The CAP and the American Society of Clinical Oncology (ASCO) collaborate in producing guidelines to improve testing accuracy and reduce the substantial risks associated with false positive and false negative results. Laboratory assays for HER2 and Estrogen Receptor (ER) and Progesterone Receptor (PgR) are essential in selecting patients for anti-HER2 and hormonal therapy.
Several published reports and laboratory survey results allowed the Expert Panel to evaluate the observed frequency of less common HER2 testing patterns, their apparent prognostic and predictive value when retrospectively analyzed within clinical trial data sets, and the critical need to understand the underlying distribution of HER2 IHC test results in cases that are submitted for additional testing (eg, by ISH) by a reference laboratory. The Expert Panel also wished to clarify one of its 2013 recommendations that led some laboratories to adopt the use of multiple alternative chromosome 17 probe testing as the sole strategy to resolve equivocal HER2 test results by ISH, despite limited evidence on analytical and clinical validity.
- Revision of the definition of immunohistochemistry (IHC) 2+ (equivocal) as invasive breast cancer as “Weak to moderate complete membrane staining observed in >10% of tumor cells.”1
- Repeat HER2 testing on a surgical specimen if the initially tested core biopsy is negative is no longer stated as mandatory: “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.”1
- Classification of the less common patterns (defined as ISH groups 2 to 4 which are approximately 5% of cases) observed when performing dual-probe ISH testing for breast cancer and the optimal algorithm for the diagnostic approach. (HER2/chromosome enumeration probe 17 [CEP17])
- ISH group 2 (HER2/CEP17 ratio ≥ 2.0; average HER2 copy number < 4.0 signals per cell),
- ISH group 3 (HER2/CEP17 ratio <2.0; average HER2 copy number ≥ 6.0 signals per cell),
- ISH group 4 (HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 4.0 and < 6.0 signals per cell).
- More rigorous interpretation criteria for ISH and concomitant IHC review for dual-probe ISH groups 2 to 4 is required to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The concomitant review is recommended to be performed in the same institution to ensure parallel interpretation and quality of the two assays. (Refer to the Algorithms–Figures 1-6 in the manuscript.)
- Pathologists should review associated IHC results as part of interpreting single-probe ISH results.
- The Expert Panel also preferentially recommends the use of dual-probe instead of single-probe ISH assays, although it recognizes that several single-probe ISH assays have regulatory approval in many parts of the world.
General Fixation and Processing Questions for HER2 and ER/PgR
Breast specimens that will be subject to ER/PgR and HER2 testing should be fixed in neutral buffered formalin for a minimum of six hours and a maximum of 72 hours. This fixation time begins when the specimen is initially placed in formalin (not when the specimen is sectioned during gross examination) and ends when the cassettes are no longer in formalin. This is not an absolute exclusion criterion. For specimens fixed longer than 72 hours for HER2 or ER and PgR in which negative test results are obtained, the report should state that prolonged fixation could be a possible cause for the negative result, and alternative testing methods should be considered (eg, FISH for HER2; gene expression assay for ER). For HER2 testing, labs should also consider confirming by FISH any specimen fixed longer than 72 hours that is not Score 3 by IHC.
No. For all cases in which the fixation time is within the recommended interval specified in the current ASCO/CAP guidelines for HER2 and ER/PgR testing (6 to 72 hours for ER and PgR and HER2), laboratories can append a standard statement to their reports that fixation time was in compliance with ASCO/CAP guidelines. However, laboratories will be required to put a disclaimer in any report in which the fixation time is outside those parameters. In addition, for cases with fixation times outside the recommended intervals in which a negative test result is obtained, the report should state that prolonged fixation could be a possible cause for the negative result and alternative testing methods should be considered (e.g. FISH for HER2; gene expression assay for ER). For HER2 testing, labs should also consider confirming by FISH any specimen fixed longer than 72 hours that is not Score 3 by IHC. It is also acceptable to test another sample from the same patient for these factors in these situations rather than using alternative testing methods on the same sample.
No. Refrigeration delays fixation, which has a detrimental effect on immunostaining. The testing guidelines require that specimens that will be subject to HER2, ER, or PgR testing be placed in formalin less than one hour after the tumor is removed from the patient; any further delay in fixation is now considered unacceptable.
In addition to placing in fixative as soon as possible, the guidelines also recommend slicing the specimen at regular intervals to ensure adequate fixation throughout. Since most cases also require assessment of specimen margins, institutions must develop procedures to ensure proper handling of breast excision specimens. As with any other intraoperative consultation, a pathologist (or other appropriately trained person under the direct supervision of a pathologist) must be available to handle these specimens.
