#1034 - Best Practices for IHC Detection and Interpretation of

ER, PR, and HER2 Protein Overexpression in Breast

Cancer

Richard W. Cartun, MS, PhD Andrew Ricci, Jr, MD

Department of Pathology Hartford Hospital

Hartford, CT 06102 (860) 545-1596

Richard.Cartun@hhchealth.org

Speaker Disclosure In the past 12 months, I have not had a significant financial interest or other relationship

with the manufacturer(s) of the product(s) or provider(s) of the

service(s) that will be discussed in my presentation.

Hartford Hospital

Hartford, Connecticut

Patient treatment for breast cancer depends on:

• Histologic type and grade • Tumor size • Lymph node status • Estrogen (ER) and progesterone

receptor (PR) status • HER-2/neu status (protein

overexpression and/or gene amplification)

• Gene expression testing (Oncotype DX)

This image cannot currently be displayed.

Do we have a problem with IHC predictive marker testing?

• Problems with ER and PR testing conducted between 1997 and 2005 in the Newfoundland and Labrador (Canada) health care systems.

• Repeat IHC testing performed by central testing laboratories in the United States has shown poor concordance with results from community hospitals (especially with HER2 testing).

Is there a problem with IHC predictive marker testing? (cont.)

• Unsatisfactory results with proficiency testing

programs in the United States. • Genomic Health (Oncotype DX) now reports

ER, PR, and HER2 scores because some oncologists don’t trust results from their own pathology laboratories (or laboratories at other medical institutions).

Invasive Breast Carcinoma

Estrogen Receptor Protein

Breast Cancer Consultation Case - HER2 Positive?

Outside HER2 IHC Repeat HER2 IHC

Ductal carcinoma in situ

“3+” HER2 protein overexpression

IHC Assay “Total Test Concept” • Preanalytic

Specimen type, acquisition, transport time Fixation (type and time) Tissue processing, type, and temperature Tissue sectioning

• Analytic Antigen retrieval Testing protocol, control selection Reagent validation Technical staff training/certification Laboratory certification

• Postanalytic Control evaluation Interpretation of results Reporting of results Pathologist, experience, and CME

Taylor CR: Arch Pathol Lab Med 2000;124;945-951

Should we be doing ER, PR, and HER2 IHC testing on

diagnostic biopsy tissue or wait for the excisional specimen?

HER2 Heterogeneity

HER2 IHC (-) clone HER2 IHC (+) clone

Khoury T, Sait S, Hwang H, et al.: Delay to formalin fixation

effect on breast biomarkers. Mod Pathol 2009;22:1457-1467.

“We recommend not to delay formalin fixation for more than 1 hour and do not store specimens

overnight at 4 degrees C.”

Goldstein NS, Hewitt SM, Taylor CR, et al.: Recommendations for improved standardization of immunohistochemistry. Appl

Immunohistochem Mol Morphol 2007;15:124-133.

“Tissue should be fixed in 10% neutral-buffered formalin.”

Inadequate Fixation

Yes, we can re-process tissue, but ......

Wolff AC, Hammond ME, Schwartz JN, et al.: American Society of Clinical

Oncology/College of American Pathologists guideline recommendations

for human epidermal growth factor 2 testing in breast cancer. Arch Pathol

Lab Med 2007;131:18-43 “… breast tissue should be

fixed for a minimum of 6 hours and no longer than 48 hours”

Arber D: Effect of prolonged formalin fixation on the

immunohistochemical reactivity of breast markers. Appl

Immunohistochem Mol Morphol 2002;10:183-186.

“The IHC reactivity of some breast prognostic markers is reduced, but

only after extensive fixation that may not be clinically relevant.”

Tong LC, Nelson N, Tsourigiannis J, et al.: The effect of prolonged

fixation on the IHC evaluation of ER, PR, and HER2 expression in

invasive breast cancer: A prospective study. Am J Surg Pathol

2011;35:545-552. “… fixation for limited periods beyond 72 hours does not result in a reduction in assay sensitivity in the determination

of ER, PR, or HER2 IHC status.”

HER-2/neu protein overexpression

48 Hours fixation 16 Days fixation

Minimum Fixation Time for Needle Core Biopsy Specimens?

ER SMM

(4.5 Hours)

Maximum Fixation Time?

HER2 IHC HER2 FISH

(1.5 Years)

Hartford Hospital Fixation Policy • Needle core biopsies must be fixed for a

minimum of 4.5 hours. This includes 0.5 hours pre-processor fixation and 4.0 hours of fixation on the tissue processor.

• All other specimens must be fixed for a minimum of 6 hours.

• Excisional specimens arriving after 3:30 PM are held for overnight fixation (following slicing).

Documentation of Fixation Time

Documentation of “Formalin Contact Time”

“Best Practices” for Breast Biospecimen Collection

• Minimize “cold” ischemic time to under one hour for excisional specimens.

