Agilent PD-L1 IHC 22C3 User Manual

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

FDA-approved for in vitro diagnostic use

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

For countries outside of the United States, see the local KE YTRUDA product label for approved indications and expression cutoff values to guide therapy.

Table of Contents

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Intended Use 04 Introduction 06 PD-L1 Overview 08 PD-L1 IHC 22C3 pharmDx Overview 10 Kit Configuration (SK006) 11 Technical Considerations 12

Specimen Preparation 12 In-house Control Tissue 12 Optional Additional In-house Control: Tonsil Tissue 13 Tissue Processing 13

PD-L1 IHC 22C3 pharmDx Staining Procedure 14 Technical Checklist 17 Slide Evaluation 18

General Considerations 18 Specimen Adequacy 18 Evaluating Controls 19 Slide Evaluation Flowchart 23

Combined Positive Score 24

Definition of Combined Positive Score (CPS) 24 CPS Numerator Inclusion and Exclusion Criteria 24 Determining Combined Positive Score 25 Suggested Methods 27

Interpretation of CPS 30

Identifying Patients With Urothelial Carcinoma for Treatment 31 PD-L1 IHC 22C3 pharmDx Testing Scheme 32

Reporting Results 33 Combined Positive Score Summary and Examples 34

Key Considerations in Scoring PD-L1 IHC 22C3 pharmDx 34 Stained Specimens Image Guide for Interpretation of PD-L1 IHC 22C3 pharmDx 35 Staining in Urothelial Carcinoma CPS < 10 Case Examples 49

 CPS≥10CaseExamples 53

Near Cut-off Case Examples 59 (CPS Range of Greater Than 1 but Less Than 10) Near Cut-off Case Examples 63 (CPS Range of Greater Than or Equal to 10 but Less Than or Equal to 20)

Artifacts 66 Troubleshooting Guide 71 Clinical Performance Evaluation 72 References 74

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Intended Use

For in vitro diagnostic use.

PD-L1 IHC 22C3 pharmDx is a qualitative immunohistochemical assay using Monoclonal Mouse Anti-PD-L1, Clone 22C3 intended for use in the detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC), gastric or gastroesophageal junction (GEJ) adenocarcinoma, cervical cancer and urothelial carcinoma tissues using EnVision FLEX visualization system on Autostainer Link 48.

Non-Small Cell Lung Cancer (NSCLC) PD-L1 protein expression in NSCLC is determined by using Tumor Proportion Score (TPS), which is the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. The specimen should be considered

tohavePD-L1expressionifTPS≥1%andhighPD-L1expressionifTPS≥50%.

PD-L1 IHC 22C3 pharmDx is indicated as an aid in identifying NSCLC patients for treatment with KEYTRUDA for expression cutoff values guiding therapy in specific clinical circumstances.

(pembrolizumab). See the KEYTRUDA® product label

Gastric or Gastroesophageal Junction (GEJ) Adenocarcinoma PD-L1 protein expression in gastric or GEJ adenocarcinoma is determined by using Combined Positive Score (CPS), which is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. The specimen should be considered to

havePD-L1expressionifCPS≥1.

PD-L1 IHC 22C3 pharmDx is indicated as an aid in identifying gastric or GEJ

adenocarcinoma patients for treatment with KEYTRUDA

(pembrolizumab).

Cervical Cancer PD-L1 protein expression in cervical cancer is determined by using Combined Positive Score (CPS), which is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. The specimen should be considered to have PD-L1 expression

ifCPS≥1.

PD-L1 IHC 22C3 pharmDx is indicated as an aid in identifying cervical cancer

patients for treatment with KEYTRUDA

(pembrolizumab).

Urothelial Carcinoma PD-L1 protein expression in urothelial carcinoma is determined by using Combined Positive Score (CPS), which is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. The specimen should be considered to have PD-L1

expressionifCPS≥10.

