Specification Sheet

Specification Sheet
PD-L1 IHC 22C3 pharmDx
SK006
50 tests for use with Autostainer Link 48
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) tissue using
EnVision FLEX visualization system on Autostainer Link 48. PD-L1 protein expression is determined by using Tumor Proportion Score
(TPS), which is the percentage of viable tumor cells showing partial or complete membrane staining. The specimen should be
considered PD-L1 positive if TPS ≥ 50% of the viable tumor cells exhibit membrane staining at any intensity.
PD-L1 IHC 22C3 pharmDx is indicated as an aid in identifying NSCLC patients for treatment with KEYTRUDA® (pembrolizumab).
Summary and explanation
Binding of the PD-1 ligands, PD-L1 and PD-L2, to the PD-1 receptor found on T cells, inhibits T cell proliferation and cytokine
production. Up-regulation of PD-1 ligands occurs in some tumors and signaling through this pathway can contribute to inhibition of
active T-cell immune surveillance of tumors. KEYTRUDA® (pembrolizumab) is a humanized monoclonal antibody that binds to the PD-1
receptor and blocks its interaction with PD-L1 and PD-L2, releasing PD-1 pathway-mediated inhibition of the immune response,
including the anti-tumor immune response. In syngeneic mouse tumor models, blocking PD-1 activity resulted in decreased tumor
growth.
Principle of procedure
PD-L1 IHC 22C3 pharmDx contains optimized reagents and protocol required to complete an IHC staining procedure of FFPE
specimens using Autostainer Link 48. Following incubation with the primary monoclonal antibody to PD-L1 or the Negative Control
Reagent (NCR), specimens are incubated with a Linker antibody specific to the host species of the primary antibody, and then are
incubated with a ready-to-use visualization reagent consisting of secondary antibody molecules and horseradish peroxidase molecules
coupled to a dextran polymer backbone. The enzymatic conversion of the subsequently added chromogen results in precipitation of a
visible reaction product at the site of 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.
Materials provided
PD-L1 IHC 22C3 pharmDx (Code SK006) is for automated staining using Autostainer Link 48.
Each kit includes 19.5 mL of PD-L1 primary antibody (approximately 3µg/mL protein concentration) and contains the reagents
necessary to perform 50 tests in up to 15 individual runs. The materials listed below are sufficient for 50 tests (50 slides incubated with
Primary Antibody to PD-L1 and 50 slides incubated with the corresponding Negative Control Reagent, 100 slides in total). The number
of tests is based on the use of 2 x 150 µL per slide of each reagent except DAB+ and Target Retrieval Solution.
The kit provides materials sufficient for a maximum of 15 individual staining runs.
Quantity
1 x 34.5 mL
Description
Peroxidase-Blocking Reagent
PEROXIDASE-BLOCKING
REAGENT
Buffered solution containing hydrogen peroxide, detergent and 0.015 mol/L sodium azide.
1 x 19.5 mL
Primary Antibody: Monoclonal Mouse Anti-PD-L1, Clone 22C3
MONOCLONAL MOUSE
ANTI-PD-L1
CLONE 22C3
Monoclonal mouse anti-PD-L1 in a buffered solution, containing stabilizing protein, and 0.015 mol/L sodium azide.
1 x 15 mL
Negative Control Reagent
NEGATIVE CONTROL
REAGENT
Monoclonal mouse control IgG antibody in a buffered solution, containing stabilizing protein, and 0.015 mol/L sodium
azide.
1 x 34.5 mL
Mouse LINKER
LINKER,
ANTI-MOUSE
Rabbit secondary antibody against mouse immunoglobulins in a buffered solution containing stabilizing protein and
0.015 mol/L sodium azide.
1 x 34.5 mL
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VISUALIZATION
REAGENT-HRP
Dextran coupled with peroxidase molecules and goat secondary antibody molecules against rabbit and mouse
immunoglobulins in a buffered solution containing stabilizing protein and an antimicrobial agent.
15 x 7.2 mL
DAB+ Substrate Buffer
DAB+
SUBSTRATE BUFFER
Buffered solution, containing hydrogen peroxide and an antimicrobial agent.
1 x 5 mL
DAB+ Chromogen
DAB+ CHROMOGEN
3,3’-diaminobenzidine tetrahydrochloride in organic solvent.
1 x 34.5 mL
DAB Enhancer
DAB ENHANCER
Cupric sulfate in water.
6 x 30 mL
EnVision FLEX Target Retrieval Solution, Low pH, 50x
EnVision™ FLEX
TARGET RETRIEVAL SOLUTION
LOW pH (50X)
Buffered solution, pH 6.1, containing detergent and an antimicrobial agent.
15 slides
PD-L1 IHC22C3 pharmDx Control Slides
CONTROL SLIDES
XXXXX
PD-L1 IHC 22C3
pharmDx
Each slide contains sections of two pelleted, formalin-fixed paraffin-embedded cell lines: NCI-H226 with moderate PD-L1
protein expression and MCF-7 with negative PD-L1 protein expression.
MCF-7: PD-L1 negative
NCI-H226: PD-L1 positive
Note: All reagents included are formulated specifically for use with this kit. In order for the test to perform as specified, no substitutions,
other than EnVision FLEX Target Retrieval Solution, Low pH, 50x (Code K8005) can be made. PD-L1IHC 22C3 pharmDx has been
tailored for use with Autostainer Link 48. Please refer to the User Guides for your Autostainer Link 48 and PT Link for further
information
Materials required, but not supplied
PT Link Pre-treatment Module (Code PT100)
Autostainer Link 48 (Code AS480)
EnVision FLEX Wash Buffer, 20x (Code K8007)
Hematoxylin (Code K8008)
Distilled or deionized water (reagent-quality water)
Timer
Positive and negative tissues to use as process controls (see Quality control section)
Microscope slides: Dako FLEX IHC Microscope Slides (Code K8020) or Fisherbrand Superfrost Plus charged slides
Coverslips
Permanent mounting medium and ancillary reagents required for mounting coverslips
Light microscope (4x–40x objective magnification)
Precautions
1. For in vitro diagnostic use.
2. For professional users.
3. This product contains sodium azide (NaN3), a chemical highly toxic in pure form. At product concentrations, though not classified as
hazardous, NaN3 may react with lead and copper plumbing to form highly explosive build-ups of metal azides. Upon disposal, flush
with large volumes of water to prevent metal azide build-up in plumbing (1).
