American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer

Abstract

ASCO and CAP collaborated to produce an evidence-based guideline on estrogen and progesterone receptor testing in breast cancer to produce optimal testing performance.

Introduction

ASCO and the College of American Pathologists (CAP) collaborated to produce an evidence-based guideline on optimal testing performance for estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer.^1,2 The purpose of the guideline is to improve the accuracy of immunohistochemical (IHC) testing and its utility in determining predictive markers for breast cancer treatment. The ASCO/CAP Hormone Receptor Testing in Breast Cancer Panel was composed of experts in pathology, medical oncology, laboratory medicine, laboratory regulation, and biochemistry as well as a patient advocate.

Overview

Testing for the presence of hormone receptors as part of breast cancer diagnosis is the standard of care and is used to guide therapy decisions. ER/PgR overexpression is associated with clinical outcomes and is an important predictive and prognostic factor. The relationship between target expression (ER/PgR) and efficacy of endocrine therapy is well established. Accurate assessment of ER/PgR status permits informed treatment decisions for targeted therapy, thereby identifying the patients most likely to benefit from endocrine therapy. Accurate test performance is crucial, yet there is evidence of wide variability in test performance and inaccurate results (falsely negative or falsely positive) of up to 20%. The production of the guideline¹ was deemed necessary to improve the status of ER/PgR testing. The guideline makes recommendations on methods of optimal test performance as well as on quality assurance. As we describe, in the case of IHC testing of ER and PgR status, there is no gold-standard assay available.

Discussion

Hormone Receptor Status and Treatment

The guideline¹ recommends that ER and PgR status be determined for all individuals with invasive breast cancers and breast cancer recurrences. The purpose of both tests is to help determine the likelihood of benefit from endocrine therapy for men and women with breast cancer. People with a recurrence of breast cancer should always receive hormone receptor (HR) testing so that treatment decisions can be made based on current biologic information. In the absence of definitive published data, the panel did not make a formal recommendation on ER testing in patients with ductal carcinoma in situ but rather suggests clinician-patient discussion. A previous ASCO/CAP guideline addressed human epidermal growth factor receptor 2 (HER2) testing in invasive breast cancer.³

HR positive is the most common breast cancer phenotype worldwide. Therefore, access to accurate and reliable ER/PgR testing and established and relatively affordable endocrine therapies could have a profound impact on breast cancer outcomes in high- and low-/middle-income countries around the globe. Endocrine therapies for women with HR-positive tumors include ovarian ablation (surgical or chemical), selective ER modulators, and aromatase inhibitors. Endocrine therapy is not indicated for women whose tumors do not express either ER or PgR. The predictive role of PgR status is less established than that for ER status, but it may provide predictive value independent of ER status. There exists a small subset of women with tumors that test ER negative/PgR positive; these women may be candidates for endocrine treatment because there is no single explanation for this finding at this time.

Biopsy

Large, preferably multiple, core biopsies of a tumor are preferred for testing if they are representative of the tumor (grade and type) at resection (Appendix Table A1, online only). If core samples are large and representative of the resection specimen, the guideline¹ recommends that these samples be used when possible rather than a surgical specimen for ER and PgR analyses, because such samples are less likely to be exposed to cold ischemia and more likely to be begin pathology processing within a short period of time.

Breast cancer tumor specimens should be fixed for a minimum of 6 hours in 10% neutral buffered formalin (NBF) and for no longer than 72 hours. Additional preparation of the specimen is described in the guideline. If the tumor comes from a remote location, it should be bisected on removal and sent to the laboratory immersed in a sufficient volume of NBF. Cold ischemia time, fixative type, and time sample placed in NBF must be recorded.

Assay and Laboratory Regulation

In the United States, tests and laboratories are regulated by the US Food and Drug Administration (FDA) and by the Centers for Medicare & Medicaid Services via Clinical Laboratory Improvement Act regulations and are accredited by bodies deemed private accreditors. These private organizations providing accreditation include CAP, the Joint Commission (formerly the Joint Commission on Accreditation of Healthcare Organizations), and COLA (formerly the Commission on Office Laboratory Accreditation).

IHC became the de facto standard method to measure ER and PgR status in formalin-fixed paraffin-embedded tissue in the 1990s. There is a single FDA 510(k)-cleared ER/PgR assay kit, although several antibodies have been cleared as individual reagents by the FDA. These include antibodies 1D5, 6F11, SP1, and ER.2.123+1D5 for ER and antibodies 1294, 1A6, and 312 for PgR.

