Learn about all myeloma happenings on the new Myeloma Crowd site: the first comprehensive site for myeloma patients and caregivers. Dr. Walter Michael Kuehl, MD, PhD Center for Cancer Research National Cancer Institute Interview date: April 25, 2014

 Dr. Kuehl, in partnership with Dr. Bergsagel, discovered that myeloma is not a single disease. This significant discovery has led to great insights about what triggers myeloma growth, how each type of myeloma acts differently and the implications this has for future treatment approaches. Dr. Kuehl found 7 translocation types divided into three groups. In this interview, he shares how that discovery was made and what "primary" and "secondary" translocations mean. He describes how myeloma progresses in three steps 1)The appearance of MGUS 2)The transition from MGUS to active myeloma and 3)Myeloma proliferation. He shares the fact that when MGUS is compared with active myeloma, there is 3x more MYC present, suggesting that MYC could trigger MGUS to move to an active myeloma stage. He describes how MYC may not cause the cell growth, but give myeloma the autonomy to grow. Many of these translocation groups have overexpressed Cyclin D, but he describes how Cyclin D may make the cells more sensitive but on it's own is not enough to spur myeloma growth. He describes how to test for your myeloma's biology and stresses that the more patients know, the better care they will receive. He also tells us that patients can contribute to the treatment of their disease and help stimulate newer approaches with their unique perspective. He shares the opinion that the day of immunotherapies has arrived and advocates going to specialists who can provide the very best in myeloma care - and that means getting a second opinion when needed. We owe a debt of gratitude for his tireless efforts that have offered great insights into this complex disease.  The live mPatient Myeloma Radio podcast with Dr. W. Michael Kuehl

Jenny: Welcome to today's episode of mPatient Myeloma Radio, a show that connects patients with myeloma researchers. We started this series to help you learn more about the latest research in myeloma so you can make your very best treatment decisions. We strongly encourage you to consider clinical trials as you think about treatment options. When we started this series, less than 5% of patients participated, and if we doubled that number, we could accelerate the pace that researchers can find cures. If you'd like to receive a weekly email about past and upcoming interviews, you can subscribe to our mPatient Minute newsletter on the homepage or follow us there on Facebook or Twitter, and we encourage you to share these interviews with your myeloma friends. We also have a new site called myelomacrowd.org that's the first all-inclusive and comprehensive site for myeloma. So please take some time to explore the links. There are listings for all relevant and very credible myeloma news sources, lists of who to follow on social media, a growing online myeloma specialist directory, current articles on relevant myeloma topics, and links to all the good being done by the various organizations and foundations in myeloma. You can also contribute what you are learning by clicking on the "Become a Contributor" link. Now, we are very honored to have with us today Dr. Walter Michael Kuehl of the National  Cancer Institute. So welcome!

Dr. Kuehl: Thank you.

Jenny: Let me give a little introduction for you before we get started. Dr. Kuehl is the Chief of Molecular Pathogenesis of the Myeloma Section in the Center for Cancer Research at the National Cancer Institute. Dr. Kuehl obtained a Harvard degree in Biomedical Sciences and a medical degree at Harvard Medical School. He performed his residency in Internal Medicine at Case Western Reserve and completed post-doctoral fellowships at the NIH and Albert Einstein College of Medicine. He joined the Department of Microbiology at the University of Virginia Medical School and attained the rank of professor before joining the NCI. As head of this group and Senior Investigator, his research has laid foundational work to identify myeloma not as a single disease but a disease of varying characteristics, or what you might think of as myeloma subtypes. With this, he also identified specific translocations and genes that drive the growth of myeloma, identified that MGUS almost always precedes myeloma, and identified specific pathways that are uniquely affected by the various types of myeloma. He's written over 150 publications, is the recipient of MMRF grants, and received the Lifetime Achievement Waldenström's Award from the International Myeloma Foundation. So Dr. Kuehl, thank you so much for your detailed work in the biology of myeloma.

Dr. Kuehl: Well, it's a pleasure to be here.

Jenny: Yes. Maybe you can explain what the Center for Cancer Research does, and give us a short overview of your focus.