4. Primary Antibody, Negative Control Reagent, Linker, and Visualization Reagent contain material of animal origin.
5. Specimens, before and after fixation, and all materials exposed to them, should be handled as if capable of transmitting infection,
and disposed of with proper precautions (2).
6. Incubation times, temperatures, or methods other than those specified may give erroneous results.
7. Reagents have been optimally diluted. Further dilution may result in loss of antigen staining.
8. The Visualization Reagent, Liquid DAB+ chromogen and prepared DAB+ Substrate-Chromogen solution may be affected adversely
if exposed to excessive light levels. Do not store system components or perform staining in strong light, such as direct sunlight.
9. Paraffin residuals may lead to false negative results.
10. Use of reagent volumes other than recommended may result in loss of visible PD-L1 immunoreactivity.
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11. Results from a small study, showed a similar dynamic range of PD-L1 expression in primary and metastatic NSCLC specimen pairs.
It is possible there may be differences in PD-L1 expression in primary tumors versus metastatic sites in the same patient.
12. Large tissue sections may require 3x150 µl of reagent.
13. As a general rule, persons under 18 years of age are not allowed to work with this product. Users must be carefully instructed in the
proper work procedures, the dangerous properties of the product and the necessary safety instructions. Please refer to Safety Data
Sheet (SDS) for additional information.
14. Wear appropriate Personal Protective Equipment to avoid contact with eyes and skin.
15. Unused solution should be disposed of according to local, State and Federal regulations.
16. Safety Data Sheet available for professional users on request.
Danger
DAB+ Chromogen: 1–5% biphenyl-3,3',4,4'-tetrayltetraammonium tetrachloride
H350
May cause cancer.
H341
Suspected of causing genetic defects.
P201
Obtain special instructions before use.
P202
Do not handle until all safety precautions have been read and understood.
P280
Wear protective gloves. Wear eye or face protection. Wear protective clothing.
P308 + P313
IF exposed or concerned: Get medical attention.
P405
Store locked up.
P501
Dispose of contents and container in accordance with all local, regional, national and international regulations.
Warning
EnVision FLEX Target Retrieval Solution, Low pH (50x): 1-5% Citric acid
H319
Causes serious eye irritation.
H411
Toxic to aquatic life with long lasting effects.
P280
Wear eye or face protection.
P273
Avoid release to the environment.
P264
Wash hands thoroughly after handling.
P305 + P351 +
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do.
P338
Continue rinsing.
P337 + P313
If eye irritation persists: Get medical attention.
P501
Dispose of contents and container in accordance with all local, regional, national and international regulations.
Storage
Store all components of PD-L1 IHC 22C3 pharmDx, including Control Slides, in the dark at 2-8 °C when no t in use on Autostainer Link
48.
Do not use the kit after the expiration date printed on the outside of the kit box. If reagents are stored under any conditions other
than those specified in this package insert, they must be validated by the user.
There are no obvious signs to indicate instability of this product, therefore, positive and negative controls should be run simultaneously
with patient specimens.
Specimen preparation
Specimens must be handled to preserve the tissue for IHC staining. Standard methods of tissue processing should be used for all
specimens.
Paraffin-embedded sections
Formalin-fixed, paraffin-embedded tissues are suitable for use. Alternative fixatives have not been validated and may give erroneous
results. Fixation time for 12-72 hours in 10% neutral buffered formalin (NBF) is recommended, however, a study with limited samples
showed fixation times of 4-168 hours in 10% NBF did not systematically alter PD-L1 detection. Fixation times of ≤3 hours may result in
variable PD-L1 detection. Specimens should be blocked into a thickness of 3 or 4 mm, fixed in formalin and dehydrated and cleared in a
series of alcohols and xylene, followed by infiltration with melted paraffin. The paraffin temperature should not exceed 60 °C. FFPE
tissue blocks which are 5 years or older may result in a loss of PD-L1 immunoreactivity.
Tissue specimens should be cut into sections of 4-5 µm. After sectioning, tissues should be mounted on Fisherbrand Superfrost Plus
slides, or Dako FLEX IHC microscope slides (Code K8020). To preserve antigenicity, tissue sections, once mounted on slides, should
be held in the dark at 2-8 °C and stained within 6 months of sectioning.
The use of PD-L1 IHC 22C3 pharmDx, on decalcified tissues has not been validated and is not recommended.
Reagent preparation
The following reagents must be prepared prior to staining:
EnVision FLEX Target Retrieval Solution, Low pH, 50x
Prepare a sufficient quantity of 1x Target Retrieval Solution, Low pH by diluting Target Retrieval Solution, Low pH, 50x 1:50 using
distilled or deionized water (reagent-quality water); the pH of 1x Target Retrieval Solution must be 6.1 ± 0.2. 1x Target Retrieval
Solution pH below 5.9 may give erroneous results. One 30 mL bottle of Target Retrieval Solution, Low pH, 50x, diluted 1:50 will provide
1.5 L of 1x reagent, sufficient to fill one PT Link tank which will treat up to 24 slides per use. Discard 1x Target Retrieval Solution after
three uses and do not use after 5 days following dilution.
Additional EnVision FLEX Target Retrieval Solution, Low pH, 50x, if required, is available as Code K8005.
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EnVision FLEX Wash Buffer, 20x
Prepare a sufficient quantity of Wash Buffer by diluting Wash Buffer 20x 1:20 using distilled or deionized water (reagent-quality water)
for the wash steps. Store unused 1x solution at 2-8 ºC for no more than one month. Discard buffer if cloudy in appearance. Refer to the
User Guide for your Autostainer Link 48 for further information.
EnVision FLEX Wash Buffer, 20x is available as Code K8007.
DAB+ Substrate-Chromogen Solution
This solution should be mixed thoroughly prior to use. Any precipitate developing in the solution does not affect staining quality.
To prepare DAB+ Substrate-Chromogen Solution, add 1 drop of Liquid DAB+ Chromogen per mL of DAB+ Substrate Buffer and mix.
Prepared Substrate-Chromogen is stable for 5 days if stored in the dark at 2-8 °C.
