HER2-FISH, Breast Cancer HER2-FISH, Breast Cancer

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HER2-FISH, Breast Cancer

The Human Epidermal Growth Factor Receptor 2 (HER2) is a central biomarker in invasive breast cancer, and the HER2 status has both important prognostic implications, and is predictive for the patient’s response to HER2-targeted treatment. According to the guidelines of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP), the HER2 status must be determined by genetic and/or protein expression analysis of breast tissue sections using immunohistochemistry (IHC) and/or in situ hybridization (ISH), respectively, see [1].

To determine the HER2/CEN-17 ratio, the total number of HER2 signals (Red) and the number of CEN-17 (Green) signals in the same 20 nuclei are identified. The total number of HER2 signals is then divided by the number of CEN-17 signals. Ratios above 2 are classified as amplified whereas ratios below 2 are classified un-amplified. Caution should be taken when intepreting on results near the cut-off (1.8 – 2.2). The operator only needs to define an overall region of interest.

Figure 1

Figure 1

Close-up of a group of nuclei including gene probes.

Figure 2

Figure 2

Same image as FIGURE 1, with identified nuclei and gene probes.

Figure 3

Figure 3

Close-up of a single nuclei with gene probes.

Figure 4

Figure 4

The same FOV as FIGURE 3, where the gene probes have been detected with a green and red cross.

Figure 5

Figure 5

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Quantitative Output variables

Four outputs are obtained from this protocol:

  • Red signal: The total number of red probe signals
  • Green signal: The total number of green probe signals
  • Nuclei: The total number of nuclei
  • Amplification R-G: Amplification level, calculated as the red signals divided by the green signals.


The image analysis algorithm for this protocol involves several steps. First, a pre-processing step identifies the stained nuclei and the gene probes. Nuclei exhibiting signals of only one color are then removed.


Human Epidermal Growth Factor Receptor 2, flourescence, in situ hybridization, ASCO/CAP guidelines, quantitative, digital pathology, image analysis


1. Wolff, A.C. et. al., American Society of Clinical Oncology/College of American Pathologists guideline recommendations for Human Epidermal GrowthFactor Receptor 2 Testing in Breast Cancer, J. Clin Oncol 2007, 25 (1), 118-45, DOI

2. Brügmann, A. et. al., Digital image analysis of membrane connectivity is a robust measure of HER2 immunostains, Breast Cancer Res Treat 2012, 132 (1), 41-9, DOI

3. Brügmann, A. et. al., Membrane connectivity as a robust measure for the HER2 IHC score. (Last updated/Accessed: 23/08-2019).

4. Laurinaviciene, A. et. al., Membrane connectivity estimated by digital image analysis of HER2 immunohistochemistry is concordant with visual scoring and fluorescence in situ hybridization results: algorithm evaluation on breast cancer tissue microarrays, Diagn. Pathology 2011, 6 (1), 87, DOI

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