What is fluorescence in situ hybridization (FISH) testing?

FISH testing, short for fluorescence in situ hybridization, is a powerful tool used in multiple myeloma to uncover hidden genetic changes in the cancerous plasma cells. FISH reports can be confusing because labs don't yet follow a standardized format. In this HealthTree University lesson, you will learn what FISH testing is, when this testing is done, what probes should be used, and how results are reported.

What is fluorescence in situ hybridization, or FISH testing?

FISH, which is an acronym for Fluorescent In Situ Hybridization, measures genetic abnormalities in the cancer cell. One of the characteristics of all cancers is that there are mutations in the cancer cells—their DNA goes haywire—and that's true for every form of cancer. In myeloma in particular, you can find changes in the myeloma cancer cell that aren't present in normal plasma cells in the bone marrow. These genetic abnormalities are one of the defining hallmarks of cancer.

Once these genetic abnormalities are identified by FISH testing, patients are stratified into standard or high-risk groups. Knowing the genetic risk helps guide treatment decisions and tailor therapy based on how aggressive the disease is likely to be.

It's also important to distinguish these genetic mutations from hereditary mutations. These mutations are specific to the myeloma cell line; they are not part of any other cells and are not inherited or passed on to children. They only exist in the myeloma cells. So please, distinguish genetic from hereditary. To learn more about somatic and germline mutations, watch the video in the Basic Genetics and Tumor Biology course.

How is FISH testing done?

Different laboratories use different techniques, but typically a probe is used to identify the genetic mutation. Probes are tiny pieces of DNA, each designed to find and attach to a specific gene or part of a chromosome. These probes are tagged with fluorescent dyes, so when the sample is placed under a special microscope, the areas where the probes attach light up in bright colors. By looking at the color patterns and locations of these glowing spots, scientists can see whether pieces of DNA are missing, duplicated, or rearranged.

These genetic mutations are not present in every single cell. Myeloma is a heterogeneous disease, meaning it is a mixture of different subclones, with multiple populations—some of which carry the mutation and some of which do not.

At what level is a genetic abnormality considered positive?

This depends on the laboratory and the threshold they use—10%, 15%, 20%—so there isn’t one universal answer. FISH testing is probably the most common genetic test done in multiple myeloma. It allows us to look at chromosomes even in cells that aren’t actively dividing, which is important because myeloma cells typically don’t survive or divide well in standard lab conditions. FISH testing overcomes this challenge by using fluorescent probes to stain the cells and see if the probe is where it’s supposed to be. Often, two probes of different colors are used to see if they are together or separated, which helps identify common genetic events in myeloma, like the 4;14 or 11;14 chromosome translocations.

What probes are used on a myeloma FISH panel?

There are specific abnormalities in myeloma cells that affect outcomes and predict poor response to treatment. These probes often involve chromosome 14, which detects changes there, as well as loss of chromosome 17. Some probes look for an increase in chromosome numbers, which can be favorable, or for multiple losses of odd-numbered chromosomes like 9, 11, 13, or 15, which are associated with adverse prognosis. The key abnormalities are chromosome 14, 17, and chromosome 1, which can have gains or losses of genetic material. Translocations involving chromosome 14, such as 4;14, 11;14, 14;16, and 14;20, are characteristic of myeloma. Gains of chromosome 1 (3 or 4 copies instead of 2) or deletions are also important markers. Other common patterns include deletion of chromosome 13 and gains of odd-numbered chromosomes like 3, 5, 7, 11, 15, 17, and 19.

Not every lab uses every myeloma probe. Limited sample material or differences between newly diagnosed and relapse patients may require selection of certain probes. However, there is a push to perform full panels so we understand the full genetic and biologic subtype of myeloma. Other translocations, such as 11;14, are important because they predict response to specific drugs or antigens like CD20. Myeloma is not one disease; it’s multiple diseases, and some translocations predict specific biology.

What type of sample is needed for FISH testing?

FISH testing is typically done on a bone marrow biopsy. It is crucial that the pathologist isolates the myeloma cells for testing so normal cells don’t dilute the results. This is especially important when the fraction of abnormal plasma cells is small. Enriched samples, often using magnetic beads or flow cytometry, allow for accurate detection of abnormalities. Patients can check their FISH report for indications like "selected" or "sorted" cells to confirm enrichment.

Should the same FISH probes be used at relapse?

Yes, whenever a bone marrow is done at relapse, it’s important to run the standard probes, because abnormalities may have been missed initially or evolved over time. If enough cells are present, all standard probes should be tested.

Who performs FISH testing?

FISH testing is usually performed on bone marrow samples obtained in clinic by a physician, physician assistant, or nurse practitioner. The samples are sent to a laboratory where pathologists or cytogeneticists perform the testing. Some machines can perform FISH, but it is typically done by a person.

What is found on the FISH report?

FISH reports vary between laboratories. Typically, the conclusion lists the abnormalities found, and the report body indicates which probes were used. Different companies produce different probes, and thresholds for calling an abnormality positive can differ. Reading FISH reports can be challenging, even for clinicians.

There is a movement to standardize FISH testing and reporting. Consensus groups, including the Cancer Genomics Consortium, recommend standardization because current variability makes it difficult for patients, advocates, and researchers to compare results or act on them. Standardized reports would clearly list the probes used, the percentage of cells showing abnormalities, and ensure consistent interpretation.

We hope you learned something new from this lesson. To better understand the laboratory technologies used to analyze genetic abnormalities in myeloma and assess your risk status, watch the videos in the Cytogenetic Testing in Myeloma course.