Activity Transcript

Shaji Kumar, MD: Welcome to this program where we will be discussing the role of cytogenetics and minimal residual disease testing in multiple myeloma. I'm Shaji Kumar. I'm a professor of hematology at Mayo Clinic in Rochester, Minnesota. Joining me today is Dr Dragan Jevremovic, who is a professor of pathology in the division of hematopathology at Mayo Clinic in Rochester, Minnesota. Clearly, a lot of things have changed in myeloma in the past decade, in particular, with significant improvements in outcomes of patients. There have been several improvements in therapy with novel agents as well as novel drug classes that have been introduced, and, more importantly, we have a better understanding of the disease biology, which has also translated into improved outcomes for these patients. However, one of the things that we have learned, especially over the past few years, is the significant heterogeneity that exists in this disease. Now we know even though we think about myeloma as one disease, there can be significant differences in terms of outcomes amongst patients with multiple myeloma based on a number of different factors that we will be talking about in a few seconds here.

And I think there are many unmet needs, particularly in terms of how we can adapt therapy for an individual patient based on the disease characteristics and understand how best we can employ the current therapies that we have in order to improve the outcomes of these patients. Equally important is to translate our improved understanding of the disease biology in order to identify new therapies for patients with multiple myeloma. As you know, the survival, even though it has improved significantly over the years, particularly in the past few years, we still see a significant divide between the patients whom we consider as having high-risk multiple myeloma and the patients with standard-risk disease. Even though both these groups have improved survival over the years and is gradually approaching the expected survival for the population, the gulf between these 2 groups still remains. So, the question that always comes up is, what drives the differences in the outcomes between the patient groups?

We know that the genetic abnormalities clearly play a major role, but there are other important prognostic factors as well that can determine the outcome of patients with multiple myeloma. And these include some of the host factors, such as the age and the performance status, the immune parameters, and so forth. But importantly, things that can change during the course of therapy, such as the depth of response and the duration of response achieved with any given therapy, can significantly impact survival outcome. Now, as we previously mentioned, myeloma, even though we think about it as a singular disease, clearly is made up of several subtypes of disorders, mostly characterized by the underlying genetic abnormalities. As you will hear from Dr Jevremovic later on, many of these abnormalities can clearly drive the outcomes, particularly the primary abnormalities that include the translocations and the trisomies. Importantly, as the disease evolves, we continue to see significant alterations from a genomic standpoint, including deletions and amplifications and monosomy as well as mutations of individual genes.

Now, these abnormalities in the genetic or the genomic abnormalities also influence the clinical presentation of these patients. For example, those patients with some of the translocations often have high levels of serum free light chains at presentation, which might lead to higher proportion of those patients presenting with renal insufficiency. So, the important question that always comes up is, how can we integrate these genomic abnormalities in order to define how a particular patient might behave in their disease course and also explore the possibility of treating these patients differently based on these underlying characteristics? One of the earliest attempts to try was the Revised International Staging System (R-ISS) that incorporated some of the high-risk translocations like 4;14 and 14;16 and 17p deletion into the staging system. But we know that there are other abnormalities, like 1q gain or amplification, which also can have significant impact on the outcomes.

As a result, there have been a second revision of the international staging system that incorporated some of the previous factors like the ISS staging system, the lactate dehydrogenase (LDH), but also added the 1q gain or amplification to that staging system. And this, along with other staging systems like the Mayo Additive Staging System, clearly reflects the understanding that these risk factors can have a cumulative impact in terms of outcomes. Now, there are other abnormalities in addition to these that we will also be talking about which can impact the outcomes, and hopefully these staging systems will continue to evolve to incorporate those chromosome abnormalities. But even beyond the chromosome abnormalities, we can have mutations involving the individual genes, like the TP53 gene or the RAS genes, which also appears to have significant impact on outcomes. And there are ongoing efforts trying to see how best we can incorporate these abnormalities into the staging or the risk stratification system.

But given that background, it is very clear as we do the initial diagnosis and risk assessment that we do a very accurate risk assessment, not only from the point of view of informing the patient of their potential disease course and outcomes, but also increasingly informing us as to what kind of therapy or therapeutic approaches we might employ for a given patient. Now we clearly use the mSMART (Stratification for Myeloma and Risk-Adapted Therapy) classification for patients with newly diagnosed multiple myeloma in order to assess their risk status, and Dr Jevremovic has been closely involved with the development of some of these risk stratification systems and it clearly depends significantly on the findings on the FISH testing that we do for all these patients, but increasingly also has started incorporating some of those mutation abnormalities.

