Upath PR (1E2) RUO Algorithm 1020752EN - 1

uPath PR (1E2) image analysis,
Breast Algorithm Guide RUO

2
Publication information
Template version Software version Revision date Change description

1.0 1.0 September 2021 Initial Algorithm Guide
y Revision history
Material number 09411054001
Copyright © 2021, Ventana Medical Systems, Inc. All rights

reserved.
Trademarks The following trademarks are acknowledged:
VENTANA, BENCHMARK, CONFIRM, ULTRAVIEW, and

UPATH are trademarks of Roche.
Other product names and trademarks are the property of

their respective owners.
Manufacturer address
Ventana Medical Systems, Inc.

1910 E. Innovation Park Drive
Tucson, AZ 85755
USA
+1 520 887 2155
+1 800 227 2155 (USA)
eLabDoc Electronic user documentation can be downloaded using

the eLabDoc e-service on DiaLog:
dialog.roche.com
For more information, contact your local affiliate or Roche

Service representative.
Roche Diagnostics
uPath PR (1E2) RUO algorithm v1.0 · 1020752EN · uPath RUO enterprise software · v1.0
3
Table of contents
Intended use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Symbols and abbreviations . . . . . . . . . . . . . . . . . . . . . . 4
About the system limitations. . . . . . . . . . . . . . . . . . . . . 6
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Overview of uPath RUO enterprise software. . . . . 8
Description of the algorithm test principles . . . . . 9
Using the algorithm workflow. . . . . . . . . . . . . . . . . 9
Opening a case. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Drawing whole tumor ROI. . . . . . . . . . . . . . . . . . . . . . . 13
Selecting tumor area . . . . . . . . . . . . . . . . . . . . . . . . 13
Excluding an area from a ROI . . . . . . . . . . . . . . . . . 14
Deleting a ROI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Image analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Description of color overlays. . . . . . . . . . . . . . . . . . 17
Performing image analysis . . . . . . . . . . . . . . . . . . . 17
Overriding the slide score manually. . . . . . . . . . . . 18
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Retriggering failed analysis . . . . . . . . . . . . . . . . . . . 19
Understanding areas to exclude from analysis . . 20
Roche Diagnostics
4 Intended use
Intended use
The uPath PR (1E2) image analysis, Breast RUO algorithm
is an adjunctive computer-assisted tool that identifies PR
negative and positive tumor cell nuclei within a
pathologist-annotated viable tumor region in images of
formalin-fixed, paraffin-embedded neoplastic breast
tissue scanned on a VENTANA DP 200 slide scanner.
For Research Use Only. Not for use in diagnostic

procedures.
Symbols and abbreviations

Product names Products and name variants for the products mentioned
in this publication.
Product names Product name variant

uPath PR (1E2) image uPath PR (1E2) RUO algorithm
analysis, Breast RUO
CONFIRM anti-Progesterone CONFIRM anti-PR (1E2)
Receptor (PR) (1E2) Rabbit antibody
Monoclonal Primary Antibody
Roche uPath RUO enterprise uPath RUO enterprise
software version 1.1.1 software
VENTANA DP 200 slide n/a
scanner
Benchmark ULTRA n/a
ultraView Universal DAB n/a
Detection Kit
y Product names
Symbols used in the publication

Symbol Explanation
o List item
u Related topics containing further information
Tip. Extra information on correct use or useful
q
hints.
r Start of a task
I Extra information within a task
f Result of an action within a task.
c Frequency of a task.
n Duration of a task.
d Materials that are required for a task.
j Prerequisites of a task.
u Topic. Used in cross-references to topics.
y Symbols used in the publication
Roche Diagnostics
Symbols and abbreviations 5
Symbol Explanation
p Task. Used in cross-references to tasks.
Figure. Used in figure titles and cross-
w
references to figures.
Table. Used in table titles and cross-references
y
to tables.
y Symbols used in the publication
Abbreviations The following abbreviations are used.
Abbreviation Definition
FFPE Formalin-fixed, paraffin-embedded
IHC Immunohistochemistry
PR Progesterone receptor
ROI Region of interest
RUO Research use only
WSA Whole slide analysis
y Abbreviations
Roche Diagnostics
6 About the system limitations
About the system limitations