Dr. Kuehl: The NCI, the National Cancer Institute, has been reorganized numerous times over the 25-year period that I've been here. Most recently, about five or six years ago -- I don't remember the exact date -- they decided to reorganize it in a new way. So it broke down into two main divisions. There's a Center for Cancer Research and then there's the Division of Cancer Epidemiology and Genetics which is focused on who gets the disease, what are the population characteristics, what are genetic factors that might predispose to the disease, and so on. That's a very small entity. The Center for Cancer Research is the much larger entity and that's divided into a clinical and a basic science part. So the clinical would do translational research. They would do some clinical trials and it would be focused on that. The basic science would focus on fundamental mechanisms not only in cancer cells but in normal cells and even in lower organisms so that we could better understand the biology of what make cells grow and survive and so on. In any case -- so that's enough about that. So I'm in the clinical part of the Center for Cancer Research, and I'm in the Genetics Branch, which is focused on cancer genetics. I want to start off by just saying that the way I started to work on myeloma is -- it turns out to be somewhat interesting. A number of years ago, one of the fellows in my lab, Leif Bergsagel, who recently was on your show, we were sitting at lunch one day and he says to me, "Why don't you study myeloma?" Leif's father was one of the absolute giants in designing some of the first treatments for myeloma. I said, "You know, the problem is it's hard to get adequate material." I said, "If we could get cell lines, then I think I would definitely start to study human myeloma." I was studying mouse tumors mostly at that point, and he being a very inventive person, he went to the computer, which I hardly even knew how to turn on a computer then, and he came up with fifty different myeloma cell lines that had been isolated but no one has studied. So as soon as he said that I said, "All right, I'm going to stop everything in my lab and we're going to start working on myeloma," which is what we did. We sat down and we said, "What's important in myeloma? What might be important in trying to understand it?" We decided based on other kinds of tumors there were two things that we want to focus on. One was translocations; chromosomal translocations that involved immunoglobulin loci, and two was MYC, because both of those had been important in mouse plasmacytomas and other kinds of tumors. So that's what my work has focused on is using that as a springboard, using the cell lines initially but always realizing the cell lines do not represent exactly what you see in the tumor. They're actually an advanced form of myeloma. They're sort of the terminal stages of myeloma. Those are the tumors that go to extramedullary sites or plasma cell leukemia, and those are what give you cell lines. So they're very advanced, but most all of the early events including the primary translocations -- and abnormalities of MYC which is a later event, usually -- are immortalized in those cell lines. So it's like an archaeological dig essentially to see what's there, and then take that information and go to look at the tumors at different stages, including premalignant MGUS even. So that's what we've done. So we started everything in the cell lines, and interestingly, for a long time there were people who were -- even though cell lines had been used for many years by many people, there are skeptics out there who always would say, "Oh, the cell lines don't mean anything. You have to study the real tumor." But the reality was that what we found in the cell lines, we've never found any major pathogenic change in a cell line that we didn't verify in a tumor. Now, that being said, cell lines do not represent the full spectrum of the disease, and there are certain types of myeloma like hyperdiploid -- which we can talk more about that later -- seem to be underrepresented among the cell lines, and the primary translocations which we started working on initially are overrepresented. I can explain reasons for why that is, but the bottom line is that the cell lines are not a perfect mimic of the tumors certainly at the various earliest stages of the disease but they give us a window into trying to understand how we got there. So that being said, our initial focus was then to focus on IgH translocations, so these are on chromosome 14, and the IgH locus has very strong regulatory elements that increase the readout of genes in the immunoglobulin locus, and what a translocation does is it moves other genes, oncogenesin this case, closer to the immunoglobulin locus on chromosome 14 so that those regulatory elements which cause high levels of RNA expression now are imposed on other genes that are brought in. A translocation, of course, is a rearrangement between two different chromosomes which bring a particular gene region on a different chromosome into the immunoglobulin domain so that it can be regulated by the immunoglobulin regulatory elements, and then you get very high levels of expression that don't change during those cell cycles. They're relatively constant in very high levels. In the process of doing that, what we determined was that we found that about -- as it turned out, about 40% of myeloma tumors have a primary IgH translocation. Now, what do I mean by "primary"? Primary it doesn't necessarily mean is the initiating event although they might well be, but what it means is that they're translocations that are commonly found both in myeloma and in premalignant MGUS. So we called those "primary" even though we can't prove that they're the first event, but they're the first event that has been identified by anyone. So there were seven translocations which are in three groups. There's a cyclin D group, the three cyclin D genes D1, D2, D3. Then there's a MAF group and there's three MAF genes; there's MAF, there's MAFB, and there's MAFA. Then finally, there's a group called the MMSET FGFR3 group, and so that's the other group. The cyclin D1 is by far the most common and the MMSET FGFR3 is second most common. The MAF and MAFB are less common and the others are relatively infrequent in the range of 1% or 2% of tumors. So one of the other things is that we got interested also in other kinds of rearrangements and there are what we call secondary rearrangements. Well, all that means is from our point of view is, number one, they tend to be present in some myeloma tumors but not in the premalignant MGUS; number two, they sometimes -- if you're lucky enough to have samples from a patient that you can observe over a period of time that you can show that those rearrangements are acquired with time, and one of the best examples of that is the MYC oncogene which we have actually and others have observed that those rearrangements come up during the progression of the tumor. But they may also come up earlier, which we can talk more about that. So at least, in a nutshell -- and the other thing that I should just point out is that primary translocations generally are very simple exchanges between two different chromosomes. They are thought to be mediated primarily by errors in normal mechanisms in B cells, in germinal center B cells or earlier cells possibly that undergo three specific DNA modifications.  So one is called VDJ recombination; that's what makes the antigen receptor part of the heavy chain or light chain in the immunoglobulin molecule. Another is somatic hypermutation which is somatically hypermutating the antigen-binding site in the antibody. That generally occurs mainly in the germinal center. The third is switch recombination, IgA switch recombination, which is involved in changing from one isotype of immunoglobulin like IgM to make other isotypes like, for instance, IgG or IgA. These three events all involve rearrangements or modification of DNA that create double-stranded breaks on the DNA and that then predisposes to translocations. So most primary translocations appear to be mediated by one of those processes. Most of those are thought to occur mainly in the germinal center, so this is consistent with the idea that a myeloma is a tumor whose normal equivalent is a long-lived plasma cell that has undergone modifications by those three processes, and it happens mainly in the germinal centers. So the thought is -- that's why we think that that is certainly a very early if not the initiating events of the tumor.

Jenny: Do you have any thoughts about what causes those primary translocations?

Dr. Kuehl: Well, not really. I think that there are errors that are rare, but what can happen is those three mechanisms can spill over into genes other than immunoglobulin locus. So they're not perfect. So two things happen. One is when you create a break, if something disrupts the rapid repair of it or the rapidly -- the normal process that occurs is hanging around and then it can pick up some other piece of a break in another piece of DNA. Secondly, those mechanisms can spill over and sometimes affect not just the immunoglobulin genes but other genes. All of these are rare events. These are rare. So in other words, most of the time things work out smoothly, but occasionally, errors are made. What we think is that the primary translocations are mediated by errors in these B cell-specific mechanisms. By contrast, I should just add that secondary translocations occur after -- beyond the germinal center when you already probably have an MGUS tumor or beyond. Those mechanisms are not on there, and lo and behold, when you look at the rearrangements there, number one, they're very complicated, we don't fully understand that, but more importantly, they don't have the hallmarks that you would expect for these B cell-specific DNA modification mechanisms. Does that make sense?

