In our fifth MCRI episode featuring the top 10 proposals for high-risk multiple myeloma, Dr. Bruno Paiva describes his study of chemo-resistant myeloma cells after therapy using new, more sensitive minimal residual disease (MRD) monitoring. With the abundance of newer therapies, myeloma doctors have an opportunity to use specific treatments for certain patients, but this requires deeper knowledge about the biology of the tumor cells and better tools to monitor response to treatment. In his study, he aims to answer important questions like: Why do high-risk patients have similar MRD response rates but inferior survival? Why are some patients MRD positive and still have great outcomes? Is it because of the biology of the chemo-resistant cell? Can this information lead to tailored treatment and better survival for high-risk patients? If all patients have several different types of myeloma cells in them at the time of diagnosis, which cells are surviving after high-dose chemo?  In a clinical trial with 100 high-risk patients, he will use the next-generation flow MRD test after chemo to study the tumor cells that remain to hopefully find an attractive target. This is the first time that a newer, more evolved MRD test will not only measure depth of response in a more sensitive way, but also look at the biology of the cells. The Myeloma Crowd Radio Show with Dr. Bruno Paiva, PhD

Jenny: Welcome to today's episode of Myeloma Crowd Radio, a show that connects patients with myeloma researchers. I'm your host, Jenny Ahlstrom, and I'm joined today by my myeloma friends including Pat Killingsworth, Gary Peterson, Cynthia Chmielewski, and Jack Aiello as co-hosts. This is the fourth and a very important series featuring the Myeloma Crowd Research Initiative. For the first time, patients, including you, are teaming up with myeloma researchers to find and fund the ideas in myeloma that could have the greatest impact for the next generation of myeloma therapies. We decided to go after high-risk myeloma for patients that have no viable options today either because they have high-risk features or relapsing or refractory to existing drugs. We need new options and if we can find a solution for high-risk patients, it's highly likely that it'll work in medium or low-risk patients. It's also common to pick up more aggressive genetic features as the disease progresses. Unfortunately, many of us become high-risk after our myeloma has relapsed multiple times or current medications become ineffective. We're now in stage three of the Myeloma Crowd Research Initiative or we call it the MCRI. In stage 1, we asked researchers around the world to submit their proposals and we received back 36 high-quality letters of intent. In stage 2, we have those proposals scored by our Scientific Advisory Board. They selected ten proposals, which they believed were the top proposals, and we are now holding Myeloma Crowd Radio shows so that you can become involved. We want you to understand the proposals, so please listen in, ask questions, read the transcript after the show is posted, and share it with your friends and family. After the full proposals are submitted, the Scientific Advisory Board and Myeloma Patient Advisory Board will together decide on a limited number to fund through patient-driven campaigns, and we will need your help to get the word out and share the very amazing work that's being done. Now, today if you'd like to donate, we have a "donate" button on the Myeloma Crowd site and you can help us start to donate for this very unique endeavor. The specific projects will come later as we allow time for these full proposals and all the shows to be completed. Now, we are very privileged today to have with us Dr. Bruno Paiva of the University of Navarra in Spain. And because Dr. Paiva is joining us from Spain, we may have a little delay until he joins us, so I'm going to give us a little introduction for Dr. Paiva. Dr. Paiva is Scientific Coordinator of the Flow Cytometry Department and CIMA Lab Diagnostics group at the University of Navarra in Spain. He teaches courses regularly on cellular biology, techniques of quantification and analysis, new treatments in hematological cancers, inflammation as it relates to the blood and minimal residual disease. The Spanish group with which he has worked has performed very extensive work appreciated by the entire myeloma community and myeloma practitioners on the flow cytometry test as it relates to minimal residual disease. He specializes in understanding the heterogeneity or biological complexity of MGUS, smoldering myeloma, active multiple myeloma and how it evolves. He is involved in both the detection and classification of myeloma and has written numerous papers on the topic, writing or speaking on the subject over 140 times. He's also been Principal Investigator on clinical trials utilizing immunotherapies including the SAR650984 anti-CD38 monoclonal antibody. He participates as ad-hoc reviewer for Blood Cancer Journal, Blood, Pathobiology, Leukemia and several other publications. He's received Premier ASH Abstract awards in 2014, 2013, 2012 and the Young Investigator Award by the IMF to name just a few. Dr. Paiva, welcome to the show.