And also, there are other factors like the plasma cell proliferation that also plays an important role. Dr Jevremovic, maybe we'll go into some of the details in terms of the practical aspects of testing patients, particularly the FISH testing and the mutational profiling, and others. Very, very important testing that you do in the lab to inform the clinicians about the risk status of a given patient.

Dragan Jevremovic, MD: Sure. Thank you Dr Kumar. When we talk about the pathologic assessment of patients with the plasma cell disorders, what clinicians usually want from us pathologists is a firm diagnosis, is a finding prognostic factor that will be relevant, and potentially even therapeutic guidance. What we have to deal with is really our tissues, blood and bone marrow, and the tools that we have at our disposal are morphology, immunophenotyping, whether it be flow or immunohistochemistry, and genetics with many different types of technologies being applied.

Prior to going into details about these types of testing, I want to emphasize one thing -- that when we do bone marrow sampling, we do consecutive draws of the bone marrow aspirate and we do a wide needle core biopsy. And so, this bone marrow aspirate is being drawn into different tubes, consecutive tubes. The first tube is usually done for morphology, the second tube for flow cytometry and/or MRD testing, the third tube for cytogenetics and FISH, and the fourth tube usually for the molecular test. One thing to keep in mind is with every draw, every consecutive draw, you get more and more hemodilution. And so, that will always impact the quality of the sample that we have.

Why do we have this type of draw? Well, morphology is still the key for many diseases, including for enumeration of plasma cells. But if you envision other types of disorders such as acute leukemia, you would really want to have the morphology being assessed from the first tube because you need to assess the percentage of blasts and you never know what you're going to get when you insert a needle into the patient.

Dr Kumar: A couple of questions now. You talked about the sequencing of samples, right? The first one going to morphology. Are there things that people really need to know out in the community or any guidance in terms of how do we direct the samples? How the quality of samples could play a major role?

Dr Jevremovic: Yes. First of all, reasonable advice is always to reposition the needle when obtaining the sample, and our nurses are well trained to do that so that we minimize hemodilution, but you're always going to get more and more hemodilution. The other thing is, you can look at some parameters by flow cytometry. You can look at some parameters such as percentage of mast cells and percentage of immature B-cell precursors or hematogones, and these could be some guidance as to whether the sample is hemodiluted or not. It has been shown that samples that have less than 0.002% of mast cells are likely to be hemodiluted. In terms of the hematogones, proposed cutoff was 0.05% of CD27-negative hematogones. But this has also been challenged because this will also depend on the type of therapy and the age of the patient. These are not easy assessments to make, but definitely something to keep in mind, that especially when you get MRD-negative results, you should pay attention to how good the sample was.

When you talk about genetics of myeloma, we have to acknowledge that it is extremely complex and as technology is advancing, it appears more and more complex. I like to think of genetics of myeloma from 2 angles, 2 separate angles. One is practical clinical implications, and at this point it is still mostly FISH probes, doing FISH on the plasma cells from myeloma patients. And then you have a pathogenesis aspect, which is at this point, with the newer technology, really restricted to the research and clinical trials.

When you talk about FISH, I always said it is a mainstay, and Dr Kumar said it's a mainstay of prognosis. For the most part, everybody at this point is doing a FISH on sorted cells. You need to enrich the bone marrow aspirate for the plasma cells. We switched from our cytoplasmic immunoglobulin staining of the cells to identify plasma cells and enrich our scoring. We switched a few years ago to the flow sorting and we now do interphase FISH, and that switch few years ago resulted in an increased number of detected positive cases. Our initial diagnostic setup usually includes break-apart IGH probe as well as break apart MYC probe and this other minus 13, plus 1q, and minus 17p. And then, after this setup, what we perform is analysis and then we first check these 5 FISH probes, and based on these results we will continue for diagnostic reflexes.

All of this is in order to save time and the resources needed because the FISH is expensive and a labor-intensive procedure. Importantly, for every follow-up patient, we only do the progression probes minus 17p, plus 1q, and break apart MYC. And so, we only perform FISH if we have enough cells, which we show by flow cytometry. If there is less than 0.1% of plasma cells by flow, we do not perform FISH because it’ll be a waste of resources. We sort the cells. We do not set up hyperdiploidy probes if our flow has resulted ploidy, and I will talk about that in a few slides because we switch from detecting hyperdiploidy by FISH to detect hyperdiploidy by flow cytometry. As I mentioned, we start with limited panel and do reflex based on the results. And for recurrent patient, we only do progression probes.