Important Notes:
•
If using an entire bottle of DAB+ Substrate Buffer, add 9 drops of DAB+ chromogen. Although the label states 7.2 mL,
this is the useable volume and does not account for the “dead volume” in the bottle.
•
The color of the Liquid DAB+ Chromogen in the bottle may vary from clear to lavender-brown. This will not affect the
performance of this product. Dilute per the guidelines above. Addition of excess Liquid DAB+ Chromogen to the DAB+
Substrate Buffer will result in deterioration of the positive signal.
Staining procedure on the Autostainer Link solution
Procedural Notes
The user should read these instructions carefully and become familiar with all components and instrumentation prior to use (see
Precautions).
All reagents should be equilibrated to room temperature (20-25 °C) prior to immunostaining. Likewise, all incubations should be
performed at room temperature.
Do not allow tissue sections to dry during the staining procedure. Dried tissue sections may display increased nonspecific staining.
All of the required steps and incubation times for staining are preprogrammed in the Dako Link software. Please refer to the User
Guides for Autostainer Link 48 and PT Link for further information on programming protocols and loading slides and reagents.
Note: The reagents and instructions supplied in this system have been designed for optimal performance when used with the
recommended reagents and materials. Further dilution of the reagents or alteration of incubation times or temperatures may give
erroneous or discordant results.
Staining Protocol
Please select the PD-L1 IHC 22C3 pharmDx staining protocol from the options in the Dako Link drop down menu.
All of the required steps and incubation times for staining are preprogrammed in the Autostainer Link 48. If the appropriate PD-L1 IHC
22C3 pharmDx protocols are not on your server please contact your local Technical Service Representative to obtain the protocols.
Step 1: Deparaffinization, Rehydration and Target Retrieval (3-in-1) Procedure
For details, please refer to the PT Link User Guide.
Set PT Link (Code PT100) Preheat and Cool to 65 °C. Set Heat to 97 °C for 20 minutes.
►Fill PT Link tanks with 1.5 L per tank of Target Retrieval Solution, Low pH, 1x working solution to cover the tissue sections.
►Preheat the Target Retrieval Solution to 65 °C.
►Immerse Autostainer racks containing mounted, FFPE tissue sections into the pre-heated Target Retrieval Solution, Low pH, (1x
working solution) in PT Link tank. Incubate for 20 minutes at 97 °C.
►When target retrieval incubation has been completed and the temperature has cooled to 65 °C, remove ea ch Autostainer slide rack
with the slides from the PT Link tank and immediately place the Autostainer rack with slides into a tank (e.g., PT Link Rinse Station,
Code PT109) containing diluted, room temperature Wash Buffer (Code K8007).
►Incubate slides in diluted, room temperature Wash Buffer for 5 minutes.
Step 2: Staining Procedure
After deparaffinization, rehydration and target retrieval (3-in-1) procedure, the Autostainer racks with slides are placed on Autostainer
Link 48. The instrument will perform the staining process by applying the appropriate reagent, monitoring the incubation time and
rinsing slides between reagents. The reagent times are preprogrammed in the Dako Link software.
Step 3: Counterstain
Slides should be counterstained for 5 minutes with Hematoxylin (Link) (Code K8008).
preprogrammed in the protocol.
The Hematoxylin incubation time is
Step 4: Mounting
Non-aqueous, permanent mounting media is required.
Note: Some fading of stained slides may occur, depending on several factors including, but not limited to, counterstaining, mounting
materials and methods, and slide storage conditions. To minimize fading, store slides in the dark at room temperature (20-25 °C).
Quality Control
Reagents in PD-L1 IHC 22C3 pharmDx have been quality controlled by immunohistochemistry using the target retrieval and staining
procedures outlined above.
Deviations in the recommended procedures for tissue fixation, processing and embedding in the user’s laboratory may produce
significant variability in results. In-house controls should be included in each staining run.
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Differences in tissue fixation, processing and embedding in the user’s laboratory may produce significant variability in results,
necessitating regular performance of in-house controls in addition to the Control Slides supplied in the kit (3). In the USA, consult the
quality control guidelines of the College of American Pathologists (CAP) Certification Program for Immunohistochemistry, see also CLSI
Quality Assurance for Immunocytochemistry, Approved Guideline (4) for additional information.
Table 1: The Purpose of Daily Quality Control
Tissue
Reagents
Purpose
Positive Control: Tissue or cells containing target
antigen to be detected. The ideal control is
weakly positive staining tissue, which may be
more sensitive in detecting reagent degradation.
Primary Antibody & Detection
System
Controls all steps of the analysis. Validates
reagents and procedures used for PD-L1 staining.
Negative Control: Tissues or cells expected to be
negative (could be located in patient tissue or
positive control tissue).
Primary Antibody & Detection
System
Detection of unintended antibody cross-reactivity
to cells/cellular components.
Control Slide supplied by Dako
Primary Antibody & Detection
System
Controls staining procedure only
Patient tissue slide
*Negative Control Reagent &
Detection of non-specific background staining.
same Detection System as used
with the Primary Antibody
*From the same species as the primary antibody, but not directed against the same target antigen. To detect non-specific antibody
binding, e.g. binding of Fc portion of antibody by the tissue.
Control Cell Line Slides (provided)
Each slide contains sections of two pelleted, formalin-fixed paraffin-embedded cell lines: NCI-H226 with moderate PD-L1 protein
expression and MCF-7 with negative PD-L1 protein expression. One control slide should be stained with the Primary Antibody to PD-L1
in each staining run. The evaluation of the Control Slide cell lines supplied in the kit indicates the validity of the staining run. They should
not be used as an aid in interpretation of patient results.
Positive Control Tissue
Controls should be fresh biopsy/surgical specimens of the same tumor indication as the patient specimen, fixed, processed and
embedded as soon as possible in the same manner as the patient sample(s). Positive tissue controls are indicative of correctly prepared
tissues and proper staining techniques. One positive tissue control for each set of test conditions should be included in each staining
run.
The tissues selected for use as the positive tissue controls should give weak to moderate positive staining so they can detect subtle
changes in assay sensitivity. Specimens processed differently from the patient sample(s) validate reagent performance only and do not
verify tissue preparation.