• Do not allow the tissue to dry out. • Fixation in 10% buffered formalin (1:10 ratio). • Slice large specimens to facilitate fixation ASAP

(hold for overnight fixation if needed). • Submit gross sections no more than 2-3 mm in

thickness. • Document “cold” ischemic time and formalin

contact time, and total time in formalin (if possible).

Needle Core Biopsy #2

“Uniform Immunoreactivity”

Needle Core Biopsy #1

“Edge Effect” Due To Drying

HER2 Protein Overexpression

Center Surface

(tissue fixed in formalin 8 hours)

Fixation Problem?

Estrogen Receptor Protein

No, tissue sectioning problem.

Estrogen Receptor Protein

Breast CA - Core Biopsy

Estrogen Receptor Protein

Tissue Sectioning Recommendations:

• 4-5 Microns in thickness • Sections should be placed in the center of

the slide (not on the “+” or “x”) • Multiple sections for biopsy specimens • Orientation (identical if possible) • Make sure slides are dry before starting

IHC testing • Always verify patient identification and

block designation • “No oven” for unstained slides send-out

Slide Protocol for Diagnostic Biopsy Specimens

• Slide 1 - H&E • Slide 2 - Unstain • Slide 3 - Unstain • Slide 4 - H&E • Slide 5 - Unstain • Slide 6 - Unstain • Slide 7 - H&E

Should “stored” unstained slides be used for IHC testing?

Estrogen Receptor Protein

“Freshly Cut” Unstained Slides

Estrogen Receptor Protein

Xie R, Chung J-Y, Ylaya K, et al.: Factors influencing the degradation of

archival formalin-fixed paraffin-embedded tissue sections. J Histochem

Cytochem 2011;59:356-365.

“… the presence of water, both endogenously and exogenously, plays a

central role in antigenicity loss.”

Shintaku IP and Said JW: Detection of ER with mAbs in routinely

processed formalin-fixed paraffin sections of breast CA; use of a

DNase pretreatment to enhance sensitivity of the reaction. Am J Clin

Pathol 1987;87:161-167. “This method offers a reliable and

reproducible alternative when tissue is not suitable or unavailable for DCC or

frozen tissue analysis……”

ER Detection (clone H222)

ER-ICA Breast CA - 1993

Non-neoplastic Breast Tissue

H&E ER

Invasive Duct Carcinoma

H&E ER

Immunohistochemical Testing at Hartford Hospital:

• All studies performed on Leica Biosystems’ Bond III automated IHC/ISH platform

• Bond Polymer Refine detection • DAB chromogen • Hematoxylin counterstain (off-line) • ER (mouse mAb clone 6F11) - Leica • PR (mouse mAb clone 16) - Leica • HER2 (rabbit mAb clone EP3) - Epitomics

ER - Clone 6F11

ER - Clone 6F11

PR - Clone 16

PR - Clone 16

HER2 - Clone EP3

HER2 - Clone EP3

Progesterone Receptor mAbs

Clone PgR636 Clone 16

HER2 clone EP3

Outside Hospital Consult

ER clone 6F11

“Personalized Antigen Retrieval” • There is no one retrieval method or time

that is optimal for all antibodies and tissues.

• Control tissues may be “over-fixed” and, as a result, require more aggressive retrieval to provide optimal reactivity.

• Reduce time for small specimens. • Reduce or increase time for specimens

from other hospitals/laboratories.

Antibody Validation: • Clinical response (unlikely to get this

information from clinicians) • Morphology and grade (low-grade tumors

are ER+ and most ER- tumors are high-grade)

• Most HER2+ tumors are high-grade • Compare results with other technologies

(FISH for HER2 and Oncotype DX for ER, PR, and HER2)

• Interlaboratory comparisons and PT useful

Fitzgibbons PL, Murphy DA, Hammond MEH, et al.:

Recommendations for validating ER and PR

immunohistochemistry assays. Arch Pathol Lab Med 2010;134:930-935.

“… will improve the accuracy of hormone-receptor testing, reduce

interlaboratory variation, and minimize false-positive and false

negative results.”

ER, PR, and HER2 in Breast CA

• ER is expressed in 70% to 95% of invasive lobular carcinomas and in 70% to 80% of invasive ductal carcinomas

• PR is expressed in 60% to 70% of invasive breast carcinomas

• HER2 is overexpressed and/or amplified in 15% to 25% of invasive breast carcinomas

• “Antibodies, detection systems, and interpretation guidelines with affect these numbers”

Validating ER/PR IHC Results

“ER stronger than PR by IHC”

Oncotype DX Test

Hammond MEH, Hayes DF, Dowsett M, et al.: American Society of Clinical

Oncology/College of American Pathologists guideline

recommendations for estrogen and progesterone receptors in breast

cancer. Arch Pathol Lab Med 2010;134:907-922.

