dako egfr pharmdx™ interpretation manual

EGFR pharmDxTM Interpretation Manual

P A T H O L O G Y

For In Vitro Diagnostic Use. FDA-approved as an aid in identifying colorectal cancer patients eligible for treatment with Erbitux® (cetuximab) and VectibixTM (panitumumab).

�EGFR pharmDx™ Interpretation Manual

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Introduction 2

EGFR overview 3n EGFR and the HER Family

n EGFR Expression in normal Tissue

The EGFR pharmDx™ Kit 5

Expression Rates and Recommended Data Tracking for EGFR pharmDx™ 6

Technical Tips for optimal EGFR pharmDx™ Performance 7n Tissue Fixation and Variables

n EGFR pharmDxTM Protocol Recommendations

n EGFR pharmDxTM Training Checklist

Quality Control 9

EGFR pharmDx™ Evaluation and Reporting 10n slide Evaluation

n steps to EGFR pharmDxTM Evaluation

Image Guide for Interpretation 12n EGFR pharmDxTM Interpretation Guidelines

staining Patterns 13n Heterogeneous staining

n Homogeneous staining

n EGFR staining of normal and benign Tissues

Factors to Consider in Evaluating EGFR pharmDx™ stains 16n non-specific background

n Possible Causes of non-specific background

Artifacts 17n overdigestion

n Post-Fixation Procedure

n Crush Artifact

n Edge Artifact

n Retraction Artifact

n Thermal Artifact

n non-Evaluable Areas of Tissue

Examples of Cancer stained with EGFR pharmDx™ 19n Colorectal Cancer

EGFR pharmDx™ Immunostaining in a Variety of solid Tumors 23

References 24n Additional EGFR Resources

n Acknowledgements

Table of Contents

Erbitux® is a registered trademark of ImClone Systems, Incorporated. VectibixTM is a registered trademark of Amgen, Incorporated.

� EGFR pharmDx™ Interpretation Manual

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Welcome to the EGFR pharmDx™ Interpretation Manual

This guide for pathologists includes key technical

histological staining and interpretation tips when using

the EGFR pharmDxTM kit. utilization of the suggestions that

follow will ensure that your laboratory achieves the quality

results expected from EGFR pharmDxTM.

The EGFR pharmDxTM Interpretation Manual objectives

are simple:

n Provide an understanding of Epidermal Growth Factor

Receptor biology.

n Give procedure recommendations to ensure the

EGFR pharmDxTM assay is performed consistently

for optimal results.

n Present a standard approach to staining and

interpretation to ensure reproducible results.

n supply pathologists with guidelines for consistent

interpretation of EGFR pharmDx™ to aid in assessing

colorectal cancer patients for Erbitux® or VectibixTM

eligibility.

n To troubleshoot the EGFR pharmDxTM kit if problems

occur.

We hope this EGFR pharmDxTM Interpretation Manual is

useful, and we encourage you to provide feedback on

how we can improve this tool. Contact your local Dako

representative with feedback (see back panel for contact

information).

EGFR pharmDxTM is an FDA-approved assay indicated

as an aid in identifying patients eligible for treatment with

Erbitux® (cetuximab), or VectibixTM (panitumumab) for

EGFR-expressing metastatic colorectal cancer.

Introduction


oVER

VIEWEGFR overview

EGFR and the HER Family

EGFR is a member of the EGF/erbb receptor family of

related growth factor receptors that includes HER2/erbb2

or neu, HER3/erbb3 and HER4/erbb4 (1).

Mouse monoclonal anti-EGFR clone 2-18C9 was selected

for its high specificity for EGFR. The specificity of clone

2-18C9 for EGFR (HER1) and lack of cross-reactivity with

the related HER family receptors were demonstrated by

immunocytochemistry and Western blotting using CHo

cells transiently transfected with vectors expressing

HER2, HER3 and HER4. Further specificity testing by flow

cytometry and Western blotting showed that clone 2-18C9

recognizes both the wild type and the EGFRvIII mutant

form of the receptor. The epitope bound by clone 2-18C9

was found to be a structural epitope in the extracellular

cysteine-rich region of the molecule spanning sub-domain

s2 and proximal to the transmembrane region (3).

Epidermal growth factor receptor (EGFR) is a 170 kDa

transmembrane receptor encoded by the human HER1

gene. The EGFR protein contains an extracellular ligand

binding domain, a transmembrane region and an

intracellular domain with intrinsic protein-tyrosine

kinase activity (see Figure 1). ligand binding of the

EGF receptor activates the EGFR tyrosine kinase

resulting in cell growth and differentiation (1).

