New blood-based tests are in development and coming closer to the myeloma clinic. Patients love the idea of reducing or eliminating the traditional bone marrow biopsy.

In this podcast, Dr. Ola Landgren shares multiple types of blood-based tests that help detect myeloma and measure minimal residual disease. Tests reviewed include mass spectrometry tests, MRD tests, flow cytometry tests and circulating tumor cell tests. 

Jenny: Welcome to today's episode of the HealthTree Podcast for Multiple Myeloma, a show that connects patients with myeloma researchers. I'm your host, Jenny Ahlstrom. We'd like to thank our episode sponsor GSK for their support of this program.

Before we get started with today's show, I'd like to remind you to join HealthTree Connect, if you haven't already. HealthTree Connect is our own social media platform that's part of the HealthTree suite of patient tools. If you participate in the HealthTree community chapter events, or even if you don't, you can connect and chat with other myeloma patients in these groups. You can find HealthTree Connect by going to healthtree.org/myeloma and then clicking on Apps and Programs in the menu bar. It's similar to Facebook in function, where you can post questions or comments, comment or react to someone else's post, or have videos or photos to post, and you can subscribe to certain groups which will put those conversations in your feed. We now have over 1700 users in HealthTree Connect.

At the end of June, we will have a phone-based app for that and our other programs, so keep an eye out for that.

Now onto our show. There are a host of new or up and coming blood-based tests that are being used to track a patient's myeloma. Now this was definitely more convenient for patients than the unfortunately dreaded bone marrow biopsy. Our show today is to learn about these different options from Dr. Ola Landgren, Head of the Myeloma Program at the University of Miami. Dr. Landgren, welcome to the program.

Dr. Landgren: Thank you very much for having me. It's a great pleasure to join here today.

Jenny: Well, thank you so much for your time. We really appreciate it. Let me give an introduction for you before we get started. Dr. Landgren is Professor of Medicine at the University of Miami and head of the program at the University of Miami. Prior to his appointment, he was head of the program at Memorial Sloan Kettering Cancer Center, professor at Weill Cornell Medical College and at the National Cancer Institute, and Karolinska University Hospital in Sweden, where he is from. He is an author on over 403 publications and has received the American Society of Clinical Investigation Award and many NCI awards, including the NCI Research Highlights Award, the Bench-to-Bedside Award, the Intramural Award and the Director’s Intramural Innovation Research Award. At the University of Miami, he's built an astounding team, truly astounding, with specific focus on the genetics of multiple myeloma to establish a risk profile for newly diagnosed patients, precursor condition work on MGUS and smoldering myeloma and so much more. I'm just so impressed with what you have done, building a program really from scratch.

Dr. Landgren: Thank you. You’re very kind, Jenny. I really appreciate it. Thank you.

Jenny: Let's get started with these different tests. I think it might be helpful to just use the same format and go through the different tests and have you help us explain what the tests are and how they work, why and when you would use them, if they're available today, and then the pros and cons of these different tests. You and I had a conversation about this, and it was so helpful for me. I thought it would be very helpful for other patients because they hear about these tests, but it's hard to keep them all straight actually. Let's start with maybe the MRD-based tests. Maybe we should start with the Binding Site test?

Dr. Landgren: Yes. First of all, I would like to say, when it comes to all these tests, I have no financial relationship. I have no favorite assays. I'm working in the research setting. I'm also working obviously in the clinical and translational setting.

When we talk about these Binding Site tests, what we are talking about in a little bit broader context is about a mass spec technology. Mass spec is something that's been around for a very long time. What happens specifically in the Binding Site, and there are other related assays as well that work in a similar manner, is that a very small amount of serum can be used. Serum comes from peripheral blood. You can take blood too. You can take out a very small amount of serum, and you can send laser beams in this mass spec machine through the sample. The way mass spec technology works is that it uses laser beams to go through different types of liquids to determine whether there are certain structures or not. You can have it looking for, in this case, different types of proteins.

When people talk about the Binding Site, how that works is that you take a sample. You typically would put it in a little plate that has many wells, and you would split the sample into several wells. Typically, people have done, say, five wells. Then you let the machine send laser beams through each of these wells, and ask the question whether it can identify abnormal proteins or not. The machine can then answer that, and it can actually do it very fast.

With these technologies, if you use a particular type of mass spec instrument that’s called MALDI-TOF mass spec machine, each well can be busted by these lasers in only ten seconds. If you have five of them as replicates, to make sure you see the same thing, we are talking about 50 seconds. It's less than a minute. It's very fast. The total time from preparing a sample, if you do a blood draw on a human being and you want to put it in this machine, there are obviously many more steps. You have to prepare the sample. You have to do many different steps. It doesn't take just 50 seconds from the blood draws, we have the results, but the actual assay in the machine is very fast.