ASCO and CAP recommend that ER and PgR testing be performed in a CAP-accredited laboratory or in a laboratory that meets the additional accreditation requirements set out within this guideline.¹ CAP will require that every CAP-accredited laboratory performing ER and/or PgR testing participate in a mandatory proficiency testing program beginning as soon as possible. The previous collaboration between ASCO and CAP addressed the technical and analytic issues related to accurate HER2 testing. A set of guidelines were issued that have had a positive impact on evaluation and treatment of women with breast cancer.³ Cancer specialists of all disciplines (eg, pathologists, surgeons, radiologists doing biopsies, radiation oncologists, and medical oncologists) are encouraged to learn about the accreditation status of laboratories that perform ER/PgR testing and about specimen handling and interpretation requirements, including the new thresholds for positive assays (1% of tumor cells).

Test Performance

The guideline¹ proposes a testing algorithm that relies on accurate, reproducible assay performance. It also specifies elements to reliably reduce assay variation (eg, specimen handling, proper use of controls, and interpretive and reporting criteria). A laboratory performing ER/PgR testing should initially validate its proposed or existing assay against a stable assay of another laboratory, using a clinically validated assay. To be considered acceptable, the results of the assay must be initially 90% concordant for positive ER or PgR and 95% concordant for negative ER or PgR with those of the clinically validated assay. (Concordant refers to the assay result.) See Assay and Laboratory Regulation for quality assurance recommendations.

Test Results

It is recommended that ER and PgR assays be considered positive if there are at least 1% positive invasive tumor nuclei in the sample on testing. The guideline¹ provides additional interpretation, recommending that clinicians consider endocrine therapy in patients whose breast tumors show at least 1% ER-positive cells and withhold endocrine therapy if the amount is less than 1%. It also recognizes that it is reasonable for oncologists to discuss the pros and cons of endocrine therapy with patients whose tumors contain low levels of ER by IHC (1% to 10% weakly positive cells) and make informed decisions based on the balance.

Required Reporting Elements for ER and PgR Analysis

First, the percent/proportion of positive cells:

The percent/proportion of invasive tumor cells staining positively should be recorded and reported; all of the tumor containing areas of the tissue section on the slide should be evaluated to arrive at this percentage. The percentage can be arrived at either by estimation or by quantification, either manually by counting cells or by image analysis (computer automated quantification). If the sample is a cytology specimen, at least 100 cells should be counted or used to estimate the percent of HR-positive tumor cells, particularly if the tumor specimen is limited and if the positive staining appears to involve only a minority of tumor cells.

Second, the intensity of staining:

The intensity of staining should be recorded and reported as weak, moderate, or strong; this measurement should represent an estimate of the average staining of the intensity of the positively stained tumor cells on the entire tissue section relative to the intensity of positive controls run with the same batch. Intensity is provided as a measure of assay quality over time and also allows for optional composite scoring. Tissue heterogeneity can sometimes be observed.

Third, an interpretation of the assay (≥1% is positive; <1% is negative or uninterpretable):

An interpretation of the assay should be provided using one of three mutually exclusive interpretations. The reader should provide an interpretation of the assay based on the following criteria:

• Receptor positive (either ER or PgR): The guideline recommends a cutoff of a minimum of 1% of invasive tumor cells positive for ER/PgR for a specimen to be considered positive. There is no agreement about a range for receptor equivocal, so this term should not be used.

• Receptor negative: Tumors exhibiting less than 1% of tumor cells staining for ER or PgR of any intensity should be considered negative based on data showing that such patients do not receive meaningful benefit from endocrine therapy. The sample should only be considered negative in the presence of appropriately stained extrinsic and intrinsic controls. Testing for any specimen lacking intrinsic elements (normal breast epithelium) that is negative on ER and/or PgR assay should be repeated using another tumor block or another tumor specimen, and the specimen should be reported as uninterpretable rather than negative.

• Receptor uninterpretable: The guideline¹ states that there are no absolute assay exclusions. Nevertheless, a result should be considered uninterpretable if the sample did not conform to the preanalytic specifications of the guideline, the sample was processed using procedures that did not conform to guideline specifications or the standard operating procedure of the laboratory, or the assay used to analyze the specimen was not validated and controlled as specified in the guideline.