EGFR Signaling Networks (2)

Figure 1

Ligand (EGF, TGF-a)

EGFRExtracellular domain

Cell membrane

Intracellular domain

Radiation or selected chemotherapy agents

Cell motility and metastasisCell adhesion, invasiveness

Growth effectsProliferation, differentiation

Angiogenesis effectsBlood vessel recruitment, invasion, metastasisGrowth arrest

or apoptosis

MEK

MAPK

Raf-1RasSOS

Tyrosinekinase

Grb2

Other enzymeor adapter


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Tissue Type Positive Tissue Element Staining and Staining Pattern

Adrenal Cortical cells (2+): Cytoplasmic

Bone Marrow none

Breast lobular epithelial cells (2+): Membrane and cytoplasmic

Brain (Cerebellum) Molecular layer (1+): Extracellular

Brain/Cerebrum none

Cervix+ basalar squamous epithelial cells (2+): Membrane

Colon** none

Esophagus basalar squamous epithelial cells (2+): Membrane

Heart – none

Kidney Tubules (1+): Cytoplasmic staining (granular)

Liver Hepatocytes (sinusoids) (3+): Membrane bile ducts (3+): Membrane and cytoplasmic

Lung+ Alveolar lining cells/basalar bronchial cells (myoepithelial cells) (2+): Membrane and cytoplasmic

Mesothelial Cells Mesothelial cells (2+): Membrane and cytoplasmic

Ovary none

Pancreas Ducts (2+): Membrane

Parathyroid none

Peripheral Nerve nerve cell processes (1+): Fibrous

Pituitary none

Prostate+ Glandular epithelial cells (2+): Membrane

Salivary Gland Ductal elements (1+): Cytoplasmic

Skeletal Muscle – none

Skin+ basalar squamous cells, adnexal structures (2+): Membrane and cytoplasmic

Small Intestine none

Spleen none

Stomach none

Testis none

Thymus none

Thyroid none

Tonsil basalar squamous epithelium (3+): Membrane and cytoplasmic

Uterus Endometrial gland epithelium (2+): Membrane and cytoplasmic Endometrial stromal cells (2+): Membrane and cytoplasmic Myometrium: none

EGFR Expression in Normal Tissue

The EGFR protein is expressed on a variety of normal cells including many epithelial cell types (4-10).

non-epithelial cell types that express EGFR include smooth muscle cells, fibroblasts and perineurium (11).

some examples of normal tissue stained with EGFR pharmDxTM are summarized in the table below. All tissues were

formalin-fixed and paraffin-embedded.

Table �. Evaluation of normal tissue staining by Dako EGFR pharmDxTM*

+ Recommended for positive control tissue (See page 9 for photomicrograph of normal positive tissue staining of cervix) – Recommended for negative control tissue* The majority of tissues tested had positive staining of fibroblasts in stromal tissue (1+, fibrous), as well as perineural fibroblasts

and myoepithelial cells. Endogenous peroxidase-induced staining of granulocytes has been observed occasionally.** Colon tissue may exhibit positive staining of enterocytes, smooth muscle cells, endothelial cells and perineurium.


Step 4Application of Labeled Polymer, HRP. Incubate 30 minutes.

Step 5Application of Substrate-Chromogen. Incubate 10 minutes.

Step 1Application of Peroxidase Block. Incubate 5 minutes.

Figure 2Kit Procedure for Automation

Step 2Application of Proteinase K. Incubate 5 minutes.

Step 3Application of Primary Antibody. Incubate 30 minutes.

The EGFR pharmDx™ Kit

The EGFR pharmDxTM assay is a qualitative immunohistochemical (IHC) kit system to

identify epidermal growth factor receptor (EGFR) expression in normal and neoplastic

tissues routinely fixed for histological evaluation. EGFR pharmDxTM specifically detects the

EGFR (HER1) protein in EGFR-expressing cells.

EGFR pharmDxTM is FDA-approved as an aid in identifying colorectal cancer patients

eligible for treatment with Erbitux® (cetuximab) or VectibixTM (panitumumab).

Following incubation with the primary monoclonal antibody to human EGFR protein,

this kit employs a ready-to-use visualization reagent based on dextran technology. This

reagent consists of both secondary goat anti-mouse antibody molecules and horseradish

peroxidase molecules linked to a common dextran polymer backbone, thus eliminating the

need for sequential application of link antibody and peroxidase conjugate. The enzymatic

conversion of the subsequently added chromogen results in formation of a visible reaction

product at the antigen site. The specimen may then be counterstained and coverslipped.

Results are interpreted using a light microscope. Control slides containing two formalin-

fixed, paraffin-embedded human cell lines with staining intensity scores of 2+ and 0 are

provided for validation of the kit reagent performance.

Two EGFR pharmDx™ Kit Configurations are Available K1492 EGFR pharmDx™ Kit for Manual Use 35 Test

K1494 EGFR pharmDx™ Kit for the Dako Autostainer/Autostainer Plus 50 Test

The EGFR pharmDx™ Kit Includes:

n Proteinase K

n Peroxidase block

n Dako EGFR pharmDxTM Monoclonal Mouse Antibody

n Mouse IgG1 negative Control Reagent

n labeled Polymer, HRP

n DAb+ substrate buffer

n liquid DAb+ Chromogen

n Dako Wash solution 10x

n Dako EGFR pharmDxTM Control slides

tissueproteins

EGFRprotein

Proteinase K

peroxidaseblock

EGFRantibody

secondaryantibody

HRP enzyme

dextranbackbone

DAB


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Clinical trials of EGFR-targeted therapies (cetuximab and panitumumab) have been performed using Dako EGFR pharmDx™-

positive results as one of the criteria for study eligibility.

