What is next generation sequencing (NGS)?

In earlier lessons of the HealthTree University course Cytogenetics Testing in Myeloma, we covered karyotyping and fluorescence in situ hybridization, FISH testing. Now let's explore a cutting edge technology: next generation sequencing, NGS. This advanced method maps the exact order of DNA bases A, T, C, and G, providing a complete genetic profile of an organism. Join us as we uncover how NGS is revolutionizing our understanding of myeloma genetics.

What is next generation sequencing? Genes are consistent DNA, and DNA is a series of nucleic acids that we summarize as GATC. Nucleic acids are the building blocks of DNA. GACT stands for G guanine, A adenine, T thymine, C cytosine. Sequencing is just a way of converting the molecule of DNA into a series of letters. We can add GACT that we can analyze on the computer.

Instruments called DNA sequencers are essential in molecular biology, genetics, and clinical laboratories for decoding the genetic material of organisms. DNA sequencers determine the precise order of the four nucleotide bases adenine, guanine, cytosine, and thymine in a DNA molecule. The process begins with obtaining a bone marrow sample and extracting the DNA. The extracted DNA is then fragmented into smaller pieces. The prepared sample is placed in the sequencer, and computers analyze the sequence. The results are displayed in graphs or plots.

How are DNA mutations found when using next generation sequencing? The best thing to do when sequencing is to compare the sequence of the tumor DNA to normal DNA from the same person, and then you look to see if there are any differences. Sometimes it's not practical or cost effective to sequence the normal DNA from the same person. Then you base your findings on the frequency of any given change. If it has never been seen in a normal person before, then you could assume that it's probably in the tumor.

If genetic tests like karyotyping and FISH are already available, why is sequencing still needed? What are whole genome sequencing and whole exome sequencing, and how do they provide deeper insights into myeloma genetics? We get 23 chromosomes from mom, 23 from dad to 46, which you can actually look at under the microscope. The pathologist can tell us, and you can also do probes for FISH, which look for specific mutations that you know of.

The limitations of those are cytogenetics is very rudimentary. It's like looking from 1000ft away. FISH gets you a little closer, but you need to know what you're looking for. It's not going to tell you abnormalities that you might not have probed for. The next generation of these tests, which are sequencing-based, aren't so specific. They look at the entire panel. You can do whole genome, which is the entire genomic profile of that cell, and exome.

The problem with the genome is that it's not all coding. Sometimes there's junk DNA in between. So there's genes and then there's filler DNA. You're going to sequence all of that, but not all of that gets made into proteins. Not all genes get made into mRNA which makes the protein. So if you really want to focus on the things that actually can potentially cause mRNA and protein changes, then you would do exome sequencing. You're not sequencing the junk DNA in between. That's why we're interested in these because it's more unbiased. You're not only asking for, say, 17p probe; you're looking at the entire chromosome 17 and other changes that could be there. You can pick up mutations that you could never pick up by cytogenetics and FISH.

For example, if you have two copies of 17p, there's no problem. But if there are mutations that affect the ability to make that protein, it's important. 17p is your brake on the cancer cell. It may look like your brakes work, but mutation testing may show that the brakes are not functional. That patient will have bad brakes, and you wouldn't have known that just with cytogenetics and FISH.

To learn more about TP53, often called the guardian of the genome, be sure to watch the TP53 lesson in the chromosomal and gene mutation course.

What's a targeted sequencing panel? Targeted sequencing means you're not doing the entire genome. You're looking for specific things. For example, at UCSF we have a myeloid next gen sequencing panel which looks for around 100 genes commonly mutated or abnormal in hematologic malignancies. It's not a kitchen sink; it's specific genes of interest. This tends to be faster and more cost-efficient than doing the entire panel.

Is sequencing standard of care in myeloma? When is next generation sequencing used in myeloma? The challenge with sequencing is that while everybody might say yes, the complexity is whether insurance will cover it or patients will face large bills. As a patient gets more heavily treated, sequencing can be submitted to understand why initial therapy didn’t work. Sequencing is also useful in extramedullary myeloma sites because these may have mutations targetable by drugs not typically used in myeloma. For example, genes like RAS and BRAF have approved drugs for other cancers.

Another use is to assess secondary malignancy risks. After transplant, drugs like lenalidomide increase the risk of secondary cancers. Some CAR-T therapies can also generate secondary bone marrow cancers like MDS or leukemia. Sequencing can detect gene abnormalities that predispose patients to these conditions and help guide thoughtful dosing decisions.

Why is next generation sequencing routinely done in other blood cancers? Leukemias often have targeted mutations like FLT3 or IDH, which guide therapy. In myeloma, this is less clear because of multi-genomic alterations and multiple clones. Sequencing is not yet standard of care in newly diagnosed myeloma patients, partly because historically there were few identifiable or targetable mutations. Sequencing has been more prominent in cancers where specific mutations have approved therapies.

Is next generation sequencing available for clinical use or just research? It varies by practice. Most hematologists do not routinely use it for multiple myeloma, though it’s common in solid tumors and leukemia. Panels may exist for hematologic malignancies or myeloma specifically, but coverage by insurance is not guaranteed. Patients can request sequencing, particularly if 17p deletion or other high-risk mutations are suspected.

Can next generation sequencing make myeloma treatment more specific or lead to precision medicine? Data is needed. Single gene targeting, like in 11;14 myeloma, has challenges because myeloma is multi-genomic. Doublet therapy may not be sufficient. Myeloma’s complexity makes NGS more challenging but potentially more informative.

To better understand the laboratory technologies used to analyze genetic abnormalities in myeloma and assess your risk status, watch the other video lessons in HealthTree University’s cytogenetics testing in myeloma course.