Dual-color chromogen In Situ hybridization (CISH) for the determination of Her2 status in breast cancer tissue sections

Abstract

Background: A factor associated with breast cancer development is the mutation of the Her2/neu gene located on chromosome 17. Chromogen in situ hybridization (CISH) in which the color is generated with an enzymatic reaction is a promising alternative detection method to fluorescence in situ hybridization (FISH) which is nowadays used in clinical practice. The resulting color is stable and therefore usable for permanent storage, the use of a normal brightfield microscope is possible and the morphology of the sample can be evaluated more easily than with a fluorescence microscope. For the visualization of Her2/neu it is however important to also detect chromosome 17 and use it as a reference chromosome. The aim of the study is: to select the best substrates considering color production, background staining, reactivity and localization. Secondly to develop a dualcolor procedure visualizing Her2/neu and chromosome 17 to examine the possibility of using CISH as a standardized method for the detection of Her2 positive tumors and finally to compare these results with FISH. Methods: Single target CISH with eight substrates for the enzyme horseradish peroxidase (PO) and six substrates for alkaline phosphatase (AP) was performed on cervical cancer cell lines with centromere 1 (C1) and 1p36 as targets. The best substrates were subjectively selected with the use of a brightfield microscope. The procedure was expanded to a dual-color detection on cells and formalin-fixed, paraffin embedded (FFPE) breast cancer tissue sections with Her2/neu and centromere 17 (C17) as targets. In this procedure the best conjugate detection systems were worked out. Results: In the single target detection procedure the substrates DAB, Vina Green and Seramun Grün for PO and Ferangi Blue and Vulcan Fast Red for AP were found to give the best color reactions. The dual-color detection on tissue sections showed strong and good distinguishable color reactions when combining the substrates Vina Green for C17 and Vulcan Fast Red for Her2. Results of CISH and FISH were comparable when using immersion oil for the detection of the fluorescent signals. Discussion and Conclusion: Vina Green for C17 and Vulcan Fast Red for Her2/neu are the best substrates considering background staining, color distinguishability and localization and give comparable results to FISH. The study gives a further step in the direction of introducing CISH as a standardized method for the detection of Her2/neu amplification.