Known positive tissue controls should only be utilized for monitoring the correct performance of processed tissues and test reagents,
NOT as an aid in formulating a specific diagnosis of patient samples. If the positive tissue controls fail to demonstrate appropriate
positive staining, results with the test specimens should be considered invalid.
Negative Control Tissue
Use a negative control tissue (known to be PD-L1 negative) of the same tumor indication as the patient specimen, fixed processed and
embedded in a manner similar to the patient sample(s) with each staining run to verify the specificity of the primary antibody and to
provide an indication of non-specific background 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 specific staining occurs in the negative control tissue, results with the patient specimens should be considered invalid.
Tonsil Control Tissue (optional)
Use human tonsil tissue fixed, processed and embedded in a manner similar to the patient sample(s) as an additional control material to
verify sensitivity, specificity and nonspecific background staining of the assay.
Strong positive staining should be detected in portions of the crypt epithelium and weak to moderate staining of the follicular
macrophages in the germinal centers. Negative staining should be observed in endothelium, fibroblasts as well as surface epithelium.
Negative Control Reagent
Use the supplied Negative Control Reagent in place of the primary antibody on a sequential section of each patient specimen to
evaluate non-specific staining and allow better interpretation of specific staining at the antigen site. The incubation period for the
Negative Control Reagent should be equivalent to that of the primary antibody.
Assay verification
Prior to initial use of a staining system in a diagnostic procedure, the user should verify the assay’s performance by testing it on a series
of in-house tissues with known IHC performance characteristics representing known positive and negative tissues. Refer to the quality
control procedures previously outlined in this section of the product insert and, in the US, to the quality control requirements of the CAP
Certification Program for Immunohistochemistry and/or CLSI Quality Assurance for Immunocytochemistry, Approved Guideline (4)
These quality control procedures should be repeated for each new antibody lot, or whenever there is a change in assay parameters.
Scoring Interpretation - NSCLC
All viable tumor cells on the entire slide must be evaluated and included in the PD-L1 scoring assessment. A minimum of 100 viable
tumor cells must be present for the specimen to be considered adequate for PD-L1 evaluation.
To successfully score PD-L1 IHC 22C3 pharmDx stained specimens, it is critical that the appropriate cells are evaluated, that the proper
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cellular localization is identified and that the staining intensity is properly interpreted. Slide evaluation should be performed by a
pathologist using a light microscope. For evaluation of the immunocytochemical staining and scoring, an objective of 10-40x
magnification is appropriate. Any perceptible membrane staining of tumor cells should be included in the scoring.
Score partial or complete cell membrane staining (≥1+) that is perceived distinct from cytoplasmic staining. Cytoplasmic staining should
be considered non-specific staining and is excluded in the assessment of staining intensity. Normal cells and tumor associated immune
cells such as infiltrating lymphocytes or macrophages should not be included in the scoring for the determination of PD-L1 positivity.
Tumor specimens stained with the NCR must have 0 specific staining and <1+ background staining.
Tumor Proportion Score (TPS) is the percentage of viable tumor cells showing partial or complete membrane staining (≥1+). The
specimen should be considered PD-L1 positive if TPS ≥ 50% of the viable tumor cells exhibit membrane staining at any intensity (i.e.
≥1+).
For each staining run, slides should be examined in the order presented in Table 2 to determine the validity of the staining run and
enable assessment of the staining of the sample tissue.
Refer to PD-L1 IHC 22C3 pharmDx Interpretation Manual for additional guidance.
Table 2. Recommended order of slide evaluation
Recommended order
Rationale
of slide interpretation
1. Control Cell Line Slide The Control Cell Line Slide stained with the PD-L1 primary antibody from PD-L1 IHC 22C3 pharmDx should
containing the PD-L1be examined first to determine that all reagents are functioning properly. The presence of a brown (3,3’positive and PD-L1diaminobenzidine, DAB) reaction product on the cell membrane is indicative of positive reactivity.
NCI-H226 (PD-L1-positive control cell line) acceptance criteria:
negative cell lines
•
Cell membrane staining of ≥70% of cells at ≥2+ average staining intensity.
•
Non-specific staining <1+ intensity.
MCF-7 (PD-L1-negative control cell line) acceptance criteria:
•
No specific staining.
•
Non-specific staining <1+ intensity.
Note that staining of a few cells in the MCF-7 cell pellet may occasionally be observed. The following
acceptance criteria are applicable: the presence of ≤10 total cells with distinct plasma membrane staining
is acceptable.
If either of the Control Cell Lines does not meet these criteria, all results with the patient specimens
should be considered invalid.
2. Positive Control
The Positive Control Tissue Slide should be examined next. This slide verifies that the fixation method and
Tissue Slide
epitope retrieval process are effective. Use intact cells for interpretation of staining results because necrotic or
(NSCLC)
degenerated cells often stain non-specifically. Presence of brown plasma membrane staining should be
observed. Non-specific staining should be ≤1+.
3. Negative Control
The Negative Control Tissue Slide should be examined after the Positive Control Tissue to verify the
Tissue Slide
specificity of the labeling of the target antigen by the primary antibody. The absence of specific staining in the
(NSCLC)
Negative Control Tissue Slide confirms the lack of kit cross-reactivity to cells/cellular components.
Alternatively, negative portions of the Positive Control Tissue may serve as the Negative Control Tissue, but
this should be verified by the user. If unwanted specific cell membrane staining occurs in the Negative Control
Tissue Slide, results with the patient specimen should be considered invalid.
4. Patient tissue slide
Examine patient specimens stained with the Negative Control Reagent from PD-L1 IHC 22C3 pharmDx next.
stained using the
Absence of cell membrane staining verifies the specific labeling of the target antigen by the primary antibody.
Negative Control
Staining occurring in the cytoplasm of the specimen treated with the Negative Control Reagent should be
Reagent
considered non-specific staining.
5. Patient tissue slide
Examine the entire slide of the patient specimen stained with the PD-L1 primary antibody from PD-L1 IHC
stained using the
22C3 pharmDx following review of all acceptable control slides. Positive staining intensity should be assessed
primary antibody
within the context of any non-specific background staining observed in the Negative Control Reagent slide
within the same run. As with any immunocytochemical test, a negative result means that the antigen was not
detected, not necessarily that the antigen was absent in the cells/tissue assayed. Refer to Summary and
Explanation, Limitations, and Performance Characteristics for specific information regarding PD-L1 IHC 22C3
pharmDx immunoreactivity. Complete circumferential and/or partial linear membrane staining of tumor cells
indicates positive PD-L1 staining. Granular staining in the cytoplasm is not considered as positive staining.