What I also should say is that what this type of approach really is looking for is not that very different from what you're looking for when you use immunofixation or SPEP tests that are standard tests and had for many, many years. It’s looking for abnormal monoclonal proteins. You can also look for free light chains. These are tests that we wouldn't normally look for, with regular blood tests, as everyone knows. The difference is that instead of using the technologies with electrophoresis or other types of nephelometers that are used, different types of established technologies, this type of approach uses the laser or the mass spec technology. What that does is that increases the sensitivity, so you can basically look for fragments of the immunoglobulins. You look for smaller pieces. That means that you are more able to identify lower levels, more sensitive, faster and a pretty neat way of doing it. That's a simple way of saying it.

Jenny: Understand. Now, why would you use it and when would you use it? We didn't really talk in the beginning that I should back up a little bit, just about overall usage. Why are these blood-based tests being further developed, instead of just using the bone marrow biopsy?

Dr. Landgren: The question is, why are these tests used instead of bone marrow biopsy? Well, as you mentioned at the beginning of the introduction here, bone marrow biopsies are quite unpleasant ways of capturing information. You have to do a biopsy. It's painful, and it's an invasive procedure.

If you can use blood, if that could replace the need for a biopsy, I think most patients, for sure, would like it, and of course, all doctors and everyone else involved. I think it's just for the feasibility and the inconvenience of biopsies, to try to go away from that. I don't think we are yet at the point where we can say that this type of technology really, truly can replace the biopsy. Because in order to come to that point, you need to generate a lot of data. You need to show that, if you're looking for minimal residual disease detection, that you have followed a large number of patients.

You have information from the bone marrow, and you have the information, for example, with this technology. You show that there is concordance. Say, if you test 100 patients and 99 of them, you get the same results, that's a very good outcome of the study. If you test 100 patients and only 50 of them are concordant, that's not going to work. That type of work is still ongoing. We don't have the formal results from comparisons between bone marrow-based MRD tests and these types of essays.

There are a lot of other details that could be discussed in the setting of, what are you actually tracking, and how does it work? What I mean by that is, if you do a bone marrow biopsy, look for cells or DNA, and you can answer yes or no detectable, where you can quantify what you see. With these proteins, you're not looking for cells, you're looking for proteins, but the proteins come from the cells.

That's important to remember, that if you have a patient that you treat with very effective therapy, let's say you can completely eradicate the cells, and you do a biopsy, and there are no cells to be found. Among the last things those cells did before they went away with the therapy was that they cranked out a lot of proteins, and protein can stay in the body for quite a long time. If you, at the same time as you do that bone marrow biopsy, also do a blood test and you look for protein, you probably will see protein. You're going to have one bone marrow testing negative, and then you're going to have a protein testing positive. If you just look at it that way, you're going to say, “Oh, these are not consistent. They're not concordant.” Well, you're not looking at the same thing, you're looking at proteins that were released by the cells that are no longer there.

That takes a lot of additional things into consideration. You have to probably look at the slopes of these. If you check the protein in a few weeks again, if they continue to decay, to go away, and it mimics the pattern of clearance, then you would say that these proteins really are not being continued to be made. They are just part of the washout, of the clearance in the body. As we hear me saying, these are a little bit complicated things. There is more research that needs to be done to really determine how the clearance patterns really look in a person who does not continue to make protein, and there is more work that has not been done yet.

Jenny: Yes, it sounds like you may have to just, like what you're saying, just see that slope over time. Is it going down? Is it gone? The Binding Site, you explained how this test works and why you would use it. The mass spec tests are considered MRD tests, correct? Because I think sometimes patients hear mass spec, and they don't really understand the context of those types of tests. They are, correct?

Dr. Landgren: Well, I think you could say yes to that, but you could also broaden the scope and say, what really makes a test qualified to be called an MRD test? You, maybe, could say that most tests could be viewed as MRD tests because really, when we talk about MRD tests, what we mean is that we use them to make sure that there is no residual disease. Could you use flow cytometry as an MRD test? Could you use sequencing-based technologies? Could you use protein-based technologies? There are so many different ways.

More than that, you could use imaging-based technologies. You heard me talking before, at another of your programs, about images that are immuno-PET-based. That's another way of going after MRD tests. Yes, the mass spec test could be put in that category.

The way I think about it is that it's basically a modern way to use mass spec technology to rule out presence, absence of residual disease in the blood. I do think the caveat I mentioned is that there is a delayed clearance. That is something that probably can be handled if there is better knowledge on the slopes or the clearance, but there's more work, as I mentioned, to be done. Already now, these technologies are probably very, very powerful. There is more work to be done for that as well, but they are probably very, very powerful for early detection or recurrences. Because if there is nothing, there is nothing, there is nothing, and then all of a sudden there is something; they are much more sensitive than any of the other blood tests that are currently available, like SPEP and free light chains.

The dilemma that this type of technology is facing is the fact that the proteins that are made are in the body after the cells have been eradicated. That is what I'm trying to highlight on the fact that you have the slope of the clearance where you could actually mathematically model and show that, if the slope goes down with a certain rate, all you see is just delayed clearance. We need to have more computational models to really do that in a reliable way.