Summary of EGFR-Expressing Percent in Colon Cancer Patients EGFR-Expressing Ratio % EGFR- 95% Confidence

Therapeutic Study ID (# expressing/# tested) Expressing Intervals

Pivotal Trial 474 / 577 82.1% 78.1 - 86.1% EMR 62202-007

Supportive Study 105 / 140 75.0% 66.9 - 83.1% IMCL CP02-0141

Prototype EGFR Study 292 / 401 72.8% 68.0 - 77.6% IMCL CP02-9923

VectibixTM/panitumumab Pivotal Trial 735 / 1004 73.2% 70.4 - 75.9% 20020408

Expression Rates and Recommended Data Tracking for EGFR pharmDx™ Immunostaining

Erbitux®/cetuximab

EGFR pharmDx™ Testing

use EGFR pharmDxTM data to determine

an average number of percent positive

colorectal adenocarcinomas.

If the average percent of positives falls within

73-82 percent, report results. Continue to use

EGFR pharmDxTM by following protocol.

Continue to monitor results.

73–82 percent positives

Review Patient Demographics

If results are <73% or >82%, review

EGFR pharmDx™ procedures.

<73% or >82% positives

Review Technical Procedure

see pages 7–9

Review Interpretation Procedure

see pages 10–22

Recommended Data Tracking


TECH

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Technical Tips for optimal EGFR pharmDx™ Performance

Tissue Fixation and Variables

Procedural deviations that are related to sample handling

and processing can affect the EGFR pharmDxTM results.

some of the variables that may affect results are as follows:

n non-representative tissue samples

n specimens drying prior to fixation

n Fixation with PreFer fixative (suitable fixatives include 10% (v/v) neutral buffered formalin, 10% (v/v) unbuffered formalin, 25% (v/v) unbuffered formalin, AFA, Pen-Fix, and bouin’s)12

n Age, pH and storage conditions of fixative

n length of fixation

n length of storage of unstained tissue sections

For accurate and consistent results one must adhere to the EGFR pharmDxTM protocol.

High-quality results can be achieved in any laboratory by following these guidelines.

Technical problems may arise in two areas, those involving sample collection and preparation of tissue in the pre-analytical

processing of the specimen and those involving the actual performance of the EGFR pharmDxTM assay itself. Technical

issues relating to the performance of the assay are generally related to procedural deviations from the EGFR pharmDxTM

protocol and can be alleviated.

EGFR pharmDx™ Protocol Recommendations

n If staining must be interrupted, slides may be kept in wash buffer following incubation of the primary antibody for up to one hour at room temperature (20–25 °C).

n specimens preserved in generally used fixatives (10% v/v neutral buffered formalin, 10% v/v unbuffered formalin, 25% v/v unbuffered formalin, AFA, Pen-Fix, and

bouin’s) are suitable for testing with EGFR pharmDxTM. use of EGFR pharmDxTM on PreFer fixed tissues may result in unsatisfactory preservation of morphology.12

n Automated staining: Dako recommends the use of EGFR pharmDxTM on a Dako Autostainer or Autostainer Plus. use of EGFR pharmDxTM on alternative automated platforms has not been validated and may give erroneous results.

n Wash buffer: Dilute the provided wash buffer 1:10 using distilled or deionized water. store unused solution at 2–8 °C no more than seven days. Discard diluted solution if cloudy in appearance. only use wash buffer supplied in the EGFR pharmDxTM kit or TbsT Wash buffer, code s3006.

n storage of Reagents: Reagent and control slides should be stored at 2–8 °C. Do not use the kit after the expiration

date printed on the outside of the kit box.

n False-negative immunostaining can be caused by degradation of the antigen in the tissue over time. specimens should be stained within two months of mounting of tissues on slides when stored at room temperature (20–25 °C).

n Proper Incubations: All incubation times must be performed according to the package insert. stay within the tolerance indicated in the package insert for all incubation times.

n For high-quality results, review the EGFR pharmDxTM Training Checklist (Table 2) prior to beginning your staining run.


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Table �. EGFR pharmDx™ Training Checklist

Customer name/Institution _______________________________________________________________________________________

Person Trained/Title _____________________________________________________________________________________________

Manual staining Run

Dako Autostainer software Version _________________________ Dako Autostainer serial number ____________________

Trainer________________________________________________________________ Date ___________________________________

EGFR pharmDxTM is a complete assay system requiring controls to ensure reproducible results.

Yes NoManual or Dako Autostainer Procedure

Control slides and kit stored at 2–8 °C?