Jenny: Yeah, it does. And is one worse than another? It's worse to have these as primary translocations or it's worse to have them as secondary?

Dr. Kuehl: Well, I don't think "worse" would be the word I would use. What I would say is that the primary translocation is what gets you started and they're found in MGUS, and as you know, MGUS is a pretty benign disease. In fact, I think even though it's a very common disease and people think about treating it, at this point people aren't treating MGUS except MGUS cases which have a high likelihood of progressing which isn't so easy, but that's something that we could talk about or you could talk about in another time. But I think what's important to realize is that if you were a 55-year-old person who had MGUS, your life expectancy is no different than if you didn't have MGUS, or it's marginally different. It's hardly noticeably different. So MGUS generally is a very benign condition. So these translocations give you that first step, and then it's the secondary events that lead you on to myeloma, one of which is -- what we think is that second event that causes you into this -- so I divide myeloma into three transitions.    The first transition is what gives you MGUS, so primary translocations or hyperdiploidy, which we can talk more about that. So that's the first transition, to get from a normal cell or pre-MGUS cell to a premalignant MGUS cell. The second transition is to go from MGUS to myeloma. Now, unfortunately that's a clinical definition, and so it's very hard to say if you had a patient with a tumor and there were no secondary manifestations like anemia or bone disease, et cetera, if you handed me those cells or any one of those cells, they would be hard-pressed to say whether that cell was myeloma or MGUS. So it's a clinical definition which means that unfortunately I can't say this cell is MGUS or this cell is myeloma most of the time. I mean, if it's an advanced myeloma, maybe I could. But in any case, what we know is that as you go from MGUS to myeloma, the myeloma tumors express about a threefold higher level of MYC on average than tumors that are MGUS. Similarly, we assume that KRAS mutations are present in myeloma but we have never found them in MGUS. So the thought is that the progression, that second transition from MGUS to myeloma, it could be mediated and requires the upregulation of MYC -- so MYC is more MYC RNA -- and that KRAS mutations could also be involved in that transition, although there's not a cause-and-effect that we can prove particularly for the KRAS. Now, there is an animal model for MYC which suggests that an MGUS-prone mouse, if you upregulate MYC in it -- which Lief I'm sure talked about when he was on your program, it's a model they developed -- that the animals now get myeloma. Before, they just get MGUS, but when you upregulate the MYC, they get myeloma. That's consistent with what we see in the human MGUS and myeloma cells, namely that MYC is upregulated. Let me just mention the third transition -- there's a lot that goes on in myeloma and many other molecular events that you can ask me about, but my third transition is the transition of going from a relatively quiescent state to a proliferative state, proliferative meaning that the cells are dividing. Myeloma and MGUS basically are cells that mostly are just sitting there. They're not in cell cycle, they're not replicating, even though to get where they are, they have to have had a lot of cycles replicating, but at a point in time, very few of the cells are in cell cycle and are synthesizing DNA or dividing. But late in the disease, you develop extramedullary myeloma or plasma cell leukemia. Typically, that's associated with an increase in proliferation. That is mediated by a variety of changes, one of which we've determined is disruption of the RB pathway beyond cyclin D dysregulation. We can talk more about that. P53 mutations also tend to happen more at that later stage of disease. So in any case, I think of that as an important transition because it's going from a relatively non-proliferative state to a now very aggressive state and more likely to create more problems for the patient. Does that make sense?

Jenny: Yes, it does, and I've read about growth factors and maybe they have something to do with that, the FGFR3 --

Dr. Kuehl: Yes. Well, growth factors -- I think one of the most important things to remember about normal plasma cells -- but it's also true for MGUS and for myeloma particularly at the earliest stages -- they're absolutely dependent on the bone marrow microenvironment. So all the cells that are in the bone marrow are cooperating with the tumor to sustain the tumor. The thought is that normally there's a niche in the bone marrow for plasma cells and certain other kinds of hematopoietic cells. The normal plasma cells are only about 0.5% to 1% of the cells in the bone marrow. An MGUS tumor can be up to 10% and a myeloma can go 30, 40, 50%, even 70% or 80%, at which time you start to see major problems with interfering with blood formation. The thought is -- but until very late in the disease, typically, the tumor is confined to the bone marrow. It's dependent on that niche. But the niche is expanded. It's much larger than it would be for a normal plasma cell. We don't really understand how that happens, but the thought is that to some extent, the tumor can become less dependent on the bone marrow even though it still has some dependence, so it can't survive very well outside of the bone marrow. There's also this idea, again, not fully proven, but there's certainly supportive evidence that the tumor reprograms the bone marrow and makes it more inviting and more effective to support the tumor. In fact, while it does it in some cases you see, even in MGUS, for example, you'll see an increase in normal plasma cells. Presumably, when it changes the niche, the tumor, it also can increase the number of normal plasma cells, although ultimately, if the tumor progresses, it crowds out the normal plasma cells which creates one of your problems with immunity in patients. So factors are very important. Now, you mentioned FGFR3. So there sort of has been a controversy a little bit about -- I mean, that was one of the first translocations that we identified and it actually is an unusual translocation in that it goes into a switch region, an IgA switch region, and it divides the regulatory elements onto the two chromosome derivatives. So one of those regulatory elements dysregulates FGFR3 which goes -- ectopically expresses very high. The other dysregulates MMSET, which is a chromatin-modifying gene. But it's clear that the MMSET is the more important of the two in the sense that every cell line and tumor that has the t(4;14) translocation, you always retain the chromosome that is dysregulated for MMSET. But in about 20% of tumors, you lose the chromosome that dysregulates FGFR3. So you might say, "Well, FGFR3 isn't important." I think that's wrong. I think it is important, but I think that it's important only in the initial stages of the disease, and other things can take its place later on. A RAS mutation, for instance, can eliminate the need for the FGFR3 and other kinds of things. There's definitely evidence again to support this idea, and I think most people would probably agree that the dysregulation of both genes is critical in the initiation of the disease. Does that make sense?