Jenny: Yes, that makes a lot of sense. Let's talk about how you would use this, like you said, to just rule out the presence or absence of residual disease in the blood. You're using it after treatment typically, right? You wouldn't need to use it prior to treatment because you have a big load that you can detect, like on IV or the regular monoclonal protein test. Is that the only time that you would use it is following a treatment and looking for the depth of remission? Or are there other times that you might use this test?

Dr. Landgren: These tests that we are talking about, also for full clarification, you mentioned the Binding Site technology. The Binding Site is now part of the Thermo Fisher Corporation. There are other technologies as well. I mentioned that Mayo Clinic has their own technology that they are commercially selling. They have a huge commercial laboratory in Rochester, Minnesota. There are other labs as well. There are other groups that are working, and we have no favorites. They're all doing a good job. They are very similar to each other, these types of technologies.

What could you use them for? Could you only use them for MRD tracking? Or could you also use these tests just in someone who is newly diagnosed? Could you use it to track someone who is on therapy? Could you use it on someone who has relapsed myeloma?

The answer is yes, you could, because these technologies can track the protein. They can identify them. What all these different companies and initiatives I mentioned, and others, are doing is they're still trying to see how they, with computer models, can use the data to quantify the size of the protein. The practical aspect is, if you have your SPEP, you have your IVs, you have your light chain assays, and here comes another assay, the mass spec; is this another assay? Or is this an assay that potentially could replace the others? It would be great if it just replaced everything. That would mean that you could have a more sensitive assay.

What needs to be done there, the remaining work that really needs to be done is the quantification components of this type of assay, meaning that you run it, and it will tell you 2.1 grams per deciliter. That needs to be further improved. Or that you say that there is a light chain concentration, with kappa, of 12.8 milligrams per deciliter, let's say. That is where the technology needs to further be developed in terms of the computational side. There is a lot of work already done. These assays I mentioned, and others, have already done a lot of work.

I think what needs to be done is to show that this is completely reproducible, if you do the same test in different locations, in different laboratories, that the results continue to show the same thing. This is typical for assay development. It takes time to do this.

Jenny: Yes, absolutely. I will be asking you about different types of tests by name sometimes. Because sometimes patients, we hear the names, and we don't understand the context or how one might be different from the other or better than the other. This is just an example, to start, and then we will move on to different tests. Hopefully, that's okay.

Dr. Landgren: Yes.

Jenny: Any pros and cons on the Binding Site test, in particular? You said it was very fast, the method?

Dr. Landgren: Yes, I think that the process is very fast. I mentioned that you can load a plate. Usually those plates are premade or used for many different types of machines. The type of machines that are running these plates, usually take plates that can have up to 96 wells in a plate. That means that you can put, I mentioned, five replicas. You can put five replicas times many patients. That would then feed into these plates. If you have a large laboratory, you can do one plate, the second, the third and fourth and so forth plates. It’s basically a very high throughput technology. If you have a very large lab, you could probably crank out the same amount of results. An SPEP, an IFE maybe would take five people. That could be done with just one person running the machine. It's very efficient technology. If you have a small number of samples, it's going to probably be about the same staff running it because you need to have people to do lower volume with either of the platforms, but the technology has its scalability potential, if you want.

From a sensitivity point of view, I think it probably is between ten and 100 times more sensitive than SPEP and IFE. Although you, in theory, would think that it could be even more sensitive, the dilemma is the fact that this is looking for fragments of immunoglobulins. You're looking for fragments of the abnormal proteins. In the blood, there are a lot of normal proteins. Every human being has a lot of albumin. We all have, also, normal immunoglobulins. From a lab perspective, what we are talking about is that there is a background noise. What we want to pick up is the signal. What we don't like is the noise. When you're starting to go to very low concentrations, and also when you look in the light-chain myelomas versus the ones with M-spikes, you run more and more into these problems, from a methodological point of view, with the noise. The technology still has some room for continued improvements, to improve some of these details, I would say, but it is a very, very powerful technology.

Cons are that I think that there is more work to be done, but that's true for many things in life and in medicine and things in general. Also, a con is the fact that you're tracking something that is not linked to here and now. It's something that is linked to what happened in the past. What I mean with that is the cells, I mentioned, if you eradicate them, you give CAR-T cell therapy, you give Dara-KRd or some very powerful therapy, teclistamab or some of the other bispecific antibodies; although the cells can be wiped out in just a month, let's say, the protein will probably be there for many months.

 If you're trying to see if you got rid of the disease, the proteins, they are going to stay in the body for a longer time because they take time to be cleared by the body. You need to have computer systems that can tell you that the rate of this decay, the decrease in concentration over time, is consistent with what you would expect to see in someone who's just clearing out a given concentration. You want to make sure that there is no additional protein being made which would indicate that there is residual disease. That is what I would call an inherent limitation with these protein assays that you are looking for something that is not linked directly. It's an indirect measure of the disease. You need to have computerized systems in order to be able to read what you're seeing, what that really means.