Guideline Recommendations • Positive for ER or PR if ≥ 1% of tumor

cell nuclei are immunoreactive • Specimen unsatisfactory if no tumor nuclei

are immunoreactive and no internal positive control present

• Large, preferably multiple core biopsies of tumor are preferred

• Samples for ER and PR testing are fixed in NBF for 6 to 72 hours

Breast CA - ER “Weak Positive”

Estrogen Receptor Protein (clone 6F11)

“Best Practices” for Interpretation:

• Print a copy of the pathology report (confirm case number/patient name and block designation; compare morphology).

• Evaluate all tissue fragments on biopsy specimens.

• Look for an internal positive control for ER/PR (benign breast tissue) if tumor cells are negative.

• Use LMW-CK (clone 5D3) to identify invasive tumor cells if not readily identified.

“Best Practices” for Interpretation:

• Use myoepithelial markers (SMM, Calponin, or p63) to confirm invasive tumor.

• Score % of positive invasive tumor cells and their intensity (weak, moderate, or strong); mark score on slide (record).

• Recommend repeat studies on excisional tumor when the biopsy specimen shows little tumor; ER/PR are negative and there is no internal control; or biopsy specimen shows a “Triple-Negative” immunoprofile.

Internal Positive Control

ER (clone 6F11)

Internal Positive Control

HER2 clone EP3

Allred DC, Harvey JM, Berardo M: Prognostic and predictive factors in breast

cancer by immunhistochemical analysis. Mod Pathol 1998;11:155-168.

“Allred Scoring System”

Allred Scoring System

Total Score (TS):

• Negative (0 or 2) • Positive (3 and higher)

• Determined by cutpoint analysis of

disease-free survival (DFS) in a study involving more than 1,900 patients separated into low and high risk groups (Clark GM, et al., Proc Am Soc Clin Oncol 1997;16:129A)

ER - 10x

5 + 3 = 8/8

PR - 10x

5 + 1-3 = 6-8/8

Fitzgibbons PL, Murphy DA, Hammond EH, et al:

Recommendations for validating estrogen and progesterone

receptor immunohistochemical assays. Arch Pathol Lab Med

2010;134:930-935. “all laboratories with validated ER and PgR IHC assays must periodically reassess the

assays to ensure that their analytic sensitivity has not drifted.”

Published Benchmarks (ER/PR):

• For women over 65 years of age, the % of negative cases should not exceed 20%

• For low-grade invasive carcinomas, the proportion of negative cases should not exceed 5%

• If the proportion of negative cases exceeds these rates, investigation is warranted.

For those of you performing IHC detection of HER2 protein

overexpression, what is your HER2 positive rate?

Ross J: Saving lives with accurate HER2 testing. Am J Clin Pathol 2010;134:183-184. “A number of experts in the field have

now agreed that a laboratory performing HER2 testing in the US patient population should have a

HER2+ rate of approximately 16% with a range of 12% to 20%.”

Lal P, Tan LK, Chen B: Correlation of HER2 status with

ER and PR and histologic features in 3,655 invasive breast

carcinomas. AM J Clin Pathol 2005;123:541-546

“Studied the inverse relationship between HER2 and ER and PR, and

correlated HER2 status with histologic features in 3,655 unselected invasive

breast carcinomas.”

Findings:

• Overall ER expression rate was 74% • Overall PR expression rate was 49% • Overall HER2 positive rate was 16% • HER2 was positive in 11% of grade 2 and

28% of grade 3 ductal carcinomas and negative in all grade 1 ductal carcinomas

• Only 3 of 357 (0.8%) lobular carcinomas were positive for HER2 (pleomorphic)

CLP/Hartford Hospital Predictive IHC Testing 2011-Present

• Case number • Block designation • Formalin contact time • Patient age • ER • PR • HER2

• FISH • Oncotype DX • Previous specimen • Comments

– Repeat on excision – Immunoprofile

confirmed – S/P neoadjuvant

chemotherapy – Treated with Herceptin

Pleural Fluid Metastatic Breast CA

H&E Direct Smear Cell Block ER-6F11

Cell Block Preparation

• Collect specimen in “RPMI or saline” • Centrifuge specimen • Pour-off supernatant • Add 4-5 drops of plasma to sediment; mix • Add 2-3 drops of thrombin (5,000 IU); mix • Mixture should clot within one minute • Add formalin (divide if necessary) • Wrap in filter paper and process

HER2 Detection in FNA Cytology

Cytology FNA vs. Formalin-Fixed Tissue ?

Decalcified Bone Marrow Core Biopsy

ER clone 6F11

The successful IHC detection and interpreation of ER, PR, and HER2 in breast cancer

occurs when there is a “Partnership” between breast

surgeons, radiologists, oncologists, and, most

importantly, the pathology staff.