Cell line control slides and all reagents warmed to room temperature (20-25 °C) prior to starting assay?

Tissues fixed in validated fixative?

specimens stained within two months of tissue mounting on slides when stored at room temperature?

Clearing solutions changed after 40 slides?

Deparaffinization and rehydration protocol followed?

Wash buffer prepared properly? Prepare sufficient quantity of Wash buffer by diluting Wash buffer 10X, 1:10 in Reagent Quality Water (deionized or distilled water).

Distilled or deionized water (not tap water) used for water washes after last alcohol bath in deparaffinization?

Regressive hematoxylin counterstains are not used?

Manual Procedure

Distilled or deionized water (not tap water) used for water bath after Proteinase K solution step and after substrate-Chromogen solution step?

Diluted Wash buffer used for all wash steps and baths (after Peroxidase-block, Primary Antibody/negative Control Reagent, labeled Polymer HRP)?

buffer bath(s) changed between each step?

Humid chamber used for Primary Antibody/ negative Control Reagent and labeled Polymer incubations?

slides placed in five-minute (±1) buffer baths between Peroxidase block and Proteinase K, negative Control Reagent steps/Primary Antibody, labeled Polymer and DAb+ substrate-Chromogen solution steps?

Proteinase K solution applied for five minutes?

Peroxidase block applied for five minutes and specimen fully covered?

Yes NoPrimary Antibody applied for 30 minutes and specimen fully covered?

labeled Polymer applied for 30 minutes and specimen fully covered?

DAb+ substrate-Chromogen prepared properly?

one drop DAb+ Chromogen to 1 ml DAb+ substrate buffer.

substrate-Chromogen solution applied for 10 minutes and specimen fully covered?

Dako Autostainer Procedure

slides placed in buffer five minutes (±1) before loading onto the Dako Autostainer?

Appropriate protocol template used?

Was the Dako Autostainer programming reviewed for accuracy?

slides rinsed with buffer between steps and double rinsed after the labeled polymer step with an additional five-minute rinse hold?

substrate-Chromogen prepared properly? Add 11 drops of liquid DAb+ Chromogen to one vial of DAb+ substrate buffer and mix.

substrate-Chromogen solution applied for two five-minute applications?

Instrumentation / Equipment

Is regular preventative maintenance performed on the Dako Autostainer?

Do you have all the necessary equipment to perform the EGFR pharmDxTM assay according to protocol?

If not, specify what is missing in comments below.

If you answered “no” to any of the above, you have deviated from protocol and should consult with your local Dako Technical support Representative for assistance.

Additional observations or comments:

________________________________________________________

________________________________________________________


Quality Control

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Controls for EGFR pharmDx™ Assay Validation

Figure 3 HT-29 positive cell line control stained with EGFR pharmDx™, 2 + moderate intensity.

Figure 4 CAMA-1 negative cell line control stained with EGFR pharmDx™.

Figure 5 Cervix positive control tissue. Basalar squamous epithelium stained positive with EGFR pharmDx™.

Figure 6 Skeletal muscle negative control tissue stained with EGFR pharmDx™.

The first quality control step for interpretation is the evaluation of

the control cell lines. Positive and negative cell lines are included

in each EGFR pharmDxTM kit (Figures 3 and 4) to validate staining

runs every time the assay is performed. Appropriate staining of

the control cell lines provides evidence that the EGFR pharmDxTM

assay is functioning properly. no membrane staining of the

CAMA-1 control cell line (0) and moderate brown complete or

incomplete membrane staining in the HT-29 control cell line (2+)

indicate that the staining run is valid. If either of the control cell

lines have staining results outside of these criteria, test slides

stained simultaneously within the same run should be considered

invalid and repeated.

The second quality control step is the positive control tissue

(Figure 5). use a positive control tissue (known to be EGFR

protein positive) 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 specific background staining. This validation is performed to

ascertain proper tissue preparation and staining techniques.

The presence of a brown reaction product at the cell membrane

is indicative of positive reactivity. one positive tissue control for

each set of test conditions should be included in each staining

run. Verify that the negative tissue control slide demonstrates

no specific reactivity. Known positive tissue controls should be

utilized only 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.

The third quality control step is the negative control tissue

(Figure 6). use a negative control tissue (known to be EGFR

protein negative) 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 specific background staining. The variety of different cell types

present in most tissue sections offers internal negative control.

If specific staining occurs in the negative control tissue, results

with the patient specimens should be considered invalid.

EGFR positive tissue elements such as nerve and fibroblasts are

present in most normal and neoplastic tissues. These positive

elements can serve as intrinsic internal controls in both patient

specimens and positive and negative control tissue.

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Unacceptable excessive staining intensity (too dark); 20x magnification. Excessive staining intensity of cell line may result in false-positive results.

EGFR pharmDx™ Evaluation and Reporting

Acceptable staining; 20x magnification.

