁed pathologist in conjunction with

histological examination 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) image analysis for Breast algorithm

may generate incorrect scores if the captured images have

abnormal staining (nuclear staining, pigmentation, etc.).

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

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) image analysis for Breast algorithm has

been trained, developed, and validated on invasive lobular,

ductal, mucinous, medullary, papillary, and micropapillary

breast carcinomas as well as a few metastatic lesions.

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

not been tested, or its safety and effectiveness validated, when

used with a personal computer (PC) from home.

The uPath HER2 (4B5) image analysis for Breast 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 RUO enterprise software that will allow the

pathologist to exclude such areas. These are discussed in more

detail in the staining characteristics section.

Limitations

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

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.

Data Security

• 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.

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

Materials needed

• uPath RUO enterprise software

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

• Breast tissue slides stained with the PATHWAY HER2 (4B5) Assay (using ultraView Universal DAB Detection Kit)

stained on the BenchMark ULTRA instrument.

• VENTANA DP 200 slide scanner

Workﬂow

1. A breast tissue sample on a glass slide is stained with the PATHWAY HER2 (4B5) Assay 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 RUO 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 RUO enterprise software

User Guide (1019288EN).

5. If the uPath HER2 (4B5) image analysis for Breast algorithm is installed (must be installed on a separate server from the uPath RUO

enterprise software and the IMS) and a 20x image is accessioned with the appropriate stain and tissue type, the uPath RUO 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 uPath RUO enterprise software viewer with a blue bar that reads

“analysis successful.”

8. The pathologist is able to select speciﬁc ROIs to score using the ROI and exclusion tools within uPath software.

9. The user is able to review the image analysis results and either accepts or performs a manual override of the score.

Staining

• Tissue preparation and staining should follow the recommendations provided in the PATHWAY HER2 (4B5) Assay method sheet.

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

PATHWAY HER2 (4B5) Assay method sheet.

• The uPath HER2 (4B5) image analysis for Breast algorithm requires use of the PATHWAY HER2 (4B5) Assay, and any additional

material or supplies listed in the PATHWAY HER2 (4B5) Assay method sheet, to stain tissues prior to analysis. The PATHWAY HER2

(4B5) Assay detects HER2 protein in FFPE breast tissue stained with ultraView Universal DAB Detection Kit on a BenchMark ULTRA

instrument. Though the PATHWAY HER2 (4B5) detects HER2 protein in FFPE breast carcinoma tissue stained on the BenchMark

ULTRA and XT instruments, the uPath HER2 (4B5) image analysis for Breast algorithm was validated using the ultraView Universal

DAB Detection Kit stained on the BenchMark ULTRA instrument.

Image Capture

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

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 User Guide (PN 1016906EN).

General Navigation: uPath RUO enterprise software

The uPath RUO 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) image

analysis for Breast algorithm only. For further information on the uPath RUO enterprise software, please refer to the uPath RUO

enterprise software User Guide.

Workﬂow for using the uPath RUO HER2 (4B5) image analysis for Breast algorithm

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

Pathologist Workﬂow

Opening a Case

Images of breast tissue stained with the PATHWAY HER2 (4B5) Assay can be accessed by double-clicking on a case, or by selecting

a case and pressing the viewer tab within the uPath RUO enterprise software (Figure 1).

Figure 1

Figure 2

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

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

Once a glass slide stained with the PATHWAY HER2 (4B5) Assay is scanned on a VENTANA DP 200 slide scanner at 20x, the image

is imported into the uPath RUO enterprise software and associated with a case. The uPath HER2 (4B5) image analysis for Breast

algorithm will automatically trigger WSA. Times to complete the WSA precomputing step depend on server speciﬁcations, image sizes

and 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 and 4). Once the image is completely analyzed via WSA in

the uPath RUO enterprise software, “analysis successful” will be displayed underneath the slide image within the viewer in the uPath

RUO enterprise software (Figure 5). Images cannot be scored prior to successful WSA completion.

