Identification of HER2 Immunohistochemistry-Negative, FISH-Amplified Breast Cancers and Their Response to Anti-HER2 Neoadjuvant Chemotherapy

I-sanna Gibbons-Fideler, MD; Hiroaki Nitta, PhD; Adrian Murillo, PhD; Gary Tozbikian, MD; Peter Banks, MD; Anil V. Parwani, MD, PhD; Zaibo Li, MD, PhD


Am J Clin Pathol. 2019;151(2):176-184. 

In This Article


This study reports our institution's experience with the infrequent circumstance of invasive BCs with discordant HER2 IHC and FISH results in a cohort of 1,107 invasive BCs. Our findings demonstrate a small percentage of patients (1.5%) with discordant HER2 IHC and ISH results, all of them negative by HER2 IHC but FISH amplified. To our knowledge, this is the first study to examine the response to anti–HER2-targeted NAC in patients with such discordant findings. This patient population showed either complete or partial response to anti-HER2 NAC. Had management relied on only IHC determinations, these patients would not have benefited from anti–HER2-targeted therapy. Although these patients accounted for a small portion of all patients with invasive BC, they did account for about 10% of total HER2-positive patients.

The concordance rate between HER2 IHC and FISH is very high, cited in the literature review of the ASCO/CAP guidelines as 95%.[9] However, this small group with discordant HER2 IHC and FISH results can indeed be identified. Several technical and other factors have been proposed as contributing to this phenomenon. The nontechnical factors include HER2 ITH, coamplification/polysomy 17 and monosomy 17, and so on.[15–26] Intratumoral heterogeneity is defined as coexistence of multiple tumor cell subpopulations with distinct HER2 amplification characteristics within the same tumor, and it has been reported in up to 40% of breast cancers.[12,15,26] Coamplification/polysomy 17 is considered present if ISH shows three or more signals with a probe targeting, and its prevalence ranges from 7% to 46%, as reported in various studies.[16–19,21–23,26] True polysomy 17 is rare in BC, and most of cases with increased CEN17 signals are actually coamplification of CEN17 and HER2. FISH with alternative probe of p53 (17p13.1), SMS (17p11.2), or RARA (17q21.2–21.3) can be used to further clarify true polysomy 17 from coamplification. However, these alternative probes are not available in our institution. Monosomy 17 is defined as loss of a chromosome 17 or loss of a portion of the chromosome 17. In clinical practice, monosomy 17-like pattern is usually used in the setting of mean CEN17 signals less than 2.0, although studies have used less than 1.5 or other values.[24,25,27] In the current study, these three factors accounted for 76.5% (13/17) of all discordant cases, including ITH in five cases, coamplification/polysomy 17 in seven cases, and monosomy 17–like pattern in four cases (total 13 cases since two cases had both ITH and coamplification/polysomy 17 and one case had both monosomy 17–like pattern and ITH). The detection of cases negative by IHC but expressing HER2 RNA suggests posttranscriptional mechanisms as an additional basis for discrepancy in relation to protein vs gene amplification detection.

In our current HER2 IHC–/FISH+ cohort, three (37.5%) of eight patients demonstrated PCR, and all eight cases with NAC showed low HER2 amplification signals or low HER2/CEN17 ratio. In our previous study with 64 HER2 IHC+/FISH+ cases, the PCR rate was 61% (39/64), which is much higher than the PCR rate in the current cohort.[12] Indeed, our previous study demonstrated that PCR was positively correlated with HER2 amplification signals.[12] HER2 ITH was identified in five of all 17 cases and three of eight cases with NAC. Our previous study demonstrated that HER2 ITH occurred in approximately 30% of HER2 IHC+/FISH+ cases.[12] All three cases with HER2 ITH showed residual tumor, confirming that HER2 ITH is negatively associated with response to anti-HER2 chemotherapy identified in our previous study.[12] Most patients in our cohort tolerated the therapy well with only mild anti-HER2 therapy-related adverse effects; however, two patients developed severe symptoms requiring emergency room visit and treatment. Therefore, the balance between benefits and adverse effects needs to be considered when given anti-HER2 therapy.

The novel HER2 GPA method merges HER2 IHC and dual ISH by superimposing the two tests on the same slide in correlation with tissue morphology. Previous studies have demonstrated excellent sensitivity and accuracy in detecting ITH with the GPA, in comparison with the traditional separate test modalities.[11–13,28]

The current study confirms this assay's advantage in detecting discordant HER2 IHC and ISH results. Dual HER2 testing using both IHC and ISH methods is not commonly used in US laboratories due to its recent development, the higher cost compared with a single method, and the complexity of implementation. However, the GPA may gain widespread use over time because of its appeal as a one-time test.

In conclusion, we have confirmed the existence of a small subset of invasive ductal carcinomas of breast reproducibly showing HER2 gene amplification without HER2 protein expression by IHC considered positive by recommended guidelines. Despite the relative absence of detectable protein among these cases, they showed partial or complete clinical response to neoadjuvant combined therapy, which included a HER2 blocking agent. Either combined separate IHC and ISH evaluation or the use of the recently developed GPA method can be used to avoid missing this uncommon situation.