Agilent PD-L1 IHC 22C3 pharmDx Interpretation Manual

PD-L1 IHC 22C3 pharmDx Interpretation Manual – Non-small Cell Lung Cancer (NSCLC)

CE-IVD–marked for in vitro diagnostic use

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

For countries outside of the European Union, 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 – NSCLC

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 Clinical Interpretation Guidelines 18

General Considerations 18 Specimen Adequacy 18 Evaluating Controls 19

Slide Evaluation Flowchart 23 Evaluate Staining and Determine Tumor Proportion Score 24

Definition of Tumor Proportion Score (TPS) 24 Evaluation of PD-L1 Staining 24 Guidelines and Methods to Determine Tumor Proportion Score 25

Scoring Guidelines 26 Suggested Methods for Determining TPS 27 Identifying Patients With NSCLC for Treatment 29 Reporting Results 31 PD-L1 Staining Characteristics 32

Key Considerations in Scoring PD-L1 IHC 22C3 pharmDx 32

Stained Specimens Image Guide for Interpretation of PD-L1 IHC 22C3 pharmDx 33 Staining in NSCLC

PD-L1 IHC 22C3 pharmDx TPS < 1% Case Examples 39 PD-L1 IHC 22C3 pharmDx TPS 0–10% Case Examples 43 PD-L1 IHC 22C3 pharmDx TPS 1–49% Case Examples 48

PD-L1 IHC 22C3 pharmDx TPS ≥ 50% Case Examples 52 PD-L1 IHC 22C3 pharmDx TPS 40–60% Case Examples 59

Artifacts 64 Troubleshooting Guide 69 Clinical Performance Evaluation 70 References 80

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

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), urothelial carcinoma, head and neck squamous cell carcinoma (HNSCC), and melanoma tissues using EnVision FLEX visualization system on Autostainer Link 48.

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.

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.

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

PD-L1 protein expression in HNSCC is determined by using CPS and/or TPS.

Companion diagnostic indications

Tumor Indication

NSCLC TPS ≥ 1%

Urothelial Carcinoma

PD-L1 Expression Level Intended Use

PD-L1 IHC 22C3 pharmDx is indicated as an

TPS ≥ 50%

aid in identifying NSCLC patients for treatment with KEYTRUDA

(pembrolizumab).*

CPS ≥ 10 PD-L1 IHC 22C3 pharmDx is indicated as

an aid in identifying urothelial carcinoma patients for treatment with KEYTRUDA (pembrolizumab).*

HNSCC CPS ≥ 1

TPS ≥ 50%

PD-L1 IHC 22C3 pharmDx is indicated as an aid in identifying HNSCC patients for treatment with KEYTRUDA

(pembrolizumab).*

* See the KEYTRUDA® product label for PD-L1 expression cutoff values and

specific clinical circumstances guiding therapy.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

Introduction

PD-L1 IHC 22C3 pharmDx is the only clinical trial-proven companion diagnostic CE-IVD–marked as an aid in identifying patients with NSCLC for treatment

with KEYTRUDA 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 non-small cell lung cancer (NSCLC) 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 highquality staining and diagnostic assessment. To help familiarize you with the requirements for scoring NSCLC 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 NSCLC specimens stained with PD-L1 IHC 22C3 pharmDx can help individual labs achieve reproducible and reliable results.

(pembrolizumab) monotherapy. This Interpretation Manual

PD-L1 IHC 22C3 pharmDx is considered a qualitative immunohistochemical assay. 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.

NSCLC tissue specimens that are tested for PD-L1 expression are scored and divided into expression levels based on a Tumor Proportion Score (TPS):

– TPS < 1%

– TPS ≥ 1%

– TPS ≥ 50%

PD-L1 expression levels are used to inform patient eligibility for treatment with KEYTRUDA monotherapy. 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 – NSCLC

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 31.

