High Concordance Between Herceptest Immunohistochemistry and ERBB2 Fluorescence in Situ Hybridization Before and After Implementation of American Society of Clinical Oncology/College of American Pathology 2007 Guidelines

Maria E Vergara-Lluri; Neda A Moatamed; Elizabeth Hong; Sophia K Apple


Mod Pathol. 2012;25(10):1326-1332. 

In This Article


Interlaboratory discordance rates for HER2 immunohistochemistry and ERBB2 fluorescence in-situ hybridization testing have been reported to be as high as 20%.[16,17] In order to address the issue of HER2 and ERBB2 test accuracy, the American Society of Clinical Oncology and the College of American Pathologists reviewed the existing literature on testing, and devised and published a guidance document in 2007 (ASCO/CAP 2007 guidelines), which included the modification of analysis of immunohistochemistry and fluorescence in-situ hybridization tests. It also contained the recommendation of 95% concordance rate for immunohistochemical testing for HER2 with another validated test such as fluorescence in-situ hybridization.[10] Since 2003, both immunohistochemistry and fluorescence in-situ hybridization tests have been performed at our institution regardless of HercepTest immunohistochemical scores. Thus, this allowed us to investigate testing concordance rates of HER2 immunohistochemical and ERBB2 fluorescence in-situ hybridization analysis utilizing the US FDA guidelines from years 2003–2007, and subsequently the ASCO/CAP guidelines for testing from years 2008–2010. The purpose of this study was to examine the merit of changing the HER2 and ERBB2 testing guidelines in order to improve concordance rates and to decrease the number of inconclusive cases (scored as 2+ on immunohistochemical analysis).

The present study indicates that our institution has excellent and identical concordance rates of 97.6% for HercepTest HER2 immunohistochemistry and PathyVysion ERBB2 gene fluorescence in-situ hybridization testing utilizing both US FDA and ASCO/CAP guidelines. These rates are in accordance with the recommendation that any two diagnostic companion tests establish a concordance rate of >95%.[10]

The accuracy of HER2 immunohistochemistry is reported to be highly dependent upon both pre-analytical and analytic factors. Although fixation times and optimal tissue handling procedures were only strictly imposed after publication of the ASCO/CAP guidelines in 2007, our identical concordance rates suggest that there is minimal to no interference by pre-analytic and analytic variables. We observed a similar lack of discordance in immunohistochemical accuracy when we changed fixative types, fixation times, and ischemia times for HER2 and other hormonal receptor studies for breast cancer.[18,19]

On the basis of the change in guidelines for scoring 3+ immunohistochemical positivity in 10–30% of tumor cells, one would expect an increase in equivocal (2+) cases as well as a decrease in positive (3+) cases given the more stringent cutoff. Middleton et al[20] reported a 64% reduction of inconclusive cases, from 10.8–3.4%, and an increase in their concordance rate from 98–98.5% after implementation of ASCO/CAP guidelines, supporting the change in ERBB2 testing guidelines. In our patient population, however, the percentage of inconclusive/equivocal cases by immunohistochemistry was 12% with US FDA guidelines and 11% with ASCO/CAP guidelines. Our study did not disclose a significant decline in inconclusive immunohistochemistry results.

Moreover, changing the fluorescence in-situ hybridization cutoff value for ERBB2 gene amplification from 2.0–2.2 based on ASCO/CAP guidelines, one would expect fewer ERBB2-amplified cases. Indeed, we did see a significant decline in cases considered to be ERBB2 amplified from 19–15% (P=0.03). Similarly, Atkinson et al[21] found 3.3% fewer ERBB2-positive cases when the new guidelines were implemented. Although the decrease in ERBB2-amplified cases was expected, the considerable decline in amplified cases may not be because of a change in fluorescence in-situ hybridization cutoff values alone. It may also be secondary to a change in our patient population; indeed, we have noticed a steady decline in ERBB2-positive breast cancers in our patient population, with a cumulative 7% reduction over the past 8 years (Table 6). This may represent an overall shift in patient populations, wherein earlier studies suggested that as many as 30% of breast cancers displayed HER2 overexpression.[3] Subsequent studies, however, have displayed a lower percentage of HER2-positive cases, from 15–25% of breast cancers to as low as 7%.[22,23]

A small number of our cases were rated as negative by HercepTest immunohistochemistry and yet demonstrated amplification by PathVysion fluorescence in-situ hybridization, thus considered as a false-negative result. Using a stepwise algorithm of performing immunohistochemistry first, then subsequent fluorescence in-situ hybridization testing based on immunohistochemistry results, a small percentage of samples would inevitably be scored as falsely negative, thereby inappropriately excluding these patients from trastuzumab (Herceptin) therapy. An estimated 3–4% of primary ERBB2 testing are falsely negative.[24] Excluding patients because of a falsely negative result would be quite disadvantageous, as the addition of Herceptin therapy can reduce the recurrence risk by half and mortality by a third in early stage breast cancer patients.[17] In our study, the overall false-negative rate is 1.9%, well below the aforementioned 3–4% false-negative rate. Following the ASCO/CAP guidelines did yield a slight decrease in the false-negative rate from 2.1–1.4%.

