Out of the darkness and into the light: bright field in situ hybridisation for delineation of ERBB2 (HER2) status in breast carcinoma

Abstract

Assessment of ERBB2 (HER2) status in breast carcinomas has become critical in determining response to the humanised monoclonal antibody trastuzumab. The current joint College of American Pathologists and the American Society of Clinical Oncology guidelines for the evaluation of HER2 status in breast carcinoma involve testing by immunohistochemistry and fluorescence in situ hybridisation (FISH). However, neither of these modalities is without limitations. Novel bright field in situ hybridisation techniques continue to provide viable alternatives to FISH testing. While these techniques are not limited to evaluation of the HER2 gene, the extensive number of studies comparing bright field in situ techniques with other methods of assessing HER2 status allow a robust evaluation of this approach. Analysis of the literature demonstrates that, when used to assess HER2 gene status, bright field in situ hybridisation demonstrates excellent concordance with FISH results. The average percentage agreement in an informal analysis of studies comparing HER2 amplification by chromogenic in situ hybridisation with FISH was 96% (SD 4%); kappa coefficients ranged from 0.76 to 1.0. Although a much smaller number of studies are available for review, similar levels of concordance have been reported in studies comparing HER2 amplification by methods employing metallography (silver in situ hybridisation) with FISH. A summary of the advancements in bright field in situ hybridisation, with focus on those techniques with clinical applications of interest to the practicing pathologist, is presented.

Figure 1

Conceptual schematic of single-colour chromogenic in situ hybridisation demonstrating bright field detection of a digoxigenin-labelled probe. The probe is recognised by an antidigoxigenin fluorescein isothiocyanate primary antibody followed by detection with an anti-fluorescein-isothiocyanate horseradish peroxidase (HRP). After addition and oxidation of diaminobenzidine, a dark brown signal is deposited at the target site.

Figure 2

Examples of HER2 detection using the chromogenic in situ hybridisation technique in breast carcinoma. (A) Demonstration of non-amplified HER2 producing 1–2 signals per nucleus. Examples of HER2 amplification where the peroxidase signal exists as either a cluster of gene copies (B) or as multiple individual gene copies (C). Original magnification ×600. Reproduced from Tanner et al with permission from the American Society for Investigative Pathology.

Figure 3

(A) Schematic of gold-facilitated in situ hybridisation (GOLDFISH) assay demonstrating recognition of the biotin-labelled probe with a biotinylated tyramide followed by strepavidin-Nanogold (Nanoprobes). The Nanogold particulate nucleating agent facilitates autometallographic deposition of gold from a solution of silver acetate in the presence of hydroquinone at the target site. (B) Example of HER2 detection using the GOLDFISH technique with demonstration of non-amplified HER2 in infiltrating ductal carcinoma producing 1–2 signals per nucleus. (C) Example of GOLDFISH technique in a breast carcinoma containing amplified HER2 that is demonstrated by multiple large confluent nuclear signals. Original magnification ×400. Reprinted from Powel et al with permission from Elsevier.

Figure 4

(A) Schematic of enzyme metallography demonstrating detection of the probe with a primary anti-hapten antibody followed by a horseradish peroxidase (HRP)-labelled secondary antibody. Enzyme-catalysed deposition of metallic silver from the silver acetate solution, in the presence of hydroquinone, then occurs at the target site. (B) Example of HER2 detection using the enzyme metallography EnzMet technique with demonstration of non-amplified breast cancer; 1–2 signals are present in each nucleus. (C) Example of breast carcinoma containing amplified HER2; multiple distinct signals are present in each nucleus. Original magnification ×400. Reprinted from Powel et al with permission from Elsevier.

Figure 5

Conceptual schematic demonstrating dual detection of HER2 and chromosome 17 by bright field double in situ hybridisation. By this technique, dual detection can be accomplished using individual single haptens. The two probes are incompatible and two rounds of target DNA denaturation, hybridisation and stringency washes are carried out sequentially. DNP, dinitrophenol.

Figure 6

Examples of HER2 and chromosome 17 detection in non-amplified (A) and amplified (B) breast carcinomas using the dual-colour dual-hapten approach. Single HER2 gene (C) and chromosome 17 polysomy (D) are demonstrated using bright field double in situ hybridisation. Magnification ×100. Reproduced from Nitta et al with author permission.