When working with uPath PR (1E2) image analysis, Breast
RUO, cited in this document as uPath PR (1E2) RUO
algorithm, be aware of the following limitations.
The uPath PR (1E2) RUO algorithm is designed to work

with the CONFIRM anti-Progesterone Receptor (PR) (1E2)
Rabbit Monoclonal Primary Antibody, cited in this
document as CONFIRM anti-PR (1E2) antibody. The test
results are only as good as the quality and accuracy of
the immunohistochemistry (IHC) slide that is imaged and
the subsequent image that is analyzed.
q The uPath PR (1E2) RUO algorithm has not been

tested, nor its safety and effectiveness validated when
used with a personal computer from home.
You must validate the staining run by examination of the

control slides to verify that the expected results have
been obtained before images from sample slides are
acquired by the uPath RUO enterprise software.
Follow the manufacturer’s recommendations for the

uPath PR (1E2) RUO algorithm, including using all the
positive and negative quality control materials for each
staining run.
Description of the CONFIRM anti-PR (1E2) Use the manufacturer’s recommendations for the
antibody CONFIRM anti-PR (1E2) antibody, including all positive
and negative quality control materials for each staining
run. If the control slides are not acceptable, restain the
tissue until acceptable results are obtained. Follow the
interpretation guidelines in the uPath PR (1E2) RUO
algorithm method sheet.
Roche Diagnostics
About the system limitations 7
Considerations for the uPath PR (1E2) RUO The uPath PR (1E2) RUO algorithm is not designed as a
algorithm standalone tool and must only be used by a qualified
pathologist in conjunction with histological examination
and proper controls.
• The uPath PR (1E2) RUO algorithm may generate
incorrect scores if the slide images exhibit abnormal
staining or are improperly scanned.
• The uPath PR (1E2) RUO algorithm was validated on
invasive ductal, lobular, micropapillary, mucinous,
pleomorphic, tubular, adenoid cystic, apocrine, and
medullary carcinomas. Samples covered grades 1 to 3,
scores of 0 to 100%, and tissue biopsies and
resections.
• The uPath PR (1E2) RUO algorithm is a tool that
identifies PR negative and PR positive tumor cell
nuclei.
• The uPath PR (1E2) RUO algorithm was trained to
determine stain intensity using a robust and
representative data set derived from pathologist
assessments.
The uPath PR (1E2) RUO algorithm may misinterpret

some non-tumor cells as tumor cells, especially larger
cells. In most cases there are many more tumor cells than
non-tumor in the annotated area, so this type of error
should not significantly impact the count.
The uPath PR (1E2) RUO algorithm may identify unstained

stromal cells and lymphocytes as negative tumor cells,
which may incorrectly decrease the reported percent PR
positivity. This is more common in high positive cases and
cases with a large lymphocyte infiltrate, such as
medullary carcinomas.
Roche Diagnostics
8 Overview
Overview
In this section
Overview of uPath RUO enterprise software (8)
Description of the algorithm test principles (9)
Using the algorithm workflow (9)
Overview of uPath RUO enterprise software

The Roche uPath RUO enterprise software version 1.1.1,
cited in this document as uPath RUO enterprise software,
is an end-to-end digital pathology software that consists
of image analysis algorithms and a user interface.
The uPath RUO enterprise software with the uPath PR

(1E2) RUO algorithm provides consistent quantitative
assessment of neoplastic breast tissue.
Multiple functions are available for ease of use while

handling a case:
• Viewing of ROIs at various magnifications.
• Adding annotation to cases.
• Taking tissue section measurements.
• Performing image analysis.
• Generating reports.
uPath PR (1E2) RUO algorithm scoring The uPath RUO enterprise software with the uPath PR
(1E2) RUO algorithm assists in the quantitative
assessment of PR protein expression in IHC stained
histologic sections from FFPE neoplastic breast tissue.
The CONFIRM anti-PR (1E2) antibody counts the total

number of target tumor cell nuclei and stratifies them by
whether they are stained or unstained. Stained cells are
also stratified by stain intensity (weak, medium, or
strong). The uPath PR (1E2) RUO algorithm then divides
the number of PR positive tumor cell nuclei by the total
number of tumor cell nuclei to generate the percent PR
positivity.
The uPath PR (1E2) RUO algorithm reports the percent PR

positivity for each ROI and an aggregate score for all
selected ROIs on that slide. You may either accept the
scores provided by the algorithm, or manually override
them.
Roche Diagnostics
Overview 9
uPath PR (1E2) RUO algorithm intensity Stained tumor cells are stratified by stain intensity and
categorized as weak, medium, or strong intensity. The
output parameter is displayed as “Weak/Medium/Strong
Intensity Cell Count” in the ROI and Slide Score Panels
within uPath. The reported numbers are cell counts that
are normalized to 100, effectively making the reported
value the percentage of stained cells that fall into each
intensity bin. This is a deviation from other cell counts in
the panels, where the cell count is the absolute cell count.
Description of the algorithm test principles

