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Fluorescence in situ hybridisation, better known as FISH, is a molecular cytogenetics technique that harnesses fluorescent probes that bind to specific nucleic acid sequences. FISH allows researchers to visualise and map genetic material on a chromosome to identify specific genes or portions of genes.

Since its discovery in the 1970’s1, FISH has revolutionised cytogenetics, ushering in a new era of molecular research. Of particular importance is its contribution to the field of oncology, where FISH has become an indispensable tool in the diagnosis, prognosis, and surveillance of many cancers.

Here, we discuss the important applications of FISH in cancer research and highlight its role as the gold standard for cancer diagnostics. Finally, we will explore how alternative cytogenetic technologies may serve to complement its application.

 

How did frogs produce FISH?

Over half a century has passed since pioneering researchers realized that in situ hybridisation (ISH) could be used to identify the positions of specific DNA sequences on chromosomes2.

Molecular cytogenetic techniques that harness ISH use labelled DNA or RNA sequences (probes). The probe hybridises to its complementary (target) sequence in a biological sample. Depending on the label, hybridised sequences can be detected by methods such as autoradiography, fluorescence microscopy, or chromogenic detection.

In a landmark paper, Gall and Pardue (1969)2 were the first to describe how radioactive copies of a ribosomal DNA sequence could be used to detect complementary DNA sequences in the nucleus of a frog egg.

Within a decade, researchers had replaced radioactive labels with fluorescent labels in hybridisation probes1, marking the transition from ISH to FISH. The rudimentary principles and steps that remain are similar to ISH where (Figure 1):

  1. Both the target DNA and the probe DNA are denatured
  2. The probe hybridises to its complementary (target) sequence
  3. Probe signals are detected

However, unlike previous ISH approaches, the use of FISH probes allow straight-forward detection of target sequences using a fluorescent microscope.

FISH and cytogenetics: schematic depicting each step involved in FISH

Figure 1: FISH and cytogenetics - schematic depicting each step involved in FISH

 

Applications: FISH-ing for answers

Since their discovery, FISH assays have become the gold standard for the diagnosis, prognosis, and surveillance of many cancer types – and for a good reason. In cancer diagnostics, FISH assays have been shown to increase detection capacity significantly compared to conventional cytogenetics.

For instance, multiple myeloma is a plasma cell cancer with very complex cytogenetic and molecular genetic aberrations. Conventional cytogenetics could detect approximately one third of cytogenetic abnormalities - but FISH has increased this to >90%3. Conventional cytogenetics requires the culturing of plasma cells and capturing them in the metaphase stage. However, plasma cells do not grow well in vitro; therefore, utilising cytogenetics may result in a false negative result. FISH does not require cell culturing and can be used on cells during interphase, thus, it overcomes this limitation.

Find out more about haematology FISH Probes for multiple myeloma

An additional advantage of FISH is its ability to offer rapid diagnostic results – a criticality when clinical decisions are pressing. For instance, expedited diagnosis permitting the immediate treatment of acute promyelocytic leukaemia (APL) is essential as fatal coagulation disorders and life-threatening haemorrhaging are a high clinical risk. In this case, FISH probes - such as OGT’s CytoCell® FAST PML/RARα (RARA) Translocation, Dual Fusion - can be used to rapidly detect the hallmark genetic abnormality i.e., a promyelocytic leukaemia/retinoic acid receptor alpha (PML-RARA) gene fusion, enabling faster treatment induction and can help to indicate the risk of relapse in APL patients.

FISH can also be used as a prognostic tool. In other words, it can be used to make predictions around the likely course and outcome of a disease. Some chromosomal abnormalities – such as loss of the TP53 (tumour protein p53) or ATM (ATM serine/threonine kinase) genes – may be indicative of a more aggressive disease4,5. Appropriate FISH probes, such as the CytoCell P53 (TP53)/ATM Probe Combination, can be used to detect these abnormalities and help inform clinical decisions.

Chromosomal abnormalities specific to certain cancers can give a good indication of treatment response and outcome. For example, the HER2 (ERBB2) Human Epidermal growth factor Receptor 2 gene, located at chromosome band 17q12, is activated and amplified in 20-30% of breast cancers. Patients harbouring this gene amplification may respond to  HER2 inhibitors such as trastuzumab (Herceptin), and consequently, diagnosis by FISH can inform treatment decisions6.

Finally, FISH assays can be used as surveillance tools to follow the effectiveness of treatment. In simple terms, an effective treatment will lead to a reduction in the number of abnormal cells seen in a patient sample, while continued detection of chromosomal abnormalities may indicate the presence of residual disease.

