Molecular markers
FISH overview
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PubMed Search: FISH[TI] fluorescence in situ hybridization[TIAB] review[PT] full text[SB]
Table of Contents
Definition / general | Essential features | Terminology | ICD coding | Types of ISH | ISH probes | Specimen types | Applications | Cell enrichment methods | Interpretation of ISH signals | Advantages versus limitations of fluorescence and bright-field ISH | Molecular / cytogenetics images | Sample FISH report | Board review style question #1 | Board review style answer #1 | Board review style question #2 | Board review style answer #2Cite this page: Caramelo A, Polónia A. FISH overview. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/molecularpathfishgeneral.html. Accessed May 31st, 2023.
Definition / general
- Detection of specific nucleic acid sequences (DNA / RNA)
- Use of a labeled complementary nucleic acid probe
- Visualization in the cell or tissue (in situ) on a slide by fluorescent microscopy
Essential features
- Detection of specific nucleic acid sequences (DNA / RNA) (foreign or native)
- Visualization of signals in the cell or tissue (in situ), by either digital imaging or manual microscopy
- Detection of numerical chromosome abnormalities (congenital or acquired aneuploidies)
- Detection of amplification / deletion / chromosomal translocation
Terminology
- ISH: in situ hybridization
- FISH: fluorescence in situ hybridization
- BISH: bright-field in situ hybridization
- SISH: silver in situ hybridization
- CISH: chromogenic in situ hybridization
- DDISH: dual hapten dual probe in situ hybridization
- PCR: polymerase chain reaction
ICD coding
- ICD-10: Q90-Q99 - Chromosomal abnormalities, not elsewhere classified
Types of ISH
- Fluorescence ISH (FISH): direct detection of a fluorescent labeled probe
- Bright-field ISH (BISH): indirect detection through an enzymatic reaction
- Silver ISH: detection of silver precipitation (black dots)
- Chromogenic ISH (CISH): detection of chromogen (e.g. red)
- Dual hapten dual probe ISH (DDISH): CISH + SISH
- Hybrid techniques
- Specific to allele PCR - FISH (Nat Protoc 2007;2:2782, Nat Methods 2008;5:877)
- Padlock probes with in situ rolling circle amplification (RCA) (Nat Methods 2010;7:395, Science 1994;265:2085)
ISH probes
- Large chromosome sequences (e.g. large regions, short arm, long arm)
- Repetitive sequences (e.g. centromeres)
- Unique sequences (e.g. specific genes, parts of genes)
Specimen types
- Cells in culture
- Paraffin block sections
- Aspirate smears
- Tissue imprints
- Liquid based preparations
Applications
- Interphase cytogenetics (analysis of chromosomes in nondividing cells)
- Detection of numerical chromosome abnormalities (congenital or acquired aneuploidies)
- Detection of gene amplification (e.g. HER2 in breast / gastric cancer) (J Clin Oncol 2018;36:2105, Arch Pathol Lab Med 2016;140:1345)
- Detection of gene or chromosomal deletion (e.g. TP53 deletion on chromosome 17p in myeloid neoplasms)
- Detection of chromosomal translocation (e.g. ALK translocation on lung cancer) (Arch Pathol Lab Med 2020 May 13 [Epub ahead of print], Arch Pathol Lab Med 2018;142:321)
- Viral infections: detection of viral genome (e.g. EBV, HPV, HIV, fungal species)
- Gene expression: detection of messenger RNA for various peptides (e.g. immunoglobulin light chains, albumin)
- Single transcript analysis: detection of point mutations, single nucleotide polymorphisms (SNPs), RNA edited transcripts, tissue specific allele expression, alternative splicing
Cell enrichment methods
- Objective: increase sensitivity of ISH for detection of cytogenetic abnormalities
- Technique: based on physical properties (size, deformability, density) and expression of specific surface antigens
- Applications: plasma cells in bone marrow (Arch Pathol Lab Med 2013;137:625), circulating tumor cells in blood samples (PLoS One 2020;15:e0237308, J Hematol Oncol 2019;12:48)
Interpretation of ISH signals
- Presence of signals (e.g. presence of EBER in EBV infection)
- Gain of signals (e.g. ERBB2 amplification in breast / gastric cancer)
- Loss of signals (e.g. loss of 1p/19q in oligodendroglioma)
- Positioning of signals
- Break apart probe: neighboring signals altered when detected at a distance, resulting in 2 separate color signals (e.g. EWSR1 translocation)
- Normally, 2 different color signals (red and green) are located nearby, sometimes creating a fused color (yellow); the abnormality exists if the 2 signals are separated
- Fusion probes: distant signals altered when detected nearby, resulting in overlap or juxtaposition of the 2 different color signals (e.g. EWSR1-FLI1 fusion)
- Normally, 2 different color signals (red and green) are separated; the abnormality exists if the 2 signals are together, sometimes creating a fused color (yellow)
- Break apart probe: neighboring signals altered when detected at a distance, resulting in 2 separate color signals (e.g. EWSR1 translocation)
Advantages versus limitations of fluorescence and bright-field ISH
- FISH requires fluorescence microscope (higher cost) versus bright-field ISH requires light microscope (lower cost)
- Higher multiplexing in FISH (> 2 probes) versus low multiplexing in bright-field ISH (≤ 2 probes)
- Low morphologic correlation in FISH versus high morphologic correlation in bright-field ISH
- Loss of signals with time in FISH slides (photographic record mandatory) versus permanent staining in bright-field ISH slides
- Reference: Diagn Pathol 2008;3:41
Molecular / cytogenetics images
Contributed by António Polónia, M.D., Ph.D., Ana Caramelo, B.Sc., Ana Ribeiro, M.D., Catarina Eloy, M.D. and João Vale, M.Sc.
Gastric cancer
Gastric cancer
Breast cancer
Breast cancer
Gastric cancer
1p/19q codeletion
Gastric lymphoma
Lymph node
Contributed by Leica Microsystems, Biosystems division
Immunoglobulin light chain
Images hosted on other servers:
Trisomy 21
UroVysion FISH test
Single molecule RNA FISH (smFISH)
In situ mutation detection of KRAS mutation
Albumin
Sample FISH report
- Example for HER2 FISH quantification in breast cancer:
- Number of neoplastic cells analyzed: at least 20 cells in 2 separate areas (at least 10/area)
- Number of HER2 signals:
- Average HER2 copy number:
- Number of CEP17 signal:
- Average CEP17 copy number:
- HER2/CEP17 ratio:
- Genomic heterogeneity: absence / presence (percentage of the total tumor population with amplification)
- Probe:
- Guidelines: ASCO / CAP 2018
- Type of fixative:
- Time to fixation (cold ischemic time): less than 1 hour
- Duration of tissue fixation: between 6 and 72 hours
- Commentary on the preanalytic conditions: the preanalytic conditions are (or are not) according to the recommendations of the ASCO / CAP 2018 guidelines for HER2 in breast cancer.
Board review style question #1
What is the pattern of a break apart rearrangement?
- A pair of different color signals together and 2 separate color signals
- 2 pairs of different color signals together
- 3 different color signals separate
- 4 different color signals separate
Board review style answer #1
A. A pair of different color signals together and 2 separate color signals
Comment Here
Reference: FISH overview
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Reference: FISH overview
Board review style question #2
What is the pattern of a fusion rearrangement?
- A pair of different color signals together and 2 separate color signals
- 2 pairs of different color signals together
- 3 pairs of different color signals together
- 4 different color signals separate
Board review style answer #2
A. A pair of different color signals together and 2 separate color signals
Comment Here
Reference: FISH overview
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Reference: FISH overview