On the other hand, an exceedingly small percentage of patients (0.2%) were scored as falsely positive, ie, rated as positive on immunohistochemistry, and yet proved to be unamplified by fluorescence in-situ hybridization. These cases of overexpression by immunohistochemistry were secondary to polysomy of chromosome 17, and not ERBB2 gene amplification. Polysomy of chromosome 17 is a well-documented cause for false-positive immunohistochemical results, accounting for up to 2.8% of HER2 immunohistochemically positive cases in one study.[25] The utility of targeted Herceptin therapy on polysomy 17 cases is unknown, and the current data regarding response to therapy is scarce at the present time. Elucidation of effectiveness of therapy in this setting is particularly important. Trastuzumab therapy is not without adverse effects and can even lead to significant cardiotoxicity, especially when combined with other chemotherapy agents, such as anthracyclines.[26] We attribute the increase in false-positive rates from 0–0.7% from 2003–2007 to 2008–2010, respectively, solely to three cases of polysomy 17, and not because of a change in test scoring as based on ASCO/CAP guidelines.

We ascribe our high concordance rates to several factors:

  1. High volume of HER2 testing.

  2. HER2 immunohistochemistry interpretation and scoring are performed by breast subspecialty anatomic pathologists only (three pathologists at our institution).

  3. One hematoxylin and eosin slide is submitted to the Cytogenetics Laboratory on all cases, and the area of invasive cancer is marked with permanent ink. This ensures that the cytogenetic technologists and clinical pathologists focus on scoring ERBB2 fluorescence in-situ hybridization in the designated areas of invasive carcinoma only, and excludes areas of normal breast tissue or ductal carcinoma in-situ component.

We recognized in our study that the most common type of discrepancy arose from immunohistochemistry-negative/fluorescence in-situ hybridization-positive cases (false negative). This is in contrast to a study by Grimm et al,[27] which showed that the most common type of discrepancy was immunohistochemistry positive/fluorescence in-situ hybridization-negative (false positive) cases because of over-interpretation of immunohistochemistry positivity. The causes of false-positive interpretational error identified in their study included cautery artifact, chatter artifact, granular staining, and over-interpretation of complete circumferential membrane staining—all of which were not problematic in our current study. Most of our false-negative cases (immunohistochemistry negative/fluorescence in-situ hybridization positive) were due to underestimating the score to 1+ rather than 2+, which is also an interpretational error rather than a technical error. In rare cases, where immunohistochemistry score was interpreted as '0' and subsequently proven to be fluorescence in-situ hybridization amplified, we speculated that a technical problem was at fault. In many of these false-negative cases, as a matter of troubleshooting, we would repeat immunohistochemical testing on the same tissue block, and additionally perform immunohistochemical analysis on a different block from the tissue containing invasive carcinoma. In the end, these cases would continue to be scored as 0 or 1+ on the repeat testing of additional samples. One of these cases was owing to monosomy 17 where fluorescence in-situ hybridization was reportedly amplified.

On the basis of our study, we would advocate performing fluorescence in-situ hybridization analysis in addition to immunohistochemistry testing to capture the small percentage of patients who may benefit from trastuzumab therapy and to exclude those who do not show true ERBB2 amplification. We did not retrospectively review immunohistochemistry slides for changes in scoring cutoff values as the concordance rates utilizing US FDA and ASCO/CAP guidelines were essentially identical. Of interest, pre-analytic variables such as fixation time and ischemic time did not alter the concordance rates between US FDA and ASCO/CAP guidelines. Additionally, the number of inconclusive 2+ immunohistochemical cases did not show a substantial decline, contrary to the intent of the change in ASCO/CAP guidelines. To our knowledge, our study is the largest study that examines the impact of the implementation of ASCO/CAP guidelines on HER2 immunohistochemistry and ERBB2 gene fluorescence in-situ hybridization testing concordance rates. At our institution, implementation of the new ASCO/CAP scoring guidelines did not significantly influence HER2 testing concordance rates nor was there a decrease in immunohistochemically equivocal/inconclusive cases.