Our flow cytometry is really a key to orienting us into the diagnostic testing for myeloma. We have this initial screening tube, which we called plasma cell proliferation assay, or PCPRO. And that tube contains the antibodies to detect plasma cells to detect their clonality with kappa and lambda and aberrant expression of antigens. But also, we use this to triage for potential MRD testing, and as I mentioned for plasma cell testing.

The other things that we get from this tube is a proliferation index or S phase of plasma cells, which is an important prognostic factor as well as ploidy, I mentioned before. We gate on CD138 and CD38 bright cells and look for loss of CD19 and CD45, and then we can detect the normal plasma cells which are polyclonal, and the clonal plasma cells. And using the 4′,6-diamidino-2-phenylindole (DAPI) stain, we can detect the quantity of DNA on this plasma cell population and we can establish the S phase. And S phase, as I mentioned, is an important prognostic factor as well as ploidy. We have shown previously that ploidy by flow cytometry could be a reasonable substitute for ploidy by FISH in terms of the overall and progression-free survival.

Dr Kumar: Now, when you do have limited samples, is there any kind of prioritization that you often do for the FISH probes? For example, you may do some preferentially over the others.

Dr Jevremovic: Yes, that's for the FISH probes, probably break apart for IGH will be the first one to set up, and deletion 17p. Again, for the most part these are done as a panel. And so if you have some knowledge -- for example, overexpression of cyclin D1 by immunohistochemistry -- you might be able to guide FISH probes into that direction.

Dr Kumar: Right. Now what if we are not unable to do the FISH on the fresh sample? Is there any way to go back and do that on the bone marrow biopsy specimens?

Dr Jevremovic: Not on the bone marrow biopsy. Bone marrow biopsy is decalcified, and the FISH is most likely going to fail in these specimens. But if you have a bone marrow clot section, then the FISH could be done on that specimen. Now remember, for that to be of clinical utility you would probably want to have more than 10% plasma cells in that specimen, because in that case you are not sorting, which means that you are kind of decreasing your sensitivity. And a lot of these FISH probes have a cutoff of 5% to 10% sensitivity. So, if you have less than 10% of plasma cells, they're likely going to fail.

Dr Kumar: Right. Now, in the patients with relapsed multiple myeloma, I'm sure the samples come back in often for doing repeat FISH testing. Do we really need to test everything, or is there a limited panel that we can go after for patients with relapsed disease?

Dr Jevremovic: Yes, our follow-up panel includes only 3 probes: deletion 17p, gain of 1q, and break apart MYC. Those are the only 3 probes because these primary genetic changes, they do not change during therapy. These stay the same. So hyperdiploid stays hyperdiploid and 4;14 stays 4;14 myeloma.

Dr Kumar: I guess it's also important to just highlight the fact that we cannot really use the FISH testing as a disease response assessment test. We'll be talking about the MRD soon, but here we are looking at a small number of cells. So the sensitivity for detecting very low numbers of cells carrying the FISH abnormality is going to be pretty low.

Dr Jevremovic: Yeah, absolutely. You should not use FISH testing as a substitute for MRD because it is not meant for that. So if you don't get it by FISH, it doesn't mean that there is no disease present there.

Dr Kumar: Now, in terms of the plasma cell enrichment, you talked about the flow sorting. Any advantages or disadvantages of flow sorting vs a magnetic bead suppression?

Dr Jevremovic: I think most institutions use magnetic beads. Flow sorting does have an advantage of being able to hone in on the aberrant plasma cells which are truly clonal, while avoiding the polyclonal background. You also get a higher purity of the sample, up to 99% vs 90% with the beads. There are certain advantages but there is also an increased cost. So, I think both are acceptable.

Dr Kumar: Yeah that's great. You talked about how the morphology might be indicative of the underlying cytogenetic abnormalities. Maybe can you expand on that a little bit?

