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


In the past few years, a continuous improvement of the accuracy of HER2 testing has been observed.[10–13] As a part of this process, the American Society of Clinical Oncology and the College of American Pathologists have recently updated the recommendations for HER2 testing in breast cancer. Besides improved technical guidelines for standardization and test performance, the expert panel modified the interpretation guidelines for both in situ hybridization and immunohistochemistry. Although the former ASCO/CAP guidelines focused more on the exclusion of false positive cases, the application of the current guidelines favors the exclusion of false negative HER2 results.

In this study, we applied the most recent ASCO/CAP guidelines for fluorescence in situ hybridization on one of the largest HER2 fluorescence in situ hybridization series in 6018 breast cancer specimens of the screening population of the HERA trial.[1] The number of patients included into the HERA trial would have differed by only five cases if the new ASCO/CAP 2013 criteria would have been applied. These cases are scored fluorescence in situ hybridization positive according to the new guidelines because the mean HER2 gene copy number was higher than 6. These five cases have been excluded from the HERA trial because the immunohistochemistry was equivocal and the fluorescence in situ hybridization HER2/CEP17 ratio was <2. The HERA fluorescence in situ hybridization data support the ASCO/CAP recommendation of receiving a HER2 targeted therapy in case the mean HER2 gene copy number is equal or higher than 6 irrespective of the ratio with up to 75% of all cases being also immunohistochemistry positive. Dowsett et al.[8] could show that the degree of HER2 amplification in HERA was not associated with therapy response. Patients with a low HER2 amplified tumor (mean HER2 gene copy number 4≤ and ≤9) had identical outcome compared with patients with highly amplified tumors (mean HER2 gene copy number >9). This observation implies that patients with a mean HER2 gene copy number between 4 and 6 might have a chance to profit from HER2-directed therapy irrespective of the ratio which is now reflected in the current ASCO/CAP guidelines.

Re-evaluation of the HERA trial data disclosed a high agreement between fluorescence in situ hybridization results obtained by HER2/CEP17 ratio and mean HER2 gene copy number (Table 4). The discrepancies were mainly caused by the definition of the equivocal range. The FDA criteria did not foresee any final equivocal result. The former ASCO/CAP 2007 guidelines defined the equivocal range based on the HER2/CEP17 ratio which results in 44 equivocal cases. The new ASCO/CAP guidelines define the equivocal range for dual-color in situ hybridization on a combination of HER2/CEP17 ratio and the mean HER2 gene copy number resulting in 113 equivocal cases (1.9%). About 14% are determined as HER2 positive using immunohistochemistry reflex testing whereas >80% of cases remain equivocal. The amount of cases to be re-investigated a second time would increase dramatically to 494 equivocal cases (8.2%) if the HER2 mean copy number would have been used alone as this is the case for single gene monocolor in situ hybridization, requiring an 11x increase in additional immunohistochemistry testing and hindering clinical decision making. The ASCO/CAP decision to combine both the HER2 ratio and the mean HER2 gene copy number to define the equivocal range for dual-color in situ hybridization seems to be a valid compromise between reducing the false negative rate and not unnecessarily enforcing a retest.

We analyzed the distribution of CEP17 signals in more detail. The percentage of tumors with a mean CEP17 count of ≥3.0 (so-called polysomy rate) and the mean absolute CEP17 per case count were assessed. All tumors negative or equivocal by HER2 ratio but positive based on the mean HER2 gene copy number showed CEP17 counts ≥3.0. Recent studies using CGH indicate that true polysomy, i.e., a gain of extra copies of the whole chromosome 17, is a rare event[14] (review in Hanna et al[5]). Complex rearrangements of chromosome 17 were often observed and the locus bound by the CEP17 probe may be amplified together with the HER2 locus or independently. In our data, increased CEP17 counts are coupled with high HER2 gene count (Table 2). Clinical evidence emerged that the CEP17 count on its own has no predictive value for HER2-directed therapy[8,15] whereas the value of the CEP17 count for response prediction of anthracyclines is still under debate.[16]

Our data (Table 2) show a high immunohistochemistry positivity rate among the cases with a high mean HER2 gene copy number and a low ratio. The average percentage of HER2 immunohistochemistry 3+ cases is higher than 66% for cases showing a mean HER2 gene copy number ≥6, irrespective of the ratio. This observation is supported by the fact that it was recently shown by image analysis that immunohistochemistry is more closely related to the mean HER2 copy number than to ratio values in 3401 investigated HERA trial cases.[17]

In summary, the retrospective application of the new ASCO/CAP 2013 guidelines on the HERA fluorescence in situ hybridization data results in only few changes of included patients compared with the FDA guideline. The in situ hybridization evaluation methods calculating the HER2/CEP17 ratio and absolute HER2 gene copy number show a high degree of concordance yet are not interchangeable. The determination of the mean HER2 gene copy number correlates more closely with the HER2 immunohistochemistry results whereas the ratio method is more powerful in discriminating positive from negative cases. Abnormal CEP17 numbers cause discrepancies between the two techniques. A high CEP17 count (≥3.0 copies) may justify preferring the absolute count as the majority of those ratio-low but count-high cases are immunohistochemistry positive.