EvaluationEvaluation

of ER/PR of ER/PR

ThroughThrough

ImmunohistochemImmunohistochemistryistry

NUDRAT JAWEDNUDRAT JAWEDBSMTBSMT

33RDRD YEAR, 5 YEAR, 5THTH SEMESTER SEMESTER

Contents

Hormone Receptors Why ER/PR Testing? Estrogen Receptors Progesterone Receptors Immunohistochemistry Procedure Of IHC Factors Affecting The Technique Conclusion

HORMONE RECEPTORS

It is a membrane receptor protein or in its interior that binds to a specific hormone.

The hormone causes many changes takes place in the cell.

Binding of hormone to its receptor often triggers the start of a biophysical signal that can lead to a further signal transduction pathways or triggers the activation or inhibition of genes.

Why ER/PR testing ?

Prognostic Factor Predictive Factor ER protein expressed in 70-80% invasive breast

carcinomas. Rate of expression increase with patient age. Less tumor tissue is required for ER/PR evaluation. Assays can be performed on archival materials. Exact location of ER/PR positive cells can be

determined (i.e. tumor Vs normal breast tissue). ER/PR positivity is associated with increased response

to antiestrogen therapies.

Estrogen Receptors

Estrogen regulates growth kinetics of epithelial linings (Breast and Endometrium).

It binds to ER (Cellular Proteins) directly stimulating proliferation and differentiation.

ER translocates the nucleus, where it can bind to promoter sites and thus regulates the expression of many other genes .

ER may be the best example of a tumor bio-marker.

Types

ERα

ERβ

Both ERα & ERβ have a highly homologous structure.

The Co-expression ERα, ERβ and PR as well as its association with indicators of low biological aggressiveness suggest that ERβ positive tumors are likely to respond to hormonal therapy.

Progesterone Receptor

Estrogen mediates part of its proliferative action of normal breast through transactivation of PR.

Progesterone is also a mitogenic stimulus for mammary epithelium.

PRs are ligand activated transcription factor members of the steroid hormone family of nuclear receptors.

In breast cancer, total PR levels are routinely measured as a guide to hormone therapy and as marker of disease prognosis together with ER.

It has two isoformsPR-A PR-B

IHC is a technique, used to identify cellular or tissue Ag with the help of Ab.

This technique is based on immuno-reactivity of Ab and chemical properties and enzyme complexes which reacts with colorless substrate (Chromogen) to produce colored end product.

Site of Ag-Ab complex is identified by direct labeling or secondary labeling method.

Now this technique is used for a wide variety of Ag. Staining not only makes sophisticated the diagnosis but also

provide valuable information about pathogenesis of particular disease.

IHC made diagnosis easier and less expensive as compare to other technique. It has become an essential part of surgical pathology.

Envision System

It is two step IHC technique. Based on advance Labeled Streptavidin-biotin (LSAB)

method. HRP-labeled polymer conjugate with secondary Ab. Extremely sensitive than other IHC techniques. It can easily detect Ag present in low concentration.

Introduction

This section shows the step by step Envision (DAKO) staining

procedure using Estrogen Receptor (ER) antibody

on Breast carcinoma.

Step by Step Envision Staining Method

Deparaffinization and Rehydration

paraffin wax coated slide

Remove paraffin wax and hydrate tissue section.

SPECIME

N

LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

Remove paraffin wax and hydrate tissue section.

Deparaffinization and Rehydration

tissue section

SPECIME

N

LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

Boiling for 50 minutes using citrate buffer

solution pH 6.0.

Rinse

Deparaffinization and Rehydration

Antigen Retrieval

SPECIME

N

LN

H20

09-0

13

ER

/PR

SPECIMEN

LN

H2

00

9-

01

3ER

/PR

Step by Step Envision Staining Method

Rinse in distilled water and wash 2 times in PBS buffer.

Deparaffinization and Rehydration

Antigen RetrievalRinse

PBS

Buffer

SPECIME

N

LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

3% hydrogen peroxide solution for 10 minutes to

inactivate endogenous peroxidase activity.

3%

Hydro

gen

Pero

xide

SPECIMEN

LN

H20

09-0

13

ER

/PR

Rinse

Deparaffinization and Rehydration

Antigen Retrieval

Block Endogenous Peroxidase

Step by Step Envision Staining Method

Rinse

Deparaffinization and Rehydration

Antigen Retrieval

Block Endogenous PeroxidaseRinse

Thorough rinse in distilled water and wash 2 times in PBS buffer.

SPECIME

N LN

H20

09-0

13

ER

/PR

PBS

Buffer

Step by Step Envision Staining Method

Optimally diluted ER antibody for 50 minutes.

SPECIME

N LN

H20

09-0

13

ER

/PR

ER

Antib

ody

Deparaffinization and RehydrationAntigen Retrieval

Block Endogenous PeroxidasePrimary Antibody

Rinse

Rinse

Step by Step Envision Staining Method

Deparaffinization and Rehydration

Antigen Retrieval

Block Endogenous Peroxidase

Primary Antibody

Rinse

Rinse

Rinse

Wash 2 times in PBS buffer.

PBS

Buffer

SPECIME

N LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

Biotinylated link antibody for 50 minutes.

