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.
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).
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.
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.
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.