• If ER and PgR status is negative in histologies commonly associated with ER- and PgR-positive results (eg, low grade tumors and tubular, lobular, and mucinous histologic types), then an optional cautionary statement should indicate that although the patient's tumor tested ER/PgR negative, tumors with the same histologic type or grade almost always test positive.

Optional reporting elements are described in the guideline. Briefly, they include a cautionary statement with negative ER/PgR results when histopathology is normally associated with positive ER/PgR results and a composite score (eg, H, Allred, or Quick score).

Special Questions

The guideline¹ also addresses two special questions. First, in view of the lack of definitive published studies on ER/PgR testing in ductal carcinoma in situ, the panel opted not to make a definitive recommendation at this time. Second, although the precise role of PgR in patient management has not been established, the guideline recommends that endocrine therapy not be withheld from women with ER-positive/PgR-negative tumors and that this status not be used to select type of endocrine therapy. Patients with ER-negative/PgR-positive tumors may also benefit from and should be considered for endocrine therapy.

Methodology

ASCO and Cancer Care Ontario conducted a systematic review of medical literature published from 1990 through May 2008 using Medline, EMBASE, and the Cochrane Database of Systematic Reviews. The companion systematic review will be published separately. Cancer Care Ontario will publish separate recommendations/guidelines based on the same systematic review because of differences in the way health care is provided and the way pathology labs operate in the United States and Canada.

The primary outcome of interest of the systematic review was the correlation of ER/PgR status and endocrine treatment benefit (disease-free, progression-free, or overall survival). The evidence available was primarily from retrospective comparative studies. No randomized controlled trials were identified that were designed to assess prospectively the technical aspects of HR testing in breast cancer, although a number of studies involved specimens (slides, tissue blocks) collected during the course of randomized trials that were used for retesting. Much of the literature identified involved variables concerning the preparation, conducting, and analysis of IHC tests. In addition, there was literature about laboratory proficiency/performance and quality assurance.

Additional Resources

A slide set and table showing markers corresponding to clinical presentations are available as Data Supplements (online only) to this article. The full and abridged versions of the guideline¹ are available at www.asco.org/guidelines/erpr. A patient guide is available at www.cancer.net/whattoknow.

Authors

The guideline¹ published in Journal of Clinical Oncology was developed and written by M. Elizabeth H. Hammond, Daniel F. Hayes, Mitch Dowsett, D. Craig Allred, Karen L. Hagerty, Sunil Badve, Patrick L. Fitzgibbons, Glenn Francis, Neil S. Goldstein, Malcolm Hayes, David G. Hicks, Susan Lester, Richard Love, Pamela B. Mangu, Lisa McShane, Keith Miller, C. Kent Osborne, Soonmyung Paik, Jane Perlmutter, Anthony Rhodes, Hironobu Sasano, Jared N. Schwartz, Fred C.G. Sweep, Sheila Taube, Emina Emilia Torlakovic, Paul Valenstein, Giuseppe Viale, Daniel Visscher, Thomas Wheeler, R. Bruce Williams, James L. Wittliff, and Antonio C. Wolff.

Appendix

Table A1.

Summary of Guideline Recommendations for ER and PgR Testing by IHC for Patients With Breast Cancer

