The Equivocally Amplified HER2 FISH Result on Breast Core Biopsy
To our knowledge, there are no universally accepted, evidence-based guidelines for how to resolve the HER2 status of tumors demonstrating equivocal amplification. The present study was based on 17 breast core biopsy specimens demonstrating invasive carcinoma with equivocal HER2 amplification, defined as an HER2/chromosome 17 centromere ratio of1.8to 2.2. Each case had a corresponding resection specimen, on which HER2 immunohistochemical and repeated fluorescence in situ hybridization analyses were performed. A definitive change in HER2 status based on the resection specimen occurred in 10 (59%) of 17 cases, with 4 patients (24%) becoming eligible for trastuzumab therapy and 6 (35%) triaged as ineligible. These results suggest that genetic and protein expression heterogeneity exists in tumors that show low-level HER2 gene copy numbers. For the purposes of uniform clinical management, HER2 status should be evaluated on a larger tumor sample if the core biopsy specimen demonstrates an equivocal result. These results support the recent American Society of Clinical Oncology/College of American Pathologists recommendations for further testing in cases with equivocal HER2 results.

The proto-oncogene ERBB2 (HER2) is amplified, with subsequent overexpression of its protein product, in 18% to 30% of breast cancers. During the past 2 decades, numerous studies have described the relationship between HER2 over-expression and prognosis, resistance to hormonal therapy, and response to various chemotherapeutic regimens. Furthermore, HER2 amplification in association with the absence of estrogen receptor expression portends a worse prognosis.

Trastuzumab (Herceptin) targets the extracellular domain of the HER2 product. This monoclonal antibody exerts a potent antitumor effect via multiple mechanisms, including the induction of apoptosis and inhibition of angiogenesis. Clinical trials proved the efficacy of trastuzumab, as mono-therapy and in combination with chemotherapy, in metastatic breast cancer that overexpressed HER2. Further subgroup analysis in these trials demonstrated a positive correlation between the degree of HER2 expression and clinical response. However, trastuzumab therapy is not without risk and significant cost. Given the predictive and prognostic significance of HER2 status, HER2 testing is, therefore, recommended for all newly diagnosed and metastatic breast tumors.

Laboratory assessment of HER2 is performed by evaluating immunohistochemical stains targeting the extracellular domain of the HER2 protein product and/or assessing the degree of HER2 amplification via fluorescence in situ hybridization (FISH). Recently, the College of American Pathologists (CAP), the American Society of Clinical Oncologists (ASCO), and the National Comprehensive Cancer Network Task Force proposed algorithms for initial HER2 testing via immunohistochemical analysis and FISH. Of interest is the inclusion of a borderline or equivocal result in these algorithms. An equivocal HER2 immunohistochemical result is defined as tumor cells showing 2+ membranous immunoreactivity out of a possible 3+ (see the "Materials and Methods" section). Equivocal HER2 amplification is defined as an HER2/chromosome 17 centromere (HER2/CEP17) ratio of 1.8 to 2.2 or as an average HER2 copy number of 4 to 6 signals per nucleus if an internal control for chromosome 17 is not used.

Although this equivocal or borderline result category made its first appearance in the 2003 proficiency surveys published by CAP, the nature of breast carcinomas showing equivocal amplification by FISH has not been described. To date, there is no universally agreed on method to reconcile a FISH result of equivocal amplification. The aim of this study was to determine whether an equivocal HER2 amplification result from a core biopsy specimen is consistently representative of the HER2 status of the overall tumor. In addition, we conducted a morphologic study of tumors showing equivocal amplification of HER2 on the core biopsy specimen.