FISH analysis of HSR type PIK3CA amplification in endometrial cancer

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• published: 11 Dec 2014
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Analysis of a HSR type phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA) amplification detected by fluorescent in situ hybridization (FISH) in endometrial cancer: The PIK3CA FISH probe (Abnova, Taipei City, Taiwan) is labeled with a “Texas Red®” fluorochrophor. Gene signals appear as red pinhead shaped dots. For copy number reference a chromosome enumeration probe for centromere of Chromosome 3 (CEP3) is labeled with a “FITC” fluorochphor. CEP3 signals appear as green pinhead shaped dots. Nuclei are counterstained with 4’,6-diamino-2-phenylindole (DAPI). Nuclear chromatin DNA is appearing in blue color. Video clip starts with microscope fluorescence filter setting for DAPI staining: 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 4 µm tissue sections. Filter change to “Texas Red®”: Distribution of FISH signals over cell nuclei of tissues can mostly be estimated for a first sight screening in the fluorescence filter spectrum of the gene probe fluorochrophor. Signals in full z-axis are taken into account. PIK3CA signals in this tumor occur predominantly in a pattern of clustered signal distribution of HSR type (homogeneously staining region). Clusters of two, three up six or seven signals signals nearby to each other, but also dislocated additional gene signals occur. Based on previously observed DAPI staining and visible gene signals, nuclei are selected for counting. Number of gene signals is determined. The three dimensional constitution of the nucleus is taken into account by a variable adjustment of optical z-axis layer. First nucleus (first white arrow) shows a tiny signal cluster of four signals tight nearby each other at one chromosomal allelic location, but only one signal on probably the other chromosome. Second selected nucleus (second arrow) shows about six or seven gene signals, partly in a “cluster” pattern of two or three signals nearby each other. Filter change to “FITC” (green): Signals of the centromere reference, the chromosome enumeration probe (CEP), are checked for signal quality. Signals in full z-axis are taken into account. CEP3 signals of the centromere reference are counted. Signals in full z-axis are taken into account. The first and second nucleus both show show signals for two centromere loci indicating presence of two chromosomes. Filter change to “Texas Red®”: Tissue is screened for additional nuclei suited for gene copy number determination. The third nucleus (third white arrow) shows a signal cluster of about five or six signals tight nearby each other and four additional distributed signals on other chromosomal other extra-chromosomal locations. The fourth selected nucleus (fourth arrow) shows four or even six gene signals, although the three or four gene signals marked by the arrow might be allelic gene copy signals of four chromatids as part of two chromosomes. Nevertheless, the another gene signals of the same nucleus appear as single point shape signals, indicating not to be copied on another chromatid. However, the nucleus shows at least four gene copies. Filter change to “FITC” (green): Signals of the centromere reference, the chromosome enumeration probe (CEP), are checked for signal quality. Signals in full z-axis are taken into account. CEP3 signals of the centromere reference are counted. Signals in full z-axis are taken into account. The third and fourth nucleus both show show signals for two centromere loci indicating presence of two chromosomes. Filter change to DAPI (blue): Chromatin stained nuclei are checked for integrity and separability. This video presentation is the intellectual property of the University of Bergen, Norway and is published for scientific research purpose in line with regarding ethical laws. Correspondence: frederik.holst@k2.uib.no