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

Materials and Methods

Patients and Tumor Samples

The presented data are derived from the screening population of the HERA study which was an international, multicenter, randomized trial that compared 1 or 2 years of trastuzumab with observation alone in patients with HER2-positive breast cancer in the adjuvant setting.[1,8] Tissue blocks or tissue slide sets from 10 859 locally pre-tested patients with positive or inconclusive HER2 results were sent to the central laboratory Targos Molecular Pathology GmbH, Kassel, Germany for either HER2 immunohistochemistry and/or fluorescence in situ hybridization analysis, depending on the request of the respective local site. On the basis of this test algorithm, a total of 5174 patient samples have been analyzed prospectively by HER2 fluorescence in situ hybridization. In addition to the prospectively obtained data, 844 additional fluorescence in situ hybridization analyses were conducted retrospectively at Targos and European Institute of Oncology (Milan, Italy) for HERA samples where an immunohistochemistry result existed without centrally confirmed fluorescence in situ hybridization.[8] Thus, a total of 6018 HERA patient samples were analyzed by HER2 fluorescence in situ hybridization. Of these, 3089 samples were also analyzed by immunohistochemistry.

Analysis of HER2 Protein and Gene Status

Immunoistochemistry (HercepTest Dako, Glostrup, Denmark) was performed according to the manufacturer's instructions, with the exception that an oil bath was used to heat the pretreatment buffer instead of a water bath. Fluorescence in situ hybridization staining was performed using the PathVysion HER2 DNA Probe Kit (PathVysion, Abbott Molecular, Des Plaines, Illinois, USA) according to the manufacturer's instructions.

HER2 Evaluation

HER2 immunohistochemistry scoring was performed according to the HercepTest guidelines (package insert, DAKO, Glostrup, Denmark) classifying a tumor as HER2 negative (Immunohistochemistry scores 0,1+), equivocal (score 2+) or positive (score 3+). Immunohistochemistry positivity was defined by strong complete membrane staining in >10% of tumor cells. Fluorescence in situ hybridization scoring was performed according to the PathVysion package insert guidelines determining the mean value of HER2 and CEP17 signals in 60 contiguous invasive tumor cells. Mean values of the HER2 gene copy number and the HER2/CEP17 ratio were calculated and patients with HER2/CEP17 ratios ≥2.0 were considered eligible for treatment with trastuzumab.

For the comparison with the cutoff definitions by ASCO/CAP guideline recommendations,[6,7] raw data of HER2 gene count and CEP17 counts per cell were used. An overview about the different thresholds is given in Table 1. The term 'polysomy' in this manuscript refers to the definition of ≥3 CEP17 signal counts per cell calculated by the arithmetic mean per case.[9] Homogenous protein expression was defined as ≥80% of tumor cells that express HER2. Focal protein expression was defined as ≤30% of tumor cells that express HER2. Consequently, HER2 expression in 31–79% of tumor cells was defined as heterogeneous protein expression.

Statistical Evaluation

Data analysis was performed using the Targos HERA database (closure 14 February 2006) by 'R' statistical programming language version 2.13.1 (available under the GNU General Public License at Mean values with s.d. were calculated for HER2 and CEP17 counts as well as HER2/CEP17 ratio.