Jenny: Yes, it does. Just overall a general comment, I think the discovery that there are these different mutations, different translocations, that you've subdivided them is really spectacular and stunning, and I would think -- from your perspective, what does it change in terms of your research so far?

Dr. Kuehl: Well, I think what it does is it I think it really demonstrated to me that myeloma really is different than molecular diseases. So there's a type of myeloma that has a cyclin D translocation and there's another one that has a MAF translocation. There's another one that has the MMSET FGFR3 translocation. Then there's another group that's called "hyperdiploid," and so what is that? Well, we don't understand that one very well, but what it is is it has extra copies of some combination of eight odd-numbered chromosomes. So you have extra copies, so three copies instead of two, typically. Sometimes you can have four copies instead of two. Again, what does that mean? We don't really know what that means but it's an absolute invariant finding. It's a class of tumors that has that, and we call those "hyperdiploid." They tend to have a better prognosis. The thought is that they are possibly more stromal cell-dependent. They are underrepresented in extramedullary tumor or plasma cell leukemia. They're underrepresented in cell lines and so they're a little bit mysterious. But one of the things that we -- one of the unifying theories that we came up with which we think is right is that there's a virtually universal dysregulation of a cyclin D gene either whether you have a translocation or you're hyperdiploid, or the occasional tumor that's not hyperdiploid and doesn't have a translocation. So most translocations are in the non-hyperdiploid tumors, most of the primary translocations, and the hyperdiploid tumors tend to not have primary translocations. But what's common is that in all tumors, you dysregulate cyclin D1, D2 or D3.

Jenny: Can you explain what those do and what they are?

Dr. Kuehl: Okay. Cyclin D is critical in regulating the proliferation of cells so that if you upregulate cyclin D, it can interact and activate the so-called retinoblastoma pathway (RB pathway). So the idea is -- for a long time people thought that all you have to do is upregulate cyclin D by -- it could be by a direct translocation, the MAF translocation -- by the way, MAF is a transcription factor for cyclin D2 so that's how that gets upregulated. In the hyperdiploid, they ectopically make biallelic -- that means both alleles, so not through a translocation or some obvious mutation in the gene itself -- upregulate cyclin D1. So virtually all -- like 95% plus of tumors have upregulated cyclin D1 or D2 and, rarely, cyclin D3. Even though normal plasma cells make cyclin D2 and D3, the cyclins that are dysregulated in a myeloma are made at a higher level than the normal plasma cells. Now, upregulating cyclin D is not sufficient though to drive the cell to rapidly proliferate because there are still checks on that. There are genes that can inhibit that interaction of cyclin D with -- I'm not going to get into detail, but they basically interfere with its action. So the thought is that, as I said before, myeloma cells and MGUS cells are relatively non-proliferative. The cyclin D doesn't seem to be pushing them into proliferation. On the other hand, I think what the upregulation of a cyclin D does is it makes the cells more susceptible to external signals and internal signals that periodically stimulate it to go into proliferation. So even though most of the cells aren't proliferating, I think that inexorably there's a slow amount of proliferation to the cells that occasionally enter the cell cycle through mechanism we don't really understand, but presumably through various extracellular factors that are stimulating the cells, and because they are making more of one of the cyclin D genes, they're more susceptible to entering into the replication pathway, making DNA and going on and dividing. When I talked about the transition 3, when its tumor becomes proliferative, now, at least in many cases, we know there's a second disrupting event in the RB pathway. That is the pathway that regulates proliferation. That can happen by either a mutation, inactivation, or RB itself which is the key molecule in this, or there are other inhibitors that basically block the effective interaction of cyclin D to be able to activate the pathway, one of which we call INK4c, or p18 is the one that -- there's actually different kinds of that for different sorts of cells but in plasma cells, the p18 is really what's important. So what we found is in proliferative tumors that about 25% to 30% of them have inactivated that gene actually by deleting it, just completely deleting it from the cell. So basically, we've developed the concept that the Cyclin D dysregulation is key for the initial event that's common to MGUS and myeloma, but it doesn't cause a lot of proliferation but probably causes, enables the cell to proliferate more than a normal cell. That later stage is you further disrupt the pathway. Now, the tumor can become more proliferative. That's the basic idea.

Jenny: I'm sorry to interrupt you. I'm wondering now that you know that cyclin D is the start but not the accelerator and the RB pathway might be the accelerator, are there things that have been found that you could shut that down early, like at the cyclin D stage?

Dr. Kuehl: Well, people try to develop inhibitors. There are inhibitors against the molecules that cyclin D interacts with which are called CDK or cyclin D kinases. There's a CDK4 and CDK6. So people have developed inhibitors against those but they're not specific for the tumor cell. In other words, they would affect all proliferating cells. It's like chemotherapy. A lot of chemotherapy is non-specific so it's targeted against proliferating cells, and as a result, there's significant toxicity associated with it. As you probably know, it interferes with your ability to create hematopoietic cells, red blood cells and white blood cells. So unfortunately, at this time there is no really good drug that can inhibit the effect of cyclin D1 or D2. I mean, even though people are trying with these CDK inhibitors -- they're trying to use them to treat -- unfortunately, they're not highly specific and there are significant side effects. Does that make sense?

Jenny: Yes, it does. And then the RB pathway, is there anything that you know that stops that at that point?

