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Demystifying the Lab: Cytogenetics

December 30, 2021

Clinical applications abound for the study of structural abnormalties in chromosomes.

The first chromosomal abnormality discovered in cancer using cytogenetics is likely still one of the best known: In 1959, David A. Hungerford, PhD, in collaboration with Peter C. Nowell, MD, discovered an abnormally small chromosome – the Philadelphia chromosome or derivative chromosome 22 – that was consistently present in samples from patients with chronic myeloid leukemia (CML).1 More than a decade later, Janet D. Rowley, MD, PhD, discovered a translocation between chromosomes 22 and 9 using quinacrine fluorescence and Giemsa staining.2

Since those initial discoveries, the field of cytogenetics has evolved to include everything from conventional karyotyping to molecular arrays and hybridization. This type of testing has shown its value in the clinic, providing practical information that helps clinicians in the diagnosis of disease, distinction between disease subtypes, treatment selection, and measurement of treatment response.

ASH Clinical News spoke with cytogeneticists and pathologists about the role of cytogenetics in managing patients with hematologic disease, the varying techniques used, and the future of this once futuristic technique.

Cytogenetics 101

Cytogenetics is the branch of genetics that studies chromosomes and the structure of DNA within the cell nucleus.

"Every time a cell divides, the chromosomes duplicate. In the metaphase of the cell cycle, all the chromosome pairs line up and are condensed, allowing one to see them under the microscope," said Tapan M. Kadia, MD, associate professor in the Department of Leukemia at The University of Texas MD Anderson Cancer Center.

These condensed chromosomes are then stained using Giemsa banding, or G-banding. G-banding allows for the recognition and identification of chromosomes based on their length and banding patterns. European scientists typically use a different staining technique called R-banding (reverse banding). The study of chromosomes using banding techniques is known as conventional cytogenetics or karyotyping analysis.

"Conventional cytogenetic analysis involves a trained pathologist looking at a picture of chromosome staining and, based on their knowledge, identifying what looks abnormal," Dr. Kadia said. This includes numerical abnormalities (such as gain or loss of a chromosome), structural abnormalities (such as a translocation, deletion, or inversion), or both.

"Karyotyping is the most common, routine form of cytogenetics," said Eric D. Hsi, MD, professor and chair of the Department of Pathology at Wake Forest University School of Medicine. "It is a tried-and-true technology and is still the workhorse in terms of routine characterization of the genome, but provides only a low-resolution characterization."

Indeed, karyotyping is looking for gross changes in chromosomal organization: big gains or losses of chromosomal materials, Dr. Hsi explained. Cytogenetic analysis using G-banding cannot resolve structural abnormalities that are small. Detection of small gains, deletions, single-base mutations, or cryptic translocations requires newer technology with higher resolution.

Conventional cytogenetics with karyotyping analysis has a resolution limited to about 5 Mb – meaning it can detect changes of greater than five million base pairs – and requires live dividing cells. It can also be time-consuming. Preparation of cells for the examination can take several days.3

Gone FISHing

"In the 1990s, we were able to get better and higher resolution analysis using something called FISH, fluorescence in situ hybridization," said Min Fang, MD, PhD, director of clinical cytogenetics at Seattle Cancer Care Alliance and professor in the clinical research division of Fred Hutchinson Cancer Research Center. She is also professor in the department of laboratory medicine and pathology at University of Washington School of Medicine. "FISH was very helpful in finding smaller chromosomal abnormalities that cannot be obtained in metaphase chromosome analysis."

FISH is much more specific and relies on exposing chromosomes to small DNA sequences called probes that have a fluorescent molecule attached, targeting the specific genomic area of interest.

"The caveat to FISH is that you can't see everything," Dr. Kadia explained. "You have to know what you are looking for."

One common use for FISH is looking for BCR-ABL1 rearrangement for monitoring patients with CML, acute myeloid leukemia (AML), or acute lymphocytic leukemia (ALL). When the pieces of chromosomes 9 and 22 break off and trade places, the ABL1 gene from chromosome 9 joins to the BCR gene of chromosome 22, forming the BCR-ABL1 fusion gene.

"A probe made for the fusion genes will tag them with fluorescence," Dr. Kadia said. "However, if you use a probe for BCR-ABL1, you would not find anything related to monosomy 5 or monosomy 7, other markers of certain leukemias."

In contrast to the 5Mb limit of karyotyping, FISH has a higher resolution and is able to detect abnormalities in smaller amounts – about 100 kb to 1 Mb. FISH analysis also can use cells in any stage of the cell cycle, not just metaphase, and does not require fresh tissue.

"Most of the time, when we use FISH, we let samples sit overnight for hybridization," Dr. Fang said. "For cases where we need immediate results, we could do a turnaround of about four hours."

More Progress on the Horizon

Testing has further evolved in the past decade, as pathologists have begun to use array-based comparative genomic hybridization (CGH) or chromosome microarray analysis (CMA), alternatively called chromosome genomic array testing (CGAT).

This method has moved cytogenetics from the microscope to the computer, Dr. Fang explained. "This technique allows simultaneous genome-wide analysis of many chromosomal abnormalities at high resolution," she said. "It combines thousands or millions of FISH probes into one chip and uses a computer method to assemble them into chromosomes for analysis."