KEYTRUDA is a registered trademark of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc.

PD-L1 IHC 22C3 pharmDx is indicated as an aid in identifying urothelial carcinoma

patients for treatment with KEYTRUDA

(pembrolizumab). See the KEYTRUDA®

product label for specific clinical circumstances guiding PD-L1 testing.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Introduction

PD-L1 IHC 22C3 pharmDx is the only companion diagnostic approved by the FDA as an aid in identifying patients with urothelial carcinoma for treatment with KEYTRUDA

This Interpretation Manual is provided as a tool to help guide pathologists and laboratory personnel in achieving correct and reproducible results in assessing PD-L1 expression in formalin-fixed, paraffin-embedded urothelial carcinoma specimens. PD-L1 expression evaluation may be used to identify patients for anti-PD-1 immunotherapy.

The manual provides detailed scoring guidelines and technical information from the PD-L1 IHC 22C3 pharmDx Instructions for Use (IFU) to ensure high-quality staining and diagnostic assessment. To help familiarize you with the requirements for scoring urothelial carcinoma stains with PD-L1 IHC 22C3 pharmDx, example cases of various PD-L1 expression levels are provided as references. These example cases and in-depth recommendations for interpretation of urothelial carcinoma specimens stained with PD-L1 IHC 22C3 pharmDx can help individual labs achieve reproducible and reliable results.

(pembrolizumab).

PD-L1 IHC 22C3 pharmDx is considered a qualitative immunohistochemical assay. PD-L1 expression in urothelial carcinoma is determined by using Combined Positive Score (CPS), which is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100.

Urothelial carcinoma tissue specimens that are tested for PD-L1 expression are scored and divided into two groups based on their Combined Positive Score (CPS):

– CPS < 10

– CPS≥10

For more details on staining and interpretation, please refer to the current version of the IFU provided with PD-L1 IHC 22C3 pharmDx, Code SK006 or visit www.agilent.com.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Assay Interpretation

The clinical interpretation of any staining, or the absence of staining, must be complemented by the evaluation of proper controls. Evaluation must be made by a qualified pathologist within the context of the patient’s clinical history and other diagnostic tests. This product is intended for in vitro diagnostic (IVD) use.

Reporting Results

To help understand what information should be reported to the treating physician, please refer to the Reporting Results section of this manual on page 33.

Photomicrographs

The included photomicrographs are of urothelial carcinoma unless otherwise noted.

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

Tissue samples supplied by Asterand Bioscience.

Tissue samples were provided by the Cooperative Human Tissue Network which is funded by the National Cancer Institute. Other investigators may have received specimens from the same subjects.

Data and tissue used in this project were provided by Tumorgenetics Ltd., Budapest, Hungary with appropriate ethics approval and through Trans-Hit Biomarkers Inc.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

PD-L1 Overview

The PD-1/PD-L1 Pathway Controls the Immune Response in Normal Tissue

The Tumor Escapes Detection by Utilizing the PD-1/PD-L1 Pathway

Anti-PD-1 Therapy Enables the Immune Response Against Tumors

Programmed death-ligand 1 (PD-L1) is a transmembrane protein that binds to the programmed death-1 receptor (PD-1) during immune system modulation. The PD-1 receptor is typically expressed on cytotoxic T-cells and other immune cells, while the PD-L1 ligand is typically expressed on normal cells. Normal cells use the PD-1/PD-L1 interaction as a mechanism of protection against immune recognition by inhibiting the action of T-cells (Figure 1). Inactivation of cytotoxic T-cells downregulates the immune response such that the inactive T-cell is exhausted, ceases to divide, and might eventually die by programmed cell death, or apoptosis.

Many tumor cells are able to upregulate the expression of PD-L1 as a mechanism to evade the body’s natural immune response. Activated T-cells recognize the PD-L1 marker on the tumor cell, similar to that of a normal cell, and PD-L1 signaling renders the T-cell inactive (Figure 2). The tumor cell escapes the immune cycle, continues to avoid detection for elimination, and is able to proliferate.