For tracking someone over time, someone who is negative, or if you want to rule out that there is no recurrence of disease, that's a whole other story. If it’s negative and it continues to be negative, that's what we want to see. That's great. These could be very early warning systems. You don't have this dilemma because once the protein is eradicated, and that's being cleared from the body, if it continues to be negative, that's what we want to see.

Jenny: Okay. Yes, makes sense, and the over time tracking makes complete sense for that test. Well, let's move on to the Sebia test. That's also a mass spec test, correct? Or no?

Dr. Landgren: Yes, it is. The Sebia technology – I want to be very inclusive here. I am a very inclusive person. I have no favorites for drugs or technology, for anything, I usually say I have no loyalty. I just want the best. I want the best for my patients. A drug company comes or a technology company comes, I tell them, always, the same thing, that you have nothing to worry about. As long as you have the best option for my patients, I will use it. Sebia is one of the good options.

There is also a technology up in Toronto, in Canada, that has been developed by the University there, Princess Margaret Hospital. There are others as well. They are different from the ones that we just talked about in the sense that they are not looking for the fragment of the whole immunoglobulins or the light chain. What these technologies are doing is that they are actually looking for peptides that are much smaller protein structures. Amino acids make peptides, and peptides make antibodies. That's the order of how these proteins are linked to each other. Everything goes down to the amino acids, the CvAT, and then you make the peptides. The peptides form structures like immunoglobulins or free light chains. These technologies are looking at that intermediate step of the peptides.

One of the key strengths with this is that you can actually, if you know the sequence of the disease for a given patient, you then know which of the peptides you're going to look for. The first technology I mentioned just screens for fragments of abnormal proteins. If you look for peptides, you can actually see, is this peptide there? Yes or no. It’s a patient-specific technology. It’s looking for smaller units of protein, if you want. Because you do it that way, that means that you don't run into this issue with the background noise because you know what you're looking for. If you see it there, then you know it's diseased. It's more precise. It probably is in the range of 100 to maybe 1000 times more sensitive than SPEP and IFE. That's fantastic.

Jenny: Yes, amazing.

Dr. Landgren: What are the strengths and weaknesses with this technology? As we hear me saying, if you have a patient-specific approach, that's fantastic. You can track. You don't have the noise. The downside with that is that, how do you know the individual patient’s sequence? Until quite recently, that has been a big hurdle with this technology. You used to have to do a bone marrow biopsy to get the sequence from sequencing. With very creative work by scientists in the field, it actually is now possible to take a very small amount of serum to determine the sequence just from the serum. You don't need to do the biopsy. You can take a small amount of serum. You run it in the technology. Then you can determine the individual patient’s sequence. Then you can ask the computer, tell me which are the ten most common peptides that are linked to these peptides, or whatever number the machines house. I think it's ten for now. That's how these technologies are developed for now. This could change in the future. Now you know what to track. You could take a very small amount. We're talking a few microliters, five or ten microliters serum, which is technically nothing. You can ask the laser in the mass spec machine, can you find this protein? Yes, no. Or these proteins. If it does, then you have positive, negative. It is a more sensitive approach. It doesn't have the issue of background. It’s much more precise.

The con with this is that you need to determine the baseline sample, but you can do it from serum. If you do a baseline in someone who is newly diagnosed or someone who still has detectable disease, you will get it out of these technology platforms that can do it in blood, in serum. The inherent con, as I mentioned before, for the mass spec in general is that you are looking for something that was made by cells that no longer may be around. You will always have this issue when you're looking for proteins, but you could also model the clearance here. If you see that the proteins go away and they follow the same trajectory as they would in a typical clearance situation, you can say there is no indication, or there's a 95% probability that this follows the slope of delayed clearance. There is no indication that there is new protein being made. I think that's where these technologies are going to land. You can also track people over time. As I mentioned, you could have early warning systems for reappearance of disease.

Jenny: Okay. I'm taking notes. Makes sense. All these different methods are very interesting. I think it's good for patients to understand so if they start hearing names of things – I didn't know that Mayo Clinic had their own test that was similar to the other tests. This is what patients are hearing randomly, and I think they need to put it in context. This is super helpful.

Can we talk about the Adaptive clonoSEQ test? Because they had the MRD test that was bone marrow-based, and now they have a blood-based test. I don't think it's FDA-approved yet, but I think it's CLIA-approved. Can you explain what that is and how it works and how it's different from the other two methods that you just explained?

