Roche Upath IVD Algorithm Guide

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

Table of Contents
Introduction1
Algorithm Summary and Explanation 2
Intended Use 3
Intended Use of Product 3
Purpose of Algorithm Guide 3
Clinical Significance 4
Test Principles 5
Limitations6
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/neu (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.

Intended Use
Intended Use of Product Purpose of Algorithm Guide
The uPath HER2 (4B5) image analysis for Breast algorithm This uPath HER2 (4B5) algorithm guide (algorithm guide) is
is intended for use as an aid to the pathologist in the semi- intended to:
quantitative detection of HER2 protein in formalin-fixed,
• Provide background information on the intended use of the
paraffin-embedded neoplastic breast tissue. When used with
uPath HER2 (4B5) algorithm, test principles, and limitations.
the VENTANA anti-HER2/neu (4B5) Rabbit Monoclonal Primary
Antibody, (VENTANA anti-HER2/neu (4B5) antibody) it is • Define the necessary materials, IT, data security, and network
indicated for use as an aid in the assessment of breast cancer requirements.
patients for whom Herceptin® (trastuzumab), KADCYLA® (ado- • Show step-wise directions for running the uPath HER2 (4B5)
trastuzumab emtansine) or PERJETA® (pertuzumab) treatments algorithm.
are being considered.
• Provide photographic images that illustrate how the uPath HER2
Note: The uPath HER2 (4B5) image analysis for Breast algorithm (4B5) algorithm should be used.
is an adjunctive computer-assisted methodology to aid in the • Provide a tool to facilitate the use of the uPath HER2 (4B5)
acquisition and measurement of images from microscope algorithm on FFPE breast sections stained with the VENTANA
glass slides of breast tissue specimens that have undergone anti-HER2/neu (4B5) antibody.
immunohistochemical staining to determine the presence of
• Provide example images of challenging cases to provide
HER2 protein. To assure the validity of image analysis scores, it
guidance on how to use the uPath HER2 (4B5) algorithm in their
is the responsibility of the pathologist to verify agreement and
evaluation.
employ appropriate controls as specified in the VENTANA anti-
HER2/neu (4B5) antibody method sheet. • Show performance characteristics of the uPath HER2 (4B5)
algorithm.
This algorithm is intended for in vitro diagnostic (IVD) use.

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.1 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.

Test Principles
The uPath enterprise software with the uPath HER2 (4B5) Criteria for intensity and pattern of cell membrane staining
algorithm employs image analysis techniques to obtain a HER2 for the uPath HER2 (4B5) algorithm’s score:
score.
Staining Pattern Score HER2 Staining
The uPath HER2 (4B5) algorithm uses pre-defined parameters to Assessment
score images of tissue stained with the VENTANA anti-HER2/neu No membrane staining is observed 0 Negative
(4B5) antibody.
Faint, partial staining of the 1+ Negative
membrane in any proportion of the
Steps involved in image analysis: cancer cells
Weak to moderate complete staining 2+ Equivocal*
• Detection of cells across the entire image. of the membrane, > 10% of cancer
cells
• Classification of cells as tumor cells or other cell types. Intense, complete staining of the 3+ Positive
membrane, > 10% of cancer cells
• Identification of stained membrane and stratification of
membrane into categories: partial or complete staining and * Recommend reflex to ISH
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.

