Chromogenic in situ hybridization (CISH) is a technique that combines in situ hybridization with immunohistochemistry to detect specific DNA sequences within tissue samples using brightfield microscopy. CISH uses nucleic acid probes labeled with enzymes that produce colored precipitates when exposed to chromogenic substrates, allowing visualization of gene amplification or rearrangements. CISH has advantages over fluorescence in situ hybridization (FISH) in being cheaper, more stable, and easier to use, though it is less sensitive than FISH. CISH is commonly used to assess HER2 gene amplification in breast cancer and detect genetic fusions in cancers.

1. CHROMOGENIC IN SITU
HYBRIDIZATION
BY- ANUBHAV AGRAWAL, 514

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

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

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

5. • Chromogenic in situ hybridization (CISH) is a cytogenetic technique
that combines the chromogenic signal detection method of
immunohistochemistry (IHC) techniques with in situ hybridization. It
was developed around the year 2000 as an alternative to fluorescence
in situ hybridization (FISH) for detection of HER-2/neu oncogene
amplification. CISH is similar to FISH in that they are both in situ
hybridization techniques used to detect the presence or absence of
specific regions of DNA.

8. 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 and
fusions, such as the fusion of ALK tyrosine kinase domain with the
promoter and 5’ region of EML4 in lung cancer.
 Apart from cancers, CISH has also been shown to be useful in detecting
human papillomavirus infections.

9. COMPARISON
• Compared to FISH- Compared to FISH, CISH has been shown to have a sensitivity of 97.5% and a
specificity of 94% for detection of HER-2/neu gene amplification. CISH is much cheaper and is easier to
use because it uses bright-field microscopes instead of fluorescence microscopes. In addition, the CISH
reagents are more stable than the FISH reagents so it is possible to store the samples and examine the same
same sample multiple times.
• Compared to IHC- A disadvantage of IHC is that it is not possible to identify false-negative and
false-positive results. In CISH, if there is no signal for the reference probe, the assay has failed.

10. CISH FISH

11. CISH IHC

12. VARIATIONS
Silver-enhanced in situ hybridization (SISH) SISH uses a similar
method as CISH, but a silver precipitate is the end product rather than a brown product. In
this 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 the
secondary antibody and HRP catalyzes the polymerization of silver in the presence of
hydrogen peroxide.
Silver metal is consequently deposited into the nuclei of the cell. Amplification of HER-2/neu
is seen as black dots.

13. DuoCISH DuoCISH is a variation of CISH that addresses the need for two different
probes 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 the
reference which, in the case of HER-2/neu amplification detection, is CEN17 (the centromere
of 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 HRP
HRP and red dye is a substrate for AP.