The uPath PR (1E2) RUO algorithm employs deep
learning and image analysis techniques to obtain a
percent PR positivity.
The uPath PR (1E2) RUO algorithm uses probability maps

to score images of tissue stained with the CONFIRM
anti-PR (1E2) antibody.
Steps in image analysis • Split image into tiles for processing by the
convolutional neural network.
• Detect cells across the entire image.
• Classify cell nucleus as tumor or other types.
• Classify tumor cell nucleus as positive or negative.
• Perform color un-mixing on positively classified tumor
cells to quantify nuclear stain intensity.
• Categorize intensity into weak, medium, strong
intensities.
• Calculate the percent PR positivity by dividing the
number of PR positive tumor cell nuclei by the total
number of tumor cell nuclei.
Tumor cell identification and scoring Cell locations and classifications are identified by parsing
probability maps generated by the deep learning network.
The classification with the highest probability determines
whether cell nuclei are classified as tumor positive, tumor
negative, or other cell types. The overlay will only identify
negative and positive tumor cell nuclei.
Using the algorithm workflow

The workflow for the uPath PR (1E2) RUO algorithm
includes the following:
• Staining with the CONFIRM anti-PR (1E2) antibody.
• Scanning with the VENTANA DP 200 slide scanner.
• Scoring with the uPath PR (1E2) RUO algorithm.
Roche Diagnostics
10 Overview
Antibody Staining The CONFIRM anti-PR (1E2) antibody identifies PR

positive tumor cell nuclei in neoplastic breast tissue.
Tissue samples are stained with the ultraView Universal
DAB Detection Kit on a Benchmark ULTRA instrument.
Follow the recommendations provided in the CONFIRM

anti-PR (1E2) antibody method sheet for tissue
preparation, staining, and review of controls.
Restain the slides if staining does not meet the guidelines

outlined in the CONFIRM anti-PR (1E2) antibody method
sheet.
Scanning The VENTANA DP 200 slide scanner is an imaging device

used to scan, digitize, compress, store, retrieve, and view
digitized images of slide specimens.
u For more information, refer to the VENTANA DP 200
slide scanner User Guide.
Software The uPath RUO enterprise software is customizable to

meet both site and pathologist needs.
u For more information, refer to the uPath RUO
enterprise software User Guide
d Make sure that the following supplies are ready before

starting this procedure:
m uPath PR (1E2) RUO algorithm
m uPath RUO enterprise software
m VENTANA DP 200 slide scanner
m Neoplastic breast tissue slides stained with the
CONFIRM anti-PR (1E2) antibody on Benchmark
ULTRA instrument with the ultraView Universal DAB
Detection Kit.
r To create and analyze slide images
1 Use a Benchmark ULTRA instrument using the

ultraView Universal DAB Detection Kit to stain a
neoplastic breast tissue sample with the CONFIRM
anti-PR (1E2) antibody on a glass slide.
2 Scan the slide with the VENTANA DP 200 slide

scanner in BIF file format at 20X magnification and at
one z-plane.
f The VENTANA DP 200 slide scanner transfers the
slide image to the image management system.
Roche Diagnostics
Overview 11
3 Create a case in the uPath RUO enterprise software

either manually in data management or automatically
through communication with the laboratory
information system.
I The uPath RUO enterprise software triggers whole
slide analysis (WSA), which automatically analyzes
the entire scanned image, and indicates in the
viewer when analysis is complete.
4 Use the ROI and Exclusion tools to annotate any viable

tumor region.
5 Review the results, and then either accept the score or

manually override it.
u Related topics
• Retriggering failed analysis (19)
Roche Diagnostics
12 Opening a case
Opening a case
Manage slide images for uPath PR (1E2) RUO algorithm
in the CASES screen.
j Make sure the following prerequisites are completed

before starting the procedure.
m You must have the pathologist role or the correct
permissions assigned before you can perform this
task.
r To open a case
1 In the CASES screen, view the cases assigned to you.
2 Choose a case by doing one of the following:

• Double-click on the desired case.
• Highlight the case and choose the VIEWER tab.
f The VIEWER screen displays the case with all the
images.
u Related topics
• Selecting tumor area (13)
Roche Diagnostics
Drawing whole tumor ROI 13
Drawing whole tumor ROI

In this section
Selecting tumor area (13)
Excluding an area from a ROI (14)
Deleting a ROI (16)
Selecting tumor area

Each ROI that you create and its analysis status is
displayed in the SLIDE PANEL. If you start to create an
annotation and decide not to continue, you can clear the
selection.
c A slide evaluation or analysis is needed.

before starting the procedure:
m The VIEWER screen is open.
task.
Roche Diagnostics
14 Drawing whole tumor ROI
r To select a tumor area
1 Choose a slide, and then magnify the image.

I The pathologist may view each ROI at various
magnifications and analyze it with the uPath RUO
enterprise software.
f The buttons in the ANNOTATION area of the
Quick Action menu bar become available (change
to bright white font).
2 Choose the ROI button, and then choose an option

from the tool bar.
f The hand cursor changes to a crosshair cursor.
3 Move the cursor over the tissue section where you

want to create the ROI, and then choose and drag the
cursor to produce the ROI.
f The ROI is created, and an annotation label above
the ROI displays the ROI name and number.
q All annotation types except a text box are

assigned a name that includes a number, such as
ROI#1, or A#1 for an arrow.
Excluding an area from a ROI

You can draw an area on a slide image to exclude it from
the ROI.
Roche Diagnostics
Drawing whole tumor ROI 15

task.
r To exclude an area from a ROI
1 Choose a slide and then magnify the image.

I All images in the case will display. Choose an
image by magnifying it or clicking on it.
f The buttons in the ANNOTATION area of the
Quick Action menu bar are activated.
2 Choose the ROI on the slide image.

I Any existing text, arrow, measurement, and
exclusions are displayed in the SLIDE PANEL.
f The selected ROI is highlighted in blue in the
SLIDE PANEL.
3 Choose the Exclusion button, and then choose an

option from the list.
4 Draw the region to be excluded on the image, and

then choose CONFIRM.
u Related topics
• Understanding areas to exclude from analysis (20)
Roche Diagnostics
16 Drawing whole tumor ROI
Deleting a ROI
You can change the ROI on a slide image.

before starting this procedure.
task.
r To delete a ROI
1 Choose on the ROI.

f The ROI annotation label pop-up is displayed.
2 In the annotation label pop-up or the STATUS PANEL

functions area, choose the icon.
I You can also delete the ROI from the ROI
DETAILS panel.
Roche Diagnostics
Image analysis 17
Image analysis
In this section
Description of color overlays (17)
Performing image analysis (17)
Overriding the slide score manually (18)
Description of color overlays

A color overlay displays after ROI Analysis is completed.
Color Overlay result
Yellow Tumor cell nucleus is positive for PR
Black Tumor cell nucleus is negative for PR
y Color overlay
The overlay disappears while moving the image. It returns

when the image is no longer selected.
Performing image analysis

The uPath RUO enterprise software allows for the
pathologist to select the ROI for each slide to be analyzed.

m You must have the appropriate privileges to perform
this task.
Roche Diagnostics
18 Image analysis
r To perform image analysis
1 Choose the whole tumor ROI by doing one of the

following:
• Choose the center of the ROI to be analyzed.
• Choose the ROI in the SLIDE PANEL.
2 For each ROI, choose the Image Analysis button

either in the SLIDE PANEL or next to the ROI.
f Results are displayed in the SLIDE PANEL: under
Slide Score and next to the ROIs.
Overriding the slide score manually

The uPath RUO enterprise software allows for the
pathologist to review all cases analyzed and change
scores if necessary.

before starting this procedure.
task.
r To override the slide score manually
1 Choose a slide for the score override, and then from

the SLIDE PANEL.
f The SLIDE SCORE secondary panel is displayed.
2 Choose the button in the Slide Score area.
3 In the Percent Positivity field, enter the score, and then

choose the CONFIRM button to save the score.
• (Optional) Enter comments in the comment field.
Roche Diagnostics
Troubleshooting 19
Troubleshooting
In this section
Retriggering failed analysis (19)
Understanding areas to exclude from analysis (20)
Retriggering failed analysis

If an error occurs while performing WSA, an analysis error
message is displayed beneath the slide image.
c As needed.