PD-1/PD-L1 interaction between tumor cells and activated T-cells (Figure 3) is a mechanistic pathway used by immunotherapeutic agents. When the tumor cell is unable to interact with the activated T-cell, the immune system remains active, helping to prevent immunosuppression.

PD-L1 IHC 22C3 pharmDx Detects PD-L1 in Urothelial Carcinoma Specimens

Detection of PD-L1 upregulation in urothelial carcinoma is a biomarker for response to anti-PD-1 therapy. PD-L1 IHC 22C3 pharmDx is the only companion

diagnostic used in the KEYTRUDA to evaluate the relationship between PD-L1 expression and clinical efficacy. KEYTRUDA is a humanized monoclonal PD-1-blocking antibody.

(pembrolizumab) clinical trial (KEYNOTE-052)

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

PD-L1 expressing cell

Inactive cytotoxic T-cell

PD-L1

PD-1

Figure 1: Inactivation of T-cells limits damage to normal tissue.

Tumor cell

Inactive cytotoxic T-cell

PD-L1

PD-1

Figure 2: Inactivation of T-cells reduces tumor cell death and elimination.

Tumor cell

Active cytotoxic T-cell

Anti-PD-1

therapy

Figure 3: Blocking the PD -1/PD- L1 interaction helps to enable active T-cells and tumor cell death and elimination.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

PD-L1 IHC 22C3 pharmDx Overview

What is PD-L1 IHC 22C3 pharmDx?

PD-L1 IHC 22C3 pharmDx is the only companion diagnostic indicated as an aid in identifying patients with urothelial carcinoma for treatment with

KEYTRUDA

PD-L1 IHC 22C3 pharmDx is a qualitative immunohistochemical (IHC) assay intended for use in the detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma tissue samples using Autostainer Link 48.

Components of PD-L1 IHC 22C3 pharmDx

PD-L1 IHC 22C3 pharmDx contains optimized reagents to perform an IHC staining procedure using a linker and a chromogen enhancement reagent (Figure 4). Deparaffinization, rehydration, and target retrieval is performed using a 3-in-1 procedure on PT Link. Following peroxidase block, specimens are incubated with the monoclonal mouse primary antibody to PD-L1 or the Negative Control Reagent. Specimens are then incubated with a Mouse LINKER, followed by incubation with a ready-to-use Visualization Reagent consisting of secondary antibody molecules and horseradish peroxidase molecules coupled to a dextran polymer backbone.

(pembrolizumab).

The enzymatic conversion of the subsequently added chromogen results in precipitation of a visible reaction product at the site of the antigen. The color of the chromogenic reaction is modified by a chromogen enhancement reagent. The specimen may then be counterstained and coverslipped. Results are interpreted using a light microscope of diagnostic quality.

Application of primary antibody. Application of Linker.

Figure 4: PD-L1 IHC 22C3 pharmDx staining procedure.

Kit Configuration

Figure 5: PD-L1 IHC 22C3 pharmDx components.

* Dr. AF Gazdar and Dr. JD Minna at NIH are acknowledged

for their contribution in developing NCI-H226 (ATCC Number: CRL-5826™)

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

PD-L1 IHC 22C3 pharmDx (Code SK006) contains reagents to perform 50 tests in up to 15 individual runs (Figure 5):

EnVision FLEX Target Retrieval Solution, Low pH (50×)

Peroxidase-Blocking

Primary antibody: Monoclonal Mouse Anti-PD-L1, Clone 22C3

Negative Control Reagent

Mouse LINKER

Visualization Reagent-HRP

DAB+ Substrate Buffer

DAB+ Chromogen

DAB Enhancer

PD-L1 IHC 22C3 pharmDx Control Cell Line Slides*

Reagent

EnVision FLEX Wash Buffer, (20×) (Code K8007) and EnVision FLEX Hematoxylin (Code K8008) are required but not included in the kit.

