Saeed Sadeghi, M.D. and Steven G. Wong, M.D.

The human epidermal growth factor receptor-2 (HER2/neu/c-erbB-2) is a transmembrane glycoprotein in the epidermal growth factor receptor family. Normal tissue expression is relatively low, typically measured 0 to 1+ on currently available immunohistochemistry tests such as HercepTest. However, in 20% to 25% of breast cancer cell lines, it is observed to be overexpressed. Early pioneering work by Slamon et al¹ demonstrated that overexpression of HER2 is found to be pathogenically driven by gene amplification and is associated with clinical outcome, such that median survival of patients is shortened from 6 years to 3 years. Studies demonstrated that HER2 status is associated with sensitivity to anthracyclines, resistance to cyclophosphamide-based regimens and resistance to hormone therapy.² Subsequently, the first humanized antibody against HER2 receptor, trastuzumab (Herceptin), was developed and studied in clinical trials.

Identification of HER2/neu receptor and subsequent development of trastuzumab has allowed for significant improvement in the clinical outcome of this subset of breast cancer patients. Use of Herceptin in both metastatic and adjuvant settings in conjunction with chemotherapy has resulted in significant increase in the disease-free survival and overall survival of these patients.^3-5 In fact, recent analysis by Dawood et al⁶ from MD Anderson has demonstrated that with the advent of Herceptin, the clinical outcome of the HER2/neu positive breast cancer patient now mirrors those who are HER2/neu receptor negative, effectively negating the poor clinical outcome which was associated with HER2/neu receptor positivity.

Given the clear benefits of Herceptin in this patient population, it is of paramount importance that this subset of patients be correctly identified. HER2 testing has undergone rigorous evaluation and several assays are available. Initial identification was done through development of an immunohistochemistry (IHC) assay for HER2 protein. This was based on the initial studies of Slamon et al who utilized this approach to distinguish positive from negative HER2 status in patients. Immunohistochemical technique involves incubation of the tissue section with a primary HER2/neu antibody. Conjugation of the secondary antibodies then allows an immunofluorescent signal which would correlate directly with the level of the protein in a specimen. The amount of staining is visualized under the microscope and a score is given. Score 0 denotes no immunostaining. Score 1+ indicates immunostaining that is weak and in less than 30% of the tumor cells. Score 2+ indicates complete membranous staining and score of 3+ indicates uniform intense membranous staining in at least 30% of cells.⁷

However, a number of factors can affect the IHC assay results, including tissue fixation and processing, reagent variability, choice of antibody used, as well as interpretation and expertise of laboratory doing the testing and processing.⁷ As a result, IHC can be inconsistent and hence inaccurate in identifying HER2 positive patients. In order for patients to be eligible for clinical trials, only patients with tumors having HER2 immunostaining scores of 2+ or 3+ were considered eligible.

Another assay method involves using fluorescence in situ hybridization (FISH) for HER2. This DNA-based assay directly assesses HER2 gene amplification and as a result may be a more accurate means of assessing HER2 positivity for at least 2 reasons: DNA is a much more stable entity than protein, and the pathogenic signature of HER2 positive disease is the amplification event as opposed to simply overexpression of the HER2 protein. In this technique, paraffin-embedded tissue sample is pretreated and digested with protease and subsequently hybridized with fluorescent-labeled probes for HER2/neu gene (HER) and alpha satellite DNA for chromosome 17 (chromosome enumeration probe or CEP). After washing off the un-hybridized probes, the amount of the hybridized probes is quantified. FISH testing is reported as amplified (HER/CEP17 ratio of > 2.0).⁸ FISH can also be done using only the HER gene probe and counting the number of positive probes per nucleus although this would not account for ploidy possibly confounding the results.

Several studies have shown that IHC can be variable in its ability to identify HER2-amplified tumors and when compared to FISH technique, discordance rates can be as high as 20%. Mass et al⁹ evaluated the influence of HER2/neu gene amplification by FISH on clinical outcomes in 3 metastatic clinical trials that had been reported by 2005. All specimens had demonstrated IHC positivity of 2+ and 3+ on clinical trials assay and were considered to be positive for FISH if the specimen had a ratio of greater than or equal to 2.0. Overall 78% of the samples were FISH positive and 22% were FISH negative and the clinical benefit of Herceptin was limited to the FISH positive group.⁹ Similarly Dybdal et al¹⁰ also looked at concordance between IHC and FISH. They demonstrated that overall concordance between FISH and IHC was 82%. Assay agreement between FISH results and specimens with immunostaining scores of 0, 1+, and 3+ was 97%, 93%, and 89%, respectively. In the IHC 2+ group, however, there was concordance only in 24% of the cases, suggesting that the bulk of IHC 2+ cases are actually FISH negative.¹⁰ In an earlier study, Press et al⁸ also compared various IHC and FISH assays that were available. Both FISH assays (Vysis and Ventano) were noted to have the greatest accuracy at 97.4% and 95.7%, respectively. Two of the IHC assays (R60 and 10H8) were nearly accurate at 96.6% and 95.7% accuracy, respectively.⁸

Based on the numerous studies assessing the validity of IHC and FISH testing for HER2, the American Society of Clinical Oncology and the College of American Pathologist have released new guidelines for the testing of HER2 status in breast cancer. IHC scores of 0, and +1 are considered negative, equivocal if 2+, and positive if 3+. HER2 FISH is reported as amplified if ratio is greater than 2.2, equivocal if 1.8 to 2.2, and negative if less than 1.8. The new guidelines also required that the laboratories demonstrate concordance of 95% between FISH + and IHC 3+ cases and FISH - and IHC 0/1+ cases.⁷

An area of concern has been the patient group in whom FISH scores fall in the equivocal range. Recommendations for this group have been to repeat the FISH test and/or do confirmatory testing with IHC. Recently it has been suggested that tissue samples with equivocal HER2 results by FISH demonstrate chromosome 17 polysomy. However, in contrast to FISH amplified cases, the polysomy 17 was not associated with high tumor grade, hormone receptor negativity or reduced disease-free survival and the tumors with polysomy 17 more resembled HER-2 negative tumors than HER-2 positive tumors.¹¹ Nevertheless, additional clinical trials are needed to investigate if polysomy 17 tumors benefit from HER-2 targeted therapy.