Dr. Kuehl: No, there are no specific drugs that can -- I mean, part of the problem here is that, as you know, there's oncogenes and there's tumor-suppressor genes. So the RB itself is a tumor-suppressor gene, so it's standing in the way of the cell going on to proliferate. So if you inactivate it by either homozygous deletion or mutation, it's very hard to replace the gene that's lost from the cell. It's the same way with this other molecule, the p18 I talked about. Again, that's a tumor-suppressor gene and it's lost from the cell, so you can't bring it back easily. So you basically need to find some other way to take advantage of the fact that that cell has lost the RB or the p18 and you -- well, the hope is that that makes the cell more sensitive to some particular agent. I mean, that's the dream, but it's tougher than that and the bottom line is there aren't good drugs that specifically target the RB pathway. So often, what's used is chemotherapy that's very toxic, it's somewhat non-specific, but with transplants, with autotransplants it's possible to overcome some of the side effects of that therapy, the blood-forming cells being the most sensitive. So that's what the transplant does is it restores blood cells that you've killed off with the therapy, and hopefully you've killed off most of the myeloma tumor cells.

Jenny: Okay. And now, I don't want to stop you from what you were talking about before. My apologies - if you can remember.

Dr. Kuehl: Oh, no, I think I -- oh, the other thing I was just going to point out is that -- so you asked me how having the different kinds of myeloma impacts research. Well, I think what's really important is that when you start looking at trials, for instance, that it's important to recognize that myeloma is multiple diseases. So a given drug might work even if it's not directly targeted against that one type of myeloma, it still might work better against one than another. In other words, for example, it might work better against the ones that have the MAF translocation than it does against the ones that have the cyclin D1, or vice versa. So I think what's important is to appreciate the fact that you're dealing here now with multiple diseases. For instance, when you do clinical trials, it's possible that if you look at the myeloma tumors that you haven't classified that you won't identify something that might give you a positive effect. Let's say in the one that have MAF translocations, so that's like 6% or 7% of tumors; so if that 5% or 6% is buried in there along with the other 95% that don't have that, you won't see the effect. I mean, you'd have to have an incredibly large study which doesn't usually happen. So I think it's very important to recognize that this is different diseases, and it's even more complicated than that in the sense that -- so we developed a classification which we call TC which is based on the translocations and Cyclin D1 expression as RNA. That holds up for both MGUS and myeloma. So it's looking at the early events, but what's important is that there are later events that happen and ultimately, that becomes important, too. So just as an example, about 40% of myeloma tumors have an N or a K RAS mutation. Now, if you had a therapy -- and there isn't one, by the way -- there's a lot of work going on here now trying to develop one -- but if you had a RAS mutation for any kind of particular myeloma tumor and you had a specific drug that targeted RAS, then the most important thing would be to identify the tumors that have the RAS mutation, and that goes on for all of the other kinds of things you see. So one of the kinds of mutations that we came upon at one point was NF-kappaB-activating mutations. So NFkB is a transcription factor that's tightly regulated and if you activate it -- which in normal plasma cells can be activated by signals from the microenvironment and in tumor cells, but as the cells become more autonomous, it turns out that it's advantageous for the tumor to pick up mutations in regulators of NF-kappaB. There are many regulators. So there are mutations in like eight or ten components of the NF-kappaB pathway and those mutations cause the upregulation and activation of NF-kappaB transcription activity. What that does is it affects genes and changes the expression of genes that favor the survival of cells mostly. So therefore, the cells are less likely to die, and this is really key even in normal plasma cells but it's done by the microenvironment. It's done by the bone marrow stromal cells that release factors that activate NF-kappaB through external signals. That's true in the MGUS and the myeloma tumors as well, but as they progress and particularly when they become very advanced, the NF-kappaB activation, a result of mutations in the cell makes that cell not dependent on the signals from the microenvironment as much. So I don't know if that makes sense, but tumors in general strive to become more and more autonomous, and as I said early on, they're completely dependent on the bone marrow microenvironment and they only can be -- in MGUS there may be 5% of the cells or less in the bone marrow. Then as you go on, they take up more and more space in the bone marrow so they have less dependence, and then ultimately they can move to extramedullary sites -- meaning outside the bone marrow, or the plasma cell leukemia -- and at that point, they no longer can rely on the signals that they got from the bone marrow. Now, they need to be able to provide their own signals. So the NF-kappaB mutations that deregulate that pathway and cause increased activity of NF-kappaB are just one example of that. I think the loss of FGFR3 is similar in that a RAS mutation, for instance, might replace FGFR3. Actually, the other thing about FGFR3 is that it's a receptor. It's fibroblast growth factor receptor 3. So it requires a ligand. It means something to stimulate a molecule, to hit it and stimulate it, and there's certain cytokines then that can -- so-called FGFs, fibroblast growth factors that can stimulate it. So that means if the FGFR3 were critical for the survival of that tumor, it would then require those signals. So it gets around that by a RAS mutation or something else. Eventually, FGFR3 becomes dispensable, although I will also say another way that it becomes actually critical is that if you have a mutation in the receptor itself that that will activate. The receptor no longer needs signals from the outside. So that happens, too.

Jenny: In the NF-kappaB pathway and the RAS mutations and some of these other mutations, how are you testing -- and the cyclin D -- how are you identifying those? Are those through the flow cytometry test, the FISH test, the gene expression profiling?

Dr. Kuehl: It's gene expression profiling. Number one, it's directed PCR and mutational analysis. It's sometimes through FISH, but I think nowadays it's more through comparative genomic hybridization. A lot of this was discovered by copy number changes and you could see homozygous loss of both copies of a gene or one copy of a gene, and then mutational analysis. Now, as you know, everything is being sequenced left and right. Although it's interesting, I think that a lot of the most important things were discovered before whole genome sequencing came along. So the whole genome or exome sequencing I think has uncovered and shown and demonstrated very clearly that there's a lot of heterogeneity within the tumor, and that some of that was not discernible by the earlier studies. But the key genes that are involved, many of them were found before whole genome sequencing, and not to say that there haven't been some new ones.

Jenny: Well, from your cell lines, right?