No. The original HER2 Testing Guidelines specified a minimum one-hour formalin fixation time for needle biopsies, but included a caveat that longer fixation is strongly recommended for these specimens. While formalin penetrates tissues at the rate of about 1mm/hour, penetration is not the same as fixation and the biochemical cross-linking that represents formalin fixation requires more time. Published studies have documented that a minimum of 6-8 hours formalin fixation is needed to obtain consistent IHC assay results for ER; fixation for less than this time has been shown to cause false negative ER staining. Because of the adverse effects of underfixation, which cannot be overcome by antigen retrieval, testing on specimens fixed for less than 6 hours is no longer acceptable. Cases in which tissues have been fixed less than 6 hours should be reported as ‘Estrogen Receptor Uninterpretable’ with an explanatory comment.
No. Fixatives other than formalin are not precluded by the guidelines. For tissue specimens, laboratories that choose to use a fixative other than neutral buffered formalin must validate that fixative’s performance against the results of testing of the same samples fixed in neutral buffered formalin and tested with the identical assay. Since cytology specimens are not ordinarily fixed in formalin such concordance studies are not practical, but labs performing testing on such specimens must document that they validated their methods and achieved acceptable concordance, perhaps by comparing staining of alcohol fixed cytology specimens with subsequently excised routinely processed, formalin-fixed, surgical pathology specimens.
The effect of rapid tissue processing protocols on predictive marker testing is unknown. Before offering such testing using any alternative method, the lab must validate that method by comparing it with testing done by standard methods (ie, the lab must test the same samples processed routinely and processed by the alternative method, and demonstrate 95% concordance for positive and negative results). Validation of reagents or equipment by vendors or manufacturers does not represent an acceptable substitute for validation done by each laboratory.
No, the 48-hour limit referred only to the former upper limit of formalin fixation. Once the tissue is processed and paraffin-embedded, there is no specified time frame for subsequent sectioning and testing; however, the interval should be as short as possible as antigen preservation is better within the block than on glass.
The latter is correct. There is no requirement that HER2 stains be done within six weeks of embedding, but labs should avoid doing HER2 stains on sections that were cut more than six weeks earlier. This also applies to positive control sections; labs should avoid using control slides that have been stored for prolonged periods after sectioning.
Interpretation and Reporting
The guideline states “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.” Does this mean that fixation <6 hours or >72 hours is a technical issue that prevents the tests from being reported as positive or negative and therefore must be called Indeterminate?
No. “Indeterminate” is to be reported if technical issues prevent one or both tests (IHC and ISH) performed in a tumor specimen from being reported as positive, negative, or equivocal. This may occur if specimen handling was inadequate, if artifacts (crush or edge artifacts) make interpretation difficult, or if the analytic testing failed. Another specimen should be requested for testing, if possible, and a comment should be included in the pathology report documenting intended action." It is at the pathologist's discretion to define inadequate specimen handling.
Laboratory Accreditation and Proficiency Testing Questions
Yes, checklist requirements regarding HER2 assay validation, specimen fixation, proficiency testing, and use of the ASCO/CAP scoring criteria for reporting results are included in the Anatomic Pathology (ANP), Cytogenetics (CYG), and Molecular Pathology (MOL) checklists. These checklists are available to CAP accredited laboratories through e-LAB Solutions or can be purchased by non-CAP accredited laboratories.
Yes. In order to be compliant with the ASCO/CAP HER2 guidelines, any laboratory that reports results of such testing must participate in an accepted PT program (see exception below). The CAP Accreditation Program requires participation in a CAP-accepted PT program.
Exception: Laboratories that interpret and report the results of HER2 testing by ISH in which the hybridization is performed at an outside laboratory should not enroll in proficiency testing for that assay due to prohibitions on proficiency testing referral by Centers for Medicare and Medicaid Services (CMS); such laboratories must perform alternative assessment. This exception does not apply to laboratories that interpret and report the results of HER2 testing by immunohistochemistry when staining is done at an outside facility.
The ASCO/CAP guidelines for HER2 testing apply only to breast carcinoma. HER2 testing on other tumor types (eg, gastric carcinoma) is not covered by these guidelines at the current time.
In order to be compliant with the ASCO/CAP ER/PgR guidelines, any laboratory that reports results of such testing on primary breast cancers must participate in a PT program (see exception below). The College’s Laboratory Accreditation Program (LAP) requires participation in a CAP-accepted PT program.
Exception: Laboratories that do ER and/or PgR staining only on tissues other than primary breast cancers (eg, other tumor types such as meningioma; for lineage determination only), are not required to enroll in proficiency testing that is specific for those analytes. Laboratories that send all primary breast cancers out to another laboratory for both staining and interpretation are not required to enroll in PT.
HER2 by Immunohistochemistry (IHC) Survey (HER2)
HER2 is an IHC Survey that provides two 10-core tissue microarray slides, twice per year. Enrollment in the HER2 Survey will satisfy LAP requirements for participation in a CAP-accepted PT program for HER2 by IHC.
HER2 by Fluorescence in situ Hybridization (FISH) Survey (CYH)
CYH is a FISH Survey that provides two unstained 5 core tissue microarray slides, twice per year. Enrollment in the CYH will satisfy the LAP requirement for participation in a CAP-accepted PT program for HER2 by FISH, interpretation and hybridization onsite activity. Laboratories that do interpretation only must perform alternative assessment.
