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What is FISH?
Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique used for detecting and locating specific DNA sequences on a chromosome or nuclei.(1) The technique combines conventional cytogenetic and molecular genetic technology to investigate a target chromosome or gene abnormality in tumor cells or tissue. FISH technology can identify whether a gain or loss of a particular chromosome or gene copy is present in the body’s cells. It can also identify whether certain genes have structural abnormalities, such as translocation and inversion that play an active role in disease initiation and progression.
“FISH has revolutionized cytogenetic analysis for clinical service in cancer, constitutional and prenatal cytogenetics applications,” said Yanming Zhang, M.D., Medical Director of the cytogenetics laboratory at Northwestern Memorial Hospital; and associate professor of pathology in the department of pathology at Northwestern University. “FISH has its important advantages and its limitations. Anyone who wants to use FISH in their lab should be aware of how it works, what the expectations are, and what are the advantages and limitations.”
The Benefits of FISH
The major benefits of FISH technology include quick results, high sensitivity and resolution, precision and accuracy, and as a result, better patient care.
“Detecting chromosome and/or gene abnormalities using FISH testing has a direct impact on patient care,” said Dr. Zhang. “If we know what chromosome abnormality is likely in a disease, we can use FISH to look for it quickly. Compared to conventional chromosome analysis that will take at least two to three days, FISH can provide results the same day. It has been a great contribution here at Northwestern University.”
In one case, Dr. Zhang had a young patient with newly diagnosed acute myeloid leukemia (AML). The physician and the patient were very anxious to see what was going on, and what kind of genetic abnormality was involved. According to Dr. Zhang, it is routine to set up a culture for chromosome analysis to look at the abnormality. But they knew it was a highly likely situation, so Dr. Zhang did a FISH analysis immediately. Within three hours, they confirmed the abnormality. The patient was told the same day, and was immediately put on an appropriate treatment protocol and had information about prognosis.
“Even after treatment, FISH is the only reliable way in some AML and myelodysplastic syndromes (MDS) patients to follow up if the previous chromosome/gene abnormality still exists. FISH results help physicians decide if the patient needs additional chemotherapy or a bone marrow transplant. It’s a quick and relatively easy technique to monitor patients in treatment, although compared to PCR, it is less sensitive.”
According to Dr. Zhang, the lab can also benefit from FISH testing. “From my experience, chromosome analysis alone can be inadequate in some bone marrow samples, such as a hypocellular bone marrow aspirate or post-chemotherapy bone marrow. In these cases, we use FISH in combination with chromosome analysis. FISH can specifically look at chromosome/gene abnormalities that are prognostically important. FISH can also increase your lab test menu and test volume, which leads to more reimbursement and revenue for the hospital.”
Detecting Chromosome Abnormalities
FISH can detect two types of chromosome abnormality: numerical and structural. For copy number changes, FISH probes can detect the gain or loss of a whole chromosome or chromosome regions. For instance, according to Dr. Zhang, gain of chromosome 8 and deletion or loss of chromosomes 5 and 7 are recurring chromosome abnormalities in myeloid disorder, such as AML and MDS, whereas trisomy 12 and deletion of the long arm of chromosome 13 are often present in chronic lymphoid leukemia (CLL). FISH can easily detect these abnormalities in leukemia cells.
FISH is also very useful for detecting microdeletions and subtelomeric aberrations in constitutional genetics applications. FISH analysis is standard to rule out trisomy 21 (Down syndrome) or other common aneuploidies in prenatal diagnosis and pre-implantation diagnosis.
In terms of structural abnormalities, FISH can detect chromosome translocation in cancer, leukemia and lymphoma. FISH can readily reveal fusions of two genes in a translocation using a fusion probe set, such as the PML/RARA fusion in acute promyelocytic leukemia (APL), or a split signal pattern using break-apart probe sets, i.e., CBFB for inv(16)/t(16;16) in AML. In combination with conventional chromosome analysis, FISH can detect multiple translocation partners and help clone those novel partner genes; for instance, multiple MLL translocations in AML and ALL.
Because FISH can quickly check for several hundred or even several thousand cells in a short period, it may detect small abnormal cell populations. Therefore it is more sensitive than cytogenetic analysis, which typically analyzes 20 metaphase cells only.