Dr. Landgren: The Adaptive Biotech clonoSEQ test is a technology that has been developed over a longer period of time. Basically, it's based on the fact that the immune cells in the body have an ability to adapt to different types of antigens that enter our body. The immune system, obviously, is a very important part of our bodies to keep us out of trouble. If there are infections or different types of threats that happen in our body, the immune system will attack it and get rid of it. The way that works is that there are certain regions of the genetic program in our bodies that can adapt and reshuffle parts of the sequences to make sure that the body matches up with a threat and then eliminate it. You can say it's very plastic. It sees something particular. It adjusts for that. It builds up more of these responses. Then it goes after that. If you sequence a lot of B cells as a part of the immune system, you will see that there are many different sequences. There are many, many, many, many of these sequences. That's basically because we are constantly exposed to different types of things. There are antigens. There are bacterias and viruses and food and everything in our environment where we live. When we are active and doing things, all those things are constantly being checked off by the immune system. What the technology that Adaptive is using is based on is this very principle that the B cells respond.

What was discovered years ago is that if you have a malignant process such as multiple myeloma, that is a process that, for an individual patient, shares a particular sequence. That by itself is very fascinating. It's probably something that we need to spend more time thinking about, because that means that there is something unique that triggered the onset of the clone of the disease in a given patient. There is something that seems to be consistent in all the cells within a given person with multiple myeloma that is unique. Then these cells have copied up many, many, many copies. If you perform more sophisticated sequencing, you can see that many of these cells look very different from each other. In fact, there are usually thousands of different mutations you can see. If you perform single cell sequencing, you can see that these mutations that you can find are not the same across these different cells.

This very sequence that I mentioned in the beginning that is unique for the B cell response is also called VDJ. That is part of this plastic part of the immune system. Although there is a huge, what's called genetic heterogeneity of the cells within a given person with myeloma, there are many different mutations in different cells that are not in other cells. They have a lot of mutations, but they are not the same. The very VDJ sequence is the same in all the cells, which is very peculiar. That means that you now have a fingerprint that is unique to the patient that is unique to the disease. What we and others have done, we have studied these signatures, and we have seen patients were treated. They were MRD-negative. Unfortunately, if there was a recurrence years later, we picked up, again, the disease, and we showed that the fingerprint of this VDJ was identical. We have seen people who went back to MRD-negativity, and unfortunately, there could be a recurrence many, many years later. Again, this signature did not change. We have followed people for over ten years, and the signature is the same. The biology of myeloma lends itself for tracking of these VDJ because once that’s there, that doesn't seem to change or go away. That's, basically, what Adaptive does.

In order to do that in a practical way, because what I said, all the B cells have their unique signature, if you just do a bone marrow biopsy of someone who is completely healthy, you will see millions of sequences, which is just reflective of how all the B cells are reacting to different things. Now if you do it on someone who's newly diagnosed with myeloma, the difference with these healthy persons that have millions of normal sequences is that the person who has myeloma has millions of sequences that represent the normal immune system but then there is one sequence that has many, many, many, millions and millions and millions of copies, and those are the myeloma cells. If you treat this person and the person is being tested at a later time point, if you didn't know what sequence to look for because you didn't perform the baseline test, after the treatment has been completed, you don't know which could be the sequence that is the one that is the clone, the disease. Even if there were just very few cells hanging around, you will not be able to tell if this is background or if it’s disease. That's why you need for these types of tests, the Adaptive clonoSEQ assay. You need to know the baseline samples. You know what to look for. If you do that, you can ask in a sample after treatment, can we still detect the sequence? Yes or no. How many copies are there? You can track it over time. Also, there are, of course, a lot more details here as well. There can be more than one sequence. There could be two sequences or three sequences. Many cases, there is only one sequence, but could be one or two or some very low number. Then you can track them over time.

Jenny: How does the Adaptive clonoSEQ blood-based test differ from the Adaptive clonoSEQ bone marrow-based test?

Dr. Landgren: The technology is the same, exactly the same. You're looking for these signatures. The only difference is that you are looking to see if you can pick up the sequence in another part of the body. Instead of putting a needle in the bone marrow, you put a needle in the blood. Now, if you ask yourself, you have someone who is newly diagnosed with myeloma, and you do a bone marrow biopsy. You have a perfect sample. You do a perfect blood draw, a perfect sample. You say, how many copies of this abnormal sequence do I find in the bone marrow per, say, one milliliter of blood as a unit? Then you do one milliliter of bone marrow. Well, the answer is that there will probably be 100 or 1000 times more copies in the bone marrow because that's where the disease is. Not all the disease will go out in the blood. What that means in practical terms is what you're looking for in the bone marrow, if you start looking for that same thing in the blood, you're making your life 100 to 1000 times more difficult as a researcher, as a clinician, because that will be between 100 and 1000 times less to look for. If you have too small of a blood volume, you may end up with a negative result when you look in the blood, which could be either because there is no disease, or it could be that there are too few copies to reliably detect with this assay. If you took out a whole liter of blood or a very large volume of blood, you probably would be able to reliably do it. Of course, no one would like to do that. We only have four and a half liters of blood in our bodies. That would just never happen.

If you're talking about strengths and weaknesses, the weakness in the blood-based sequencing is that what we're looking for is less prevalent in the blood compared to the bone marrow, but it's still more sensitive than SPEP and IFE. You could argue and say, maybe it's not that different in its sensitivity compared to, say, the MALDI-TOF. We talked about the Binding Sites and other technologies, the Mayo Clinic assay, etcetera. It's a sequencing-based technology. You don't have the issue of delayed clearance with the sequences. Maybe that could be another alternative.

