General Combined Positive Score (CPS) Training

General Combined Positive Score (CPS) Training

PD-L1 IHC 22C3 pharmDx

October 13, 2020 PD-L1 IHC 22C3 pharmDx For Training Purposes Only 1

Training Objectives


Reference: 1. PD-L1 IHC 22C3 pharmDx [package insert]. Carpinteria, CA: Dako, Agilent Pathology Solutions; 2020.

Discuss the application of Combined Positive Score (CPS)

assessment in Agilent’s PD-L1 IHC 22C3 pharmDx assay

Demystify and simplify the CPS approach so that pathologists

can be confident in their assay assessment and apply the

algorithm confidently in their patient population

Provide a self-assessment tool for pathologists to test their

CPS evaluation skills

CPS:

Gain Confidence,

Build Competence

ONE algorithm for five approved indications

with PD-L1 IHC 22C3 pharmDx1

General CPS Training Program Agenda


1

Understanding the CPS Equation

– CPS Scoring Overview

– Evaluating Specimen Adequacy for CPS

2 CPS Interactive Quiz (WebEx Poll)

3 Evaluating the CPS Denominator & Numerator (TC & IC)

4 Live Case Walkthrough (Pathotrainer)

5 CPS Exclusions, Artifacts and Controls

6 Session Wrap Up

Disclaimer

This presentation is used solely to describe the fundamental principles of interpretation and general application of Combined Positive Score (CPS) in PD-L1 IHC 22C3 pharmDx scoring only and is not to be used in lieu of a specific tumor indication’s Interpretation Manual (IM) and Instructions for Use (IFU).

Refer to the tumor indication-specific Interpretation Manual (IM) and Instructions for Use (IFU) for detailed inclusion and exclusion criteria specific to the indication to be scored.

This presentation may not be used for purposes other than scoring interpretation trainings without the prior written permission of Agilent Technologies.

Note: Photomicrograph magnification levels may appear different than indicated in respective annotations due to adjustment of image size.





CPS equation:

Important points:

• CPS determines the PD-L1 expression level of the specimen*

• Although the result of the CPS calculation can exceed 100, the maximum score is defined as CPS 100

• Scores are given in whole numbers (no fractions)


* Refer to the tumor indication-specific Interpretation Manual (IM) and Instructions for Use (IFU) for PD-L1 expression levels guiding therapy.

CPS = # PD-L1 staining cells (tumor cells, lymphocytes, macrophages)

Total # of viable tumor cells× 100

CPS Scoring Overview Denominator inclusions/exclusions


Tissue Elements Included in the Denominator Excluded from the Denominator

Tumor Cells All viable invasive* tumor cells• Any necrotic or non-viable invasive* tumor cells

• Carcinoma in situ*

Immune Cells Not included • All immune cells of any type

Other Cells Not included

• Benign cells

• Stromal cells (including fibroblasts)

• Necrotic cells and/or cellular debris

Do NOT include✗ Apoptotic tumor cells

✗ Crushed or poorly visualized tumor cells

* Refer to the tumor indication-specific Interpretation Manual (IM) and Instructions for Use (IFU) for detailed inclusion and exclusion criteria specific to the indication to be scored.

CPS Scoring Overview Numerator inclusions/exclusions



† In MICs, membrane and cytoplasmic staining are often indistinguishable due to high nuclear to cytoplasmic ratio. Therefore, membrane and/or cytoplasmic staining of MICs is included in the

CPS numerator.

‡ Adjacent MICs are defined as being within the same 20x field as the tumor. However, MICs that are NOT directly associated with the response to the tumor should be excluded.

§ Macrophages and histiocytes are considered the same cells.

Tissue Elements Included in the Numerator Excluded from the Numerator

Tumor CellsConvincing partial or complete linear membrane

staining (at any intensity) of viable invasive*

tumor cells

• Non-staining tumor cells

• Tumor cells with only cytoplasmic staining


Immune Cells

Membrane and/or cytoplasmic† staining (at any

intensity) of mononuclear inflammatory cells (MICs)

within tumor nests and adjacent supporting stroma‡:

• Lymphocytes (including lymphocyte aggregates)

• Macrophages§

Only MICs directly associated with the response to the

tumor are scored

• Non-staining MICs

• MICs (including lymphoid aggregates) associated

with ulcers or other processes not associated with

the tumor

• MICs associated with carcinoma in situ*

• MICs associated with benign structures

• Neutrophils, eosinophils, and plasma cells


• Benign cells



Fundamental Aspects of CPS

A patient specimen can have a Combined Positive Score (CPS) that is at or above the

PD-L1 Expression Level diagnostic cut-off* with:


* Refer to the tumor indication-specific Interpretation Manual (IM) and Instructions for Use (IFU) for PD-L1 expression levels guiding therapy.