m You must have Administrator privileges to perform
this task.
r To retrigger failed analysis
1 Login to the uPath RUO enterprise software.
2 Navigate to Administrator Settings and choose Job

Queue.
I The administrator settings is only available if you
have administrator privileges.
3 Choose the Failed tab.

f All slides with failed analyses are displayed.
4 Choose the slide to be analyzed then choose the Start

Job button.
f Slide moves to the Canceled/Completed tab when
done.
Roche Diagnostics
20 Troubleshooting
Understanding areas to exclude from analysis

Follow best practices in selecting ROIs. Exclude areas
from ROIs that are typically avoided when scoring
manually. If you disagree with the score given by the
uPath PR (1E2) RUO algorithm, you have the option to
override it.
Areas to exclude from analysis

Areas to exclude from analysis No overlay With overlay
Necrosis
Exclude area from analysis. It can affect
the slide score.
.
Tissue folds
the slide score.
Off-target staining
the slide score.
Ink
the slide score.
y Areas to exclude from analysis
Roche Diagnostics
Troubleshooting 21
Areas to exclude from analysis No overlay With overlay

Endogenous pigment
the slide score.
Ductal carcinoma in situ or Atypical

ductal hyperplasia
the slide score.
Normal Epithelium
the slide score.
y Areas to exclude from analysis
Lymphocyte infiltrate
Description of lymphocyte infiltrate H&E No overlay With overlay
The three corresponding images to the right
demonstrate lymphocyte infiltrate and how the uPath
PR (1E2) RUO algorithm may misidentify cells.
Exclude lymphocyte rich areas from analysis or
manually override score.
y Lymphocyte infiltrate
Roche Diagnostics
22 Troubleshooting
Roche Diagnostics

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uPath PR (1E2) image analysis,

Breast Algorithm Guide RUO

Template version Software version Revision date Change description

Material number 09411054001

Copyright © 2021, Ventana Medical Systems, Inc. All rights

Trademarks The following trademarks are acknowledged:

VENTANA, BENCHMARK, CONFIRM, ULTRAVIEW, and

Other product names and trademarks are the property of

Ventana Medical Systems, Inc.

eLabDoc Electronic user documentation can be downloaded using

For more information, contact your local affiliate or Roche

For Research Use Only. Not for use in diagnostic

Symbols and abbreviations

Product names Product name variant

Symbols used in the publication

Abbreviations The following abbreviations are used.

About the system limitations

The uPath PR (1E2) RUO algorithm is designed to work

q The uPath PR (1E2) RUO algorithm has not been

You must validate the staining run by examination of the

Follow the manufacturer’s recommendations for the

The uPath PR (1E2) RUO algorithm may misinterpret

The uPath PR (1E2) RUO algorithm may identify unstained

Overview of uPath RUO enterprise software

The uPath RUO enterprise software with the uPath PR

Multiple functions are available for ease of use while

The CONFIRM anti-PR (1E2) antibody counts the total

The uPath PR (1E2) RUO algorithm reports the percent PR

Description of the algorithm test principles

The uPath PR (1E2) RUO algorithm uses probability maps

Using the algorithm workflow

Antibody Staining The CONFIRM anti-PR (1E2) antibody identifies PR

Follow the recommendations provided in the CONFIRM

Restain the slides if staining does not meet the guidelines

Scanning The VENTANA DP 200 slide scanner is an imaging device

Software The uPath RUO enterprise software is customizable to

d Make sure that the following supplies are ready before

r To create and analyze slide images

1 Use a Benchmark ULTRA instrument using the

2 Scan the slide with the VENTANA DP 200 slide

3 Create a case in the uPath RUO enterprise software

4 Use the ROI and Exclusion tools to annotate any viable

5 Review the results, and then either accept the score or

j Make sure the following prerequisites are completed

1 In the CASES screen, view the cases assigned to you.

2 Choose a case by doing one of the following:

Drawing whole tumor ROI

Selecting tumor area

c A slide evaluation or analysis is needed.

j Make sure the following prerequisites are completed

r To select a tumor area

1 Choose a slide, and then magnify the image.

2 Choose the ROI button, and then choose an option

3 Move the cursor over the tissue section where you

q All annotation types except a text box are

Excluding an area from a ROI

j Make sure the following prerequisites are completed

r To exclude an area from a ROI

1 Choose a slide and then magnify the image.

2 Choose the ROI on the slide image.

3 Choose the Exclusion button, and then choose an

4 Draw the region to be excluded on the image, and