Limitations
The uPath HER2 (4B5) algorithm is designed to work with the threshold for a score of 1+. This may result in some cases with
VENTANA anti-HER2/neu (4B5) antibody. The test results are very few cells that have membrane staining being scored as 0
only as good as the quality and accuracy of the IHC slide that is rather than 1+. This noise threshold was implemented to ensure
imaged, and the subsequent image that is analyzed. true negative cases are being called as 0.
The pathologist must validate the VENTANA anti-HER2/neu The uPath HER2 (4B5) algorithm is designed to be sensitive to
(4B5) antibody staining run by manual microscopic examination the color brown (DAB staining) to ensure even faint membrane
of the HER2 control slides to verify that the expected results have staining is detected. For this reason, it can incorrectly score a
been obtained before images of slides are accessioned onto the tissue sample containing brown artifacts such as pigmentation,
uPath enterprise software for analysis. background staining in muscle fibers or any other artifacts that
are stained brown. There are tools within the uPath enterprise
The manufacturer’s recommendations must be followed for
software that will allow the pathologist to exclude such areas.
the VENTANA anti-HER2/neu (4B5) antibody including using
These are discussed in more detail in the staining characteristics
all positive and negative quality control materials for each
section.
staining run. If the control slides are not acceptable with manual
microscopic examination, the tissues need to be re-stained with The uPath HER2 (4B5) algorithm is designed to be sensitive for
acceptable results. 2+ cases. For this reason, the uPath HER2 (4B5) algorithm may
incorrectly score certain 1+ cases as 2+. Specifically, 1+ cases
The pathologist must follow the recommendations for VENTANA
with slightly less than 10% of tumor cells with complete staining
anti-HER2/neu (4B5) antibody interpretation.
or cases with a high amount of tumor cell cytoplasmic staining
The uPath HER2 (4B5) algorithm is designed to be used may be incorrectly scored as 2+. The pathologist may need to
by a qualified pathologist in conjunction with histological override the score in these particular types of cases. Cytoplasmic
examination, relevant clinical information, and proper controls. It staining is discussed in more detail in the staining characteristics
is not designed to be a standalone tool, and requires competent section.
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

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

Data Security
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.

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.

• 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 ultraView 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.

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).
Figure 1
A screen with all images associated with a case will appear (Figure 2).
Figure 2

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 3
Figure 4
Figure 5

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 6 Figure 8
Figure 7

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.
Figure 9
Figure 10
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 11 Figure 12

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).
Figure 13 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.
Figure 16
Figure 15
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 17

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

Manual Override of Slide Scores
Scores can be manually overriden 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 pathologist
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 to 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.”
Figure 20 Figure 21 Figure 22
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 23

Staining Characteristics
Please see the VENTANA anti-HER2/neu (4B5) antibody method sheet and Interpretation Guide.
VENTANA anti-HER2/neu (4B5) Antibody Evaluation

Breast neoplastic cells stained with the VENTANA anti-HER2/neu (4B5) antibody 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.
uPath HER2 (4B5) Algorithm Stain Evaluation

The pathologist using the uPath HER2 (4B5) algorithm should be familiar with manually scoring the VENTANA anti-HER2/neu
(4B5) antibody. The pathologist should use the freehand tool to circle the Whole Tumor Area. The pathologist should reference the
associated hematoxylin and eosin slide and negative control slide prior to using the uPath HER2 (4B5) algorithm. When selecting the
areas to be analyzed please take into consideration the limitations described in the Limitations and the Areas to Avoid sections below.
If the pathologist disagrees with the score provided by the uPath HER2 (4B5) 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.

Images of various staining patterns and expressions are provided in the subsequent Figures (24-28). HER2 expression level is reported
as a whole number (0 to 3+).
Figure 24: Scanned images of IHC stained breast tissue with a HER2 score of 1+ in uPath enterprise software; before analysis
(above) and after analysis (below). The red overlay represents cell membrane determined to be positively stained for HER2.



Borderline Cases
Figure 27: Scanned images of a 1+/2+ borderline case in uPath enterprise software; before analysis (above) and after
analysis (below). The red overlay represents cell membrane determined to be positively stained for HER2. The uPath HER2
(4B5) algorithm is designed to be sensitive for 2+ cases and may incorrectly score certain 1+ cases as 2+. Pathologists should
override the score as necessary.

Cytoplasmic Staining
Figure 28: Example of cytoplasmic staining before analysis (above) and after analysis (below). 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
types of cases.

Areas to Avoid When Using the uPath HER2 (4B5) Algorithm
When using the uPath HER2 (4B5) algorithm, pathologists should avoid scoring any regions that are typically avoided when scoring
manually. The overall score for HER2 can be overridden if the pathologist disagrees with the score given by the uPath HER2 (4B5)
algorithm.
Necrosis
Figure 29: Example of necrotic tissue before analysis (above) and after analysis (below). Pathologists should adhere to the
guidelines outlined in the VENTANA anti-HER2/neu (4B5) antibody method sheet. It is important to exclude necrotic tissue
because it can affect the way the uPath HER2 (4B5) algorithm scores the tissue sample and contribute to an inaccurate HER2
score.