Additional Recommendations for Interpretation of PD-L1 IHC 22C3 pharmDx Staining
1. A hematoxylin and eosin (H&E) stain of the tissue specimen is recommended for the first evaluation of the patient specimen. The
PD-L1 IHC 22C3 pharmDx assay and H&E stain should be performed on serial sections from the same paraffin block of the
specimen.
2. To verify cell membrane staining, use 10–40x objective magnification.
3. Various non-target elements, such as tumor associated immune cells (e.g. macrophages) and stroma may also stain positive for
PD-L1, however should not be included in the scoring.
General Limitations
1. Immunohistochemistry is a multi-step diagnostic process that requires specialized training in the selection of the appropriate
reagents; tissue selection, fixation, and processing; preparation of the immunohistochemistry slide; and interpretation of the staining
results.
2. Tissue staining is dependent on the handling and processing of the tissue prior to staining. Improper fixation, freezing, thawing,
washing, drying, heating, sectioning, or contamination with other tissues or fluids may produce artifacts, antibody trapping, or falsenegative results. Inconsistent results may be due to variations in fixation and embedding methods, or to inherent irregularities within
the tissue.
3. Excessive or incomplete counterstaining may compromise proper interpretation of results.
4. The clinical interpretation of any positive staining or its absence must be evaluated within the context of clinical presentation,
morphology and other histopathological criteria. The clinical interpretation of any staining, or its absence, must be complemented by
morphological studies and proper controls as well as other diagnostic tests. It is the responsibility of a qualified pathologist, who is
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5.
6.
7.
8.
familiar with the antibodies, reagents and methods used, to interpret the stained preparation. Staining must be performed in a
certified, licensed laboratory under the supervision of a pathologist who is responsible for reviewing the stained slides and assuring
the adequacy of positive and negative controls.
Tissues from persons infected with hepatitis B virus and containing hepatitis B surface antigen (HBsAg) may exhibit non-specific
staining with horseradish peroxidase (5).
Reagents may demonstrate unexpected reactions in previously untested tissue types. The possibility of unexpected reactions even
in tested tissue types cannot be completely eliminated due to biological variability of antigen expression in neoplasms, or other
pathological tissues. Contact Dako Technical Support with documented unexpected reactions.
False-positive results may be seen due to non-Immunological binding of proteins or substrate reaction products. They may also be
caused by pseudoperoxidase activity (erythrocytes) and endogenous peroxidase activity (cytochrome C) (5).
The reagents and instructions supplied in this system have been designed for optimal performance. Further dilution of the reagents
or alteration of incubation times or temperatures may give erroneous or discordant results.
Product-specific limitations
1. False-negative results could be caused by degradation of the antigen in the tissues over time. Specimens should be stained within
six months of mounting of tissues on slides when stored in the dark at 2-8°C.
2. For optimal and reproducible results, the PD-L1 protein requires target retrieval pre-treatment when tissues are routinely fixed
(neutral buffered formalin) and paraffin embedded.
3. Do not substitute reagents from different lot numbers of this product, or from kits of other manufacturers. The only exception is the
EnVision FLEX Target Retrieval Solution, Low pH 50x, which, if required, is available as Code K8005.
4. Stained control cell lines should be used only for validation of the staining run and should not be used to score the staining reaction
in tissue sections.
5. Use of Dako PD-L1 IHC 22C3 pharmDx on tissues with fixatives other than formalin has not been validated.
Clinical performance evaluation
The clinical benefit of PD-L1 IHC 22C3 pharmDx was investigated in a multicenter, open-label, randomized clinical study conducted to
assess the safety and efficacy of KEYTRUDA in patients with advanced NSCLC. Patients were PD-L1 positive by a research assay, and
had progression of disease following treatment with platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor
aberrations had disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA. Assessment of
tumor status was performed every 9 weeks. The major efficacy outcome measures were ORR (according to RECIST 1.1 as assessed
by blinded independent central review) and duration of response (6).
To evaluate the clinical utility of PD-L1 IHC 22C3 pharmDx, archived clinical study samples were retrospectively tested at a U.S based
reference laboratory with PD-L1 IHC 22C3 pharmDx. KEYTRUDA® (pembrolizumab) demonstrated a robust overall response rate with a
clinically meaningful duration of response in NSCLC patients with positive PD-L1 expression as determined by PD-L1 IHC 22C3
pharmDx.
Based on the research assay, a total of 223 NSCLC patients were enrolled in the study. Out of the 223 patients, tumor tissue from 220
patients was retrospectively tested with the PD-L1 IHC 22C3 pharmDx test. Specimens from 61 patients were positive for PD-L1
expression (≥50% of viable tumor cells exhibiting membrane staining at any intensity) and samples from 104 patients were negative for
PD-L1 expression (<50% of viable tumor cells exhibiting membrane staining at any intensity).
The baseline characteristics for this population of 61 patients with positive PD-L1 expression by PD-L1 IHC 22C3 pharmDx included:
median age 60 years (34% age 65 or older); 61% male; 79% White; and 34% and 64% with an ECOG performance status 0 and 1,
respectively. Disease characteristics were squamous and non-squamous (21% and 75% respectively); M1 (98%); brain metastases
(11%); and one (26%), two (30%), or three or more (44%) prior therapies. The mutation status among patients was EGFR (10%), ALK
(0%), or Kras (16%).
Efficacy results for NSCLC are summarized in Table 3.
Table 3: Response to KEYTRUDA® (pembrolizumab) in Previously Treated NSCLC Patients with PD-L1 tumor proportion score
≥50%
Endpoint
N=61
Overall Response Rate
ORR %, (95% CI)
41% (29, 54)*
Complete Response
0%
Partial Response
41%
Response Duration
Median in months (range)
Not reached (2.1+, 9.2+)
†
84%
% ongoing
* In patients with a PD-L1 tumor proportion score <50% (n = 104), ORR
was13% (8, 22)
† Includes 11 patients with ongoing responses of 6 months or longer
Among the 61 patients 25 patients (41%) experienced a partial response. Of these 25 patients, 21 (84%) had an ongoing response at
the time of the data analysis cut-off date. At that time, the duration of response was between 2.1 and 9.2 months for all 25 responders.