Link

Antib

ody

Deparaffinization and RehydrationAntigen Retrieval

Block Endogenous PeroxidasePrimary Antibody

Rinse

Rinse

Rinse Biotinylated Secondary Antibody

Rinse

SPECIME

N LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

Deparaffinization and Rehydration

Antigen Retrieval

Block Endogenous Peroxidase

Primary Antibody

Biotinylated Secondary Antibody

Rinse

Rinse

Rinse

Rinse

Wash 2 times in PBS buffer.

PBS

Buffer

SPECIME

N LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

Deparaffinization and Rehydration

Streptavidin HRP

Antigen Retrieval

Block Endogenous Peroxidase

Primary Antibody

Biotinylated Secondary Antibody

Rinse

Rinse

Rinse

Rinse

HRP conjugated streptavidin for 50 minutes.

HRP

Stre

ptav

idin

SPECIME

N LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

Rinse

Wash 2 times in PBS buffer.

PBS

Buffer

SPECIME

N LN

H20

09-0

13

ER

/PR

Deparaffinization and Rehydration

Streptavidin HRP

Antigen Retrieval

Block Endogenous Peroxidase

Primary Antibody

Biotinylated Secondary Antibody

Rinse

Rinse

Rinse

Rinse

Step by Step Envision Staining Method

DAB Chromogen

Deparaffinization and Rehydration

Antigen Retrieval

Block Endogenous Peroxidase

Primary Antibody

Biotinylated Secondary Antibody

Streptavidin HRP

Rinse

Rinse

Rinse

Rinse

Rinse

DAB chromogen for 5 minutes.

DAB

Chrom

ogen

SPECIME

N LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

Wash 2 times in PBS buffer and rinse in distilled water.

Deparaffinization and Rehydration

Antigen Retrieval

Block Endogenous Peroxidase

Primary Antibody

Biotinylated Secondary Antibody

Streptavidin HRP

DAB Chromogen

Rinse

Rinse

Rinse

Rinse

Rinse

Rinse

Distilled

Wat

er

SPECIME

N

LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

Counterstain in Harris’s Hematoxylin for 1 minute.

Hematoxylin Counterstain

Hem

atox

yli

n

Harris

’s

Hemat

oxylin

Deparaffinization and Rehydration

Antigen Retrieval

Block Endogenous Peroxidase

Primary Antibody

Biotinylated Secondary Antibody

Streptavidin HRP

DAB Chromogen

Rinse

Rinse

Rinse

Rinse

Rinse

Rinse

SPECIME

N

LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

Thorough wash, 1 Dip in Ammonia water again washing in tap water to “blue” the nuclei.

Hematoxylin Counterstain

Deparaffinization and Rehydration

Antigen Retrieval

Block Endogenous Peroxidase

Primary Antibody

Biotinylated Secondary Antibody

Streptavidin HRP

DAB Chromogen

Rinse

Rinse

Rinse

Rinse

Rinse

Rinse

Rinse

Tap

Wat

er

SPECIME

N

LN

H20

09-0

13

ER

/PR

Step by Step Envision Staining Method

Dehydrate and Coverslip with aqueous resins.

Deparaffinization and Rehydration

Antigen Retrieval

Block Endogenous Peroxidase

Primary Antibody

Biotinylated Secondary Antibody

Streptavidin HRP

DAB Chromogen

Rinse

Rinse

Rinse

Rinse

Rinse

Rinse

Hematoxylin CounterstainRinse

Aqueous Mount

DPX

SPECIME

N

LN

H20

09-0

13

ER

/PR

FACTORS AFFECTING THE TECHNIQUE

Pre-Analytical Variables Fixation Time

Min. 6-8 hrs Max. 48 hrs• Goldstein et al (Am J Clin Pathol 120:86-92, 2003)• ASCO-CAP Guidlines

Fixation should start < 30 min after surgical removal.

Fixative Type10% Neutral Buffered Formalin is Highly Recommended

• Ab reagents and IHC methodologies have been optimised for formalin fixed tissue.

Contd.

Analytical Variables Automated Vs Manual Procedure Antibody and Titre

• Primary Ab Clones• Mouse Monolonal 1D11, 1D5

• Rabbit Polyclonal• Rabbit Monoclonal SP1

• Concentrate Vs RTU Ag Retrieval

• HIER(Heat Induce Epitope Retrieval)• Enzyme Methodology

Detection Kit (Secondary Ab)

INTERPRETATION

Assess the invasive tumor only Staining Pattern Intensity, % of Positive cells Internal Negative Control Positive Control Pit-falls: Edge Effect, Retraction

Artifact.

QC/QA Measures

Controls are essential, use with each run. Use internal control (Non-neoplastic epithelium). Use External control (Positive Cancer with non-

neoplastic epithelium, ideally with high and low levels of ER)

Commercial controls, Cell lines available. Routine audits of results

Conclusion

ER/PR through IHC is widely validated as a predictive factor in breast cancer.

IHC reaction is influenced by tissue fixation and processing.

Retrieval may improve IHC but it does not recover tissue from autolysis or overfixation.

The choice of primary Ab, New rabbit monoclonal antibodies are commercially available.

New Detection system available with accurate and nonspecfic staining.

A quality control of pre-analytical, analytical and post analytical phases of IHC is recommended in order to optimize results.