HER2 by Brightfield in situ Hybridization Survey (ISH2)
ISH2 (HER2 brightfield ISH Survey-CISH (chromogenic in situ hybridization) and SISH (silver in situ hybridization) provides two 5-core tissue microarray slides in duplicate, twice a year. Enrollment in the ISH2 Survey will satisfy the LAP requirement for participation in a CAP-accepted PT program for HER2 by CISH, interpretation and hybridization onsite activity. Laboratories that do interpretation only must perform alternative assessment.
ER and PgR by Immunohistochemistry (PM2)
PM2 is an IHC Survey that provides four 10-core microarray slides, two for ER and two for PgR, twice per year. Enrollment in PM2 is required for CAP-accredited laboratories.
At this time, CAP is the only accepted PT provider for HER2, ER and PgR.
Image analysis can be an effective tool for improving interpretation consistency; however, the pathologist is responsible for ensuring that the result provided by image analysis reflects measurement of invasive carcinoma only. The pathologist must select or confirm the area of analysis to ensure that the appropriate area is scored.
Image analysis equipment, just as other laboratory equipment, must be calibrated and subjected to regular maintenance and internal quality control evaluation. Image analysis procedures must be validated before implementation. Refer to the CAP guideline, Validating Whole Slide Imaging for Diagnostic Purposes in Pathology, for more information.
Laboratories that do HER2 or ER/PgR staining by IHC and use in-house image analysis for interpretation and reporting are required to enroll in an IHC-based PT program and report the results following the usual testing and reporting methods used.
Laboratories that interpret and report the results of HER2 or ER/PgR testing by IHC in which staining and image analysis are performed at an outside laboratory are required to enroll in PT but must ensure that they only receive back the stained PT slide or an image of the stained PT slide. The laboratory must ensure that the outside laboratory does not send back any quantitative image analysis data as that would constitute PT Referral by CMS which can have serious consequences. As noted above, image analysis is a useful tool, but pathologists should also be able to manually score the slide without the use of quantitative image analysis.
All labs participating in PT must provide results for all PT challenges regardless of specific methods of testing used. If the PT program includes manual scoring of virtual slides or images (in addition to actual tissue challenges), every lab must provide manual scoring results for these challenges even if they normally only interpret glass slides or report results by quantitative image analysis.
Yes. Laboratories that interpret HER2, ER, or PgR slides stained by another facility must enroll in a CAP-accepted PT program and report the results of their interpretation. Since CAP is currently the only accepted HER2/ER/PgR PT provider, such labs must enroll in CAP’s HER2 and/or PM2 Surveys. You must send the unstained Survey slides to the outside facility for immunohistochemical staining, and report the results of your interpretation of the stained slides.
Yes. All laboratories that perform and/or interpret HER2 or ER/PgR testing are required to enroll in a CAP-accepted PT program (see exception below). Laboratories that send materials to another facility for staining by IHC and image analysis are required to enroll in an appropriate IHC-based PT program. For the tissue challenges in the HER2 and PM2 Surveys, the laboratory should send the slides to the outside facility for staining only; do not request quantitative image analysis at the outside facility even if this is routinely done for patient testing. Doing so could be considered PT Referral and result in severe sanctions by CMS. You must report the results of manual scoring for these PT slides. The PT Referral prohibition does not apply to staining and image analysis that are both performed in house.
Exception: Laboratories that do such testing only on tissues other than primary breast cancers (eg, other tumor types such as meningioma; for lineage determination only) are not required to enroll in proficiency testing that is specific for those analytes. Laboratories that send all primary breast cancers to another facility for both staining and interpretation are not required to enroll in PT.
In the CAP’s HER2 and PM2 Programs, all results of PT challenges are reported using manual scoring. There is currently no separate reporting by quantitative image analysis. All laboratories must provide results using the scoring systems outlined in the PT kit instructions and stained tissue challenges, even those that normally
No. Proficiency testing only applies to laboratories that perform and/or interpret the assays, not to those that simply report the results that are performed and interpreted by an outside laboratory. Labs must enroll in PT if they provide a professional interpretation, even if they are using an outside laboratory for staining and/or image analysis.
Only the results of the laboratory are reported to the PT provider. The laboratory is not required to provide responses from each pathologist for every PT challenge. The challenges must be integrated into the routine laboratory workload and analyzed using the same personnel and systems as for patient samples. Thus, if multiple pathologists routinely report HER2 or ER/PgR results in your lab, PT challenges must be rotated among the pathologists to participate in scoring these challenges.
- Wolff AC, Hammond ME, Hicks DG, et al: Reply to E.A. Rakha et al. J Clin Oncol 33:1302-4, 2015
- 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. Arch Pathol Lab Med. 2018;142(11):1364-1382.