Figure 4

Figure 3

Figure 5

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

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 (Figure 7)

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

Figure 7

Figure 8

Figure 6

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

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) image analysis for Breast 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 Freehand exclusions, 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 do

the following:

• 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.

Figure 10

Figure 9

Figure 11 Figure 12

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.

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

HER2 (4B5) Image Analysis

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, select the Image Analysis button

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

Figure 15

Figure 17

Figure 16

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 will also hide this information.

Once the HER2 Analysis is complete, the result will appear 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.

Figure 13

Figure 14

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

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 membrane determined to be positively stained for HER2. When grabbing the image

(pressing the left mouse button and moving the image) the overlay will disappear (Figure 19). When the mouse button is released, the

overlay reappears (Figure 18).

Figure 18

Figure 19

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

Manual Override Slide Score Entry

Scores can be manually overridden by clicking on the Slide Score ﬂip-out icon within the Slide Panel next to the Slide Score

(Figure 16). The Slide Score ﬂip-out will appear (Figure 20). Selecting the edit button (Figure 20) on the Slide Score ﬂip-out allows

the user to type in a manual score (Figure 21). The Comments ﬁeld allows notes regarding the case and/or decision to override the

automated score. For HER2, scores of 0-3+ may be manually entered. Upon entry of a Manual Override score, select the conﬁrm

notiﬁcation (Figure 22). A conﬁrmation message will appear, select “Yes”.

The score within the Slide Panel will now reﬂect the manual override score. The image analysis score provided next to the ROI(s)

will no longer be present (Figure 23). The user will have the option to re-analyze the image by pressing the bar graph button

(Figure 13, Figure 14).

Figure 21 Figure 22

Figure 23

Figure 20

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

Please see the PATHWAY HER2 (4B5) Assay method sheet and Interpretation Guide.

HER2 (4B5) Evaluation

Breast neoplastic cells labeled with the PATHWAY HER2 (4B5) Assay are evaluated for total percentage, completeness and intensity

of tumor cell membrane staining in order to generate a HER2 score as described in the HER2 Scoring Table in the Test Principles

section above. The immunohistochemical staining in breast cancer is membranous, and may be expressed homogeneously or

heterogeneously throughout the neoplasm. Tumor cell cytoplasmic staining is not factored into the HER2 score. Membrane staining

can have partial or circumferential pattern. An isotype-matched negative control antibody is used to evaluate the presence of

background in test samples.

HER2 Algorithm Stain Evaluation

The pathologist using the uPath HER2 (4B5) image analysis for Breast algorithm should be familiar with manually scoring the

PATHWAY HER2 (4B5) Assay. The pathologist should use the freehand tool to circle the whole tumor area and reference the

associated H&E and negative control slide prior to using the uPath HER2 (4B5) image analysis for Breast algorithm. The pathologist

should reference the associated H&E and negative control slide prior to using the uPath HER2 (4B5) image analysis for Breast

algorithm. When selecting the areas to be analyzed, please take into consideration the limitations described in the Limitations and

the Areas to Avoid sections. If the pathologist disagrees with the score provided by the uPath HER2 (4B5) image analysis for Breast

algorithm, the pathologist should manually override the score.

Non-evaluable cases include, but are not limited to, cases with insufﬁcient viable tumor, unacceptable morphology, and interfering

background. Breast cancer cases with sufﬁcient viable tumor cells (as determined by the scoring pathologist) and no interfering

background on the HER2 IHC slide are acceptable for evaluation.

Staining Characteristics

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

Images of various staining patterns and expressions are provided in the subsequent Figures (24-26). HER2 expression level is reported

as a whole number (0-3+).

Figure 24: Scanned images of IHC stained breast tissue with a HER2 score of 1+ in uPath RUO enterprise software;

before analysis (above) and after analysis (below). The red overlay represents cell membrane determined to be

positively stained for HER2.

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

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Roche uPath RUO User manual

Roche uPath RUO User manual

Roche uPath RUO User manual

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