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FISH in molecular cancer diagnostics: the whats and whys?

Fluorescence in situ hybridization (FISH) – a relatively new cytogenetic technique - is a DNA hybridization-based technique that generally uses directly-labeled fluorescent DNA probes to target specific chromosomal locations within the nucleus, resulting in colored signals that can be detected using a fluorescent microscope. Alternatively, FISH probes can be indirectly labeled with reporter molecules that are subsequently detected by fluorescent antibodies or other affinity molecules.

FISH is applied to detect genetic aberrations including

  • Characteristic gene fusions or translocations
  • Characteristic gene rearrangements
  • Partial or complete loss of chromosome
  • Presence of an abnormal number of chromosomes in a cell


Hence FISH can be applied to diseases covering genetic etiologies as well as cancer –both hematological and solid tumors. Additionally FISH can be applied to basic research applications like gene mapping or in discovery based assays like elucidation of novel oncogenes. Furthermore it can be used to aid in novel biomarker discovery. FISH has now been expanded to screen/analyze whole genome in one-go (in single experiment) using multicolor whole chromosome probe techniques like multiplex FISH or spectral karyotyping.

General applications of FISH:


  • specific gene or chromosomal abnormalities in genetic diseases
  • specific gene or chromosome abnormalities in cancer
  • characterization of marker chromosomes or biomarker genes
  • monitoring disease progression
  • pre-natal or pediatric diagnostic cases
  • monitoring success of bone marrow transplantation
  • prognostication outcomes in course of treatment
  • as predictive biomarkers in order to stratify cancer patients

Research Methods:

  • new non-random abnormalities (M-FISH or SKY)
  • gene mapping
  • 3D chromosome organization in interphase nuclei
  • new sites of translocations or amplifications

Advantages of FISH in molecular diagnostics: 

  • FISH is a highly sensitive and specific cytological assay.
  • Speed (faster scoring), relative simplicity and robustness are 3 FISH attributes that are beneficial to cancer diagnostics.
  • Conventional cancer cytogenetic tests are performed on dividing cells (metaphase chromosome) for which culturing of cells is required – can take up to 2 weeks. FISH circumvents this problem as it can assay non-dividing cells (interphase chromatin). This is particularly useful in slowly dividing initial stages of some cancer cells.
  • Small resolution techniques like chromosome banding cannot detect regions of small chromosomal rearrangements and deletions to the extent that FISH does.
  • This visually appealing technique provides an intermediate degree of resolution between DNA analysis and chromosomal investigations.
  • High concordance between gene expression and amplification status has been observed in multiple FISH-related studies.
  • The quantitative interpretation of the results is relatively straightforward and concordance rates among observers are higher than for IHC in some studies.
  • FISH can detect circulating tumor cells or circulating DNA in body fluids like urine (urinary cytology) through noninvasive procedures, as illustrated in urothelial cancer patients.
  • FISH is amenable to a wide variety of cell types and tissues including paraffin-embedded tissue.
  • Tissue morphology and gene amplification are complementary approaches to molecular cancer diagnostics. Confirmatory evaluation of genomic aberrations via FISH – apart from morphological assessments via IHC is critical for cancer diagnostics, prognostics and clinical management cases. This will ensure most appropriate tailor-made approach – precision medicine.

Current shortcomings/Disadvantages:

  • False positive rates are usually in the range 1%-5% of scored nuclei.
  • Specific and reliable predictive biomarkers to chemotherapy and other treatment regimens for solid tumors are relatively rare.
  • Robust and specific markers or probes for individual cancer indications particularly specific stages and grades in solid tumors are rare. Both can be circumvented by coupling cancer genome database expansion and mining on one hand and tumor profiling on the other hand.
  • Automation needed to process and analyze interphase FISH assays to reduce the labor-intensiveness and optimize time.
  • Owing to limitations in resolution, FISH analysis may fail to detect microdeletions smaller than 190 kb.
  • Interphase (and metaphase) FISH and to a lesser extent SKY and M-FISH can only detect known genetic aberrations, providing the specific probe is available. FISH cannot serve as a screening test for chromosomal rearrangements since most FISH techniques can only detect known imbalances.

Topics: BioGenex, Cancer research, FISH, Fluorescent in situ, Cancer diagnostics

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