Dr Jevremovic: Well, there is this particular myeloma with translocation of 11;14 or cyclin D1 IGH gene translocation. And so, this cytogenetic abnormality is the most common individual abnormality in myeloma in about 15% to 20% of newly diagnosed myelomas. What is interesting is that these cells are often small and have more of a lymphoplasmacytic morphology, less like mature plasma cells. Here in this slide, in A, you see that these are plasma cells from a myeloma. In B, you see plasma cells from lymphoma with plasmacytic differentiation.

And then in C here, this is the myeloma with the translocation 11;14 at the bottom, and you see how these cells actually look more like lymphoma than myeloma cells. In addition, they can be CD20 positive and have a weak PAX 5 positive. And it can be difficult to distinguish these from a lymphoma. So, sometimes we need to really do immunophenotyping to be sure what we are dealing with. Luckily for us from the diagnostic perspective, these plasma cells, they are truly plasma cells. They do express CD138 and very bright CD38, they lose expression of CD19 and CD45. They're still negative, and so they're like true myeloma cells. They do not phenotype like a lymphoma, but morphologically it can be challenging.

Dr Kumar: That's great. Now obviously the FISH testing is important, but increasingly we know that the myelomas can be genomically very complex with a lot of mutations and particularly new mutations that can appear with clonal evolution. And obviously there's advanced technology that can be used, but how commonly are we using mutation profiling in the clinic and how easy is it to get that done?

Dr Jevremovic: As we mentioned before, the genetics of myeloma is super complex, probably like genetics of many other cancers. And if we use the tools that really show us at the nucleotide-level changes, we see that the genetics is super, super complex. And so, this mutation analysis done by, for example, whole genome sequencing can detect so many different single variants as well as the insertions and deletions and could be used for prognosis. But for the most part we do not use that in a clinical practice. What we use and most other people use are small next-generation sequencing panels with a limited number of genes. And the reason for that is multiple: it is cost, it is the time, the effort that is needed to analyze whole genome. And so these small panels do give us some insight into the pathways which are mutated mitogen-activated protein (MAP) kinase pathway, nuclear factor-kappa B (NFκB) pathway. And so these are what we use in a general clinical practice.

Dr Kumar: That's great. And obviously the important question that comes up in addition to prognosis, how are we going to use this information to treat the patient in front of us? And I think the concept of precision medicine or individualized medicine has been around for a long time, particularly in the setting of cancers, and it's slowly making its way into myeloma as well. And one good example of that is, of course, the translocation 11;14 that we use that we talked about, where we clearly have known for some time these patients can... the finding can have a prognostic value with an outcome that is kind of in between standard risk and high risk. But more importantly, what we have learned over the past few years is the preferential activity of a drug called venetoclax when used in patients with myeloma with a translocation 11;14. What we know from a molecular standpoint is that patients with translocation 11;14 tend to be high expressors of BCL-2 in their myeloma cells, and making them uniquely sensitive to the BCL-2 inhibitor venetoclax.

When you combined with dexamethasone or with bortezomib we clearly see a lot more synergy as well. And this does translate into significant response rates in patients with relapsed multiple myeloma. Now based on those initial phase 1 and phase 2 studies, there was a large phase 3 trial that was done that had looked at the impact of adding venetoclax to bortezomib and dexamethasone, which demonstrated an improved progression-free survival in patients with relapsed myeloma. But there was a negative impact on the overall survival with the addition of venetoclax. Subsequent follow-up and more detailed studies clearly demonstrates that the patients who benefit the most are those people who have a translocation 11;14 abnormality. And in those people, there does not seem to be any negative impact so far.

Now, there are other ways or the other findings that can also guide our therapy. For example, the 4;14 translocation patients, which is considered as a poor prognostic marker. These patients are often treated with a proteasome inhibitor-containing combination, often given an early autologous stem cell transplant, and often receive 2-drug maintenance therapy. But also there are some exciting potential therapies around the corner that are going through clinical trials which can inhibit the MMSET, which again is a driver for the abnormalities that we see in patients with the 4;14 translocation. So these are currently in clinical trials, and hopefully these kind of individualist therapies will increase the importance of getting the FISH testing and how we act upon those results.

Now similarly, there are other studies that are ongoing looking at specifically at patients with 17p deletion. And there are clinical trials like the myeloma MyDRUG trial, that is a purely genomic-driven clinical trial that is sequencing all patients at the time of relapse. And these are focused on patients with functional high-risk myeloma, which are patients relapsing very early after an initial therapy. And then based on the underlying abnormality, these patients get different kinds of therapy which adds a targeted agent to the standard-of-care myeloma backbone therapy. So, clearly, Dragan, a lot of the things that you're doing in the lab trying to prognosticate and identify these patients are going to have increasingly important role in terms of how we will treat these patients.