Dr. Paiva: Thank you so much. I'm sorry for the slight delay. I had some technical difficulties, but I think now we are all set.

Jenny: Well, perfect! We are very thrilled to have you. On the call live, we have Cynthia. We have Jack. Let me bring on Gary. Gary, you should be on.

Dr. Paiva: Good afternoon to all.

Gary: Yes, I am.

Jenny: For those who are joining live, we as co-hosts, we'd ask you to keep your microphone on mute unless you're ready to ask your question. Dr. Paiva, maybe you can give us first some background about the study of minimal residual disease, why it's important in myeloma, what you've learned so far. I know this is background because we definitely want to get to your research and what you're studying now, but if you could give us a little background, I think it'd be very helpful for patients.

Dr. Paiva: Absolutely, plus it's really an imagined field, which is quite natural. In fact, the implementation of new biomarkers takes several years. If we look, for example, to other diseases such as acute leukemia, the concept of MRD, which is now fully established, took a long time. In multiple myeloma, unfortunately until five or ten years ago, there weren't many therapeutic options. The options available were not tremendously effective and therefore, complete remissions were rather scanty. Only a few patients achieved deep levels of remission. Consequently, also survival was limited at the time. Nowadays, as we are probably all aware, there are many more very effective options to treat multiple myeloma. We've produced nowadays a much higher rate of complete response, which for both younger transplant patients as well as for elderly non-transplant candidates are now in the range of 50%. Therefore, you can see the dramatic evolution and the fortunate evolution on the treatment of the disease. We can also see a dramatic evolution in our understanding of the biology of the myeloma plasma cell, also the biology of normal cells, for example, the chemo-resistant stem cells. We also see a great improvement in patient's differential diagnoses. We now have better tools to differentiate between treatments requiring myeloma, ultra high-risk myeloma, smoldering myeloma, high-risk smoldering myeloma, MGUS, also better tools to predict the risk of a newly diagnosed myeloma patient, discriminating between those with standard versus high-risk disease. But in contrast and for many years now, there has been no evolution in how we measure response to therapy. While before there was really no rationale to improve the sensitivity of the techniques used to monitor this costly therapy, nowadays given the efficacy of the novel agents and more complex treatment schemas, it becomes mandatory to measure a response to therapy using very sensitive tools in order to understand and to identify those patients that are truly responding to chemotherapy and achieve deep levels of remission than those patients which are more chemo-resistant. The identification of both will be crucial as we move along into precision medicine and we'll try to avoid both the under as well as over-treatment. Here lies the importance of minimal residual disease.

Jenny: Yeah. I know that International Myeloma Foundation has the Black Swan Research Initiative is supportive of the minimal residual disease testing.  I didn't fully understand the importance of minimal residual disease testing until I talked to Dr. Landgren and he was saying how it will tailor treatment and allow more of the precision medicine to say, "Okay, if you haven't responded, this is why," and then you can customize treatment for different individuals.

Dr. Paiva: Absolutely. That's the final goal imperative of all the years of intensive research. Many groups, I'll say in particular, but not the only one, the Spanish Myeloma Group is really devoted to MRD development. After developing the technology that at the time was pretty much below sensitive, it emerged as one of the most important prognostic factors -- in our experience, the most relevant prognostic factor -- and now we are in the second half of this, much in which after the initial development of the techniques to monitor MRD, we are now making a final fine-tuning to really optimize the sensitivity of the methods, also to standardize methods for a global and wide application of MRD technology, ultra sensitive MRD technology, and this is not anymore the plain or simple prognostic marker, but also a diagnostic tool to really try to optimize the management of myeloma patients. It should be emphasized that again, precision medicine or treatment therapies can never be decided on a single biomarker, rather in a combination or a constellation of biomarkers that provide complemented information, but we certainly believe that now with more sensitive and standardized MRD techniques, MRD can become indeed the next biomarker to help optimizing the management, the clinical management of myeloma patients.

Jenny: Perfect. Can you explain what the MRD test originally was looking at and then how it's evolved, what it's looking at now, and explain the evolution of that test?