One important advantage of FISH technology is that FISH can be performed on non-dividing or terminally differentiated cells. As a result, FISH can be used in detecting chromosome and/or gene abnormalities in interphase cells, such as in non-dividing CLL cells, and also in paraffin-embedded tissue sections for various chromosome abnormalities, including Her2/neu in breast cancer, and ALK gene aberrations in non-small cell lung cancer.
In addition, FISH technique can directly be combined with morphology observation or immunophenotyping in leukemia and lymphomas to define chromosome and gene abnormalities in certain lineages and differential stages. It also significantly improves FISH detection sensitivity and enables to correlate gene copy numbers with protein expression level and proliferation level of tumor cells. Combined FISH and immunophenotyping with CD138 or kappa/lambda staining is commonly performed in multiple myeloma to detect recurring chromosome abnormalities, such as IGH translocations, and deletion of P53.
The National Human Genome Research Institute identifies three main types of FISH probes, each of which has a different application:
1. Locus specific probes bind to a particular region of a chromosome or gene. This type of probe is most useful for detecting structural abnormalities, such as the BCR and ABL1 genes for the t(9;22) in CML and ALL, and the break-apart MYC probes for MYC abnormalities in high-grade B cell lymphoma.
2. Centromeric specific probes are generated from alpha and beta repetitive sequences found in the centromere region of each chromosome. The majority of 24 human chromosomes have their unique centromere specific probes. These probes can be used to simply determine copy number of chromosomes, such as trisomy 21 for Down syndrome, trisomy 8 in MDS, or trisomy 12 in CLL, etc.
3. Whole chromosome or chromosome arm/band specific painting probes are actually collections of smaller probes, each of which binds to a different sequence along the length of a given chromosome. Using multiple probes labeled with a mixture of different fluorescent dyes, the resulting full-color map of the chromosome is known as a spectral karyotype (SKY) or multiple-FISH (M-FISH). These types of probes are particularly useful for examining complex structural chromosomal abnormalities, enabling visualizing all 24 chromosomes on one image. However, it is not commonly used in clinical application because it requires specific computer software programs and various filters.
“We have more than 45 FISH tests/probes in use for leukemia, lymphoma and solid tumors,” said Dr. Zhang. “For clinical tests, we are using more locus-specific probes like genomic bacterial artificial chromosome (BAC) probe.”
He hopes there will be more and more FISH probes available. Often times Dr. Zhang will contact his supplier to make requests and suggestions on new probes. Supplier companies usually value this insight and use it in research and development.
“There are a lot of new discoveries about novel chromosome genomic abnormality in cancer. Sometimes, we know we have an abnormality, but we just don’t have a probe to confirm or follow up. I hope there will be more probes available for clinical service to help with diagnosis, treatment and prognosis for disease,” he said.
Bringing FISH In-House
Dr. Zhang recommends that if you want to bring FISH technology in-house for clinical service, lab directors should perform systematic validation of the method and probes, intensive practice to guarantee proficiency, have extensive experience with various cases and samples to assure accurate interpretation, and detail documentation of all procedures and results to meet the accrediting agencies’ criteria. Lab directors must have hands-on experience with the basic technology:
1. Know the procedure involved — prepare the slide and probe, denature and
hybridization, wash and scope analysis.
2. Know what is required for technical validation, create a very detailed validation plan,
and know how to determine if the results are accurate. It is important to set up a
positive and a negative control in parallel in the test, and compare with outside results
to see whether your results are comparable.
3. Make sure you have a full understanding of the advantages and limitations of
4. A cut-off value for reporting a positive abnormal result is based on adequate case
numbers of the same sample types.
5. Spend a lot of time training technologists.
6. Be aware of unusual signal patterns due to complex genomic abnormalities.
Advantages: FISH is a quick and relatively straightforward test with high efficiency of hybridization, and has a higher sensitivity and specificity than conventional chromosome analysis. FISH does not require metaphase cells so it can be performed in non-dividing cells such as paraffin embedded tissue sections. FISH can be directly combined with morphology observation and immunophenotyping and can be adapted for automated systems.