Characteristic	Recommendation	Comments
Optimal algorithm for ER/PgR testing	—Positive for ER or PgR if finding of ≥ 1% of tumor cell nuclei immunoreactive	—These definitions depend on laboratory documentation of the following:
	—Negative for ER or PgR if finding of < 1% of tumor cell nuclei immunoreactive in presence of evidence that sample can express ER or PgR (positive intrinsic controls seen)	1. Proof of initial validation in which positive ER or PgR category is 90% concordant and negative ER or PgR category is 95% concordant with clinically validated ER or PgR assay
	—Uninterpretable for ER or PgR if finding that no tumor nuclei are immunoreactive and that internal epithelial elements present in sample or separately submitted from same sample lack any nuclear staining	2. Ongoing internal QA procedures including use of external controls of variable ER and PgR activity with each run of assay, regular assay reassessment, and competency assessment of technicians and pathologists
		3. Participation in external proficiency testing according to proficiency testing program guidelines
		4. Biennial accreditation by valid accrediting agency
Optimal testing conditions	—Large, preferably multiple, core biopsies of tumor are preferred for testing if representative of tumor (grade, type) at resection	—Specimen should be rejected and repeated on separate sample if any of following conditions exist:
	—Interpretation follows guideline recommendation	1. External controls not as expected (scores recorded daily show variation)
	—Accession slip and report must include guideline-detailed elements	2. Artifacts involve most of sample
		—Specimen may also be rejected and repeated on another sample if:
		1. Slide has no staining of included normal epithelial elements and/or normal positive control on same slide
		2. Specimen decalcified using strong acids
		3. Specimen shows ER-negative/PgR-positive phenotype (to rule out false-negative ER assay or false-positive PgR assay)
		4. Sample has prolonged cold ischemia time or fixation duration < 6 or > 72 hours and is negative on testing in absence of internal control elements
		—Positive ER or PgR requires ≥ 1% of tumor cells immunoreactive; both average intensity and extent of staining reported
		—Image analysis is desirable method of quantifying percentage of immunoreactive tumor cells
		—H, Allred, or Quick score may be provided
		—Negative ER or PgR requires < 1% of tumor cells with ER or PgR staining
		—Interpreters have method to maintain consistency; competency documented regularly
Optimal tissue handling requirements	—Time from tissue acquisition to fixation should be as short as possible; samples for ER and PgR testing are fixed in 10% NBF for 6-72 hours; samples should be sliced at 5 mm intervals after appropriate gross inspection and margins designation and placed in sufficient volume of NBF to allow adequate tissue penetration; if tumor comes from remote location, it should be bisected through tumor on removal and sent to laboratory immersed in sufficient volume of NBF
	—As in ASCO/CAP HER2 guideline, storage of slides for > 6 weeks before analysis is not recommended3
	—Time tissue is removed from patient, time tissue is placed in fixative, duration of fixation, and fixative type must be recorded and noted on accession slip or in report
Optimal internal validation procedure	—Validation of any test must be done before test is offered; described in separate article on testing validation (Fitzgibbons et al, manuscript in preparation)
	—Validation must be done using clinically validated ER or PgR test method
	—Revalidation should be done whenever there is significant change to test system, such as change in primary antibody clone or introduction of new antigen retrieval or detection system
Optimal internal QA procedures	—Initial test validation (described in Fitzgibbons et al, manuscript in preparation)
	—Ongoing quality control and equipment maintenance
	—Initial and ongoing laboratory personnel training and competency assessment
	—Use of standardized operating procedures including routine use of external control materials with each batch of testing and routine evaluation of internal normal epithelial elements or inclusion of normal breast sections on each tested slide wherever possible
	—Regular, ongoing assay reassessment should be done at least semiannually (as described in Fitzgibbons et al, manuscript in preparation); revalidation is needed whenever there is significant change to test system
	—Ongoing competency assessment and education of pathologists
Optimal external proficiency assessment	—Mandatory participation in external proficiency testing program with at least two testing events (mailings) per year
	—Satisfactory performance requires at least 90% correct responses on graded challenges for either test
	—Unsatisfactory performance requires laboratory to respond according to accreditation agency program requirements
Optimal laboratory accreditation	—On-site inspection every other year with annual requirement for self-inspection
	—Reviews laboratory validation, procedures, QA results and processes, results, and reports
	—Unsuccessful performance results in suspension of laboratory testing for ER or PgR

NOTE. Data adapted.¹

Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; QA, quality assurance; NBF, neutral buffered formalin; CAP, College of American Pathologists; HER2, human epidermal growth factor receptor 2.

Authors' Disclosures of Potential Conflicts of Interest

The authors indicated no potential conflicts of interest.

Author Contributions

Conception and design: M. Elizabeth H. Hammond, Daniel F. Hayes, Antonio C. Wolff, Sarah Temin

Administrative support: Antonio C. Wolff, Pamela B. Mangu, Sarah Temin

Provision of study material or patients: M. Elizabeth H. Hammond, Antonio C. Wolff

Collection and assembly of data: M. Elizabeth H. Hammond, Daniel F. Hayes, Antonio C. Wolff, Pamela B. Mangu

Data analysis and interpretation: M. Elizabeth H. Hammond, Daniel F. Hayes, Antonio C. Wolff

Manuscript writing: M. Elizabeth H. Hammond, Daniel F. Hayes, Antonio C. Wolff, Sarah Temin

Final approval of manuscript: M. Elizabeth H. Hammond, Daniel F. Hayes, Antonio C. Wolff, Pamela B. Mangu, Sarah Temin