Dr. Paiva: Absolutely. Originally, MRD testing was performed mostly with three different techniques, multiparameter flow cytometry that looks into live plasma cells and discriminates between the normal plasma cells which all of us have in our bone marrow as well as in secondary lymphoid tissues, peripheral blood, versus in myeloma plasma cells, which have a different phenotype. The way we discriminate between normal versus myeloma cells is according to the presence of the different phenotypes, aberrant antigen expression in the surface, as well as inside the myeloma plasma cell. Alternatively, there's also molecular PCR that is capable of detecting the DNA of patient's bone marrow samples. It's capable of detecting the clonotypic sequence of the monoclonal immunoglobulin that characterizes all myeloma plasma cells from every single patient. It's like the passport of the myeloma plasma cell. Now, both techniques have been there for myeloma, as well as for other hematological malignancies for now more than 10 or perhaps 15 years, PCR in particular, the most specific PCR approach, which is called ASO PCR. Allele-Specific Oligonucleotide PCR has perhaps been more sensitive than multiparameter flow, but is a handicap, the drawback of applicability. Because myeloma is an end-stage B cell disorder, it's also characterized by a higher rate of somatic hypermutations and this clearly limits the applicability of ASO PCR to monitor MRD in most myelomas. My contrast multi-parameter flow from 10 or 15 years is much more applicable to virtually all the patients, but was less sensitive. Nevertheless, all the available data -- and we now have more and more data and consistent data -- using both low sensitive flow and ASO PCR showed that both strategies as well as clear prognostic value even when performed among patients in complete response. In complete response, theoretically you don't have the disease anymore, but by using more sensitive measures, residual disease can still be identified. Now, more recently, there has been a dramatic evolution in both approaches. First, ASO PCR has moved from a single monitoring of unique clonotypic immunoglobulin sequence into high throughput sequencing or next generation sequencing. This allows to monitor MRD by molecular approaches with the same sensitivity, perhaps even slightly higher as compared to ASO PCR, but the applicability -- since now we measure all the immunoglobulins present in bone marrow samples, the applicability is much higher in the range of 90% of the patients. Regarding flow cytometry, we have moved from measuring only four proteins at the same time into eight or ten proteins, simultaneously antigens, and we have moved from measuring 200,000 cells into five million up to ten million cells. This means that the flow is now much more specific, much more sensitive. It has moved from a sensitivity of 10^-4. That is the identification of a tumor cell within 10,000 normal cells up until 10^-6, which is the identification of one tumor cell in one million normal cells. Plus, with the availability of better cytometers to measure again eight or ten antigens simultaneously, this means that the technique is applicable to virtually all myeloma patients. So this has been one technological revolution. There is a second one that should come in the next three to five years, which is to implement, envision to either flow or molecular techniques that measures MRD in the bone marrow to make synergism. These techniques are the imaging techniques such as head CT or whole body MRI that could measure minimal residual disease in other sites of the bone marrow that could not be evaluated in a single bone marrow estimate, as well as extramedullary disease. Altogether, this means that even though so far many studies from different cooperative groups in different countries have shown the great clinical value of MRD studies, perhaps we still don't fully understand and realize the clinical significance of MRD in myeloma since we now have much better tools to monitor sensitive response to treatment.

Jenny: Okay, so in the evolution, it sounds like you're combining the flow cytometry with the PCR test, which I understand are the gene expression profiling tests. Is that correct?

Dr. Paiva: Not exactly. During the spectrum of the evolution of the disease, many different techniques or tools should be employed according to the specific clinical questions, for example, initially karyotyping, then FISH, then gene expression profiling. Now, deep targeted sequencing or panels of mutations are being employed or already employed to determine the risk of the patients at the baseline when the patient is newly diagnosed. By contrast, these types of techniques have been more used to monitor response. Then again, different techniques are complementary along the spectrum of the disease.

Jenny: Okay, so it's more of a monitoring tool during the course of treatment.

Dr. Paiva: It's a diagnostic tool. It's also used at the initial screening. For example, even when there is only a suspicion of a monoclonal gammopathy and we need to establish clonality in the bone marrow or to detect circulating tumor cells in peripheral blood or to make a differential diagnosis between, for example, two different monoclonal gammopathies as it could be myeloma or Waldenstrom Macroglobulinemia, so there is an application for flow at the beginning, as well as particularly during treatment to assess response.