Dr. Kuehl: Yes. I mean, almost all of the abnormalities that one sees you can find in one or another cell line, and what you're talking about mostly is the prevalence or frequency of things. So you might find that some particular thing -- well, it's like the inactivation of p18. So cells lines are proliferative, and so about 30% of the cell lines have homozygously inactivated p18 mostly by homozygous deletion. In the tumors it's only about 5%, but if you pull out the tumors that are proliferative -- which you can do by looking at what genes they make and so on or were there other ways to do it -- now, all of a sudden you see those homozygous deletions of p18 that are similar to what you see in the cell lines. Does that make sense?

Jenny: It does. Can I ask the question a little bit more about testing? I know there's a new testing -- I think it's from the binding site called the heavy/light chain test. Since these are heavy chain translocations, is that test important? Explain if that test is relevant or what that test does.

Dr. Kuehl: No, I don't think it's related to translocations. I mean, I'm not that up on that test, but what that test is doing -- so I shouldn't talk too much about it. I should know more about it but I'm not fully up on it, but I think basically, the idea is that there was a light chain assay. There's a free light chain assay which is very sensitive, and adding the heavy chain in, it just increases your sensitivity because now you're looking at a specific subgroup of the heavy chain. There are different kinds of heavy chains, and now, if you -- there's what are called different isotypes, so there's alpha heavy chains for IgA, there's mu heavy chains for IgM, there's gamma heavy chains for IgG and there's four different kinds of gamma, and there's delta chains for IgD, and there's epsilon chains for it. So what happens is if you look at all of the IgG at the same time, for example, you're looking at four different kinds of IgG for the four different gamma chains. There's IgG1, 2, 3 and 4. So by having them look at the single heavy chain, you can develop more sensitive assays because now you've just narrowed the window of what you're looking at. Does that make sense?

Jenny: Yeah, it does.

Dr. Kuehl: It doesn't have anything to do with translocations per se.

Jenny: That makes sense. So I know Dr. Bergsagel talked about MYC, and being able to find MYC was only because there were more detailed tests available. Before, you couldn't even test for it. So are you finding new things with the deeper level of testing that's available now?

Dr. Kuehl: Well, so let me just tell you the MYC thing real quickly. I think I told you that he and I talked about this a lot and we decided that MYC was really important and that we should look at it. At that time, it wasn't known that there are rearrangements of MYC. It was very hard -- there weren't good methods for looking at rearrangements. So we decided to look for mutations in MYC and we found some mutations, but they turned out to be germline polymorphism, so normal alleles. That was disappointing, but we decided that, "Well, since we have these mutations and we can distinguish the two alleles, let's look at that. "What we found in our cell lines was that when we found mutations that distinguish the  maternal and paternal MYC alleles, they only expressed one in almost all cases. So then we decided to develop FISH assays to look for MYC rearrangements and they turned out to be very complicated. They had not been picked up for the most part before. So we then found them there, and then another group came in and looked at MGUS and they saw very few in MGUS. So the idea was that these were secondary events. We also saw heterogeneity in myeloma tumors, and then when Leif developed the mouse model, it wasn't thought that that was going to create myeloma. It was an MGUS-prone strain and it was thought it was going to create a different disease, but it turned out it created myeloma. So upregulating the MYC created that, and then when you looked again at the gene expression profiling, MYC was up higher. Then recently, what we've took advantage of is a combination of CGH initially -- comparative genomic hybridization -- that looks at copy number changes, and we saw -- it was amazing because about a third to 40% of myeloma tumors, now full-blown myeloma tumors -- and I think that's important, not necessarily the earliest stages of myeloma tumor -- I'm not talking about MGUS now -- had copy number changes in the MYC region and they were pretty scattered. Then in addition, there were FISH analyses for rearrangements involving IgH, immunoglobulin heavy chain and light chain loci, that show that there are even more. So somewhere in the range of 40%, probably 50% of myeloma tumors where you can get enough sample to study have MYC rearrangements. So we now think that MYC is the most common abnormality in myeloma. We think that MYC upregulation, which can be biallelic, doesn't require a rearrangement, but the rearrangements happen and we know some of them are happening during progression, but we know also that some of them -- it's very likely but this remains to be proven -- are causing that transition from MGUS to myeloma. That's our theory, anyway. So there are studies going on now that the MMRF has sponsored to look at whole genome sequencing and the preliminaries out there is they're finding these MYC rearrangements there also now, and it's a little bit discrepant with previous studies that were done in France using FISH analyses. They found lower levels. The theory now -- for me, anyway -- is what MYC is doing is it's not causing increased proliferation. That's very clear. There's no correlation of MYC expression and proliferation. Instead, what I think it's doing is it's increasing autonomy of the tumor and it's enabling the tumor to go from a small tumor to a larger tumor outside of the -- beyond the normal bone marrow niche; still in the bone marrow but now, instead of being a few percent of the cells in the bone marrow, it's now 10, 20, 30, 40, 50%. I think a MYC rearrangement is giving it a degree of autonomy, and the reason that I think that the French group did saw a lower instance is they were looking at -- probably their samples are skewed towards an earlier stage of the disease. I mean, it's a way of -- but anyway, the bottom line is that MYC is obviously a key, I think, in the pathogenesis of myeloma and distinguishes it from MGUS in that sense, and that it's hard to target but there are these promising agents now that suggest that you might be able to target MYC. I think that's gradually happening. We'll see if it pans out.

Jenny: We have a few caller questions that I know people are going to want to ask at the end so I want to leave some time for that, but I guess my question would be for you, with this information about MYC and all this detail -- and this is such deep detail about myeloma, I'm just so impressed with it, to be able to subdivide it like that and understand all the pathways that it's doing -- where do you go from here?