The patients with response durations of 2.1 and 9.2 months were both ongoing at the time of data cutoff, denoted by appending a + sign
to those values in the table. Median DoR is calculated based on the number of events and their timing and had not been reached at the
data cut-off date.
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P03951_02/SK00621-5/2015.09 p. 7/12
Non-clinical performance evaluation
Analytical Sensitivity
Analytical sensitivity of PD-L1 IHC 22C3 pharmDx was tested on 127 unique cases of non-small cell lung carcinoma (NSCLC) FFPE
specimens staged I to IV using a manufactured production lot. Assessment of PD-L1 expression demonstrated staining across a range
of 0-100% positive tumor cells and 0-3 staining intensity.
Repeatability/External Reproducibility
The Repeatability/External Reproducibility of PD-L1 IHC 22C3 pharmDx was evaluated at Dako and three external testing sites. The
performance data are provided in Table 4 and Table 5. For the repeatability studies performed at Dako, negative percent agreement
(NPA), positive percent agreement (PPA) and overall agreement (OA) were determined as shown in Table 4. Since the repeatability
studies resulted in 100% agreement, confidence intervals were calculated using the Wilson Score method, based on the number of
independent pair-wise comparisons.
For external reproducibility studies, the average negative percent agreement (ANA), average positive percent agreement (APA) and
overall percent agreement (OA) were determined as shown below in Table 5. Average agreements were calculated since no natural
reference exists in reproducibility parameters such as site and observer. Confidence intervals for the average agreements were
computed using a percentile bootstrap method.
(Note that the repeatability studies were not analyzed using bootstrap confidence intervals, since the aforementioned cannot be
computed on data with zero discordance.)
Table 4: Repeatability of PD-L1 IHC 22C3 pharmDx tested at one site
Repeatability Study
Diagnostic Cutoff
Study Design
Each of 16 NSCLC specimens (10
PD-L1-negative and 6 PD-L1-positive) with a
range of PD-L1 IHC expression was tested on
each of six Autostainer Link 48 instruments.
Inter-operator
≥50%
Each of 16 NSCLC specimens (10
PD-L1-negative and 6 PD-L1-positive) with a
range of PD-L1 IHC expression was tested using
six analysts on one Autostainer Link 48
instrument.
Inter-day
≥50%
Each of 16 NSCLC specimens (10
PD-L1-negative and 6 PD-L1-positive) with a
range of PD-L1 IHC expression was tested on six
non-consecutive days on the Autostainer Link 48
instrument.
Inter-lot
≥50%
Each of 16 NSCLC specimens (8
PD-L1-negative and 8 PD-L1-positive) with a
range of PD-L1 IHC expression was tested with
three replicates and each of three reagent lots on
the Autostainer Link 48 instrument.
Intra-run
≥50%
Each of 16 NSCLC specimens (10
PD-L1-negative and 6 PD-L1-positive) with a
range of PD-L1 IHC expression was tested with six
replicates within a run on the Autostainer Link 48
instrument.
Intra-day
≥50%
Each of 16 NSCLC specimens (10
PD-L1-negative and 6 PD-L1-positive) with a
range of PD-L1 IHC expression was tested on two
runs within a day, repeated over three days, on the
Autostainer Link 48 instrument.
NPA= Negative Percent Agreement; PPA= Positive Percent Agreement; OA=Overall Agreement
Inter-instrument
(128206-001)
≥50%
% Agreement (95% CI)
≥50% cut-off:
NPA 100% (92.9-100%)
PPA 100% (88.6-100%)
OA 100% (95.4-100%)
≥50% cut-off:
NPA 100% (92.7-100%)
PPA 100% (88.6-100%)
OA 100% (95.4-100%)
≥50% cut-off:
NPA 100% (92.9-100%)
PPA 100% (88.6-100%)
OA 100% (95.4-100%)
≥50% cut-off:
NPA 100% (92.6-100%)
PPA 100% (92.6-100%)
OA 100% (96.2-100%)
≥50% cut-off:
NPA 100% (92.9-100%)
PPA 100% (88.6-100%)
OA 100% (95.4-100%)
≥50% cut-off:
NPA 100% (88.3-100%)
PPA 100% (82.4-100%)
OA 100% (92.4-100%)
P03951_02/SK00621-5/2015.09 p. 8/12
Table 5: Reproducibility of the PD-L1 IHC 22C3 pharmDx tested at three external sites
Reproducibility
Diagnostic Cutoff
Study
Study Design
Inter-site
≥50%
Each of 36 NSCLC specimens (21 PD-L1-negative and
15 PD-L1-positive) with a range of PD-L1 IHC expression
was tested on five non-consecutive days. Inter-site
analysis was performed between three sites on a total of
2700 pair-wise comparisons.
Intra-site
≥50%
Each of 36 NSCLC specimens (21 PD-L1-negative and
15 PD-L1-positive) with a range of PD-L1 IHC expression
was tested on five non-consecutive days at each of three
study sites. Intra-site analysis was performed for three
sites on a total of 1080 pair-wise comparisons.
Inter-observer
≥50%
Scoring of 62 NSCLC specimens (30 PD-L1-negative and
32 PD-L1-positive) with a range of PD-L1 IHC expression,
stained with PD-L1 IHC 22C3 pharmDx, was performed
by three pathologists, one at each of three study sites, on
three non-consecutive days. Inter-observer analysis was
performed between three sites on a total of 1674 pairwise comparisons.
Intra-observer
≥50%
Scoring of 62 NSCLC specimens (30 PD-L1-negative and
32 PD-L1-positive)with a range of PD-L1 IHC expression,
stained with PD-L1 IHC 22C3 pharmDx, was performed
by three pathologists, one at each of three study sites, on
three non-consecutive days. Intra-observer analysis was
performed for three sites on a total of 558 pair-wise
comparisons.