Jenny: Okay. All right. That makes sense. So your study -- go ahead. Cynthia, do you have a question?

Cynthia: Yeah, I have a couple of questions. Can I butt in? Because I still have to return to a meeting.

Jenny: Sure. Go ahead.

Cynthia: Okay. My understanding, doc, is that you're developing this device that's going to be much more sensitive in testing MRD than any other device that's already available on the market. Is that correct?

Dr. Paiva: Indeed. The assay, it's not fully devised, but it was an optimization of the already available devices. And so far in one and a half to two years of research between different groups, certainly under the umbrella of projects or research programs such as the Black Swan Research Initiative, now those techniques are much more sensitive, so sensitivity is already there.

Cynthia: And then this device will then be readily available in labs throughout different countries that many clinicians can use to test for MRD negativity and positivity. Is that correct?

Dr. Paiva: It is as we believe that MRD monitoring would become more and more sequenced in multiple myeloma given the higher and higher rates of complete response after initial therapy, as well as after salvage therapy for relapsed/refractory patients. We believe that MRD will become more and more implemented. This is of course to be used worldwide.

Cynthia: Okay. Now, when I was reading your letter of intent, it seemed like there were different groups of people and that high-risk multiple myeloma people who after treatments still have MRD positive results in your testing are more likely to progress than standard risk multiple myeloma people who still have MRD positivity in the results. Is that correct?

Dr. Paiva: Yes.

Cynthia: So what you're trying to do is look at the minimal residual disease clones that are left over after treatment and compare those clones from the people who have high-risk disease to standard risk disease to see what makes their clones more aggressive than the other people's clones?

Dr. Paiva: You are correct. There are different objectives in this letter of intention, research program, but bottom line, there are two clear and well-defined objectives. The first is focusing on high-risk patients that reached MRD negativity. We want to understand, what is the clinical significance for high-risk patients to reach MRD negativity with ultra sensitive techniques? Does this mean that high-risk patients reaching these levels of remission will have a similar outcome as compared to standard risk patients also reaching MRD negativity? If this is true, can MRD negativity become the surrogate marker for improved survival in high-risk patients? If this is true, can MRD negativity become a clinical endpoint for high-risk patients? If this is true, can MRD negativity become a surrogate marker for prospective determination of the efficacy of novel agents before waiting for a prolonged turnover of survival to see differences between two treatment arms, for example, a randomized clinical trial, so this is a clear objective in high-risk patients reaching MRD negativity. Alternatively or conversely, there is a second well-defined objective, which in fact arises from our own observations, which is that patients with standard and high-risk disease with the same level of persistent minimal residual disease after therapy, theoretically the outcome should be identical because they have achieved the same level of response, but it is not. And unfortunately, patients with high-risk disease diagnosis have inferior outcomes even though they have the same number of residual cells after therapy. This is the main reason why in the next couple of years, we want to study the biology of the resistant cells after initial therapy. It's important to emphasize that -- and this is really for the first time ever -- we don't want to study only diagnostic cells. We don't want to study cells at the moment of relapsed and refractory. We want to study those cells, those very cells that are capable of resisting to have some therapy. We want to understand the biological differences of these cells between standard versus high-risk patients in order to understand why in the later outcomes unfortunately are still inferior.

Cynthia: Okay, so for the resistant cells of those high-risk myeloma patients that you tested for MRD, you're going to start probing them and looking for some biomarkers that might be on those cells that maybe people who don't have high-risk myeloma or have leftover disease don't have those biomarkers.

Dr. Paiva: Yes. As Jenny mentioned at the beginning, this is where really next generation flow and next generation sequencing or immunotherapeutic and molecular technologies really merge and combine value. One of the advantages of monitoring MRD with flow is that we cannot only monitor the disease, but we can also sort with high accuracy those very few residual cells. After sorting, we can perform molecular assays on those cells also using next generation sequencing methods, and that is for low cell numbers. And by comparing the genetic signature of these cells after therapy with the genetic signature of the cells from the same patient, but at the moment of diagnosis, we might understand or we might get the information on the genes or signaling pathways that are crucial for those cells to resist chemotherapy. If we can identify such genes for signaling pathways, this would mean that we may get really crucial and unique information to overcome chemo resistance to standard and backbone myeloma therapy.

