#1034 - best practices for ihc detection and ... · correlation of her2 status with er and pr and...
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#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
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.