Application of Visualization Reagent. Application of DAB+ Substrate

Chromogen Solution.

Application of DAB Enhancer.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Technical Considerations

Technical problems related to PD-L1 IHC 22C3 pharmDx may arise and can be attributed to two factors: specimen collection and preparation prior to performing the test, and the actual performance of the test itself. Technical problems are generally related to procedural deviations and can be controlled and minimized through training and, where necessary, clarification of the product instructions.

Specimen Preparation

In-house Control Tissue

Specimens must be handled to preserve the tissue for immunohistochemical staining. Determine intact tumor morphology and the presence of sufficient tumor cells for evaluation. Use standard methods of tissue processing for all specimens.

Differences in processing and embedding in the user’s laboratory may produce significant variability in results. Include positive and negative in-house control tissue in each staining run, in addition to the PD-L1 IHC 22C3 pharmDx Control Cell Line Slide.

Select positive and negative control tissue from fresh specimens of the same tumor indication as the patient specimen. Fix, process, and embed the control tissue in the same manner. Control tissues processed differently from the patient specimen validate reagent performance only and do not verify tissue preparation.

The ideal positive control tissue provides a complete dynamic representation of weak-to-moderate staining of tumor cells and tumor-associated mononuclear inflammatory cells (MICs: lymphocytes and macrophages). The negative control tissue should demonstrate no staining in tumor cells and only a few staining immune cells.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Optional Additional In-house Control: Tonsil Tissue

Tissue Processing

Tonsil stained with PD-L1 should be pre-screened to exhibit strong staining in portions of the crypt epithelium and weak-to-moderate staining of the follicular macrophages in the germinal centers. PD-L1 expression of the endothelium, fibroblasts, as well as the surface epithelium should be negative.

Formalin-fixed, paraffin-embedded tissues have been validated for use. Block specimens into a thickness of 3 mm or 4 mm, fix in formalin and dehydrate and clear in a series of alcohols and xylene, followed by infiltration with melted paraffin. The paraffin temperature should not exceed 60 °C. Feasibility studies

onNSCLCtissuesampleswereperformedwithfixationin10%neutralbuffered

formalin for 12–72 hours. Fixation times of 3 hours or less should not be used for PD-L1 assessment. The use of PD-L1 IHC 22C3 pharmDx on decalcified tissues or tissues processed with other fixatives has not been validated and is not recommended.

Cut tissue specimens into sections of 4–5 µm. After sectioning, tissues should be mounted on Dako FLEX IHC microscope slides (Code K8020) or Fisherbrand Superfrost Plus slides, and then placed in a 58 ± 2 °C oven for 1 hour. Store tissue sections in the dark at 2–8 °C (preferred) or at room temperature in the dark up to 25 °C to preserve antigenicity, and stain within the time period given in the IFU for each temperature condition.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

PD-L1 IHC 22C3 pharmDx Staining Procedure

The PD-L1 IHC 22C3 pharmDx reagents and instructions have been designed for optimal performance. Further dilution of the reagents, alteration of incubation times, temperatures, or materials may give erroneous results. All of the required steps and incubation times for staining are pre-programmed in the DakoLink software.

Reagent Storage

Store all components of PD-L1 IHC 22C3 pharmDx, including Control Cell Line Slides, in the dark at 2–8 °C when not in use.

Reagent Preparation

Equilibrate all components to room temperature (20–25 °C) prior to immunostaining. Do not use after the expiration date printed on the outside of the package.

EnVision FLEX Target Retrieval Solution, Low pH

Dilute EnVision FLEX Target Retrieval Solution, Low pH, (50×) 1:50 using distilled or deionized water (reagent-quality water). One 30 mL bottle of concentrate provides 1.5 L of working solution, which is sufficient to fill one PT Link tank. Discard 1× EnVision FLEX Target Retrieval Solution, Low pH after 3 uses or 5 days after dilution.

