FISH – Detection of Gene Sequences

Fluorescent in situ hybridization (FISH) is a powerful diagnostic and discovery tool for identifying the genomic status of samples, particularly oncology tissues. FISH involves the use of fluorescence probes to detect specific gene sequences within chromosomes and can be used to detect different kinds of mutations such as gene amplifications, deletions and translocations (fusions and breaks). Viral (RNA) sequences can also be detected. FISH can be applied to Formalin Fixed Paraffin Embedded (FFPE) and Flash Frozen (FF) samples, although FFPE is preferred due to superior morphology.

 

Using FISH to detect different types of mutations

Gene Amplifications – In a fraction of patients with breast cancer, the HER2 protein is overexpressed. This is due to HER2 gene amplification.

Gene Deletions – Deletion of the AMT gene can be associated with mantle cell lymphoma, a subtype of non-Hodgkin’s lymphoma.

Gene Translocations – Translocations can fuse the coding sequences of two genes together to generate potent oncogenes. For example, the BCR-ABL fusion protein encoded by the chimeric gene is associated with chronic myelogenous leukemia (CML). Gene translocations can also be detected using break-apart probes. Abnormalities in the ROS1 gene can lead to non-small cell lung carcinoma.

FISH can also be used to detect viral sequences within cells – e.g. HPV (human papillomavirus).
fish1

Diagrammatic representation of the different signaling patterns specific to gene amplification, deletions and translocations. Specific patterns are visualized and interpreted based on probe design.

 

HER2 overexpression due to HER2 gene amplification

There are commercially available FISH probes against an ever-increasing number of genes. Once such example is Her2.  In a fraction of patients with breast cancer, the HER2 protein is overexpressed due to HER2 gene amplification. The image below shows FISH analysis of HER2 gene expression in formalin fixed, paraffin-embedded samples of breast tumor from four donors. HER2 amplification status of a sample can be quantified by calculating the HER2 to CEN-17 ratio. Further, specimens with a ratio equal or above 2 are considered HER2 gene amplified.

fish2

FISH analysis of HER2 gene expression in formalin fixed, paraffin-embedded samples of breast tumor from 4 donors. The probe mix consists of a mixture of Texas Red-labelled DNA probe against HER2 gene (which is located on chromosome 17) and a fluorescein (green)-labelled probe targeted at the centromeric region of chromosome 17. The upper panels (D1 and D2) show normal expression – 2 green and 2 red signals per cell. The lower panels (D3 and D4) show HER2 amplification whereby there is a clear increase in the red signal.

 

Custom FISH Probe Design and Validation

If probes to the target gene are not available, a custom-made probe can be designed in house and validated by our expert scientists.

In this instance, probes were designed to bind to two regions in close proximity that spanned a break-apart mutation. Thus, overlapping signals indicated a wild-type gene, and non-overlapping signals indicated the presence of a translocation mutation.

b4lastlast

Overlap of red and green fluorescent signals results in a yellow signal.

 

In the example below, custom-made break-apart probe was validated in positive and negative control cell lines. A cohort of 60 non-small cell lung carcinoma samples were then screened in order to identify samples with the translocation mutation. In this instance, frozen sections of tissues were used. No mutations were identified (right hand panel), most likely due to the low frequency of the mutation.

FISH Break-Apart Mutation Lung Cancer

Wild-type cells exhibited the expected adjacent/overlapping red and green signals (left hand panel), whilst the cell line that contained the translocation mutation showed separation of the red and green signals (central panel). No mutations was identified in the cohort of donor tissues(right hand panel, representative section).

 

in situ Hybridization – Answering Your Research Questions

  • Precise location of mRNA in human tissue
  • More sensitive target and biomarker validation using the branched chain approach for ISH
  • Custom assay development and testing for commercially-available ISH probes
  • Ease of design of custom target probes versus creation of specific, selective antibody
  • Localization of multiple mRNA targets in the same tissue section
  • Localization of mRNA and protein expression in the same tissue section
  • Comparison of mRNA and protein localization for the same target
  • Antibody validation
  • Different targets

 

For further information, or to discuss a specific study, contact us at advantage@asterandbio.com.