Dr. Kuehl: Well, here's what I think. I think knowledge is a great thing. I think the more you know about things, the better chance to be able to do things. I'm a deep believer in basic science in trying to understand how things work, what is there, because I don't think you can predict where the next lead is going to come from and I think that doing those basic studies gives you the chance. That being said, I think that the amazing thing that's happened in the last ten years is the development of drugs like the lenalidomide, thalidomide, Velcade, and so on. They're targeting the plasma cell. They're not targeting a specific kind of myeloma generally although they might be more active in one type of myeloma than another, but they're targeting what I would call the "plasma cell-ness" of the tumor. So I think that those kinds of therapies, if we could understand them better, are really important. I think another target is the interaction of the myeloma cell at the bone marrow environment which is pretty much of a mystery. We really don't have a firm understanding but I think that's really important. And I think the third place that's really important is immune therapies. I think that the day of immune therapies has arrived and there are definitely tumors where immune therapy is very effective, and there's a good reason to believe that immune therapies might be useful in myeloma. I mean, I think it's important to remember myeloma cells are plasma cells, and if you could wipe out all of the plasma cells in your body including the myeloma cells, that wouldn't be a disaster. You can live without plasma cells. You can do antibody injections and so on. I mean, not that it's ideal. That's better than if you had a lung tumor. You can't knock out the lung. So anyway, I think that there are many options for therapies. I think targeted therapy is a great idea but I think single target therapies just generally haven't worked, and I think there's a lot of work going on trying to identify ways of targeting multiple targets at the same time. That's kind of my view of this, but the future is so hard to predict and I never would bet against science and medicine coming up with new therapies. I think it's just hard to predict when they're going to be there, but I think they will be.

Jenny: Well, that's, I guess, my recommendation for patients is if you are educated and you understand the biology of your own disease, then when something new comes out, you can say, "Oh! Well, I sort of qualify for that, and that might be appropriate for me because I have this particular translocation or I know that I'm hyperdiploid," or whatever.

Dr. Kuehl: That's absolutely true. I think it's like patients can actually contribute to the treatment of their disease because when they ask questions, they can stimulate people to think, and they have great vested interest more so than anyone else in their disease. I'm very serious about this. I mean, there are examples of patients who've come up with ideas that researchers and clinicians have used that have been beneficial. So I think that patients need to understand their disease, and they can definitely make a contribution to improving the treatment of the disease.

Jenny: Well, I think that's the whole theory behind this program is that patients can be major contributors, because researchers are brilliant and they're amazing and they're doing this wonderful work, but unless we participate by helping them speed things along -- I mean, it could take twenty years to do something that could have been done in ten, or could have been done in five or two.

Dr. Kuehl: Yeah, we all have blind spots. You know the story about the elephant and the people feeling the different parts of it? Well, we all have a perspective, and the patients have an important perspective, there's no doubt.

Jenny: Well, thank you. I would like to open it up for caller questions.

Dr. Kuehl: Sure.

Jenny: So we have a caller. If you have a question for Dr. Kuehl, call 347-637-2631 and press 1 on your keypad. Okay, so our first caller question, go ahead.

Caller: Hi, Doctor. This is [Caller]. How are you? Hi, Jenny. I have a question. I was very interested in what you were discussing about Cyclin D1. I'm smoldering so I'm really just trying to learn as much as I can. In my situation, I'm wondering if there's any other process that could result in the upregulation of Cyclin D1 other than the primary translocation t(11;14)? I have Cyclin D1 on my immunohistochemistry. They found up to 15% total plasma cells on the core sample, 6% in the aspirate sample -- this is a two-year-old bone marrow biopsy -- but I had a normal FISH. So in theory, from what I understand, we should have found the translocation t(11;14) in my FISH. I reached out to a pathologist who ran a plasma cell labeling index for me as a courtesy on this actual sample and the results were 0%, so he explained to me that I had a very low proliferation rate but stated that my cells were expressing Cyclin D1 "like crazy" -- that was what he basically explained to me; I needed the layman's terms, of course -- yet 97% of my cells were ID'd as aberrant on the eight-color flow. So I guess depending upon these risk progression models, since I am in the smoldering phase -- that's what I kind of try to focus on -- the flow indicated that I had a higher risk of proliferating and progressing. So at this point I'm two years into this, two years and a couple of months, and my M spike and my abnormal free light chain ratio have been relatively stable. I'm really just trying to kind of grasp anything, like at this point, what do I need to know about my biology in order to move forward and be smart about this.

Dr. Kuehl: Okay, so let's take your first question which is about the Cyclin D. So hyperdiploid tumors can make Cyclin D. Now, usually it's lower than if you have a translocation. There's a clear-cut difference, a cut-off, and it can go to quite low levels but it can be moderately high. So it's possible -- I don't know if they know whether your tumor is hyperdiploid or not, but the fact that they didn't detect a translocation could mean that you have a hyperdiploid tumor that happens to be making a relatively high amount of Cyclin D. Another possibility is that when they do the test -- this would be very rare -- but the test they do for Cyclin D probably is looking for IgH cyclin D1 translocations. But rarely, it's possible that you could have an IgL translocation. That's the light chain or lambda or kappa --

Caller: Oh, that's the first time I've ever heard of that.

Dr. Kuehl: Well, it's very rare. Not only that, but the other thing that I should say is that it is my belief that the translocations that are so-called primary translocations, sometimes the genes that are involved in primary translocations can also come up in secondary translocations. That is they didn't come up as primary. They came up as secondary. It's my belief -- and this is based on our studies on MYC where they're not all Ig; many of them are not Ig but they involve a limited number of other loci -- there's no test for those now. So it's possible that you would have a Cyclin D -- that they've missed the translocation. It may not be Ig or whatever. Now, that being said, just with respect to your disease, I think that -- it sounds like you're getting the flow cytometric test that stratifies patients and they're telling you that you're in the group that's more likely to progress.

Caller: Exactly, yes.