EnVision FLEX Wash Buffer

Dilute EnVision FLEX Wash Buffer, (20×) 1:20 using distilled or deionized water (reagent-quality water). Store unused 1× buffer at 2–8 °C for no more than one month. Discard if cloudy in appearance.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

DAB+ Substrate-Chromogen Solution

Add 1 drop of DAB+ Chromogen per mL of DAB+ Substrate Buffer and mix. Prepared DAB+ Substrate-Chromogen is stable for 5 days if stored in the dark at 2–8 °C. Mix the DAB+ Substrate-Chromogen Solution thoroughly prior to use. Any precipitate developing in the solution will not affect staining quality.

– If using an entire bottle of DAB+ Substrate Buffer, add 9 drops of DAB+

Chromogen. Although the DAB+ Substrate Buffer label states 7.2 mL, this is the usable volume and does not account for the “dead volume” of DAB+ Substrate Buffer in the bottle

– The color of the DAB+ Chromogen may vary from clear to lavender brown.

This will not affect the performance of the product. Dilute per the guidelines above. Adding excess DAB+ Chromogen to the DAB+ Substrate Buffer results in deterioration of the positive signal

Controls to Assess Staining Quality

The following quality controls should be included in each staining run:

– One PD-L1 IHC 22C3 pharmDx Control Cell Line Slide stained with

the primary antibody

– Positive and negative in-house control tissues stained with the

primary antibody

– Subsequent sections of each patient specimen stained with the

Negative Control Reagent

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Deparaffinization, Rehydration, and Target Retrieval

Use PT Link to perform a Deparaffinization, Rehydration, and Target Retrieval 3-in-1 procedure.

– Set Preheat and Cool to 65 °C, and set Heat to 97 °C for 20 minutes

– Fill PT Link tanks with 1.5 L per tank of 1× EnVision FLEX Target Retrieval

Solution, Low pH working solution to cover the tissue sections

– Preheat the Target Retrieval Solution, Low pH to 65 °C

– Immerse Autostainer racks containing mounted, FFPE tissue sections into

the preheated Target Retrieval Solution, Low pH in PT Link tank. Incubate for 20 minutes at 97 °C

– When incubation has been completed and the temperature has cooled to

65 °C, remove each Autostainer slide rack with slides from the PT Link tank and immediately place the slides into a tank (e.g., PT Link Rinse Station, Code PT109) containing room temperature 1× EnVision FLEX Wash Buffer working solution

– Leave Autostainer rack with slides in room temperature 1× EnVision FLEX

WashBufferfor5minutes

Staining and Counterstaining

– Place the Autostainer rack with slides on the Autostainer Link 48

– Ensure slides remain wet with buffer while loading and prior to initiating the

run. Dried tissue sections may display increased non-specific staining

– Select the PD-L1 IHC 22C3 pharmDx protocol. The instrument performs the

staining and counterstaining procedures by applying the appropriate reagent, monitoring the incubation time, and rinsing slides between reagents

– Counterstain slides using EnVision FLEX Hematoxylin, Code K8008

Mounting

Use non-aqueous permanent mounting media. To minimize fading, store slides in the dark at room temperature (20–25 °C).

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Technical Checklist

Use the checklist below to ensure correct usage of PD-L1 IHC 22C3 pharmDx:

Customer Name/Institution

Name and Title

Autostainer Link 48 Serial Number Software Version

Regular preventive maintenance is performed on the Autostainer Link 48 and PT Link?

PD-L1 IHC 22C3 pharmDx is used before the expiration date printed on the outside of the box?

All PD-L1 IHC 22C3 pharmDx components, including Control Cell Line Slides, are stored in the dark at 2–8 °C?

