How is MRD testing done?

Measurable residual disease or minimal residual disease?

MRD There are different methods in which it's tested. There are two common methods that you will see: next generation sequencing and next generation flow. The next generation sequencing method is the one that's FDA approved, and one of the companies that approved it is the Clono-seq method through Adaptive Biotechnologies.

The next gen sequencing method is looking at a baseline bone marrow sample which has myeloma cells in it and looking at a clone ID of those cancer cells, looking at the immunoglobulin rearrangement signature.

So that signature can be unique as an ID to every patient, and that is the one that's followed over time to see if it's there. That sequence, that clonal sequence, is what's being followed.

Whereas with next generation flow, this is a method using flow cytometry. Flow cytometry is a method of quantifying different cell types in the blood or any plasma.

What it does is it takes millions of cells from the bone marrow aspirate, or it could even be done from peripheral blood. But we don't do that for MRD testing. When we take this bone marrow aspirate and take all the cell fractions from it and run it through a flow cytometer, it's able to tell us the different cell types in that population.

What we are able to tell is how many of these are plasma cells and how many are abnormal plasma cells. It uses 10 to 12 colors, meaning 10 to 12 proteins on the surface of these cells to identify which category or which type they fit into.

This method is not FDA approved, but it requires the institute or the cancer center where you're getting your treatment to have this method because it has to be a fresh sample.

As soon as the bone marrow is done, that sample needs to go to the lab, and they need to run it through the flow cytometer. It cannot be done on a frozen sample. So when a fresh sample is used, they're able to quantify the abnormal plasma cells in it.

The other difference is that the Clono-seq method has a sensitivity of one in 1 million cells, so one in ten raised to the power of six, whereas flow cytometry, given it's usually ten colors, is about one in ten to the power of five or one in 100,000 cells.

There is a little bit of difference in the sensitivity of these two tests. As flow cytometry gets better and we use more colors to detect these proteins, it can be as good as one in 1 million as well. It just depends on where it's being tested and what method they're using.

Another difference is that, like I said, next generation sequencing requires a baseline sample and the clone ID, whereas next generation flow doesn't require a baseline sample.

So if somebody never tested this at the baseline, you can still look for this in a follow-up sample. Often, if the next generation sequencing with Clono-seq has not been done in the beginning, it is possible to do this.

With a frozen sample or a past sample, if it's available, by sending it too. So you could ask your doctor about that because it's a standardized test, it's much easier to do at all centers, whereas flow cytometry requires a center to have the expertise to do it.

There's also another company, InVivoScribe, that also does a test similar to the Clono-seq or next generation sequencing that some institutions use.

Can a baseline sample be obtained at relapse if the sample done at diagnosis is unavailable?

I think it can be done at a relapse. It basically needs to be done on a sample where there are enough myeloma cells to detect what the sequence is, and then it can be used when there's barely any myeloma detectable to look for MRD. But we cannot look for MRD if we don't know what it is when there is enough disease. So we have to have one sample with enough disease to find the clonal ID.

How accurate is MRD testing?

The test is as good as, you know, the technique in which it's done and things like that. So if you think about next generation sequencing and next generation flow, they're both using a bone marrow biopsy sample, the bone marrow biopsy, and aspirate.

So the bone marrow was done in a place where it was a little bit of a patchy distribution and we couldn't find any cells, it may look falsely negative compared to if another site has more disease. However, mostly bone marrows are not that patchy that you would see really nothing in one place and a lot in another place.

So overall, you know, if it is negative, I think we can feel pretty confident that it is because it's looking at one in a million cells. We're looking at a lot of cells.

What are the technologies that are currently approved by definitions for MRD testing?

Per the International Myeloma Working Group criteria, the bone marrow test would be the platform, and the sensitivity level is one cell in a hundred thousand to 10 to the minus five. That's how the definition is written. There are two types of technologies that are in the guideline. One is flow cytometry, and the other one is next generation sequencing. As long as these platforms have a sensitivity of one cell in 100,000, being able to rule that out.

One myeloma cell in a hundred thousand, you can declare an MRD negativity. Technically, the next generation sequencing is about ten times more sensitive than flow cytometry. So the sequencing tests can rule out one cell in a million, and the long-term progression-free survival is better if the patient is 10 to the minus 6 negative versus 10 to the minus 5 negative.

MRD can be measured in several ways with different sensitivities. We used to only measure it by flow cytometry. And that was a little bit difficult because different labs would do it differently.

Now we have certain labs that are doing it very well and can detect as little as one in a million cells. Not all labs can do that. But the advantage is, you can look at that at any bone marrow sample. What at our institution we're doing more is next generation sequencing. This uses the actual DNA clone that is specific to a patient's myeloma and identifies it in a diagnostic sample and then uses that later as a comparison to see if any clones are still residual, for example, after a patient has undergone transplant.

So this is really important for us because it's a way to get a very exact reading. Unfortunately, there is a certain percentage of patients where that index bone marrow sample is not available, and then we're not able to look at MRD by that way. But it also has a sensitivity of at least one in a million cells. So when we can get that test done, then we try to do that.

If we can't do it, we do flow cytometry. No matter what, we try to have an informed discussion with our patients about what it means.

What are the pros and cons of next generation sequencing versus next generation flow?

So the two types of MRD testing we have are next generation sequencing (NGS) and flow cytometry. They're different technologies.

Next generation sequencing in large part is done right now by a single company in a standardized way. And with flow cytometry, there are standardized flow cytometry methods, and there are also flow cytometry methods that are developed by individual institutions.

They both have their pluses and minuses.

In terms of sensitivity, currently, meaning how low can we get?

It looks like next generation sequencing (NGS) is a little bit more sensitive.

Flow cytometry has its benefits in that you don't need former samples to identify the sequence, and it can be done without those samples.

The other thing that's good about flow cytometry is that it can potentially be done at individual institutions.

The good thing about NGS is that it's a much more standardized method.