Rather than metaphase chromosomes, this technique uses slides arrayed with small segments of DNA. This DNA is then compared with a reference genome to identify differences.

CMA/CGAT can detect changes greater than 1 Mb at low resolution or changes as small as 10Kb at high resolution.

Which Test, and Why?

With a wider selection of cytogenetic tests available, the million-dollar question is which one to use, according to Dr. Fang.

"At our institution, we designed standard pathway testing dependent on the disease. Without that built-in guidance, it could get very complicated," she said.

Under the testing pathway, physicians submit a sample for testing and the suspected diagnosis. The pathologists and the cytogeneticists decide on the appropriate test after considering a wide variety of factors such as the clinical condition suspected, sample type and volume available, and family history.

For example, if acute promyelocytic leukemia (APL) with t(15;17) translocation is suspected, immediate FISH is appropriate, Dr. Fang said.

"APL is typically very acute and time sensitive," she explained. "You want results quickly and don't want to wait for cultured cells. With FISH, we could do a four-hour hybridization and return the results to the physician so they can quickly decide how to go about treating their patient."

FISH might also be employed in malignancies such as chronic lymphocytic leukemia or multiple myeloma, where the karyotype analysis often shows no abnormalities, Dr. Fang said. A FISH panel that specifically looks at the genes and chromosomes frequently altered in these disorders can help pinpoint the disease very easily.

Diagnosis, Prognosis, Treatment

When talking to a patient about cytogenetics testing, Raajit K. Rampal, MD, PhD, clinical director of the Leukemia Service at Memorial Sloan Kettering Cancer Center, uses simple language to explain what the test is, how to interpret it, and what results will mean for their care.

One test that he commonly discusses with patients is for eosinophilia, a workup associated with myeloproliferative neoplasms. "We see specific rearrangements that not only confirm diagnosis, but guide us toward treatment," Dr. Rampal said. Detection of the FIP1L1/PDGFRA fusion gene, for example, would support the initiation of imatinib.

Dr. Hsi cited Burkitt lymphoma as another example. Here, cytogenetic testing is used to look for the classic t(8;14)(q14;q32) chromosomal translocation involving the MYC gene on chromosome 8 and the typical IGH gene on chromosome 14. Variant translocations involving MYC and other known partner genes also can be identified via karyotyping.

"Since it is such an aggressive and rapidly growing lymphoma," Dr. Hsi said, "looking for this abnormality can help confirm this diagnosis and allow for immediate appropriate therapy."

Regarding prognosis, patients with myelofibrosis who are found to have del7 or inv3 will likely have poor prognosis. "Cytogenetics are often factored into prognostic scoring models that help us further stratify patients based on risk of progression," Dr. Rampal said.

Finally, cytogenetics may also aid in developing new targeted treatment for patients with certain hematologic malignancies. In patients with ALL or AML, a variety of KMT2A (MLL) rearrangements have been described and are associated with aggressive disease and poor clinical outcomes.

"We now have investigational drugs called menin inhibitors that may therapeutically target these MLL translocations," Dr. Kadia said. "In the future, in addition to many of the other standard chromosome panels, I think it will become mandatory to test whether a patient with acute leukemia has an MLL translocation."

"We have to work toward expanding availability or access to FISH and other newer techniques … Cytogenetics is an area where we have to constantly innovate."

—Tapan M. Kadia, MD

The Future of Cytogenetics

Cytogenetics and molecular genetics are both fields of genetic study, but each has a different focus. Molecular genetics is the study of genes at the DNA level, whereas cytogenetics is the study of chromosomes.

"These are all genetic tests, but chromosomal abnormalities are big changes, with large sections broken off, missing or moving or sticking to another chromosome," Dr. Kadia said.

According to Dr. Fang, the traditional boundary between cytogenetics and molecular genetics is blurring as the technologies evolve. In fact, the American Board of Medical Genetics and Genomics no longer has separate certification programs for cytogenetics and molecular genetics; they have been combined into a single Laboratory Genetics and Genomics program.

Conventional karyotyping is a mature technology, Dr. Hsi said. If improvements remain to be made, it will be on the molecular side.

"With more FISH probes or better application of next-generation sequencing assays we will find more mutations, copy number changes, or translocations in the different diseases we treat," Dr. Hsi said.

In addition, the incorporation of more automation into the process could go a long way toward improving workflow and laboratory efficiency.

"Cytogenetics overall is a heavily labor-intensive type of technology," Dr. Fang said. "In recent years, many automated tools have been introduced into cytogenetics labs that have improved workflow and reduced labor costs, but it is not yet enough."

As those automated processes are developed and refined, Dr. Kadia emphasized the importance of maintaining high-level training for pathologists on both new techniques and conventional karyotyping.

"We also have to work toward expanding the availability or access to FISH and other newer techniques," he said. "Cytogenetics is an area where we have to constantly innovate." â€”By Leah Lawrence


  1. Fox Chase Cancer Center. The Philadelphia chromosome: history and implications for the future. Accessed June 7, 2021.
  2. Rowley JD. A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature. 1973;243:290-293.
  3. Eurofins. Biomnis. Constitutional cyto-and molecular genetics: karyotyping, FISH and CGH array. Accessed June 6, 2021.

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