Photomicrographs

The included photomicrographs are of NSCLC unless otherwise noted.

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

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

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 NSCLC Specimens

PD-L1 upregulation in NSCLC is a biomarker for response to anti-PD-1 therapy. PD-L1 IHC 22C3 pharmDx was the only companion diagnostic used in the

KEYTRUDA and KEYNOTE-042) to evaluate the relationship between PD-L1 expression and clinical efficacy. KEYTRUDA is a humanized monoclonal PD-1-blocking antibody.

(pembrolizumab) clinical trials (KEYNOTE-010, KEYNOTE-024,

The PD-1/PD-L1 Pathway

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

PD-L1 expressing cell

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

Tumor cell

Inactive cytotoxic T-cell

PD-L1

PD-1

Inactive cytotoxic T-cell

PD-L1

PD-1

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

Tumor 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.

Active cytotoxic T-cell

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

PD-L1 IHC 22C3 pharmDx Overview

What is PD-L1 IHC 22C3 pharmDx?

PD-L1 IHC 22C3 pharmDx is the only clinical trial-proven companion diagnostic indicated as an aid in identifying patients with NSCLC for treatment with

KEYTRUDA qualitative immunohistochemical (IHC) assay intended for use in the detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) NSCLC tissue samples using EnVision FLEX visualization system on 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) monotherapy. PD-L1 IHC 22C3 pharmDx is a

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.

Application of Primary Antibody. Application of Linker.

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

Kit Configuration (SK006)

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

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™)

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*

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

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

Reagent

Application of Visualization Reagent. Application of DAB+ Substrate-

Chromogen Solution.

Application of DAB Enhancer.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

Technical Considerations

Technical problems related to PD-L1 IHC 22C3 pharmDx may arise and can be attributed to two areas: 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. The ideal negative control tissue gives no staining on tumor cells but contains tumor-associated macrophages/immune cells which may express PD-L1 and offer an internal positive control.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

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 up to 25 °C to preserve antigenicity, and stain within 6 months of sectioning.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

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 1 month. Discard if cloudy in appearance.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

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 – NSCLC

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 – NSCLC

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 NSCLC 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 slides?

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

Specimens are stained within 6 months of sectioning when stored in the dark at 2–8 °C (preferred) or at room temperature 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 – NSCLC

Clinical Interpretation Guidelines

General Considerations

Specimen Adequacy

PD-L1 IHC 22C3 pharmDx evaluation should be performed by a qualified pathologist using a light microscope. Details of the PD-L1 IHC 22C3 pharmDx interpretation guidelines are reviewed on page 26. Before examining the patient specimen for PD-L1 staining, it is important to examine the controls to assess staining quality.

PD-L1 expression 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 – NSCLC

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. Do not use the Control Cell Line Slide as an aid in interpretation of patient results.

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 – NSCLC

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

– Non-specific staining < 1+ intensity

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

Do not use the Control Cell Line Slide as an aid in interpretation of patient results.

Positive and Negative In-house Control Tissue (NSCLC)

Examine the positive in-house NSCLC 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 tumor cell membrane staining (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 (10× magnification).

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

The ideal NSCLC negative control tissue demonstrates no staining on tumor cells but contains tumor-associated macrophages/immune cells that express PD-L1 and offer an internal positive control (Figure 10). 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.

Figure 10: Ideal negative in-house control tissue demonstrating lack of staining of tumor cells (10× 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 – NSCLC

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).

Examine the patient specimens stained with the NCR to determine if there is any non-specific staining that may interfere with interpreting the PD-L1 stained slide.

Figure 12: Ideal negative in-house control tissue stained with NCR (20× magnification).

NCR-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 – NSCLC

Tissue Block

3 serial sections are

cut/prepared

One section is stained with

H&E (H&E Patient Specimen)

Is H&E slide acceptable?

(≥ 100 viable tumor cells)

Repeat staining run with a

deeper cut in the block

or a new patient specimen

Yes

Control Cell Line

Slide acceptable?

