Bright-field microscopy for HER2 gene assessment: not just DISH-ful thinking?

The human epidermal growth factor receptor gene ERBB2 (HER2) is located on chromosome 17 and amplified in approximately 20% of invasive breast carcinomas. 1,2 Due to the availability of effective anti-HER2 therapies, the prognostic significance of HER2 gene amplification, and the predictive value of HER2 status with regard to response to chemotherapy, laboratory testing for protein overexpression and/or amplification is critical for clinical decision making in individual patients. Testing of HER2 on all newly diagnosed invasive breast carcinoma is standard of care. Although there is no gold standard for assessing HER2 status in breast cancer , immunohistochemistry (IHC) for protein expression and fluorescence in situ hybridization (FISH) for HER2 gene copy number are the preferred assays. 3 Although no test is perfect, clinical utilization depends on a number of factors, including test accuracy but also consistency of results, as well as ease of performance and interpretation. Due to early standardization, consistency in reporting, and only a small number of equivocal results, the FISH assay became the preferred method for assessing HER2 gene status. 4,5 FISH is also used as the reflex test in IHC equivocal (2+ score) cases. FISH on IHC equivocal cases can identify a small percentage of amplified cases but also aneuploidy and other difficult to assess abnormalities of chromosome 17. Despite its widespread use, the FISH assay does have some drawbacks. FISH is considered a morphology-based assay, but the fluorescent nature of the assay results in less than optimal histologic details (compared with bright-field microscopy), so the area of invasive tumor generally needs to be marked on a parallel H&E-stained section. The procedure itself takes 3 days to complete, and the signals fade over time. To overcome the delay in reporting FISH results and to have better histologic details present on tissue section, researchers developed bright-field in situ hybridization assays. Chromogenic in situ hybridization or CISH uses diaminobenzidine as the chromogen and therefore results in brown signals. Although some studies have shown excellent correlation between CISH and FISH, 6-8 the HER2 signals on CISH slides are sometimes not very discrete and difficult to count due to background staining. Moreover, with the first-generation CISH assays, the centromeric probe for chromosome 17 (CEP17) and the HER2 probe had to be hybridized and visualized on different tissue sections, complicating assessment for aneuploidy and determination of the HER2/CEP17 ratio. The second-generation in situ hybridization assays use an enzyme-linked probe to deposit …