ANA=Average Negative Agreement; APA=Average Positive Agreement; OA=Overall Agreement
% Agreement (95% CI)
≥50%:
ANA 90.3% (84.4-95.2%)
APA 85.2% (75.6-92.9%)
OA 88.3% (81.4-94.3%)
≥50%:
ANA 91.9% (88.8-94.8%)
APA 87.6% (82.5-92.2%)
OA 90.2% (86.3-93.7%)
≥50%:
ANA 92.6% (87.8-96.7%)
APA 92.8% (88.1-96.8%)
OA 92.7% (88.1-96.8%)
≥50%:
ANA 96.4% (94.0-98.5%)
APA 96.5% (94.3-98.6%)
OA 96.4% (94.3-98.6%)
Normal tissues: Plasma membrane staining was observed on immune cells and cells of epithelial origin. Cytoplasmic staining was
noted in some cell types but was not recorded as positive staining. Table 6 summarizes Monoclonal Mouse Anti-PD-L1, Clone 22C3
immunoreactivity on the recommended panel of normal tissues. All tissues were formalin-fixed and paraffin-embedded and stained with
PD-L1 IHC 22C3 pharmDx according to the instructions in this package insert. There were no unexpected results observed in cell types
or tissue types tested. The observed staining was consistent with the reported literature for PD-L1 IHC expression in normal tissues (7,
8).
Table 6: Summary of PD-L1 IHC 22C3 pharmDx normal tissue reactivity
Tissue Type
Positive Plasma Membrane
Positive Cytoplasmic Staining:
(# tested)
Staining: Tissue Elements
Tissue Elements
Adrenal (3)
0/3
1/3 Medullary cells
Bone marrow (3)
3/3 Megakaryocytes
3/3 Megakaryocytes
Breast (3)
0/3
0/3
Cerebellum (3)
0/3
0/3
Cerebrum (3)
0/3
0/3
Cervix (3)
1/3 Epithelium
0/3
Colon (3)
2/3 Macrophages
0/3
Esophagus (3)
0/3
0/3
Kidney (3)
1/3 Tubular epithelium
0/3
Liver (3)
1/3 Macrophages
0/3
1/3 Heptatocytes
Lung (3)
3/3 Alveolar macrophages
0/3
Mesothelial cells (2)
0/2
0/2
Muscle, cardiac (3)
0/3
0/3
Muscle, skeletal (3)
0/2
0/2
Nerve, peripheral (3)
0/3
1/3 Connective tissue/vessels
Ovary (3)
0/3
0/3
Pancreas (3)
0/3
0/3
Parathyroid (3)
1/3 Glandular epithelium
0/3
Pituitary (3)
1/3 Anterior hypophysis
1/3 Anterior hypophysis
1/3 Posterior hypophysis
1/3 Posterior hypophysis
Prostate (2)
2/2 Epithelium
0/2
Salivary gland (3)
0/3
0/3
Skin (3)
0/3
0/3
Small intestine (3)
0/3
0/3
Spleen (3)
2/3 Macrophages
0/3
Stomach (3)
2/3 Lymphocytes
1/3 Gastric glands
1/3 Gastric glands
Testis (3)
0/3
0/3
Thymus (3)
3/3 Medullary epithelium
0/3
Thyroid (3)
0/3
0/3
Tonsil (3)
3/3 Crypt epithelium
0/3
2/3 Germinal center
(macrophages)
Uterus (3)
0/3
0/3
(128206-001)
Non-specific
Staining
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/2
0/3
0/2
0/3
0/3
0/3
0/3
0/3
0/2
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
P03951_02/SK00621-5/2015.09 p. 9/12
Neoplastic tissues: Plasma membrane staining was observed on immune cells and cells of epithelial origin. Cytoplasmic staining was
noted in some cell types but was not recorded as positive staining. Table 7 summarizes Monoclonal Mouse Anti-PD-L1, Clone 22C3
immunoreactivity on a panel of neoplastic tissues. All tissues were formalin-fixed and paraffin-embedded and stained with PD-L1 IHC
22C3 pharmDx according to the instructions in this package insert. There were no unexpected results observed in the tumor specimens
tested. The observed staining was consistent with the reported literature for PD-L1 IHC expression in neoplastic tissues (7-10).
Table 7: Summary of PD-L1 IHC 22C3 pharmDx neoplastic tissue reactivity
Tumor Type
Adenocarcinoma
Adrenocortical carcinoma
Astrocytoma
Basal cell carcinoma
Carcinoma
Chondrosarcoma
Chordoma
Embryonal carcinoma
Ependymoma
Glioblastoma
Hepatoblastoma
Hepatocellular carcinoma
Islet Cell tumor
Interstitialoma
Leiomyosarcoma
Lymphoma
Anaplastic Large Cell
Diffuse B-cell
Hodgkin
Non-Hodgkin
Medulloblastoma
Medullary carcinoma
Melanoma
Meningioma
Mesothelioma
Neuroblastoma
Neurofibroma
Osteosarcoma
Pheochromocytoma
Primitive Neuroectodermal Tumor
(PNET)
Renal Cell carcinoma
Papillary
(128206-001)
Location
PD-L1 positive/total
N=159
0/1
0/2
0/7
Appendix
Breast, DCIS
Breast, invasive ductal
Breast, invasive ductal metastatic to lymph
node
Cervix, endocervical type
Colon
Colon, metastatic to liver
Colon, mucinous
Esophagus
Gallbladder
GI, metastatic to lung
Head & neck, hard palate
Lung
Ovary
Ovary, endometrioid
Ovary, mucinous
Ovary, serous
Pancreas
Pancreas, ductal
Prostate
Rectum
Salivary/parotid gland
Small intestine
Stomach
Stomach, mucinous
Thyroid, follicular
Thyroid, follicular-papillary
Thyroid, papillary
Uterus, clear cell
Uterus, endometrium
Adrenal
Cerebrum
Skin
Nasopharyngeal, NPC
Bone
Pelvic cavity
Testis
Brain
Brain
Liver
Liver
Pancreas
Colon
Rectum
Small intestine
Soft tissue, chest wall
Bladder
0/1
0/5
0/1
0/1
0/1
1/5
0/1
0/1
1/4
0/1
0/1
0/1
0/1
0/2
0/3
0/5
0/4
0/2
0/2
0/6
0/1
0/1
0/1
0/3
0/1
0/3
0/1
0/3
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/5
0/1
0/1
0/1
0/1
0/1
0/1
Lymph node
Lymph node
Lymph node
Lymph node
Brain
Thyroid
Rectum
Nasal cavity
Brain
Peritoneum
Retroperitoneum
Soft tissue, lower back
Bone
Adrenal
0/1
0/4
2/2
1/1
0/1
0/1
0/1
0/1
0/2
0/1
0/1
0/1
0/2
0/1
Retroperitoneum
0/1
Kidney
0/1
0/1
P03951_02/SK00621-5/2015.09 p. 10/12
Tumor Type
Clear Cell
Rhabdomyosarcoma
Seminoma
Signet Ring Cell carcinoma
Small cell carcinoma
Spermatocytoma
Squamous Cell carcinoma
Synovial Sarcoma
Thymoma
Transitional Cell carcinoma
Location
Kidney
Soft tissue, embryonal
Prostate
Retroperitoneum
Testis
Metastatic colon signet ring cell carcinoma
to ovary
Colon
Lung
Testis
Metastatic esophageal squamous cell
carcinoma to lymph node
Cervix
Esophagus
Head & neck
Lung
Skin
Uterus
Pelvic cavity
Mediastinum
Bladder
Kidney
PD-L1 positive/total
N=159
0/6
0/1
0/1
0/1
0/2
0/1
0/1
0/1
0/2
0/1
2/5
0/7
0/2
1/2
0/2
0/1
0/1
1/1
0/6
0/1
Troubleshooting
Table 8: Troubleshooting
Problem
1. No staining of slides
Probable Cause
1a. Programming error.