Dr. Kuehl: Well, what I would say is what you want to do is you want to make sure that you get careful follow-up. There are trials out there now where they're starting to do trials on patients that have smoldering who are in this high-risk group and they are starting to include some of those patients on trials. When you enter a trial, that is the patient's choice. So you want to apprise yourself of what those trials are and what the status of them is. Find out from the people who are doing these tests on you, do they recommend that you would do that trial? And then there are a variety of places that are doing these trials now. Some of them Mayo Clinic, I think. I think some here at the NIH. Ola Landgren has done some. He's moving to Sloan Kettering so I know he's doing some of that. So I think just make sure you get the best possible medical care, that you get good follow-up, and then make sure you read about these things so that you can ask intelligent questions of your doctor. That's really important.

Caller: Now, what about additional cytogenetic testing? Do I need to pursue that? Should I have another bone marrow biopsy? Do I need gene expression profiling to bring better clarity to my situation? What would you think?

Dr. Kuehl: Well, you know, I'm biased in this regard. I believe more information is better. So my feeling is more information can't hurt; well, I mean, apart from having your bone marrow taken. But in all seriousness, I think the more you know, the better, and because they think that that is important. That being said, it isn't necessary that at this point in time that it's going to make a lot of difference because maybe there aren't treatments right now, but things may change. So I think having that information, I don't think it's a big deal, and if you can get it done and your insurance and whatnot pays for it, I would do it. That's what I would do if I were in your situation.

Caller: All right, thank you for that. Thank you so very much.

Dr. Kuehl: Okay.

Jenny: Okay, thanks for your question. Our second caller, go ahead with your question?

Caller: Hi. That was an awesome answer. It kind of feeds into the question that I have because the question was really what do I do differently as a patient if I know my disease biology? And that was like a very good case in point about what I might know and what I might do. I've listened to the show and we've talked a lot about disease biology, but that was like the first example of what I might do differently now that I know it. Can you give us other examples about the importance of understanding this disease biology and how that really affects the treatment path?

Dr. Kuehl: Well, I think that preliminarily, myeloma is divided into high-risk and low-risk. So high-risk would be -- and this is based a lot on cytogenetics because those are the tests that have been available. So high-risk is t(4;14) translocation, it's MAF translocations, it's p53 mutations, and sometimes chromosome 13 abnormality, although that's thought to be mainly because that's associated with MAF and FGFR3. Then you have the low-risk. The low-risk are hyperdiploid and Cyclin D1. Now, usually it's not routine to do studies for hyperdiploidy, and so mostly people have done studies for translocations. They've done p53 that look for loss of allele of p53 which is associated with progression of the disease. Chromosome 13 loss is done although that isn't as important if you do the t(4;14) and MAF translocations. But I think that's important to know. Again, what I would say though, it depends who you're working with. I think as a patient, you want to know what's going on. You want to push your doctors. You want to ask them questions. You could ask me and I'd say, "If I were..." People come and ask me. Periodically, they'll call me. They've read something I wrote and they'll say, "Can you give me some advice?" and one of the things I would say is -- I don't treat patients but I have a philosophy, and I also know where I would go if I had myeloma, and I think what I would say is, "You want to get the best possible treatment. You want to know as much about the disease as possible. I think it's important to have as much information so that when..." More information can never hurt. Obviously, there are limits on cost and et cetera, et cetera, but  I think what I said to the previous patient I would say the same thing to you. I mean, I think  -- learn about the disease, talk to people, and ask questions of your doctor, and if you're not satisfied, get another opinion. Don't be afraid ever to get another opinion. Any good doctor would never discourage you from getting a second opinion. I don't know if that's helpful, but anyway, that's --

Caller: Yeah, it's a great answer. I think [Caller] and Jenny may be the most informed patients on the planet. So I applaud what you guys are doing and keep up the great work.

Dr. Kuehl: Well, yes. I think follow the MMRF. They're great. I think that they just made such an incredible impact. Kathy Giusti is out of sight as far as I'm concerned. So I think there's a lot to learn. I mean, I'm not putting down any other organization, but I'm very impressed with what they've done. It's focused on research which I think is really key.

Caller:  That's great. Well, thank you.

Dr. Kuehl: Okay.

Jenny: Okay, and we have one -- I know we're over time so I'll ask this quickly, but we have one write an email question and it said, "Has the NCI ever considered putting together patient data similar to how that MMRF CoMMpass study has done, but on a larger scale?"

Dr. Kuehl: Not that I know of. I don't know that that's necessary. I think what the MMRF has done, I don't think the NCI is going to do better than they've done. I think what they've done is spectacular.

Jenny:  I’ve  heard that.

Dr. Kuehl: And I think that the whole landscape of myeloma research has just changed since they got involved. I still remember years ago that they assembled all the myeloma grants at NIH, and I thought that one of the amazing things was -- there were very few, and there were very few actually on myeloma itself, but one of them is that it turned out when I looked at the list, there were numbers of melanoma in it which, as you know, is a skin tumor. So I said to someone, "You know we've really made it in myeloma when people are no longer confused about melanoma and myeloma." Anyway, I think the MMRF is doing the job.

Jenny: Well, I heard from several researchers that they love their researcher gateway and all the data that comes from their compiled data. So I applaud what they're doing. It's really amazing work.

Dr. Kuehl: Yes, there's just no doubt about it.

Jenny: Well, Dr. Kuehl, thank you so much for joining us today. We really appreciate you taking the time to help us understand the biology, why it's important to us, and then what we do with that information. Thank you for helping us identify the fact that all myeloma is not the same so we can get to more personalized care.

Dr. Kuehl: It's a pleasure, and I've always said that I get a great deal of satisfaction when a patient calls me and asks my opinion. I always tell them, "Well, my opinion probably is not worth much more than I charge for it," which is nothing, but I hope that's not strictly true. But I'm really glad to be able to do this, and if it's helpful, it's great.

Jenny: It's very helpful to patients so we really appreciate you taking the time.

Dr. Kuehl: Okay.

Jenny: Well, thank you for listening to another episode of Innovation in Myeloma. Join us next week for our next mPatient interview as we learn about how we as patients can help drive to a cure for myeloma by joining clinical trials.