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.