1b. Lack of reaction with DAB+
Substrate-Chromogen Solution (DAB)
1c. Sodium azide in wash buffer.
1d. Degradation of Control Slide
2a. Weak staining of specimen slides.
2a. Inappropriate fixation method used.
2b. Weak staining of specimen slides
or of the positive cell line on the Dakoprovided Control Slide.
3. Excessive background staining of
slides.
2b. Inadequate target retrieval.
3a. Paraffin incompletely removed.
4. Tissue detached from slides.
3b. Slides dried while loading onto
Autostainer Link 48.
3c. Nonspecific binding of reagents to
tissue section.
4. Use of incorrect microscope slides.
5. Excessively strong specific staining.
5a. Inappropriate fixation method used.
Suggested Action
1a. Verify that the PD-L1 IHC 22C3 pharmDx program
was selected for programming of slides.
1b. Verify that DAB+ Substrate-Chromogen Solution
was prepared properly.
1c. Use only Dako Wash Buffer (Code K8007).
1d. Check kit expiration date and kit storage
conditions on outside of package.
2a. Ensure that only approved fixatives and fixation
methods are used.
2b. Verify that the 3-in-1 pre-treatment procedure was
correctly performed.
3a. Verify that the 3-in-1 pre-treatment procedure was
correctly performed.
3b. Ensure slides remain wet with buffer while loading
and prior to initiating run.
3c. Check for proper fixation of the specimen and/or
the presence of necrosis.
4. Use Dako FLEX IHC Microscope Slides (Code
K8020), or charged slides (such as Fisherbrand
Superfrost Plus).
5a. Ensure that only approved fixatives and fixation
methods are used.
5b. Use only Dako Wash Buffer (Code K8007).
6. This is normal and does not influence staining.
5b. Inappropriate wash buffer used.
6. Target Retrieval Solution is cloudy
6. When heated the Target Retrieval
in appearance when heated.
Solution turns cloudy in appearance.
NOTE: If the problem cannot be attributed to any of the above causes, or if the suggested corrective action fails to resolve the problem,
please call Dako Technical Support for further assistance. Additional information on staining techniques and specimen preparation can
be found in Dako Education Guide: Immunohistochemical Staining Methods (11) (available from Dako).
References
1.
2.
3.
4.
5.
6.
7.
8.
Department of Health, Education and Welfare, National Institutes for Occupational Safety and Health, Rockville, MD. “Procedures
for the decontamination of plumbing systems containing copper and/or lead azides.” DHHS (NIOSH) Publ. No. 78-127, Current 13.
August 16, 1976.
Clinical and Laboratory Standards Institute (formerly NCCLS). Protection of Laboratory Workers From Occupationally Acquired
Infections; Approved Guideline – Third Edition. CLSI document M29-A3 [ISBN 1-56238-567-4]. Clinical and Laboratory Standards
Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087 – 1898 USA, 2000.
Herman GE, Elfont EA. The taming of immunohistochemistry: the new era of quality control. Biotech & Histochem 1991; 66:194.
Clinical and Laboratory Standards Institute (formerly NCCCLS). Quality assurance for Immunocytochemistry; Approved guideline.
CLSI document MM4-A (1-56238-396-5) CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA; 1999.
Omata M, Liew C-T, Ashcavai M, Peters RL. Nonimmunologic binding of horseradish peroxidase to hepatitis B s surface antigen: a
possible source of error in immunohistochemistry. Am J Clin Path 1980; 73:626.
Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung
cancer. New Eng J Med 2015; 372:2018-28.
Okazaki T, Honjo T. PD-1 and PD-1 ligands: from discovery to clinical application. International Immunol 2007(19); 7:813.
Brown JA, Dorfman DM, Ma F-R, Sullivan EL, Munoz O, Wood CR, et al. Blockade of Programmed Death-1 Ligands on Dendritic
Cells Enhances T Cell Activation and Cytokine Production. J Immunol 2003;170:1257.
(128206-001)
P03951_02/SK00621-5/2015.09 p. 11/12
9.
Cooper WA, Tran T, Vilain RE, Madore J, Seliger CI, Kohonen-Cornish M, et al. PD-L1 expression is a favorable prognostic factor
in early stage non-small cell carcinoma. Lung Cancer 2015; 89:181.
10. Chen B, Chapuy B, Ouyang J et al. PD-L1 Expression Is Characteristic of a Subset of Aggressive B-cell Lymphomas and VirusAssociated Malignancies. Clin Cancer Res 2013; 19:3462-3473.
11. Taylor CR and Rudbeck L. Education Guide: Immunohistochemical Staining Methods. Sixth Edition. Dako, Carpinteria, California;
2013.
Edition 09/15
(128206-001)
P03951_02/SK00621-5/2015.09 p. 12/12
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