Inconsistent Results With Different Secondary Reflex Assays for Resolving HER2 Status

Carlynn Willmore-Payne; Kristy Damjanovich-Colmenares; Alexandra V. Pasi; Theresa L. Werner, MD; H. Evin Gulbahce, MD; Erinn Downs-Kelly, DO; Katherine B. Geiersbach, MD


Am J Clin Pathol. 2016;146(5):618-626. 

In This Article

Abstract and Introduction


Objectives: Guidelines suggest that secondary reflex testing may be useful for resolving HER2 status in breast cancers with equivocal results by both immunohistochemistry (IHC) and in situ hybridization (ISH). We compared two reflex ISH assays and a polymerase chain reaction (PCR) assay for this application.

Methods: Twenty-nine breast cancers with equivocal IHC and ISH results were retested two ways: (1) ISH using differentially labeled probes targeting ERBB2 (HER2, 17q12) and either RAI1 (17p11.2) or ORC4 (2q22.3–2q23.1) in two separate assays and (2) real-time quantitative PCR amplification of ERBB2 and a control locus (EIF5B, 2q11.2).

Results: Results of the HER2/RAI1 and HER2/ORC4 ISH assays were concordant for 21 (72%) cases, and results of all three secondary reflex assays were concordant for only 18 (62%) cases. Result discrepancies between the two ISH readers were observed for cases close to the cutoff threshold.

Conclusions: Use of different control loci for ISH is associated with discordant results, and PCR is more likely to classify cases as nonamplified, possibly due to contamination with nontumor cells. While resolution of HER2-equivocal results is desirable from a clinical perspective, different secondary reflex assays yield different results, and the correlation of these results with clinical outcomes is unknown.


ERBB2 (commonly known as HER2 or HER2/neu) gene amplification and overexpression occur in 15% to 20% of breast cancers and are associated with shorter overall survival.[1] Because patients with HER2 overexpression or gene amplification are candidates for targeted therapy, HER2 testing is important in the clinical management of patients with breast cancer.[2–6] Current American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines for HER2 testing designate two alternative methods for HER2 testing, immunohistochemistry (IHC) and in situ hybridization (ISH).[7,8] Results for IHC and ISH are interpreted as negative, equivocal, or positive according to ASCO/CAP guidelines. Institutions vary in their testing approaches; a commonly used testing algorithm is IHC followed by ISH for equivocal cases that show only moderate HER2 immunostaining (2+) by IHC. ISH is the primary HER2 testing method for other laboratories, followed by IHC testing for those cases with equivocal results by ISH. According to current guidelines, breast cancers with a mean HER2 copy number per cell of at least 4.0 but less than 6.0 (with HER2/centromere ratio below 2.0 for dual-probe assays) are classified as equivocal.[7,8] These cases comprise a significant proportion of total cases evaluated by ISH; since the implementation of the 2013 ASCO/CAP guidelines, laboratories have reported ISH-equivocal rates of 4% to over 14%.[9–15] While the case mix may differ between institutions based on testing referral patterns, leading to substantial differences in the ISH-equivocal rate, the equivocal rate appears to have increased for many laboratories since the implementation of the new guidelines. This presents a problem for laboratories in following up on cases where HER2 status remains unresolved following both IHC and ISH testing. Current ASCO/CAP guidelines recommend resolution of HER2 status in cases with equivocal results, first by employing the alternative testing method (IHC for ISH-equivocal cases and vice versa); core biopsy specimens with equivocal results should also be followed up with repeat testing on the resection specimen.[7,8] For cases that are equivocal by both IHC and ISH ("double equivocals"), follow-up ISH testing with a noncentromeric control probe on chromosome 17 has also been suggested as a method to resolve HER2 status.[7,8] However, additional follow-up testing on these "double-equivocal" samples increases health care costs and can cause delays in clinical decision making.

Several different secondary reflex ISH assays have been described for resolving breast cancers with equivocal HER2 results.[15–17] Alternative (non-ISH) tests are also available for determining HER2 status, including polymerase chain reaction (PCR)–based assays.[18–26] Data are currently lacking on the comparative clinical performance of different secondary reflex tests on breast cancers that are HER2 equivocal by both IHC and ISH according to the 2013 ASCO/CAP guidelines. We compared the performance of two different reflex ISH assays and a real-time PCR assay for resolving HER2 status in a series of HER2-equivocal breast cancers.