Tissue Folds
Figure 30: Example of tissue folds before analysis (above) and after analysis (below). Pathologists should adhere to the
guidelines outlined in the VENTANA anti-HER2/neu (4B5) antibody method sheet. It is important to exclude tissue folds
because they can affect the way the uPath HER2 (4B5) algorithm scores the tissue sample and contribute to an inaccurate
HER2 score.

Background Staining
Figure 31: Example of background staining before analysis (above) and after analysis (below). Pathologists should adhere to
the guidelines outlined in the VENTANA anti-HER2/neu (4B5) antibody method sheet. It is important to exclude background
staining because it can affect the way the uPath HER2 (4B5) algorithm scores the tissue sample and contribute to an inaccurate
HER2 score.

Ink
Figure 32: Example of ink before analysis (above) and after analysis (below). Pathologists should adhere to the guidelines
outlined in the VENTANA anti-HER2/neu (4B5) antibody method sheet. It is important to exclude ink because it can affect the
way the algorithm scores the tissue sample and contribute to an inaccurate HER2 score.

Endogenous Pigment
Figure 33: Example of endogenous pigment before analysis (above) and after analysis (below). Pathologists should adhere to
the guidelines outlined in the VENTANA anti-HER2/neu (4B5) antibody method sheet. It is important to exclude endogenous
pigment because it can affect the way the algorithm scores the tissue sample and contribute to an inaccurate HER2 score.

Ductal Carcinoma in situ (DCIS)
Figure 34: Example of DCIS before analysis (above) and after analysis (below). Pathologists should adhere to the guidelines
outlined in the VENTANA anti-HER2/neu (4B5) antibody method sheet. It is important to exclude DCIS because it can affect
the way the algorithm scores the tissue sample and contribute to an inaccurate HER2 score.

Performance Characteristics
The performance of the uPath HER2 (4B5) algorithm was evaluated through method comparison, inter-reader agreement, intra-
reader agreement, inter-scanner agreement, and intra-scanner agreement studies. All analysis was performed using the uPath HER2
(4B5) algorithm on the uPath enterprise software. Participating pathologists followed the guidelines outlined in this algorithm guide
throughout the studies.
Method Comparison
A method comparison study was conducted to compare scores using the uPath HER2 (4B5) algorithm image analysis (IA) to scores
of the same cases by the same 3 pathologists using manual read (MR). A total of 529 evaluations from each method were included
in the comparison. The ground truth (GT), determined by a consensus panel of pathologists, none of whom served in the reader
pathologist role, was used as the reference for calculating positive percent agreement (PPA), negative percent agreement (NPA), and
overall percent agreement (OPA) for both IA and MR. The difference in PPA, NPA and OPA between IA and MR are also provided to
demonstrate the concordance between IA and MR scores. Results from the study demonstrate the non-inferiority in performance of
uPath HER2 (4B5) algorithm IA to that of pathologist MR, where performance of each method was measured against a GT reference.
Table 3. Agreement: Manual Read vs. Image Analysis

MR Status
GT Status IA Status Concordance Discordance Total
Concordance 250 6 256
Discordance 6 6 12
Total 256 12 268

Positive
MR PPA n/N (%) (95% CI) 256/268(95.5) (92.9, 97.8)
IA PPA n/N (%) (95% CI) 256/268(95.5) (92.9, 97.4)
Difference of PPA (IA - MR) n/N (%) (95% CI) 0/268(0.0) (-3.0, 2.6)
Discordance 3 9 12
Total 248 13 261

Negative
MR NPA n/N (%) (95% CI) 248/261(95.0) (92.3, 97.5)
IA NPA n/N (%) (95% CI) 249/261(95.4) (93.1, 97.7)
Difference of NPA (IA - MR) n/N (%) (95% CI) 1/261(0.4) (-1.1, 1.9)
Discordance 9 15 24
Total 504 25 529

Overall
MR OPA n/N (%) (95% CI) 504/529(95.3) (93.5, 97.2)
IA OPA n/N (%) (95% CI) 505/529(95.5) (93.6, 97.2)
Difference of OPA (IA - MR) n/N (%) (95% CI) 1/529(0.2) (-1.5, 1.7)
Note: 2-sided 95% confidence intervals were calculated using the percentile bootstrap method.
Note: GT=Ground Truth, IA=Image Analysis, MR=Manual Read, CI = Confidence Interval
Note: Concordance/discordance refers to the MR or IA result agree/disagree with GT.
Note: This analysis includes all 3 reader pathologists’ results.

