FISH Analysis of ESR1 Amplification in Breast Cancer - A Video Illustration

Analysis of estrogen receptor alpha gene (ESR1) amplification detected by fluorescent in situ hybridization (FISH) in breast cancer:

The ESR1 FISH probe (ZytoVision GmbH, Bremerhaven, Germany) is labeled with a “SpectrumGreen” fluorochrome. Gene signals appear as green pinhead shaped dots.

For copy number reference a chromosome enumeration probe for centromere of Chromosome 6 (CEP6) is labeled with a “SpectrumOrange” fluorochrome. CEP3 signals appear as orange pinhead shaped dots.

Nuclei are counterstained with 4’,6-diamino-2-phenylindole (DAPI). Nuclear chromatin DNA appears in blue color.

The video clip was taken using an Olympus IX51 microscope at 100´ magnification, an Olympus XM10 digital camera (1,376 ´ 1,032 pixels) and Olympus cellSens imaging software.

Analysis starts with microscope fluorescence filter setting for fluorochrome “SpectrumGreen”:

Distribution of FISH signals over cell nuclei of tissues can mostly be estimated by a first sight screening in the fluorescence filter spectrum of the gene probe fluorochrome. Signals in full z-axis are taken into account.

Filter change to “SpectrumOrange”:

Signals of the centromere reference, the chromosome enumeration probe (CEP), are checked for signal quality.

Filter change to “SpectrumGreen”:

An area with nuclei showing distinguishable gene signals respectively signal clusters (e.g. signal cluster marked by white arrow in minute 01:27) is selected to determine copy number. Signals in full z-axis are taken into account.

Filter change to “SpectrumOrange”:

Signals of the centromere reference, the chromosome enumeration probe (CEP), are checked for signal quality. Signals in full z-axis are taken into account.

Filter change to DAPI (blue):

Chromatin stained nuclei are checked. Sufficient integer and separable nuclei are selected to determine gene and centromere copy number. Full z-axis is taken into account.

It has also to be taken into account, that a complete integrity of nuclei can not be expected or guaranteed for most nuclei analyzed, since a risk of truncation of nuclei due to tissue slide preparation, is always given in 4µm tissue sections.

Filter change to “SpectrumGreen”:

Gene signals are counted. The three dimensional constitution of the nucleus is taken into account for analysis by variable adjustment of optical z-axis layers.

Filter change to “SpectrumOrange”:

CEP3 signals of the centromere reference are counted. Signals in full z-axis are taken into account.

References

Singer, C.F. et al. Estrogen receptor alpha (ESR1) gene amplification status and clinical outcome in tamoxifen-treated postmenopausal patients with endocrine-responsive early breast cancer: An analysis of the prospective ABCSG-6 trial. in ASCO Annual Meeting Vol. 30 suppl; abstr 10501 (J Clin Oncol 30, 2012 (suppl; abstr 10501), 2012).

Moelans, C.B., Holst, F., Hellwinkel, O., Simon, R. & van Diest, P.J. ESR1 Amplification in Breast Cancer by Optimized RNase FISH: Frequent but Low-Level and Heterogeneous. PLoS One 8, e84189 (2013).