Impact of Updated HER2 Testing Guidelines in Breast Cancer—Re-evaluation of HERA Trial Fluorescence in Situ Hybridization Data

Oliver C Stoss; Andreas Scheel; Iris Nagelmeier; Hans-Ulrich Schildhaus; Thomas Henkel; Giuseppe Viale; Bharat Jasani; Michael Untch; Josef Rüschoff


Mod Pathol. 2015;28(12):1528-1534. 

In This Article

Abstract and Introduction


Recently the American Society of Clinical Oncology and the College of American Pathologists have updated their clinical practice guidelines for HER2 testing in breast cancer. In order to evaluate these new recommendations, we have re-assessed the HER2 status of 6018 breast cancer cases of the screening population for the HERceptin adjuvant (HERA) trial that were originally centrally tested by fluorescence in situ hybridization based on the FDA-released test guidelines. According to the most recent 2013 ASCO/CAP recommendations, 3380 (56.2%) cases were classified as HER2 positive compared with 3359 (55.8%) applying the HERA/FDA scheme and 3339 (55.5%) applying the 2007 ASCO/CAP guidelines. Twenty-one cases switched from negative (HERA/FDA scheme) to positive (2013 ASCO/CAP guidelines). This group is characterized by a mean HER2 gene copy number of ≥6.0, polysomy or co-amplification of CEP17 with an average CEP17 count of 5, and with HER2 receptor overexpression in 75% of cases. On the basis of the HER2 gene copy number alone, we observe 494 cases (8.2%) that are in the equivocal range. Most of these cases (>80%) were also nondecisive by immunohistochemistry (score 2+) irrespective of whether ratio was <2.0>. The number of equivocal cases that would require HER2 reflex testing decreases to 113 (1.9%) if in addition to the HER2 gene copy number also the ratio of HER2 and CEP17 copy numbers is considered via dual-color in situ hybridization. The combination of applying the HER2 mean gene copy number as well as the HER2/CEP17 ratio to define equivocal test decisions by fluorescence in situ hybridization as proposed by the current ASCO/CAP guidelines appears to be a more optimum approach to adopt in order to avoid or minimize reporting of false negative results. Using the mean HER2 gene copy number alone for decision making results in a significant increase of equivocal cases.


Accurate testing of HER2/neu protein level by immunohistochemistry and/or HER2 gene copy-number status by in situ hybridization is a prerequisite for effective anti-HER2 therapy in breast and gastric cancer.[1,2] Thereby, determination of HER2 status is an integral part of the diagnostic work-up for sub-typing breast cancer and for clinical therapy decision making.[3]

Most of the previous HER2-directed therapy studies were based on HER2 positivity criteria described in FDA-approved test kits employed ('FDA criteria'). The cutoff value of 2.0 was established during certification of the PathVysion dual-color fluorescence in situ hybridization kit in 2002.[4] Accordingly, in most of the approved dual-color in situ hybridization tests patients are eligible for HER2-directed treatment if the HER2/CEP17 ratio is ≥2.0 ( The positivity cutoff for the mean HER2 gene copy number was initially set at >4.0 by the Inform Oncor monocolor fluorescence in situ hybridization test which was approved in 1997 for prognostic purposes ( Later on, FDA approved a monocolor chromogenic assay (SpoT-Light, Invitrogen, USA) for predictive purposes and herein the cutoff for the mean HER2 gene copy number was set at >5 ( If HER2 immunohistochemistry tests were used, positivity was defined by intense ring-shaped immunostaining in >10% of tumor cells (HercepTest, Dako, Denmark) (reviewed in Hanna et al[5]). In 2007, ASCO/CAP recommendations were published which defined a HER2/CEP17 ratio of >2.2 as HER2 positive and also introduced an equivocal range of 1.8–2.2.[6]

Recently, ASCO/CAP have recommended new cutoff thresholds for definition of positivity for both immunohistochemistry and in situ hybridization,[7] based on the refinement of the former 2007 criteria. Most importantly separate cutoffs for mono- and dual-color in situ hybridization are given. Tumors with a HER2/CEP17 ratio ≥2.0 and those with a mean HER2 gene copy number ≥6.0 are considered eligible for HER2-directed therapy. Thereby cases with a ratio ≥2.0 are considered HER2 positive irrespective of the mean HER2 gene copy number putting this criterion as the most decisive. In addition, much more emphasis is put on quality assurance issues such as the consideration of histopathological breast cancer subtypes and tumor grading.

In this study, we revisited the primary HER2 testing data of the HERceptin adjuvant (HERA) screening population[1,8] with regard to FDA and ASCO/CAP scoring criteria and to the concordance of fluorescence in situ hybridization HER2/CEP17 ratio and the mean HER2 gene copy number.