Slide Evaluation

slide evaluation should be performed by a pathologist using a light microscope. EGFR pharmDxTM stains a variety of normal

and neoplastic tissues. observed EGFR staining patterns are heterogeneous or homogeneous depending on the tissue

and/or tumor type. Heterogeneity includes various intensity areas within a single neoplasm. The staining can show a range

of 0–3+ staining intensity. Cell staining patterns can also be heterogeneous including membrane staining, cytoplasmic

staining or both. When only cytoplasmic staining is present, in the absence of membrane staining, the result is negative.

The performance characteristics of EGFR pharmDxTM make the visualization of four staining intensity levels possible from

0 to 3+. EGFR staining at the cellular level has been observed on both the membrane and in the cytoplasm at all intensity

levels.

Dako recommends the following interpretive approach in the assessment of EGFR Immunostaining.

All assessments are to be made on the tumor region of the specimen.

Steps to EGFR pharmDx™ Evaluation

1 Evaluate the control cell lines to validate the assay staining run.

Cell lines should not be used as an interpretive aid.

2 Evaluate the positive and negative tissue control slides to validate

proper tissue preparation, staining techniques and assay

performance.

3 An H&E stained slide of the test tissue is useful for comparison.

The neoplasm may not be easily appreciable on the EGFR pharmDxTM

stained slide.

4 Evaluate the EGFR-stained section for identification of neoplasm at

low power, 4x magnification. 3+ intensity staining of tumor cells is

easily identified at 4x magnification.

5 observe cells that stain brown, move to a higher power (10x

magnification) to confirm the staining. In general, most cases should

be obvious at 10x magnification.

6 If the staining pattern is an artifact, it should be disregarded for

evaluation. Find another representative area(s). Refer to page 17

for “Interpreting Artifacts.”

7 If membrane staining at 10x is not obvious, use 20x or 40x

magnification to assess staining pattern (membranous versus

cytoplasmic). Confirmation of EGFR pharmDxTM staining at 20x

magnification may be useful in those neoplasms with abundant

cytoplasmic staining. some tumors have several populations of

cells with different intensities of EGFR membrane staining.

8 If there is no tumor staining and normal elements are not staining,

review control slides of that staining run to confirm that appropriate

levels of EGFR expression are observed. If positive elements in the

control slides are negative, repeat the staining run.

Examples of Acceptable and Unacceptable HT-29 Positive Cell Line Staining with EGFR pharmDx™

Unacceptable weak staining intensity (too light); 20x magnification. Weak staining intensity of cell line may result in false-negative result.

��EGFR pharmDx™ Interpretation Manual

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Table �. EGFR pharmDx™ Pathology Report Form

Patient name_______________________________________ Collection Date _____________________________________

ordering Physician _________________________________ Received Date _____________________________________

ordering Facility ____________________________________ Report Date ________________________________________

Medical Record # ___________________________________ lab Reference # ____________________________________

speciman ID # _____________________________________ Tumor source ______________________________________

Date of birth _______________________________________ Patient Gender _____________________________________

Description

Patient Result

EGFR protein Positive Negative

EGFR pharmDxTM is indicated as an aid in identifying

colorectal cancer patients eligible for treatment with

Erbitux® (cetuximab) or VectibixTM (panitumumab).

EGFR pharmDx™ staining Results

These definitions of positive and negative results are in accord with published literature12, but may require modification in specific contexts.

Clinical Trials

several clinical trials of EFGR-targeted therapies

(cetuximab and panitumumab) have been performed.

Patients whose tumors had EFGR expression as

demonstrated using the Dako EFGR pharmDxTM assay

were eligible for study enrollment. The response rate

for EGFR-negative patients and patients with EGFR-

positive staining in less than one percent of tumor cells is

unknown as no such patients were present in the clinical

drug trials. Tumors with EGFR-positive staining in ≥1% of

their cells are considered EGFR expressing with regard

to the current EFGR-targeted therapy indications for use.

Deparaffinized tissue and appropriate control tissue

sections are stained using the FDA-approved Dako

EGFR pharmDxTM immunohistochemistry kit.

Tumors should be reported as EGFR positive or EGFR

negative using membrane staining as the evaluable

structure. A tumor cell is EGFR positive if it exhibits

any membrane staining above background, whether

or not it is completely circumferential. A tumor with no

membrane staining above background in any tumor

cell is reported as an EGFR-negative tumor.

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Report to Treating Physician Definition

EGFR-Negative Tumor Absence of membrane staining above background in all tumor cells.

EGFR-Positive Tumor EGFR-positive staining is defined as any IHC staining of tumor cell membranes above

background level; whether it is complete or incomplete circumferential staining.

Staining Intensity Percent of Tumor Cells Staining

1+, 2+, or 3+ >0%

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Image Guide for Interpretation

Colorectal adenocarcinoma, no staining, 0 staining intensity; 10x magnification.

Colorectal adenocarcinoma, no staining, 0 staining intensity; 20x magnification.