BOTH PD-L1 staining tumor

cells and tumor-associated

MICs combined

Only PD-L1 staining

tumor cells

Only PD-L1 staining

tumor-associated MICs

Evaluating Specimen Adequacy


Evaluating Specimen AdequacyH&E

A serial H&E stained section from the same paraffin

block of the specimen is important to evaluate

prior to CPS evaluation of the patient PD-L1

stained slide in order to:

✓ Identify adequacy of fixation

✓ Identify poor slide preparation or sectioning artifacts

✓ Identify areas of necrosis

✓ Identify areas of inflammation not directly associated

with tumor

✓ Identify areas of carcinoma in situ or apoptotic tumor


Evaluating Specimen AdequacyPD-L1 stained slide

• There must be at least 100 viable invasive tumor cells* present in the PD-L1 stained slide for the

specimen to be considered adequate for evaluation

• Non-specific staining must be ≤ 1+ staining intensity and must not obscure any specific staining

• The following pre-analytical variables can result in a non-evaluable specimen. A specimen with any

of these pre-analytical variables should not be considered adequate for evaluation:

– Poor sample fixation

– Poor slide preparation

– Improper storage of cut tissue sections

– Processing artifacts



CPS Interactive Quiz (Poll)



ESCC specimen stained with PD-L1 primary antibody (20× magnification).

CPS Quiz


HNSCC specimen stained with PD-L1 primary antibody (20× magnification).

CPS Quiz



CPS Quiz



CPS Quiz



CPS Quiz


Gastric carcinoma specimen stained with PD-L1 primary antibody (20× magnification).

CPS Quiz

Evaluating the Denominator – Inclusions and Exclusions


Evaluating the Denominator – Inclusions and Exclusions

Do NOT include

✗ Apoptotic tumor cells

✗ Crushed or poorly visualized tumor cells


Tissue Elements Included in the Denominator Excluded from the Denominator

Tumor Cells All viable invasive* tumor cells• Any necrotic or non-viable invasive tumor cells*


Immune Cells Not included • All immune cells of any type


• Benign cells






Evaluating the Numerator – Identifying PD-L1 Staining Tumor Cells


membrane

cytoplasmic



Tissue

ElementIncluded in the Numerator

Tumor CellsConvincing partial or complete linear

membrane staining (at any intensity)

of viable invasive* tumor cells

ESCC specimen stained with PD-L1 primary antibody exhibiting perceptible and convincing

linear membrane staining of tumor cells that is distinct from cytoplasmic staining at

20× magnification (arrows).

* Refer to the tumor indication-specific Interpretation Manual (IM) and Instructions for Use

(IFU) for detailed inclusion and exclusion criteria specific to the indication to be scored.

Tumor cells with perceptible and

convincing membrane staining at

20× magnification must have:

• Partial or complete staining

• Staining at any intensity




Key takeaways

1. Tumor cells with perceptible and convincing membrane staining at 20× must have:

• Partial or complete linear membrane staining

• Staining at any intensity

2. Tumor cells with only cytoplasmic staining are not PD-L1 staining cells unless

convincing linear membrane staining is also present


Evaluating the Numerator –Tumor-associated Mononuclear Inflammatory Cells (MICs)



20× Rule: To determine which MICs are considered to be

adjacent to the tumor, place the edge of the tumor mass, or the

tumor nest, in the center of the 20× field

Evaluating the Numerator –Identifying PD-L1 Staining Tumor-associated MICsThe 20× rule

Tissue


Immune

Cells

Membrane and/or cytoplasmic* staining

(at any intensity) of mononuclear

inflammatory cells (MICs) within tumor

nests and adjacent supporting stroma†:

• Lymphocytes (including lymphocyte

aggregates)

• Macrophages‡

Only MICs directly associated with the

response to the tumor are scored.

