ASCO/CAP clinical practice guideline focused update: HER2 testing in breast cancer

Topic	2013 recommendations	2018 focused update recommendations
Specimens to be tested	All newly diagnosed patients with breast cancer must have a HER2 test performed. Patients who then develop metastatic disease must have a HER2 test performed in a metastatic site, if tissue sample is available.	No change.
Optimal algorithm for HER2 testing	Must report HER2 test result as positive for HER2 if: IHC 3+ based on circumferential membrane staining that is complete, intense. ISH positive based on: Single-probe average HER2 copy number ≥6.0 signals/cell. Dual-probe HER2/CEP17 ratio of ≥2.0; with an average HER2 copy number ≥4.0 signals/cell. Dual-probe HER2/CEP17 ratio of ≥2.0; with an average HER2 copy number <4.0. Dual-probe HER2/CEP17 ratio of <2.0 with an average HER2 copy number ≥6.0 signals/cell. Must report HER2 test result as equivocal and order reflex test (same specimen using the alternative test) or new test (new specimen, if available, using same or alternative test) if: IHC 2+ based on circumferential membrane staining that is incomplete and/or weak to moderate and within >10% of the invasive tumor cells or complete and circumferential membrane staining that is intense and within ≤10% of the invasive tumor cells. ISH equivocal based on: Single-probe ISH average HER2 copy number ≥4.0 and ≤6.0 signals/cell. Dual-probe HER2/CEP17 ratio of <2.0 with an average HER2 copy number ≥4.0 and ≤6.0 signals/cell. Must report HER2 test result as negative if a single test (or both tests) performed show: IHC 1+ as defined by incomplete membrane staining that is faint or barely perceptible and within >10% of the invasive tumor cells. IHC 0 as defined by no staining observed or membrane staining that is incomplete and is faint or barely perceptible and within ≤10% of the invasive tumor cells. ISH negative based on: Single-probe average HER2 copy number <4.0 signals/cell. Dual-probe HER2/CEP17 ratio of <2.0 with an average HER2 copy number of 4.0 signals/cell. Must report HER2 test result as indeterminate if technical issues prevent one or both tests (IHC and ISH) from being reported as positive, negative, or equivocal. Conditions may include: Inadequate specimen handling. Artifacts (crush or edge artifacts) that make interpretation difficult. Analytic testing failure. Another specimen should be requested for testing to determine HER2 status. Reason for indeterminate testing should be noted in a comment in the report.	The revised definition of IHC 2+ (equivocal) is invasive breast cancer with "weak to moderate complete membrane staining observed in >10% of tumor cells." It is now stated that, on the basis of some criteria (including a tumor grade 3), "If the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may be ordered on the excision specimen..." If a case has a HER2/CEP17 ratio of ≥2.0 but the average HER2 signals/cell is <4.0, a definitive diagnosis will be rendered based on additional work-up. If not already assessed by the institution or laboratory performing the ISH test, IHC testing for HER2 should be performed using sections from the same tissue sample used for ISH, and the slides from both ISH and IHC should be reviewed together to guide the selection of areas to score by ISH (local practice considerations will dictate the best procedure to accomplish this concomitant assessment): If the IHC result is 3+, diagnosis is HER2 positive. If the IHC result is 2+, recount ISH by having an additional observer, blinded to previous ISH results, count at least 20 cells that include the area of invasive cancer with IHC 2+ staining: If reviewing the count by the additional observer changes the result into another ISH category, the result should be adjudicated per internal procedures to define the final category. If the count remains an average of <4.0 HER2 signals/cell and the HER2/CEP17 ratio is ≥2.0, diagnosis is HER2 negative with a comment. If the IHC result is 0 or 1+, diagnosis is HER2 negative with a comment. If a case has an average of ≥6.0 HER2 signals/cell with a HER2/CEP17 ratio of <2.0, formerly diagnosed as ISH positive for HER2, a definitive diagnosis will be rendered based on additional work-up. If not already assessed by the institution or laboratory performing the ISH test, IHC testing for HER2 should be performed using sections from the same tissue sample used for ISH, and the slides from both ISH and IHC should be reviewed together to guide the selection of areas to score by ISH (local practice considerations will dictate the best procedure to accomplish this concomitant review): If the IHC result is 3+, diagnosis is HER2 positive. If the IHC result is 2+, recount ISH by having an additional observer, blinded to previous ISH results, count at least 20 cells that include the area of invasion with IHC 2+ staining: If reviewing the count by the additional observer changes the result into another ISH category, the result should be adjudicated per internal procedures to define the final category. If the HER2/CEP17 ratio remains <2.0 with ≥6.0 HER2 signals/cell, diagnosis is HER2 positive. If the IHC result is 0 or 1+, diagnosis is HER2 negative with a comment. If the case has an average HER2 signals/tumor cell of ≥4.0 and <6.0 and the HER2/CEP17 ratio is <2.0, formerly diagnosed as ISH equivocal for HER2, a definitive diagnosis will be rendered based on additional work-up. If not already assessed by the institution or laboratory performing the ISH test, IHC testing for HER2 should be performed using sections from the same tissue sample used for ISH, and the slides from both ISH and IHC should be reviewed together to guide the selection of areas to score by ISH (local practice considerations will dictate the best procedure to accomplish this concomitant review): If the IHC result is 3+, diagnosis is HER2 positive. If the IHC result is 2+, recount ISH by having an additional observer, blinded to previous ISH results, count at least 20 cells that include the area of invasion with IHC 2+ staining: If reviewing the count by the additional observer changes the result into another ISH category, the result should be adjudicated per internal procedures to define the final category. If the count remains an average of ≥4.0 and <6.0 HER2 signals/cell with a HER2/CEP17 ratio of <2.0, diagnosis is HER2 negative with a comment. If the IHC result is 0 or 1+, diagnosis is HER2 negative with a comment.