Limitations: FISH is a targeted test and will only tell you what the probes target. “In CML patients, FISH can quickly confirm the BCR/ABL1 fusion, but you have no idea if there are additional abnormalities that might be indicative of disease progression. FISH interpretation must be in combination with chromosome study,” said Dr. Zhang. FISH is limited to those abnormalities that can be detected with currently available probes, and usually only one or few abnormalities can be assessed simultaneously. FISH is less applicable for detecting deletion or loss of targets than assessing gain of chromosomes or gene copies – if the abnormal clone is small. FISH requires fluorescence microscopy and an imaging analysis system.
For a lab to run FISH analysis, bench work training including slide preparation, putting on probes, and denaturing slides and other bench work is relatively easy. For PET tissue FISH, it is relatively challenging and will take more time. More importantly, technologists should learn to recognize normal and abnormal signal patterns, and be aware of what kind of probe they are using.
“For numeric abnormality, they need to look for one copy, two copies or even three or more copies,” says Dr. Zhang. “For structural abnormality such as translocation, they need to know what kind of probes they are using, i.e. fusion probes or break-apart probes. Technologists need to be familiar with typical signal patterns, but also with unusual signal patterns, such as single fusion in dual fusion probe testing.”
Dr. Zhang spends a lot of time in the dark room with his staff to show them positive and negative slides. “If signals on a slide are weak, they should repeat the slide because that will affect results. They must have the skill to judge whether the signals are qualified for analysis. Training should be thorough so technologists are familiar with both bench work and scope analysis in the dark room. They need good training so the results will be reliable,” said Dr. Zhang.
Beyond this training, Dr. Zhang stresses that quality control and troubleshooting skills are also very critical. “Protocol must be written in advance and well-studied, so your staff knows procedures precisely.”
FISH and Personalized Medicine
FISH-based diagnostic tests offer clinicians a standardized, clinically validated method to identify patients who are more likely to benefit from a new therapy. Because FISH is very targeted, it helps determine a treatment that is tailored to a patient’s unique genetic profile.
Since the technology works especially well for identifying genetic markers in solid tumors, cancer diagnostics are one of the fastest growing FISH applications. According to Dr. Zhang, FISH is used in cancer diagnostics in three areas:
1. To help make a diagnosis, such as the BCR/ABL fusion in CML.
2. To follow up on treatment response and disease status (evaluate remission or relapse).
3. To predict disease prognosis.
“If a patient has a non-small cell lung cancer with an ALK gene translocation or inversion, he or she is eligible and will likely respond to a specified treatment with a tyrosine kinase inhibitor,” said Dr. Zhang. “The FISH technique can quickly confirm if the patient is eligible for that particular treatment. In cases of chronic myelogenous leukemia (CML), we can confirm quickly with FISH so the patient can receive TK inhibitors immediately. We know that the earlier we start chemotherapy, the better. If you can confirm such genetic aberrations, the patient will respond better to a certain treatment and the prognosis is much better.”
The Future of FISH
In Dr. Zhang’s experience, more and more markers are being discovered in personalized medicine for treatment and prognosis. He believes that FISH will play a bigger role in terms of making diagnosis and for following up in disease progression. Personalized medicine in cancer will be expanded and additional FISH probes will be available.
“Before FISH technology became available, cytogenetic analysis would take much longer to get the same results. In addition, chromosome analysis may not be straightforward or conclusive due to poor chromosome morphology or suboptimal samples. FISH can give a result quickly,” said Dr. Zhang.
In Dr. Zhang’s cytogenetics lab, he reports that FISH is performed in about 70 percent of the leukemia cases that were performed for chromosome analysis. In more than half of these cases, multiple FISH probes were used, mainly in patients with CLL or myeloma, and some patients with MDS. For his staff, FISH has become a very essential part of cytogenetic analysis and will only continue to expand.
“Now genomic microarray and next-generation sequencing can perform whole genome screening for genomic gain or loss, but FISH is still helpful in certain cases to confirm the abnormality,” said Dr. Zhang. “Even microarray has become a first-tier test in constitutional cytogenetics, FISH is still useful to further clarify some abnormalities discovered by microarray and to detect balanced translocations.”