* In MICs, membrane and cytoplasmic staining are often indistinguishable due to high

nuclear to cytoplasmic ratio. Therefore, membrane and/or cytoplasmic staining of MICs

is included in the CPS numerator.

† Adjacent MICs are defined as being within the same 20× field as the tumor.

However, MICs that are NOT directly associated with the response to the tumor

should be excluded.

‡ Macrophages and histiocytes are considered the same cells.

20× field with edge

of tumor in middle 20× field with tumor

focus in middle

Evaluating the Numerator –Identifying PD-L1 Staining Tumor-associated MICs


Tissue


Immune

Cells






aggregates)

• Macrophages‡



HNSCC specimen stained with PD-L1 primary antibody exhibiting staining of tumor-associated

lymphocytes (arrows) (20× magnification).




† Adjacent MICs are defined as being within the same 20× field as the tumor. However,

MICs that are NOT directly associated with the response to the tumor should be excluded.


Note: Immune cells may have smaller nuclei than tumor

cells; therefore, a larger number of MICs can occupy the

same area as a smaller number of tumor cells.

Evaluating the Numerator –Identifying PD-L1 Staining Tumor-associated MICsMacrophages


Tumor-associated macrophages with any PD-L1 staining (cytoplasmic and/or membrane,

partial or complete) at any intensity should be included in the CPS numerator (arrows)

(20× magnification). Note: Image above depicts esophageal adenocarcinoma.




† Adjacent MICs are defined as being within the same 20× field as the tumor. However,

MICs that are NOT directly associated with the response to the tumor should be excluded.


Tissue


Immune

Cells






aggregates)

• Macrophages‡




ESCC specimen stained with PD-L1 primary antibody exhibiting staining of multinucleate

giant cells (arrows) (20× magnification).

Some histiocytes may be multinucleate

and each multinucleate cell should be

each counted as one cell. The same

rule should be applied for inclusion of

macrophages in the numerator.

Evaluating the Numerator –Identifying PD-L1 Staining Tumor-associated MICsMultinucleate giant cells

Evaluating the Numerator –Identifying Immune Cells Excluded from the Numerator


Immune Cells

• Non-staining MICs

• MICs (including lymphoid aggregates) associated with ulcers and other processes not

associated with the tumor

• MICs associated with carcinoma in situ*

• MICs associated with benign structures

• Neutrophils, eosinophils, and plasma cells

Tissue Elements Excluded From the Numerator



Transition to Pathotrainer

- DP Case Walkthrough

1. Navigate to www.mypathotrainer.com

2. Log on with User Name and Password (provided via email)

3. Click on CPS General Training Session

4. Click on section titled “Walkthrough”

http://www.mypathotrainer.com/

Identifying Other Cells Excluded from the CPS


ESCC specimen stained with PD-L1 primary antibody exhibiting staining of benign

epithelial cells (red arrows) and associated MICs (black arrows), both of which should

be excluded from the score (20× magnification).

Identifying Other Cells Excluded from the CPS Benign cells


Tissue

Element

Excluded from Numerator

and Denominator

Other Cells• Benign cells



Benign cells (PD-L1 staining and

non-staining) should be excluded

from the score.


Tissue

Element


and Denominator




ESCC specimen stained with PD-L1 primary antibody exhibiting PD-L1 staining stromal cells

which should be excluded from the score (arrows) (20× magnification).

Identifying Other Cells Excluded from the CPS Stromal cells

Stromal cells exhibiting PD-L1 staining

should be excluded from the score.


Tissue

Element


and Denominator




ESCC specimen stained with PD-L1 primary antibody exhibiting staining of necrosis;

necrosis staining should be excluded from the score (20× magnification).

Identifying Other Cells Excluded from the CPS Necrotic cells and/or cellular debris

Necrotic cells and/or cellular debris

staining should always be excluded from

the score.

Artifacts to Exclude


Artifacts to Exclude


ESCC specimen stained with PD-L1 primary antibody exhibiting

non-specific staining; non-specific background staining

(red arrows) should be excluded from the score. Weak nuclear

staining is also present and should be ignored (black arrows)

(20× magnification).