Cynthia: That sounds exciting. Thank you so much.

Jenny: It sounds like in your first aim that one of the objectives is -- you said typically patients have to wait two to three years to see if a new therapy is going to work for them because you enroll people in a clinical trial and you have to wait for the time frame for the trial to be over, and then to wait to see if their disease progresses. But what you're saying is if you have the right testing and you can predetermine who will respond and who will not respond to a new therapy potentially, you can cut that time short. Is that correct?

Dr. Paiva: Yes, indeed. This is really one of the objectives of really pushing forward the field of MRD, is to really make possible -- and then again, both academia, also pharmaceutical industry, as well as regulatory agencies such as the FDA or the European agency are really working together to see if there is indeed or not a role for MRD as a tool to really make possible and available new and effective drugs for patients before time. This means that we need to show a clear correlation between death and response, MRD negativity versus MRD positivity, and the differences in outcome. So far, this has been really established concerning myeloma again by different groups using different techniques. So now, I think the time has come to really implement these new and high sensitive techniques respectively in clinical trials, so then we demonstrate the added value and opt for it in a few years now to come to really again help with the management of patients with multiple myeloma.

Jenny: Just a follow-up question because when you said it had evolved, this flow cytometry tests have evolved and you can go from now four proteins -- you were saying four proteins to eight proteins, what type of proteins are you looking at and how have these types of proteins been detected before? Were they like RNA sequencing or is this a totally new type of capability for this assay?

Dr. Paiva: No. Many of those -- well, what we do with the flow is to measure single cells that are in suspension in a cellular fluid and we analyze the morphology of all those single cells, as well as for the presence or absence of antigens, proteins in the surface or in the cytoplasm of every single cell. So with this technology, we usually combine proteins, antigens that allow us to identify a cell population of interest such as plasma cells if the sample is from a patient with multiple myeloma, and we combine identification markers, proteins, with other markers that allow for the discrimination between a normal versus a myeloma plasma cell because a myeloma plasma cell has a different genomic profile. A different genomic profile will lead into a different RNA profile, and a different RNA profile will lead into a different protein expression profile, which means that often the myeloma cell expresses antigens in the surface or lacks antigens in the surface that's commonly present in normal plasma cells. By looking into every single cell and combining the information of the morphology of the cell, plasma cell identification markers as well as markers that are either absent or present in tumor cells were capable of discriminating one tumor cell within now almost one million normal cells. This is the concept of MRD monitoring by flow in myeloma.

Jenny: And it's taken quite some time to get to that point.

Dr. Paiva: Well, things never turned as we would like, but in the '90s, at the beginning of the '90s, there was a lot of technological development I'll say particularly from both the Spanish and the UK leads groups. They have really established at the time a four-color or up to six-color assay. Then this assay was prospectively incorporated in different clinical trials. At the time also -- because this was now seven or eight years ago -- the clinical trials that did not really incorporate the best trial that we have available today, nevertheless already from those trials, it could be clearly seen that MRD was one, if not the most important prognostic marker. And that after seeing those results that were reproduced in different centers and in different trials, this led into the second half of technological development in which again the way we do MRD nowadays is completely different from the way we did it in the past. I can tell you, now we monitor more than 200 to 300 myeloma patients only at this institution and much more in other institutions with this new assay. And believe me when I tell you that the amount of information that we can now see has no point of comparison -- is not comparable to what we have seen before, which means that we are extremely confident and hopeful on a new clinical meaningful of what we are doing in the laboratory.

Jenny: Excellent. Gary, you had some questions about outcomes between MRD negative, high and low-risk patients. Do you want to go ahead and ask those questions?

Gary: Sure, Jenny. Doctor, thank you very much. One thing I was wondering is the eight-color flow cytometry. Is that the same one that's used in Arkansas?