Pathologist Reproducibility Studies
The pathologist reproducibility studies for the uPath HER2 (4B5) algorithm consisted of 529 evaluations based on 180 individual breast
cancer cases (interpreted by three qualified pathologists) for inter-reader agreement and 118 evaluations based on 40 individual
breast cancer cases (each case interpreted three times by the same qualified pathologist) for intra-reader agreement.
Table 4. Inter-Reader Agreement of HER2 Status

Modal Result Agreement
Method Reader Reader Result Positive Negative Total Measurement [a] % (n/N) 95% CI [b]
Positive 259 9 268 PPA 95.6(259/271) (93.5, 97.4)***
Overall Negative 12 249 261 NPA 96.5(249/258) (94.6, 98.4)***
Total 271 258 529 OPA 96.0(508/529) (94.7, 97.4)***
Positive 81 0 81 PPA 89.0(81/91) (80.9, 93.9)**
Reader 1 Negative 10 86 96 NPA 100.0(86/86) (95.7, 100.0)**
Total 91 86 177 OPA 94.4(167/177) (89.9, 96.9)**
WTA IA
Positive 88 7 95 PPA 98.9(88/89) (93.9, 99.8)**
Total 89 86 175 OPA 95.4(167/175) (91.2, 97.7)**
Positive 90 2 92 PPA 98.9(90/91) (94.0, 99.8)**
Total 91 86 177 OPA 98.3(174/177) (95.1, 99.4)**
WTA=Whole Tumor Area, IA=Image Analysis, CI = Confidence Interval

[a] PPA = positive percent agreement; NPA = negative percent agreement; OPA = overall percent agreement.
[b] The confidence intervals were calculated using the percentile bootstrap method (***) or the Wilson method (**)
Note: Only observations with non-missing evaluation were included in this analysis.
Table 5. Intra-Reader Agreement of HER2 Status

Method Read Reader Result Positive Negative Total Measurement [a] % (n/N) 95% CI [b]
Positive 69 0 69 PPA 95.8(69/72) (91.7, 100.0)***
Overall Negative 3 46 49 NPA 100.0(46/46) (92.3, 100.0)**
Total 72 46 118 OPA 97.5(115/118) (94.8, 100.0)***
Positive 21 0 21 PPA 87.5(21/24) (69.0, 95.7)**
Read 1 Negative 3 16 19 NPA 100.0(16/16) (80.6, 100.0)**
Total 24 16 40 OPA 92.5(37/40) (80.1, 97.4)**
WTA IA
Positive 24 0 24 PPA 100.0(24/24) (86.2, 100.0)**
Total 24 15 39 OPA 100.0(39/39) (91.0, 100.0)**
Positive 24 0 24 PPA 100.0(24/24) (86.2, 100.0)**
Total 24 15 39 OPA 100.0(39/39) (91.0, 100.0)**

[b] The confidence intervals were calculated using the percentile bootstrap method (***) or the Wilson method (**).

Scanner Reproducibility Studies
The scanner reproducibility studies for the uPath HER2 (4B5) algorithm consisted of 360 evaluations based on 120 individual breast
cancer cases. They were scanned by a trained user for 3 scans on each of the 3 different VENTANA DP 200 slide scanners at 20X and
analyzed using the uPath HER2 (4B5) algorithm. The scores were compared across scanners.
Table 6. Inter-Scanner Agreement of HER2 Status