Colorectal adenocarcinoma, membrane staining, 1+ staining intensity; 20x magnification.






EGFR pharmDx™ Interpretation Guidelines

Dako emphasizes that interpretation of EGFR pharmDxTM should be performed within the context of the pathologist’s past

experience and best medical judgment. This guide will highlight examples of EGFR pharmDxTM positivity and negativity,

different staining intensities and areas of interpretation that are potentially problematic for EGFR pharmDxTM users.

Examples of EGFR pharmDx™ Staining Intensity Figure 7


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staining Patterns

Figure 8 Colorectal adenocarcinoma, example of heterogeneous positive staining; 10x magnification.

Figure 9Colorectal adenocarcinoma, example of leading edge heterogenous positive staining; 10x magnification.

Figure 10 Colorectal adenocarcinoma, example of homogeneous positive staining; 20x magnification.

The Dako EGFR pharmDxTM stains a variety of normal and neoplastic tissues. observed EGFR staining patterns are

heterogeneous or homogeneous depending on the tissue and/or tumor type.

Heterogeneous Staining

Heterogeneity includes various staining intensities within

a single neoplasm. The staining can show a range of

0–3+ staining intensity. Cell staining patterns can also be

heterogeneous, including membrane staining, cytoplasmic

staining or both.

Homogeneous Staining

Cancers with EGFR protein expression can also exhibit

homogeneous staining patterns.


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Figure 11 Colon, example of perineural fibroblast staining; 40x magnification. Perineural fibroblasts are found in most tissues and serve as a good internal positive control.

normal and benign tissues, some of which are

summarized in Table 1, exhibit specific EGFR staining.

These can serve as useful internal controls. staining of

normal tissue elements should be excluded from the

EGFR pharmDxTM evaluation.

EGFR Staining of Normal and Benign Tissues

EGFR is expressed in a variety of normal cells.

These include but are not limited to:

n esophageal squamous epithelial cells

n hepatocytes

n mesothelial cells

n fibroblasts

n tonsillar and cervical squamous epithelial cells

n ductal and myoepithelial cells of the breast

n pulmonary alveolar epithelial and bronchial basal cells

n prostatic luminal cells

n ductal elements of the salivary gland

n endometrial gland and stromal cells

n normal colonic surface epithelial cells may or may not display weak positive staining of EGFR

n smooth muscle cells


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Figure 12 Colorectal adenocarcinoma infiltrating the liver. Example of positively stained normal hepatocytes and negative tumor cells; 10x magnification. The sample is negative.

Figure 15 Needle biopsy from liver exhibiting strong tumor staining; 10x magnification. The sample is positive.


Needle Biopsy from LiverHepatocyte Staining in the Liver



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Factors to Consider in Evaluation of EGFR pharmDx™ stains

Figure 16 Colorectal adenocarcinoma, example of endogeneous peroxidase staining in granulocytes; 20x magnification. The sample is negative.

Non-specific Background

non-specific background staining is defined as diffuse,

non-specific staining of tissue elements. It may be caused

by a variety of factors including both biologic activity and

technological processes.

Possible Causes of Non-specific Background

Biologic Activity

n Pseudoperoxidase activity (erythrocytes) and endogenous peroxidase activity (granulocytes) may result in non-specific background staining due to decomposition of H2o2 within the substrate.

Technological Processes

n use of fixatives other than those recommended (see page 7)

n Incomplete deparaffinization of tissue sections prior to staining

n use of alternative buffers (use kit-supplied buffer or equivalent wash buffer, code s3006)

n Incomplete rinsing of reagents from slides

n Inappropriate drying of slides during staining procedure (use a humid chamber when assay is performed manually)

Staining of Granulocytes


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TsFigure 18 Colorectal adenocarcinoma, example of lymphoid nodule overdigested; 10x magnification.

Figure 19 Colorectal adenocarcinoma, example of lymphoid nodule overdigested; 20x magnification.

Figure 17 Colorectal adenocarcinoma, example of overdigestion; 20x magnification.

Interpreting Artifacts

Overdigestion

EGFR pharmDxTM includes pretreatment by means of a

proteolytic enzyme digestion step. Tissue sections may

occasionally be overdigested, causing disruption of cell

membranes and overall tissue architecture. Run the assay

with careful attention to the duration of the proteolytic

digestion step.

Note

If overdigestion is a persistent problem with tissues fixed

in 10% (v/v) neutral buffered formalin, tissue sections may

be post-fixed in 10% (v/v) neutral buffered formalin for 10

minutes after deparaffinization. see procedure right:

Post-Fixation Procedure(Validated for tissues originally fixed in 10% (v/v) nbF)

1. Deparaffinize sections and immerse in reagent quality water.

2. Immerse slides in a 10 percent neutral buffered formalin for 10 minutes.

3. Rinse slides twice in deionized or distilled water.

4. Continue with the EGFR pharmDxTM staining procedure.

Crush Artifact

Compression of tissue around the periphery of specimens,

especially biopsy specimens can produce non-specific

staining of the tissue components in addition to the

membranes of neoplastic cells. The appearance of this

staining artifact is similar to those produced by tissue

edge staining artifacts.