Dr Jevremovic: Yes, I agree. And soon I would imagine that you guys will stop using us pathologists and just use the molecular profiling and without people.

Dr Kumar: I doubt that's going to happen because we do need the complete picture, and the morphology, as you already said, plays an important role as well. So, maybe switching gears a little bit, I think the second important factor that drives outcomes and potentially can drive therapeutic decisions is the minimal residual disease status of the therapy. Now the MRD negativity is not a new concept in myeloma. It's been around for a while, as with other hematologic malignancies, and there are different methodologies that can be employed in order to detect minimal residual disease after therapy, as you will hear in a couple minutes.

But what we know in myeloma is that the deeper the response to therapy, the longer it'll take for the disease to bounce back. And that may seem very intuitive. And what we really want to see is whether these therapies can take the disease to such a low level that the other mechanisms are able to hold the tumor down. Now, there have been multiple meta-analyses that have been done that have clearly demonstrated that the MRD negativity is associated with a better progression-free survival as well as better overall survival. Now, clearly there are many different ways that you can approach or detect MRD negativity or establish it and I will maybe hand this over to you, Dragan, to see if we can just go over some of those methods and the pros and cons.

Dr Jevremovic: Yes, thank you. The 2 main methods that have surfaced are really next-generation flow cytometry and the next-generation sequencing. The allele-specific quantitative polymerase chain reaction (qPCR) has kind of fallen out of favor in the community. So the VDJ (variability, diversity, and joining) sequencing and the flow cytometry are pretty much what everybody's doing. They each have their pluses and minuses. You can probably go somewhat deeper in the response with the next-generation sequencing. At the same time, you need to know exactly what you're looking for. So you have to have the original sample. Flow cytometry, you can go pretty deep, close to 10^-6. And you do not need the original sample to know that this is MRD positive. On the other hand, you do need fresh specimens, so it has to be done within 24 to 72 hours. So, there are pluses and minuses and people choose which method they prefer. We have shown that in our hands we can do very close to 10^-6 in a direct comparison with the next-generation sequencing (NGS). So our flow is pretty much as good as NGS.

Dr Kumar: Yeah. The big question that's going to come up is, how are we going to use this information to change therapy? Right now, it's very clear that this is prognostically very useful, particularly in patients with high-risk disease. And I think the clinical trials that are ongoing are looking at utilizing the MRD status to either increase the intensity of therapy or decrease it based on whether they're positive or negative and also determine what is the ideal duration of therapy. Because at the end of the day, unless we are able to change the treatment and improve the outcome of a patient, the value of MRD negativity remains limited as a prognostic tool. There are multiple different clinical trials that are ongoing right now. We have heard the results of some of these trials, like the MASTER trial, which looked at the MRD guided deescalation.

There's a lenalidomide maintenance trial called DRAMMATIC that is looking at deciding when to discontinue maintenance based on the MRD status. So, clearly all this technology has made the myeloma treatment paradigm quite different, and it's very interesting to hear from Dr Jevremovic about all the advances that have been made from a laboratory testing standpoint, which all have very important implications beyond just having new therapies. I think it's fair to conclude that the identification of genetic abnormalities will remain a cornerstone of prognostication and increasingly therapeutic decision-making in patients with multiple myeloma.

I think we are going to see more and more of the individualized therapies going forward for patients with multiple myeloma, both in the newly diagnosed and relapsed setting. And all the technology associated with the detection of MRD negativity will continue to improve, I am certain. And with the prospective clinical trials providing evidence for a role for that in treatment decision-making, this will become a centerpiece of the myeloma treatment paradigm. And I'm sure that we are going to see a lot more advances in the testing field as well. And Dr Jevremovic, what do you think the future looks like for laboratory testing for multiple myeloma?

Dr Jevremovic: Well, I think we are going to be going deeper and deeper in terms of sensitivity. I think that is going to be more and more individualized. So a combination of mutation profile with the FISH abnormalities. And then finally, I think circulating plasma cells will also be a key important factor in future for detecting myeloma cells and possibly even doing the genetic analysis on them.

Dr Kumar: Wonderful. Thank you, Dr Jevremovic, for joining today. And I would like to thank the audience for listening in.

This transcript has not been copyedited.