Cish

CHROMOGENIC IN SITU HYBRIDIZATION

BY- ANUBHAV AGRAWAL, 514

CONTENTS

• Introduction• Procedure • Comparison to similar techniques• Services in medical studies• Modifications and variations

INTRODUCTION

In Situ Hybridization (ISH) is a technique that allows for precise localization of a specificsegment of nucleic acid within a histologic section. The underlying basis of ISH is thatnucleic acids, if preserved adequately within a histologic specimen, can be detectedthrough the application of a complementary strand of nucleic acid to which a reportermolecule is attached.Visualization of the reporter molecule allows to localize DNA or RNA sequences in aheterogeneous cell populations including tissue samples and environmental samples.Riboprobes also allow to localize and assess degree of gene expression .

RNA in situ hybridization - KRT5 and housekeeping gene in human melanoma FFPE tissue section -visualized under brightfield and fluorescence microscope.

• Chromogenic in situ hybridization (CISH) is a cytogenetic techniquethat combines the chromogenic signal detection method ofimmunohistochemistry (IHC) techniques with in situ hybridization. Itwas developed around the year 2000 as an alternative to fluorescencein situ hybridization (FISH) for detection of HER-2/neu oncogeneamplification. CISH is similar to FISH in that they are both in situhybridization techniques used to detect the presence or absence ofspecific regions of DNA.

MEDICAL APPLICATION

CISH is frequently applied to assess gene amplification, such as HER-2/neu status in breast cancer samples.

CISH is also used for detection of chromosomal rearrangements andfusions, such as the fusion of ALK tyrosine kinase domain with thepromoter and 5’ region of EML4 in lung cancer.

Apart from cancers, CISH has also been shown to be useful in detectinghuman papillomavirus infections.

COMPARISON

• Compared to FISH- Compared to FISH, CISH has been shown to have a sensitivity of 97.5% and aspecificity of 94% for detection of HER-2/neu gene amplification. CISH is much cheaper and is easier touse because it uses bright-field microscopes instead of fluorescence microscopes. In addition, the CISHreagents are more stable than the FISH reagents so it is possible to store the samples and examine the samesame sample multiple times.

• Compared to IHC- A disadvantage of IHC is that it is not possible to identify false-negative andfalse-positive results. In CISH, if there is no signal for the reference probe, the assay has failed.

CISH FISH

CISH IHC

VARIATIONS

Silver-enhanced in situ hybridization (SISH) SISH uses a similarmethod as CISH, but a silver precipitate is the end product rather than a brown product. Inthis method, a probe tagged with dinitrophenol (DNP) binds to the target sequence.A primary anti-DNP antibody is then added followed by a secondary antibody conjugated to

HRP. Silver acetate, hydroquinone, and hydrogen peroxide are subsequently added to thesecondary antibody and HRP catalyzes the polymerization of silver in the presence ofhydrogen peroxide.Silver metal is consequently deposited into the nuclei of the cell. Amplification of HER-2/neu

is seen as black dots.

DuoCISH DuoCISH is a variation of CISH that addresses the need for two differentprobes on the same slide.It is a well-established technique for HER-2/neu amplification detection even though it is

sometimes reported to be less effective than FISH. In this technique, one probe binds thereference which, in the case of HER-2/neu amplification detection, is CEN17 (the centromereof chromosome 17) while the other probe binds the target sequence which is HER-2/neu.DuoCISH combines both FISH and CISH in that it converts signals from FISH probes to

chromogenic substrates. It works on the principle that blue cyanine dye is a substrate for HRPHRP and red dye is a substrate for AP.