Dr. Paiva: Well, certainly and in fact, we were involved a couple of years ago and we are in close collaboration with Arkansas, as well as other institutions in the United States. They used it and they had implemented a quite sensitive, eight-color method. The method we are using now is very recent. It is the final product of the last two years of research. Therefore, it is not yet fully used in all different laboratories for most Europe and US, as well as other continents. Our goal is really -- and of course, being continually reasonable and rational -- we know that it is impossible for every single center to adopt the same affect, but it is certainly our goal after demonstrating the added value of what we have produced in the last two years that a significant number of myeloma centers, top myeloma centers, as well as small myeloma hospitals can really adopt these strategies, which in our opinion is very cost-effective. It does not need the sample diagnoses, which sometimes may be a limiting factor and again allows for a great sensitivity to measure response assessments. The Arkansas group, as well as other groups, we now could say periodically meets in workshops and work in meetings in which we discuss briefly the way we do flow and we really make an attempt for many different groups to adopt the same and standardized flow method.

Gary: I understand that you've done some remarkable things with flow cytometry. The Spanish group has just been amazing with regard to that. Is it true that you taught Dr. San Miguel everything he knows?

Dr. Paiva: Quite the opposite. I've been very fortunate to have a boss like Dr. San Miguel, which he's not only a boss, but a mentor, a friend -- well, a true friend. Despite all of these attempts, unfortunately in seven or eight years that we have been together, I still haven't learned anything that he has to teach me, but I try hard every single day.

Gary: Well, he's one of the ones that I consider the great eight of all myeloma specialists, so I hold him in the very highest of regards. Now, in your presentation, you've mentioned -- or proposal -- you've mentioned that MRD negative high and low-risk patients have similar outcomes. Did I understand that correctly?

Dr. Paiva: Indeed and that was a very positive surprise we had recently. Like I mentioned throughout this radio show before using the four-color assay, which was less sensitive, we saw that what both us and the UK groups, we have seen that the outcome of high-risk patients reaching MRD negativity was in between the outcome of standard risk patients either in MRD negative or MRD positive. It was more or less in the middle of the two standard risk groups of patients.

Gary: Okay. Now, here's going to be the clincher for you.

Dr. Paiva: Let me just get through this idea. We now have data from a more recent Spanish trial that started to enroll patients in 2010 in which already there, we started to implement eight colors. We didn't measure at that time the same number of millions and millions of cell that we measure nowadays. Let's say that the sensitivity or the assay that was implemented in the 2010 trial was right in the middle between the four-color flow and the flow we are using today. Nowadays, we have a median follow-up for that particular clinical trial. It is now in the range of three years, so it's still a relatively short turn-over, but it's very promising. And again, emphasizing on the short follow-up, what we are seeing just by incrementing the number of colors, we are seeing that the outcome of MRD negative patients irrespectively of the risk by FISH cytogenetics is completely super-imposeable. This was a very exciting and promising result.

Gary: So that's for four-color or eight-color?

Dr. Paiva: So in 2010, we were already using eight colors.

Gary: Okay. This is kind of the clincher question. Of the high-risk patients that have achieved MRD negative status, is there a treatment plan which has the best outcomes?

Dr. Paiva: I have a hard time understanding your question. Let me see if I understood. Is the treatment algorithm according to the MRD stages of high-risk patients? Correct?

Gary: Or if you have a high-risk patient and you give them treatment Plan A or treatment Plan B -- and let's say A is RVD, transplant, maintenance and treatment Plan B is dual transplant plus maintenance, does one treatment plan provide better results? For example, I know that you guys did something -- I thing it was the Spanish group who did something -- I think it was RVD and then you had CT -- it alternated old drugs right after the other and came up with some pretty great results. I'm not sure if it was your group or it was another group, but I saw it at ASH. I was wondering if there is a plan or some kind of a treatment plan. Nobody has come up with the plan that resolves high-risk disease, so is there anything that gives a better response that you've seen given your experience?

Dr. Paiva: Well, that's a very, very good question, perhaps one of the most important. Let me tell you that the study that you are describing, the alternating study that incorporated all or the majority of the effective and available agents for the treatment of elderly myeloma patients was indeed the study that I was just describing that we used the eight-color method and we received super-imposable results for high-risk and standard risk patients with MRD negativity, so it was that specific clinical trial. Now, regarding your question, which again is a critical question, I'm deeply sorry and being completely honest. Unfortunately, there is no answer. The reason why there is no answer is because we need to be extremely cautious on why we moved from a laboratory biomarker into a diagnostic tool or a treatment decision tool. A treatment decision tool is equally important as a new drug, equally important. This means that now that we have further increased the sensitivity of our methods, now that we are standardizing methods, now is really the time to implement such methods in all possible clinical trials to do the biology, to address the biology questions regarding MRD in order to collect prospective data from clinical trials to more patients receiving different treatments, chemotherapy in order to address your question. It’s hard to address the question because it's really addressing the question without having consistent data.