✗ Tissue artifacts exhibiting staining should always be excluded from the CPS

Non-specific staining Edge Artifact

Cervical cancer specimen stained with PD-L1 primary antibody

exhibiting edge staining which should be excluded from the score

(20× magnification).


Esophageal adenocarcinoma specimen stained with

PD-L1 primary antibody exhibiting crush artifact; crush artifact

should be excluded from the score (20× magnification).

ESCC specimen stained with PD-L1 primary antibody exhibiting poor

tissue fixation which should be excluded from scoring (20×

magnification).

Crush artifact Poor tissue fixation

✗ Tissue artifacts exhibiting staining should always be excluded from the CPS✗ Tissue that is poorly fixed should be excluded from scoring

CPS: Artifacts to Exclude

Evaluating Controls


Evaluating Controls

The following four required tissue controls should be evaluated prior to CPS evaluation of patient

PD-L1 stained slide:

Tonsil tissue may be used as an additional optional control. Tonsil tissue controls should be used only

in conjunction with the required controls and should not be used independently.

If unwanted staining occurs in any of the controls, results with the patient specimen should be

considered invalid. Refer to the following slides for details regarding the acceptable staining for

each of the required controls, and for the additional optional tonsil tissue control.


Positive In-house

Control Tissue

Negative In-house

Control Tissue

Negative Control

Reagent (NCR)

Control for Patient

Specimen

Control Cell Line

(CCL) Slide


PD-L1 IHC 22C3 pharmDx Instructions for Use

Control Cell Line

Slide adequate?

Positive control

tissue adequate?

Patient specimen

stained with

Negative Control

Reagent

adequate?

Negative control

tissue adequate?

Patient specimen

stained with

primary antibody

exhibiting ≥ 100

viable tumor cells

Yes Yes Yes Yes

No No

Repeat

staining run

Repeat

staining run

Repeat

staining run

Repeat

staining run

Repeat staining run

with a deeper cut in

the block or a new

patient specimen

Scored by a

pathologist

Provide case report

No No No No

One section is

stained with H&E

(H&E Patient

Specimen)

Is H&E slide

adequate? (intact,

well-preserved)

Yes

Evaluating Controls Recommended order of slide evaluation

ESCC Positive In-house Control Tissue stained with PD-L1 primary antibody demonstrating a complete dynamic representation of weak-to-moderate staining of tumor cells and MICs.

ESCC Negative In-house Control Tissue stained with PD-L1 primary antibody demonstrating lack of staining of tumor cells and MICs.*

20× 20×

Evaluating ControlsPositive & negative in-house tissue control examples


* Because prevalence of PD-L1 expression on immune cells is high, a few staining immune cells are acceptable. Examine the negative in-house control tissue to determine the expected staining.

Wrap Up

October 13, 2020 PD-L1 IHC 22C3 pharmDx For Training Purposes Only PR7000_

You will receive the digital version to use as a reference.

Each picture leads to the indication webpage, where you can find:

• Interpretation Manual

• Interpretation Training Program

• Online Atlas of Stains (for CPS indications: Urothelial Carcinoma, Gastric or GEJ adenocarcinoma, and HNSCC)

October 13, 2020 PD-L1 IHC 22C3 pharmDx For Training Purposes Only

PD-L1 IHC 22C3 pharmDx Universe of Indications

44

You can also refer to:

https://www.agilent.com/en-us/pd-l1-ihc-22c3-pharmdx-overview

Useful Approaches to Denominator Evaluation

Learn how to efficiently and reproducibly evaluate the CPS denominator:

https://youtu.be/Ttl6PFjDdog

October 13, 2020 PD-L1 IHC 22C3 pharmDx For Training Purposes Only PR7000_

https://www.youtube.com/watch?v=Ttl6PFjDdog

PD-L1 IHC 22C3 pharmDx For Training Purposes Only 46 October 13, 2020

Thank You

Publication Acknowledgements

Tissue samples supplied by BioIVT Asterand®.

The data and biospecimens used in this project was provided by US Biolab Corporation, Inc, Rockville, MD, USA with appropriate ethics approval and through Trans-Hit Biomarkers Inc.

The data and biospecimens used in this project was provided by SageBio LLC, Sharon, MA, USA with appropriate ethics approval and through Trans-Hit Biomarkers Inc.


Documents

General Combined Positive Score (CPS) Training