Jenny: Yes, lots of nuances with the different tests. That's why I think it's very helpful for you to help us understand what they are and pros and cons. No delayed clearance issue. Myeloma doesn't always leach out into the blood, or does it?

Dr. Landgren: Well, the sequences will be detectable in the blood, I would say, probably 99.9% of patients at diagnosis. I actually think it does, but there are a lot of other granular things that didn't go through. If you're trying to characterize the details of the disease, if you are not only looking for these VDJ that are like genomic fingerprints for the immune trigger, if you're looking for other things, if you're looking for particular genes and mutational signatures or copy number variations of things, then the question becomes much more complicated because you could ask the question, are the cells, all the DNA that ends up in the blood, are those representative of what's in the bone marrow? What I mean by that is, if you think about, you have millions of different cells sitting in the bone marrow, what are the ones that are ending up in the blood like? Do they look like any random sample? Or are there particular subsets that are the ones in the bone marrow that are more prone to go out in the blood? No one knows the answer to that. Because we don't know that, I think you have to be careful and say, if you profile the disease in the blood with more disease-specific tests, do you know for sure that this is representative of the bone marrow? That's a whole other question that goes way beyond MRD testing. That's more molecular profiling.

Jenny: Right. I think you'll have to see that over time, I guess, with more utility of these and more data and things like that.

Dr. Landgren: Yes.

Jenny: Now this is a next generation sequencing. You explained how that’s just like a person's personal signature. How does that differ from the flow-based, like EuroFlow or other flow-based – I know a lot of facilities have their own flow-based MRD testing. What's the difference between those, and what are some pros and cons with the flow-based MRD testing?

Dr. Landgren: The flow-based assays are very different from sequencing in the sense that they are not looking for the inside of the cells. They look for the outside of the cells. All the cells in our bodies have a nucleus, and that's the center of the cell. They have the cytoplasm, and they have their sources. In the cytoplasm, inside are a lot of functions that float around and do a lot of things. One thing that is very well-known in myeloma, which is true in every cell, but in myeloma is particularly important, for example, inside the cytoplasm, we have the proteasomes. You have a lot of other things that have functions that help the cell function and do what it's supposed to do. On the surface of every cell in the body, there are many different proteins that are attached there. We don't fully understand how all these things work, but part of the discovery that has been done for years shows that the cells could use some of these proteins to basically navigate in the body. Certain proteins would take cells to certain locations. They can use that to communicate with each other. They can trigger functions between different types of cells. It's a very fascinating and very sophisticated combination of things that these cell proteins do.

When we're talking about flow-based MRD testing, it’s basically a way to run a sample through a machine when you are looking at individual, one cell at a time, markers of these proteins. If you have spent time studying this, which a lot of people have done over the years, you know that myeloma cells do express a protein that's called CD38, which happens to be the same target for isatuximab and daratumumab, for example. They usually express CD138, but they also express a lot of other markers. There is something called CD56. They usually do not but they can express CD20. They usually do not express CD45. They may or may not express CD67, and so forth, and so forth, and so forth. If you take the sample in the person who has myeloma, and you look for these markers in one single person, what you will see is that many of these markers are present, and many of these markers are not so present, just a little bit present. You will also see that within a given person, some cells, not all of them, have certain markers and some don't. That makes it very complicated if you're trying to use a technology that looks for markers on the surface, and use that as a way to determine if there is residual disease or not, because what happens if the cell you're looking for does or does not express the particular protein you're now after?

People have then spent a lot of time thinking about it. We have worked on this also for many years. The EuroFlow, it really started in the UK, with a group in Leeds in the UK. Other groups have worked on it as well, the NCI group, the Sloan Kettering group, the Mayo group. A lot of groups have worked on this. What people have agreed to is that you need to have at least ten different markers to look for. If you only look for one marker, if that is positive, it's positive, but if it's negative, it could either be really true negative, or it could be false negative. If you have at least ten markers, if they are all negative, that is negative. Basically, a way to rule out the false negatives. If you have those ten markers, then you can reliably rule out. You also need to look through a large number of cells. You cannot just look through, say, 10,000 cells and say, “I didn't find any. There is nothing.” People have said, you need to look through a certain number of cells. It's in the range of a few million cells, between one and three million, in that type of range.

Now we're basically talking about enforcing rules for how to use technology that's basically available in literally every hospital these days. All places have flow machines. When you use flow cytometry, it’s more a matter of which markers you look for and how you do it. Because if you do the wrong markers, or you don't do enough markers, you don't do the right number of cells. If you read negative, what about false negatives? You need to really reliably be able to say that this is done the right way. That's why there's been a lot of controversy in the field. The European group in Spain patented this, and they called it EuroFlow. They call it by other names. They wanted it to sound like next generation sequencing. They call it next generation flow. All it is is ten-color flow cytometry that was standardized. Other groups call it other things. It's basically a more standardized, very sensitive way of doing flow cytometry. These days, we have the same technology as Sloan Kettering has. It’s a 12-color technology. You could have 20 or 50 colors, but what we and others for now believe is that if you add beyond the 12 markers, that is not going to be more sensitive, unless we come up with some new insights.