The staining intensity of cell membranes within compressed

tissue is frequently greater than similar appearing cells in

regions of normal architectural tissue arrangements.


Figure 20 Colorectal adenocarcinoma, example of edge artifact; 10x magnification.

Edge Artifact

Frequently, increased staining is observed around

the periphery of the tissue specimen, known as the

“edge effect.” Edge artifacts are commonly the result

of inappropriate pre-analytic handling of the tissue. The

edge effect represents fixation artifact or tissue drying

prior to fixation. usually the staining artifact is limited to a

thin rim of stained cells with an abrupt termination to the

staining reaction. often the method of surgical extraction

is the cause (see Crushing Artifact section).

Tissue section edge staining artifacts are common if there

are significant tissue section irregularities. Thick tissue

sections may mimic edge artifacts and can be corrected

by recutting the tissue block to produce a uniform, thin,

3-5 µm thick section that is devoid of folds and wrinkles.

When the peripheral positive reaction is only at the edge

of the tissue section, evaluation of EGFR staining should

exclude tumor cells within the region’s edge artifact.

Retraction Artifact

stromal retraction around tumor cell glands can create

clefts where pooled antibody can non-specifically stain.

Thorough washing after the primary antibody incubation

step may prevent this reaction. Retraction space staining

manifests itself as a hemicircumferential reaction around

the periphery of the gland.

Thermal Artifact

Thermal electrocautery may alter nuclei and cell

membranes. Dako recommends that the evaluation

of EGFR pharmDxTM staining be performed on tissue with

no or minimal thermal electrocautery artifacts.

Non-Evaluable Areas of Tissue

Areas of stained slides that should not be evaluated include

dissociated, free-floating groups or aggregates of neoplastic

cells, necrotic cells, and damaged areas of the tissue

section (torn sections, folded or wrinkled areas, etc.).

AR

TIFA

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Examples of Cancer stained with EGFR pharmDx™

Colorectal Cancer

Figure 25 Colorectal adenocarcinoma, example of H&E; 40x magnification.


Figure 24 Colorectal adenocarcinoma, example of negative staining; 20x magnification.

Figure 21 Colorectal adenocarcinoma, example of poorly differentiated carcinoma; 2+ staining intensity; 20x magnification.

Figure 22 Colorectal adenocarcinoma, example of well-differentiated carcinoma; 3+ staining intensity; 10x magnification.

Examples of Negative EGFR pharmDx™ Immunostaining

EGFR pharmDx™ Interpretation Manual ��

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Figure 26Colorectal adenocarcinoma, example of negative staining; 40x magnification.

�0 EGFR pharmDx™ Interpretation Manual

Colorectal Cancer

Figure 32 Colorectal adenocarcinoma, example of weak membrane staining, 1+ staining intensity; 20x magnification.


Figure 29 Colorectal adenocarcinoma, example of cytoplasmic staining with no membrane staining; 20x magnification. EGFR positive cells can be observed to exhibit cytoplasmic and membrane immunostaining. When only cytosplasmic staining is present the immunostaining is interpreted as negative.

Figure 30 Colorectal adenocarcinoma, example of cytoplasmic staining with no membrane staining; 20x magnification. EGFR positive cells can be observed to exhibit cytoplasmic and membrane immunostaining. When only cytosplasmic staining is present the immunostaining is scored as negative.

Examples of Weak to Moderate EGFR Immunostaining



Co

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Figure 36 Colorectal adenocarcinoma, example of weak to moderate positive membrane staining, 2+ staining intensity; 40x magnification.



Figure 38 Colorectal adenocarcinoma, example of moderate positive membrane staining, 2+ staining intensity; 40x magnification.


Figure 34 Colorectal adenocarcinoma, example of weak positive staining, 1+ staining intensity; 40x magnification.

Colorectal Cancer


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Figure 40 Colorectal adenocarcinoma, example of strong positive membrane staining, 3+ staining intensity; 20x magnification.





Colorectal Cancer

Examples of Moderate to Strong EGFR Immunostaining



IMM

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EGFR pharmDx™ Immunostaining in a Variety of solid Tumors

EGFR is expressed in a number of solid tumors.

The EGFR pharmDxTM assay is a qualitative

immunohistochemical (IHC) kit system useful in

identifying epidermal growth factor receptor expression

in normal and neoplastic tissues routinely fixed for

histological evaluation.

Results from tissue specimens stained using EGFR

pharmDxTM for purposes other than Erbitux® or VectibixTM

assessment have no known clinical utility.


REF

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References

1. sliwkowski MX, lofgren JA, lewis GD, et al. nonclinical studies addressing the mechanism of action of Trastuzumab (Herceptin). 1999; seminoncol 26:60-70, (suppl 12).