Method Scanner Scanner Result Positive Negative Total Measurement [a] % (n/N) 95% CI [b]
Positive 230 0 230 PPA 98.3(230/234) (95.7, 100.0)***
Overall Negative 4 126 130 NPA 100.0(126/126) (97.0, 100.0)**
Total 234 126 360 OPA 98.9(356/360) (97.2, 100.0)***
Positive 77 0 77 PPA 98.7(77/78) (96.2, 100.0)***
Scanner 1 Negative 1 42 43 NPA 100.0(42/42) (91.6, 100.0)**
Total 78 42 120 OPA 99.2(119/120) (97.5, 100.0)***
WTA IA
Positive 76 0 76 PPA 97.4(76/78) (92.3, 100.0)***
Total 78 42 120 OPA 98.3(118/120) (95.0, 100.0)***
Positive 77 0 77 PPA 98.7(77/78) (96.2, 100.0)***
Total 78 42 120 OPA 99.2(119/120) (97.5, 100.0)***

[b] The confidence intervals were calculated using the percentile bootstrap method (***) or the Wilson method (**). Since each scanner had 3 scanning sessions
pooled, the bootstrap method is recommended for all confidence interval calculations. If a 100% agreement is observed, the Wilson method is recommended since the
bootstrap method will not provide reasonable confidence intervals.
Note: For each scanner, the 3 scanning sessions were pooled in this analysis.
Table 7. Intra-Scanner Agreement of HER2 Status

Method Scanner Scanner Result Positive Negative Total Measurement [a] % (n/N) 95% CI [b]
Positive 229 1 230 PPA 99.1(229/231) (97.9, 100.0)***
Overall Negative 2 128 130 NPA 99.2(128/129) (97.4, 100.0)***
Total 231 129 360 OPA 99.2(357/360) (97.8, 100.0)***
Positive 77 0 77 PPA 98.7(77/78) (96.2, 100.0)***
Total 78 42 120 OPA 99.2(119/120) (97.5, 100.0)***
WTA IA
Positive 75 1 76 PPA 100.0(75/75) (95.1, 100.0)**
Scanner 2 Negative 0 44 44 NPA 97.8(44/45) (93.3, 100.0)***
Total 75 45 120 OPA 99.2(119/120) (97.5, 100.0)***
Positive 77 0 77 PPA 98.7(77/78) (96.2, 100.0)***
Total 78 42 120 OPA 99.2(119/120) (97.5, 100.0)***

[b] The confidence intervals were calculated using the percentile bootstrap method (***) or the Wilson method (**). Since each scanner had 3 scanning sessions
pooled, the bootstrap method is recommended for all confidence interval calculations. If a 100% agreement is observed, the Wilson method is recommended since the
bootstrap method will not provide reasonable confidence intervals.
Note: For each scanner, the 3 scanning sessions were pooled in this analysis.

Troubleshooting
Retriggering Failed Analysis

If an error occurs while performing WSA in uPath enterprise software, “analysis error” will be displayed underneath the slide image in
a red bar (Figure 35).
Figure 35
To retrigger an analysis within the uPath enterprise software, begin by logging in as an administrator (Figure 36).
Figure 36
Once in Administrator Settings, click Job Queue (Figure 37).
Figure 37

Selecting the Failed tab will display all slides with failed analyses. Select the slide that needs to be analyzed to enable the Cancel Job
and Start Job buttons (Figure 38).
Figure 38
Selecting Start Job will initiate the analysis and “Job scheduled successfully” will be displayed at the top of the screen in a green bar, if
successful (Figure 39).
Figure 39

The slide image will now appear in the Pending page. Once in this page, it is possible to rearrange jobs in order of priority if there are
multiple images in the queue (Figure 40).
Figure 40
After a few seconds, the top slide image will then move to the In-Progress page and display a progress bar. It is not necessary to
remain on this page to perform the analysis (Figure 41).
Figure 41

When a slide analysis is completed it will move to the Canceled/Complete page (Figure 42). In the uPath Viewer, “analysis successful”
will now be displayed under the slide image (Figure 43) and all analytical functionality relevant to the slide will be available (Figure 41).
Figure 42
Figure 43