The pro here is that technology is available everywhere. There are agreements on how to do it. It's very fast. You can have a sample in the morning, and you have the results in the afternoon. The downside is that it takes a lot of bandwidth to do all the things I told you, and you need to really stick to the same principle over and over and over and over again. It technically may be probably ten times less sensitive than sequencing, but there are benefits with this technology also. If you really do it very, very well, and you have high volume, and you know what you're doing, you could argue that this is also a good test to have.

Jenny: This is only bone marrow-based, or is this blood-based also?

Dr. Landgren: It’s the same exact thing as I spoke about for the Adaptive clonoSEQ. You could apply it to anything. You can look into blood, but the same principle applies that there is much less disease in the blood. If you have a larger volume, you could compensate for it. You could also say I recognize that it’s less sensitive, but it's still more sensitive than SPEP and IFE.

Jenny: Okay, makes sense. Okay, we have one more test to talk about, and then I'll open it up for caller questions. It's a lot. Circulating tumor cells, can you explain what that test is? Because that's a blood-based test and we're hearing about it on the research side. I don't know if people are using it in the clinic yet. Maybe you can explain what that is. I know Menarini is one of the companies, but there might be others that we can talk about.

Dr. Landgren: Yes. As the name indicates, circulating tumor cells, it’s looking for cells that have made their way into the bloodstream, and you can look for them there. Menarini is one of the systems. They already have an FDA clearance for solid tumors, and I think they're working also to get them approved in other areas. This is a more sophisticated way of profiling cells than just looking for the VDJ. I mentioned the word fingerprint before where you can look to see if you have the clone's signature in the baseline sample. You track it over time. You can say yes-no, or you can see, number of copies. With this type of technology, we do circulating tumor cells. You can actually sequence the entire cell. When people talk about doing whole genome sequencing or single cell sequencing in the bone marrow, what people are doing is that you can run the whole code, or you can identify, characterize the whole code. If you do whole genome sequencing, you take a sample, you extract the DNA, run it through the sequences, you get the sequence. If you do single-cell, you do the same thing, but you do it for individual cells. The first approach is what people also call bulk sequencing where you get everything from a given sample, while the single cell is for individual cells. They have a lot of pluses and minuses, and we probably need to spend a few more hours talking about them, which we don't have.

Jenny: This is a very extensive topic.

Dr. Landgren: When you’re looking for tumor cells in the blood, you're looking for individual cells. As you hear me saying, this gives you much, much more information. Let's say that you can identify that there are mutations that are okay, and NRAS and BRAF mutations, or there are a lot of markers that you can identify in a given sample. You heard me saying before, when I was talking about, when you asked me, do all patients have detectable disease in the blood? I said yes, but I also said, we don't really know if the blood sample is representative of the bone marrow. A potential caveat with these technologies is that what you find in the blood, we don't know if that really captures the whole picture of what's going on in the bone marrow. That's something we don't know. Another limitation with these technologies, similar to all the other technologies, is that you have much less of the disease in the blood. If you basically do 1,000 patients with these technologies, in the bone marrow, and then you do those same 1,000 cases, in the blood; at diagnosis, you’d probably find the disease in the bone marrow and the blood in the vast majority of cases. If you treat and look for residual disease in the bone marrow, and you do it in the blood, there will be a high proportion of those cases in the blood that's going to be negative, and it will probably be false negative. The same is true if you use these technologies to track disease in the blood in patients with precursor disease, with monoclonal gammopathy or smoldering myeloma.

Dana Farber has shown in their PROMISE study, in multiple presentations, that they have up to 50% of patients that have no information in some of their presentations because the assay doesn't work, always, when you have low levels. I'm sure this is going to get better. I know they and others and we also are working on this to see how these technologies can get better and better by enriching the samples for what you want to look for in the cells. This is also technology development. I'm sure we're going to come to a point where we will be able to use these technologies, also, in the blood for the majority of patients with precursor disease.

Jenny: I guess my final question would be, how do you look at these as a researcher, but also as a clinician in your practice, using these or not using these, and where do you see that headed in the next two to three years?