ISH rejection criteria	Test is rejected and repeated if: Controls are not as expected. Observer cannot find and count at least two areas of invasive tumor. >25% of signals are unscorable due to weak signals. >10% of signals occur over cytoplasm. Nuclear resolution is poor. Autofluorescence is strong. Report HER2 test result as indeterminate as per parameters described.	No change.
ISH interpretation	The pathologist should scan the entire ISH slide before counting at least 20 cells or use IHC to define the areas of potential HER2 amplification. If there is a second population of cells with increased HER2 signals/cell and this cell population consists of >10% of tumor cells on the slide (defined by image analysis or visual estimation of the ISH or IHC slide), a separate counting of at least 20 nonoverlapping cells must also be performed within this cell population and reported. For brightfield ISH, counting requires comparison between patterns in normal breast and tumor cells because artifactual patterns may be seen that are difficult to interpret. If tumor cell pattern is neither normal nor clearly amplified, test should be submitted for expert opinion.	The pathologist should scan the entire ISH slide before counting at least 20 cells or use IHC to define the areas of potential HER2 amplification. If there is a second population of contiguous cells with increased HER2 signals/cell and this cell population consists of >10% of tumor cells on the slide (defined by image analysis or visual estimation of the ISH or IHC slide), a separate counting of at least 20 nonoverlapping cells must also be performed within this cell population and reported.
Acceptable (IHC and ISH) tests	Should preferentially use an FDA-approved IHC, brightfield ISH, or FISH assay.	No change.
IHC rejection criteria	Test is rejected and repeated or tested by FISH if: Controls are not as expected. Artifacts involve most of sample. Sample has strong membrane staining of normal breast ducts (internal controls).	No change.
IHC interpretation criteria	Should interpret IHC test using a threshold of >10% of tumor cells that must show homogeneous, dark circumferential (chicken wire) pattern to call result 3+, HER2 positive.	No change.
Reporting requirements for all assay types	Report must include guideline-detailed elements except for changes to reporting requirement and algorithms defined in this table.	No change.
Optimal tissue handling requirements	Time from tissue acquisition to fixation should be as short as possible; samples for HER2 testing are fixed in 10% neutral buffered formalin for 6 to 72 hours; cytology specimens must be fixed in formalin. Samples should be sliced at 5- to 10-mm intervals after appropriate gross inspection and margin designation and placed in a sufficient volume of neutral buffered formalin. Any exceptions to this process must be included in the report.	No change.
Optimal tissue sectioning requirements	Sections should ideally not be used for HER2 testing if cut >6 weeks earlier; this may vary with primary fixation or storage conditions.	No change.
Optimal internal validation procedure	Validation of test must be performed before test is offered.	No change.
Optimal initial test validation	Laboratories performing these tests should be following all accreditation requirements, one of which is initial testing validation. The laboratory should ensure that initial validation conforms to the published 2010 ASCO/CAP recommendations for IHC testing of ER and PgR guideline validation requirements with 20 negative and 20 positive for FDA-approved assays and 40 negative and 40 positive for LDTs. This requirement does not apply to assays that were previously validated in conformance with the 2007 ASCO/CAP HER2 testing guideline, and those who routinely participate in external proficiency testing for HER2 tests, such as the program offered by CAP. Laboratories are responsible for ensuring the reliability and accuracy of their testing results, by compliance with accreditation and proficiency testing requirements for HER2 testing assays. Specific concordance requirements are not required.	No change.
Optimal monitoring of test concordance between methods	Refer to text following under "Optimal laboratory accreditation".	No change.
Optimal internal QA procedures	Should review and document external and internal controls with each test and each batch of tests: Ongoing quality control and equipment maintenance. Initial and ongoing laboratory personnel training and competency assessment. Use of standardized operating procedures including routine use of control materials. Revalidation of procedure if changed. Ongoing competency assessment and documentation of the actions taken as a part of the laboratory record.	No change.
Optimal external proficiency assessment	Participation in and successful completion of external proficiency testing program with at least two testing events (mailings) a year. Satisfactory performance requires at least 90% correct responses on graded challenges for either test. Unsatisfactory performance will require laboratory to respond according to accreditation agency program requirements.	No change.
Optimal laboratory accreditation	Onsite inspection every other year with annual requirement for self-inspection. Reviews laboratory validation, procedures, QA results and processes, results, and reports. Unsatisfactory performance results in suspension of laboratory testing for HER2 for that method.	No change.

ASCO: American Society of Clinical Oncology; CAP: College of American Pathologists; HER2: human epidermal growth factor receptor 2; IHC: immunohistochemistry; ISH: in situ hybridization; CEP17: chromosome enumeration probe 17; FDA: US Food and Drug Administration; FISH: fluorescent in situ hybridization; ER: estrogen receptor; PgR: progesterone receptor; LDT: laboratory developed test; QA: quality assurance.

From: Wolff AC, Hammond, EH, Allison KH, et al. Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update. J Clin Oncol 2018; 36(20):2105-2122. Reprinted with permission. Copyright © 2018 American Society of Clinical Oncology. All rights reserved.

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ASCO/CAP clinical practice guideline focused update: HER2 testing in breast cancer