All PD-L1 IHC 22C3 pharmDx components, including Control Cell Line Slides, are equilibrated to room temperature (20–25 °C) prior to immunostaining?

Yes No

Appropriate positive and negative control tissue from urothelial carcinoma are identified?

Tissues are fixed in neutral buffered formalin?

Tissues are infiltrated with melted paraffin, at or below 60 °C?

Tissue sections of 4–5 µm are mounted on Dako FLEX IHC Microscope Slides or Fisherbrand Superfrost Plus charged slides?

Specimens are oven-dried at 58 ± 2 °C for 1 hour?

Specimens are stained within the time period(s) given in the IFU when stored in the dark at 2–8 °C (preferred) or at room temperature in the dark up to 25 °C?

EnVision FLEX Target Retrieval Solution, Low pH is prepared properly? pH of 1× Target Retrieval Solution must be 6.1 ± 0.2.

EnVision FLEX Wash Buffer is prepared properly?

DAB+ Substrate-Chromogen Solution is prepared properly?

Slides are counterstained with EnVision FLEX Hematoxylin?

The Deparaffinization, Rehydration, and Target Retrieval 3-in-1 procedure is followed using PT Link?

Slides remain wet with buffer while loading and prior to initiating run on Autostainer Link 48?

The PD-L1 IHC 22C3 pharmDx protocol is selected on Autostainer Link 48?

Do you have all the necessary equipment to perform the PD-L1 IHC 22C3 pharmDx according to protocol? If not, specify what is missing in comments below.

Additional observations or comments:

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Slide Evaluation

General Considerations

Specimen Adequacy

PD-L1 IHC 22C3 pharmDx evaluation should be performed by a qualified pathologist using a light microscope of diagnostic quality. Details of the PD-L1 IHC 22C3 pharmDx interpretation guidelines are reviewed on page 30. Before examining the patient specimen for PD-L1 staining, it is important to examine the controls to assess staining quality.

PD-L1 interpretation is best assessed by requesting 3 serial tissue sections (H&E, PD-L1 stain, and NCR stain) so that if the H&E is first assessed and is acceptable, IHC staining of the remaining 2 serial sections is likely to be acceptable.

Each PD-L1 IHC 22C3 pharmDx is configured with Control Cell Line Slides that should be included in each IHC run. Guidelines on interpreting the Control Cell Line Slide are reviewed to the right. In-house control tissue slides should also be assessed with every IHC run.

Confirm the Presence of at Least 100 Viable Tumor Cells

A hematoxylin and eosin (H&E) stained section is recommended for the evaluation of specimen adequacy. PD-L1 IHC 22C3 pharmDx and the H&E staining should be performed on serial sections from the same paraffin block of the specimen.

A minimum of 100 viable tumor cells must be present in the PD-L1 stained slide for the specimen to be considered adequate for PD-L1 evaluation.

Instructions for Patient Specimens With Less Than 100 Viable Tumor Cells

Tissue from a deeper level of the block, or potentially another block, could have a sufficient number of viable tumor cells for PD-L1 IHC 22C3 pharmDx testing.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Evaluating Controls

Figure 6: Each Control Cell Line Slide contains sections of cell pellets with positive and negative PD-L1 expression.

PD-L1 IHC 22C3 pharmDx Control Cell Line Slide

Examine the PD-L1 IHC 22C3 pharmDx Control Cell Line Slide to determine that reagents are functioning properly. Each slide contains sections of cell pellets with positive and negative PD-L1 expression (Figure 6). Assess the percentage of positive cells and the staining intensity. If any staining of the Control Cell Line Slide is not satisfactory, all results with the patient specimens should be considered invalid.

Evaluate the overall staining intensity using the following guide:

0 Negative

1+ Weak intensity

2+ Moderate intensity

3+ Strong intensity

Positive Control Cell Pellet

The following staining is acceptable for the PD-L1 positive cell pellet (Figure 7):

– Cellmembranestainingof≥70%ofcells

– ≥2+averagestainingintensity

– Non-specific staining < 1+ intensity

Figure 7: Positive cell pellet with acceptable staining of PD- L1 IHC 22C3 pharmDx Control Cell Line Slide (20× magnification).