Dr. Landgren: Our take, absolutely, as I said before, I have no favorites. I have no intention whatsoever to tell anyone else what to do. I can share with you what we do. I have been in Miami for about two and a half years. When I came here, my goal was, and it still is, to make sure we are one of the top three myeloma centers in the US within five years of our start. We have done about two and a half years. What we are doing this year is that we are actually implementing all these technologies that we have talked about today, both the MALDI-TOF, the monocyclic peptides, and also the circulating tumor cells. We want to make these available for all patients that come to our center. We want to use them for research purposes, and we ultimately want to use them for clinical tracking of patients. We are opening up a large laboratory as we speak, where we have all these platforms going. We are installing all these instruments right now. We will be able to track patients. What I want to do is to see how these assays perform in parallel, and see where they really are useful. The proof will always be in the pudding. If you have a lot of data, if you have a lot of data points, you can compare the sequencing from the blood with the proteins in the blood, and you can also compare it with bone marrow testing. That is our approach. I think there are other groups as well that are thinking about or doing similar work.

Jenny: I think that's the only way that we get to better utility, because you're gathering data and generating that with your patients. Again, I'm just so impressed with what you've been building. Your team is amazing. Everybody has a different and unique specialty. I think this folds in very nicely also with the genetics work that you're doing, to determine risk factors and potential prognosis based on some of these factors. I just think what you're doing is amazing.

Dr. Landgren: Thank you. You’re way too kind. It's just a lot of hard work.

Jenny: It is a lot of hard work. We're glad that you're doing it. Okay, I'd like to open it up just for a few minutes of caller questions. Go ahead with your question.

Caller: Hello. Dr. I have a question. Is there ever a case for using blood biopsies or liquid biopsies instead of bone marrow biopsy to diagnose a patient when MM is suspected? Let's say in the elderly population, people who may have osteoporotic compression fractures, limited mobility, is there ever a case for doing the liquid biopsy exclusive of the bone marrow biopsy in early stages?

Dr. Landgren: That's a very good question you're asking. The question is, could you replace the conventional work-up with the bone marrow biopsy and just do a blood-based test? I assume that you're thinking about the circulating tumor cell test I was talking about.

Caller: Yes, I heard.

Dr. Landgren: I do think, theoretically, we could already do that to date, because you can see if you have abnormal cells. You can determine if they are cloned or not. You can look for the signature and so forth. There are a lot of things that the tests currently will not be able to answer. It, for example, will not answer, how many cells are there? Is the patient we're talking about, say, carrying 15 or 20% plasma cells, or is the patient having up to 90% of plasma cells? Or any number in between. I just made these up right now. Also, there are other things that you wouldn't know in the bone marrow microenvironment. You just only know if there is disease, and you can profile the disease. You can look for, technically, all the gains and losses and translocations, fissions that genetics would do. You could also do sequencing of it. Collectively, you could characterize it, but you don't have those other numbers. I think the field is probably not ready to do this because we are so used to having access to all this information. I think that work that is ongoing and will be continued to be done, most likely would generate data that will give people more confidence that you can probably go this route. There's basically more development work that is more clinical, to prove that this could be a useful way. I think you are on to something important, but there needs to be more work before we can really replace. We're not there yet.

Caller: Thank you. Thank you very much.

Jenny: Appreciate it so much. Okay, another caller, go ahead with your question.

Caller: Yes, thank you so much, Dr. I have, actually, a two-part question regarding the VDJ and normal sequence that is found in MM patients. The question is, is this the same abnormal VDJ sequencing in the hematopoietic stem cells that are used for transplant? If yes, would that then suggest doing an allogenic transplant instead of an autologous transplant for high-risk patients?

Dr. Landgren: That's another very good question. What you're asking me is if you do a biopsy in multiple myeloma, you determine the VDJ signature of the clone, which is the sequence that has the most copies, is that the same sequence that you find in the stem cells in the same person? The answer is no. If you profile the stem cells, you do not see the same sequence. For sure, you do not see it as a peak. I say that with a little bit of a caveat because no one really knows what is the true origin of multiple myeloma. Could there be a very low number of stem cells that are still there that give rise that are not plasma cells? I guess it's theoretically possible that there could be some cells there in the background that could give rise, but if you collect stem cells from the peripheral blood, or even if you do it the old way, from the bone marrow, if you do it the new way with regular leukapheresis, you take out the cells; if you sequence them for VDJ, you will not see the same peak as you do with the myeloma cells. That would not be a justification by itself to say that the allogeneic transplant is the right way. I think usually people would say that allogeneic transplant should only be reserved for research studies because there is no convincing data that is superior to autologous. Of course, you have all the CAR-T cells coming, and other therapies as well. That's a much longer conversation, but the short answer to your question is, no, it's not there in the majority of those cells.

Caller: Thank you.

Jenny: Okay, great question. Since we're at time, I think we'll close. Dr. Landgren, thank you so much for joining us today and explaining this very complicated topic. I think we'll end up hearing more about these blood-based tests and liquid biopsies. I know patients are looking forward to them very significantly. We're just very grateful for you to join us today. Thank you so much.

Dr. Landgren: Thank you very much for having me, Jenny. It's always a pleasure working together with you. You do fantastic with your whole foundation. Thank you so much.

Jenny: Thank you. Thanks to our listeners for listening to the HealthTree Podcast for Multiple Myeloma. Join us next time to learn more about what's happening in myeloma research and what it means for you.