 

A fish out of water? Why FISH remains the go-to for cancer research

While FISH represents a robust, easily applicable technique with short turnaround times (TAT), it does have some limitations. For example, designing new assays can be a time-consuming process, where each stage of the protocol are probe- and sample-specific7. As such, multiple probes and precious clinical samples may be required to optimise clinically-validated products.

To address this, companies such as OGT offer an innovative custom probe design and manufacture service. By utilising CytoCell’s proprietary BAC clone collection of more than 220,000 clones, OGT can produce fully quality-assured custom FISH probes for virtually any sequence in the entire human genome.

With this wide availability of FISH probes comes a wealth of diagnostic and research opportunities; including the detection of small chromosomal changes that are too small to be detected by conventional methods. Indeed, chromosomal microdeletions, that are usually <5Mb, evade identification by light microscopy and karyotyping but can be detected by FISH.

Microdeletions, or sub-microscopic deletions, are classically associated with constitutional diseases however, they can also be seen as acquired abnormalities in some cancer types. OGT’s CytoCell TMPRSS2/ERG Deletion/Breakapart probe, for example, can be used to detect a gene fusion resulting from a sub-microscopic deletion that is estimated to occur in over 50% of prostate tumours8

With companies such as OGT expanding probe availability and helping to streamline the assay development and optimisation workflow, it is certain that FISH is here to stay. Nevertheless, exploring other technologies, such as NGS, can offer additional or complementary insight in the field of cancer research.

 

Combining FISH and NGS

While FISH remains an indispensable tool in cancer research, more modern techniques can be used to reveal unique and valuable information that can support FISH in cancer diagnostics, prognostics, and surveillance. Over recent years, one of the most notable developments in genetic disease research is the use of next-generation sequencing (NGS) technology.

NGS can capture a large amount of genomic information about cancer. In turn, it provides an opportunity to cast a wide net when many diverse mutations are indicative of disease.

Find out more about NGS for haematological cancer and solid tumours

Since FISH and NGS provide different advantages and have the potential to deliver unique genomic information, their utility is not mutually exclusive. For example, in diagnostics, FISH can provide rapid results and provides a valuable technique for the first round of testing and in time-critical scenarios. When negative FISH results demand further testing, NGS offers an approach to profile a larger genetic space.

 

FISH and cytogenetics: Here to stay!

Combining FISH with other techniques such as NGS, is just one approach to overcoming some of its limitations. However, advances in FISH technology, including automated workflows and streamlined probe design and manufacture, are serving to strengthen its position as the gold standard for detecting many genetic aberrations in cancer.

Whether your needs are off-the-shelf or custom, OGT can provide high-quality FISH probes optimised for your application. Discover a wide range of CytoCell FISH probes optimised for haematological malignancies and the assessment of genetic aberrations in solid tumour samples. If you can’t find what you’re looking for, at OGT we’re here to help with our CytoCell myProbes® - a custom FISH probe design and manufacture service.

 

References

  1. Rudkin, G. T. & Stollar, B. D. High resolution detection of DNA–RNA hybrids in situ by indirect immunofluorescence. Nat. 1977 2655593 265, 472–473 (1977).
  2. Gall, J. G. & Pardue, M. L. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. U. S. A. 63, 378–383 (1969).
  3. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. (IARC, 2008).
  4. Rossi, D. et al. Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia. Blood 121, 1403–1412 (2013).
  5. Artmut, H. et al. Genomic Aberrations and Survival in Chronic Lymphocytic Leukemia. http://dx.doi.org/10.1056/NEJM200012283432602 343, 1910–1916 (2009).
  6. Kauraniemi, P. et al. Effects of Herceptin treatment on global gene expression patterns in HER2-amplified and nonamplified breast cancer cell lines. Oncogene 23, 1010–1013 (2004).
  7. Huber, D., Voith von Voithenberg, L. & Kaigala, G. V. Fluorescence in situ hybridization (FISH): History, limitations and what to expect from micro-scale FISH? Micro Nano Eng. 1, 15–24 (2018).
  8. Saramaki, O. & Visakorpi, T. Chromosomal aberrations in prostate cancer. Front. Biosci. 12, 3287–3301 (2007).
  9. Halder, A., Jain, M., Chaudhary, I., Gupta, N. & Kabra, M. Fluorescence in situ hybridization (FISH) using non-commercial probes in the diagnosis of clinically suspected microdeletion syndromes. Indian J. Med. Res. 138, 135 (2013).

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