Yes

Positive control

tissue acceptable?

Yes

Negative control

tissue acceptable?

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 acceptable?

Yes

Patient specimen stained with

primary antibody exhibiting

≥ 100 viable tumor cells?

Scored by

Pathologist

Provide case report

Repeat

staining run

Repeat

staining run

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

Evaluate Staining and Determine Tumor Proportion Score

Definition of Tumor Proportion Score (TPS)

The Tumor Proportion Score is the percentage of viable tumor cells showing

partial or complete membrane staining at any intensity (≥ 1+) relative to all viable

tumor cells present in the sample.

TPS is defined accordingly:

TPS (%) =

# PD-L1 staining cells (tumor cells)

× 100

Total # of viable tumor cells

Table 1: TPS Numerator Inclusion/Exclusion Criteria for NSCLC

Tissue Elements Included in TPS Scoring for NSCLC Excluded from TPS Scoring for NSCLC

Tumor Cells Convincing partial or complete cell

membrane staining (at any intensity) of viable tumor cells

Immune Cells Not included Exclude any staining of immune cells,

Other Cells Not included Exclude any staining of:

Exclude any cytoplasmic staining

such as:

– Mononuclear inflammatory cells

(large lymphocytes, monocytes, pulmonary macrophages)

– Plasma cells

– Neutrophils

–  Normal cells adjacent to tumor cells

–  Stromal cells (fibroblasts)

–  Necrotic cells and/or cellular debris

–  Anthracotic pigment

Evaluation of PD-L1 Staining

Score partial or complete cell membrane staining (≥ 1+) of tumor cells

that is perceived distinct from cytoplasmic staining. Cytoplasmic staining should be considered non-specific staining and is excluded in the assessment of staining intensity.

Score only viable tumor cells. Exclude any staining of immune cells, such as mononuclear inflammatory cells (large lymphocytes, monocytes, pulmonary macrophages), plasma cells, and neutrophils. Exclude any

staining of normal cells adjacent to tumor cells, stromal cells (fibroblasts),

necrotic cells and/or cellular debris, as well as anthracotic pigment.

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

Guidelines and Methods to Determine Tumor Proportion Score

– At low magnification, examine all well-preserved tumor areas. Evaluate

overall areas of PD-L1 staining tumor cells, keeping in mind that partial

membrane staining or ≥ 1+ membrane staining may be difficult to see at low

magnification. Ensure there are at least 100 viable tumor cells in the sample

– At higher magnifications, including 10×, 20×, and 40×, observe all tumor areas

with and without cell membrane staining

– At this stage of working with multiple magnifications, primary analysis involves:

Distinguishing tumor cells from tumor-associated immune cells

Determining PD-L1 staining and non-staining tumor areas

Determining partial and complete membrane staining (≥ 1+) of tumor cells

– Calculate the Tumor Proportion Score by evaluating the percentage of PD-L1

staining tumor cells relative to all viable tumor cells present in the specimen

Note: Carefully consider the overall tumor area without any perceptible and

convincing cell membrane staining

Make Sure to Exclude Immune Cells and Necrotic Tissue From Scoring

The following considerations can help distinguish tumor cells from immune cells:

– Immune cells may have smaller nuclei than tumor cells

– Macrophages may contain pigmented particles in their cytoplasm

– Macrophages may have a scattered distribution. Pulmonary macrophages

are present in the alveolar space

PD-L1 IHC 22C3 pharmDx Interpretation Manual – NSCLC

Scoring Guidelines

The TPS determines the PD-L1 expression levels of the specimen. See the table below for scoring guideline examples.

Table 2: TPS and PD-L1 Expression Levels

TPS Expression Level Image (20× magnification)

< 1% No PD-L1 Expression

≥ 1% PD-L1 Expression

≥ 50% High PD-L1 Expression

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