References
1. Hsu JL, Hung MC. The Role of HER2, EGFR, and Other Receptor Tyrosine Kinases
in Breast Cancer. Cancer Metastasis Rev. 2016;35(4):575-588.
2. Dickson RB, and Lippman ME. Genes, Oncogenes, and Hormones. Boston: Kluwer Academic Publishers; 1992.
3. Herceptin (Trastuzamab) [Package Insert]. EMEA (European Medicines Agency). http://www.ema.europa.eu/docs/

en_GB/document_library/EPAR_-_Product_Information/human/000278/WC500074922.pdf. Published 01/03/2010.
4. Roche PC. Immunohistochemical stains for breast cancer. Mayo Clin Proc. 1994;69: 57-58.
5. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of Chemotherapy Plus a Monoclonal Antibody against HER2
for Metastatic Breast Cancer That Overexpresses HER2. N Engl J Med. 2001;344(11):783-792.
6. Swain SM, Kim SB, Cortés J, Ro J, Semiglazov V, Campone M, Ciruelos E, Ferrero JM, Schneeweiss A,
Knott A, Clark E, Ross G, Benyunes MC, Baselga J. Pertuzumab, trastuzumab, and docetaxel for HER2-
positive metastatic breast cancer (CLEOPATRA study): overall survival results from a randomised,
double-blind, placebo-controlled, phase 3 study. Lancet Oncol. 2013 May;14(6):461-71.
7. Hurvitz SA, Dirix L, Kocsis J, Bianchi GV, Lu J, Vinholes J, Guardino E, Song C, Tong B, Ng V, Chu YW, Perez EA.
Phase II randomized study of trastuzumab emtansine versus trastuzumab plus docetaxel in patients with human
epidermal growth factor receptor 2-positive metastatic breast cancer. J Clin Oncol. 2013 Mar 20;31(9):1157-63.
8. von Minckwitz G, Huang CS, Mano MS, et al. Trastuzumab Emtansine for Residual Invasive
HER2-Positive Breast Cancer. N Engl J Med. 2019;380(7):617-628.
9. Moasser MM, Krop IE. The Evolving Landscape of HER2 Targeting in Breast Cancer. JAMA Oncol. 2015;1(8):1154-1161.
10. Hudis CA. Trastuzumab--Mechanism of Action and Use in Clinical Practice. N Engl J Med. 2007;357(1):39-51.

Contact Information
Roche Diagnostics GmbH

Sandhofer Strasse 116
D-68305 Mannheim
Germany
+800 5505 6606
dialog.roche.com
© 2020 Ventana Medical Systems, Inc. and Roche Diagnostics

International, Inc. All rights reserved.
VENTANA, BENCHMARK, ultraView, UPATH, and the VENTANA

logo are trademarks of Roche. All other trademarks are the property
of their respective owners.
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Roche Upath IVD Algorithm Guide

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uPath HER2 (4B5) image analysis for

Breast Algorithm Guide

Algorithm Summary and Explanation 2

Intended Use of Product 3

Purpose of Algorithm Guide 3

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

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

uPath HER2 (4B5) Algorithm Stain Evaluation 18

Pathologist Reproducibility Studies  31

Scanner Reproducibility Studies  32

The uPath enterprise software is an end-to-end digital pathology

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

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

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

In vitro diagnostics for the evaluation of HER2 status in breast

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

weak, medium, or strong intensity staining.

• Computation of the HER2 score by combining cell classification,

How HER2 image analysis algorithm identifies tumor cells

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

• Identified tumor cells are classified as stained using detected

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

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

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

human intervention throughout the analysis process.

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

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

The uPath HER2 (4B5) algorithm identifies DAB stained

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

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

To avoid infection by malicious software or unauthorized access

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

• Make sure that other computers and services on the network

• Customers are responsible for the security of their local area

• Restrict physical access to the instrument and all attached IT

• Make sure that instrument backup and archive files are

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

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

• The default operating system (OS) configuration provided with

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

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

Materials Required but not Provided

• VENTANA DP 200 slide scanner

Warnings and Precautions

2. For professional use only.

6. WSA automatically analyzes the entire scanned image.

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

General Navigation: uPath enterprise software

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

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

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

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

Drawing Whole Tumor ROI(s): Deletion

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

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

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

Figure 20 Figure 21 Figure 22

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

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

uPath HER2 (4B5) Algorithm Stain Evaluation

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

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

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

Algorithm Summary and Explanation 2

Intended Use of Product 3

Purpose of Algorithm Guide 3

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

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

uPath HER2 (4B5) Algorithm Stain Evaluation 18

Pathologist Reproducibility Studies 31

Scanner Reproducibility Studies 32