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Negative Control Cell Pellet

For the PD-L1 negative cell pellet, the following staining is acceptable (Figure 8):

– The majority of cells should demonstrate no staining. Note: The presence of

10 or fewer cells with distinct cell membrane staining is acceptable

– Any background staining is less than 1+ staining intensity

Figure 8: Negative cell pellet with no staining of PD -L1 IHC 22C3 pharmDx Control Cell Line Slide (20× magnification).

Positive and Negative In-house Control Tissue (Urothelial Carcinoma)

Examine the positive in-house urothelial carcinoma control tissue to determine that the tissues are correctly prepared and reagents are functioning properly. The ideal positive control tissue provides a complete dynamic representation of weak-to-moderate staining of tumor cells and tumor-associated mononuclear inflammatory cells (MICs) (Figure 9). If staining of positive in-house control tissue is not satisfactory, all results with the patient specimen should be considered invalid.

Figure 9: Ideal positive in-house control tissue (20× magnification).

The ideal negative control tissue should demonstrate no staining on tumor cells and immune cells (Figure 10). However, because prevalence of PD-L1 expression on immune cells is high, staining immune cells are acceptable. Examine the negative in-house control tissue to determine the expected staining. The variety of different cell types present in most tissue sections offers internal negative control sites; this should be verified by the user.

If unwanted staining occurs in the in-house control tissues, results with the patient specimen should be considered invalid.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Figure 10: Ideal negative in -house control tissue demonstrating PD -L1 positive expression of CPS < 1 (20× magnification).

Optional Control Tissue

In addition to the Control Cell Line Slide and in-house control tissues, FFPE tonsil may also be used as an optional control specimen. Tonsil stained with PD-L1 should exhibit strong membrane staining in portions of the crypt epithelium and weak-to-moderate membrane staining of the follicular macrophages in the germinal centers (Figure 11).

PD-L1 expression of the endothelium, fibroblasts, and the surface epithelium should be absent.

Figure 11: Tonsil stained with PD -L1 primary antibody exhibiting strong membrane staining in portions of the crypt epithelium (A) and weak-to-moderate membrane staining of follicular macrophages in the germinal centers (B) (10× magnification).

Do not use in-house control tissue as an aid in interpretation of patient results.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Negative Control Reagent (NCR)

Examine the slides stained with the NCR to identify non-specific background staining that may interfere with PD-L1 staining interpretation, making the specimen non-evaluable. Satisfactory performance is indicated by the absence of staining (Figure 12).

Figure 12: Ideal negative in-house control tissue stained with Negative Control Reagent showing no specific staining (20× magnification).

Negative Control Reagent stained slides indicate non-specific background staining and allow for better interpretation of patient specimens stained with the primary antibody.

Slide Evaluation Flowchart

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Urothelial Carcinoma

Tissue Block

3 serial sections are

cut/prepared

One section is stained with

H&E (H&E Patient Specimen)

Is H&E slide adequate?

(intact, well-preserved,

urothelial carcinoma)

Yes

Control Cell Line

Slide adequate?

Yes

Positive control

tissue adequate?

Yes

Negative control

tissue adequate?

Sections of 4–5 µm thickness

are mounted on glass

microscope slides

Repeat

staining run

Repeat

staining run

Repeat

staining run

Figure 13: Recommended order of slide evaluation.

Yes

Patient specimen stained with

Negative Control Reagent

adequate?

Yes

Patient specimen stained with

primary antibody exhibiting

≥ 100 viable tumor cells?

Scored by

Pathologist

Repeat staining run

with a deeper cut in

the block or a new

patient specimen

Provide case report

Repeat

staining run

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