2. Harari PM, Huang s-M. Modulation of Molecular Targets to Enhance Radiation. 2000; Clin Cancer Res 6:323.

3. Pii K, Andersen FG, Jensen s, spaulding b. Characterization of a new monoclonal antibody, clone 2-18C9, for the measurement of Epidermal Growth Factor Receptor expression in solid tumors. Am Assoc Canc Res 95th annual meeting, orlando, Fl Mar 27–31 2004; Abst #5029.

4. Coussens l, yang-Feng Tl, liao y-C, Chen E, Gray A, McGrath, J seeburg PH, libermann TA, schlessinger J, Francke u, levinson A, ullrich A. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. science 1985; 230: 1132.

5. yamamoto T, Ikawa s, Akiyama T, semba K, nomura n, Miyajima n, saito T, Toyoshima K. similarity of protein encoded by the human c-erb-b-2 gene to epidermal growth factor receptor. nature 1986; 319:230.

6. schechter Al, Hung M-C, Vaidyanathan l, Weinberg RA, yang-Feng Tl, Francke u, ullrich A, Coussens l, The neu gene: An erbb-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. science 1985; 229:976.

7. Gusterson b, Cowley G, smith JA, ozanne b. Cellular localisation of human epidermal growth factor receptor. Cell biol Intl Rpts 1984; 8(8):649.

8. Gullick WJ. Prevalence of aberrant expression of the epidermal growth factor receptor in human cancers. br Eed bull 1991; 47(1):87.

9. sainsbury JRC, Farndon JR, sherbet GV, Harris Al. Epidermal-growth-factor receptors and oestrogen receptors in human breast cancer. lancet 1985; 1(8425):364.

10. ozanne b, Richards Cs, Hendler F, burns D, Gusterson b. over-expression of the EGF receptor is a hallmark of squamous cell carcinomas. J Pathol 1986; 149:9.

11. Werner MH, nanney lb, stoscheck CM, King lE. localization of immunoreactive epidermal growth factor receptors in human nervous system. J Histochem Cytochem 1988; 36:81.

12. Atkins D, Reiffen KA, Tegtmeier Cl, Winther H, bonato Ms and störkel s. Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: Variation in staining intensity due to choice of fixative and storage time of tissue sections. J Histochem Cytochem 2004; 52:893.

Additional EGFR Resources

n Hong WK, ullrich A. The role of EGFR in solid tumors and implications for therapy. onColoGy bIoTHERAPEuTICs, Volume 1, number 1, 2000.

n Cunningham D, Humblet y, siena s, Khayat D, bleiberg H, santoro A, bets D, Mueser M, Harstick A, Van Custem E. Cetuximab (Erbitux®) in combination with irinotecan or as single agent in patients with EGFR-expressing, irinotecan-refractory metastatic colorectal cancer (study EMR 62202-007 ‘bonD’). AsCo Abstract no. 1012, 2003.

n Mendelsohn J. Epidermal Growth Factor Receptor Inhibition by a Monoclonal Antibody as Anticancer Therapy. Clinical Cancer Research 1997; Vol. 3; 2703-2707.

n ogiso H, Ishitani R, nureki o, Fukai s, yamanaka M, Kim JH, saito K, sakamoto A, Inoue M, shirouzu M, yokoyama s. Crystal structure of the Complex of Human Epidermal Growth Factor and Receptor Extracellular Domains. Cell 2002; Vol. 110; 775-787.

n Erbitux® (cetuximab) Package Insert

n VectibixTM (panitumumab) Package Insert

n EGFR pharmDx™ Package Insert

n spaulding DC, spaulding bo. Epidermal Growth Factor Receptor Expression and Measurement in solid Tumors. seminars in oncology 2002; Vol 29, number 5, suppl 14 (october); 45-54.

n nahta R, Hortobagyi Gn, Esteva FJ. Growth Factor Receptors in breast Cancer: Potential for Therapeutic Intervention. The oncologist 2003; 8:5-17.

n nicholson RI, Gee JMW, Harper ME. EGFR and cancer prognosis. European Journal of Cancer 2001; 37: s9-s15.

n Ciardiello F, Tortora G. Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. European Journal of Cancer 2003; 39: 1348-1354.

n Artegea Cl, baselga J. Clinical Trial Design and End Points for Epidermal Growth Factor Receptor-targeted Therapies: Implications for Drug Development and Practice. Clinical Cancer Research 2003; Vol 9, 1579-1589.

n Goldstein ns, Armin M. Epidermal Growth Factor Receptor Immunohistochemical Reactivity in Patients with American Joint Committee on Cancer stage IV Colon Adenocarcinoma. CAnCER 2001; Volume 92, number 5; 1331-1346.

Acknowledgements

Dr. neal Goldstein, Dr. James Thompson, Dr. Kenneth bloom, Prof. stephan störkel and betsy spaulding contributed by offering their expertise and many images that can be seen throughout this manual.

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