Gary: I guess my point would be is in that trial, as I recall -- one of the things that was interesting is in that trial where you sequenced these drugs, the difference between a high-risk and low-risk group was nonexistent as far as outcomes, which is remarkable.

Dr. Paiva: Indeed. And again, emphasizing that the follow-up of the study still need time, so definitely more mature data will be needed to really make a final statement on that particular analysis, but yes, this trial is very, very safe. Other clinical trials are really showing that the outcome between high-risk and standard risk patients -- well, the poor outcome of high-risk patients is not yet fully overcome, but it is being ameliorated. This means that -- and this is my personal point of view, that as therapy becomes more and more effective, the way we call high-risk now may be different from the way we will call high-risk in the future, plus this will also mean that perhaps -- and everything is complementary, but perhaps more important is risk stratification of diagnosis because therapy has become so much effective, but perhaps more important than that would be really a sensitive monitoring of this positive therapy. To make a final argument on your question and again, really sending the message that as we move along very slowly, as it needs to be done, we cannot rush into precision medicine. We need to ask very safe questions. For example, the research program that was more or less described in the letter of intention would take place for example in a clinical trial. For example, it's being conducted by a Spanish group in which, for the first time ever, we ask very precise clinical questions regarding MRD, which is not at the beginning of the treatment, but it's varying the treatment after a period of maintenance therapy. We ask the questions of whether or not maintenance therapy can be stopped according to the patient's MRD status. So patients that are MRD negative will stop maintenance, whereas patients that continue to be MRD positive, they will continue to receive maintenance therapy. This will allow us to understand these sensitive MRD assessments not performed one single time, but performed throughout the patient's treatment after induction, after transplant, after consolidation, and then after maintenance, and using sensitive techniques, then perhaps change the duration of maintenance, so then really emphasizing on the prospective nature of the clinical questions as well as to be very cautious on the clinical questions as possible using a laboratory biomarker.

Gary: Okay. Well, doctor, thank you so much. One other thing is that you said that if you're not MRD negative, there are clones left over and you're going to take a look at those clones whether it's in high or low-risk.

Dr. Paiva: Yes.

Gary: And evaluate those clones to see what the difference in those clones are. This is actually a question from the Dallas Myeloma Group, the multiple myeloma support group there who asked me to ask this question, and that is, how will this evaluation lead to new treatments?

Dr. Paiva: This is a very pertinent question, a very good question. By understanding the genomic signature of those cells that resist after therapy and then again the importance of really studying the patients longitudinally -- because if we only study dead cells alone, the information would be limited. We need to compare the MRD cells with the diagnostic baseline cells before the patient is entered in therapy to really understand the genes that were deregulated, as well as the genetics of clonal selection by looking into mutations.. Now, if we understand and there is consistent data from patient to patient suggesting that for a particular treatment schema, there is always in the significant number of patients, deregulation, downregulation or upregulation of a given gene or a given pathway, this means that we can then move for example into in vitro systems using myeloma cell lines to manipulate those genes that we identified in the precious patient samples, developed in vitro myeloma cells with the signature of MRD cells, and then test in vitro how to overcome the chemo-resistant profile of those MRD cells either by testing new drugs, targeting a new gene that was so far not targeted in myeloma, or by combining the many different and available agents.

Gary: Thank you, doctor. Jenny, I think there's someone else ready to ask some questions. Is that correct, Jack?

Jenny: Jack, you have a couple of questions.

Gary: I've got one more, but only if I have time.

Jenny: Jack, would you like to go ahead with your questions?

Jack: Yes, I'm on the line. I really appreciate this conversation, although it's quite difficult and I apologize if I'm asking similar questions to what have already been asked. I understand with respect to the eight-antigen or color markers that you're using for flow, are they the same? Have we standardized those eight now? Are they the same that go with the Black Swan Initiative that different institutions now are being trained to use those same eight markers?

Dr. Paiva: Indeed. The platform or the assay we are currently doing today is the assay that we helped develop within the Black Swan Research Initiative.

Jack: Great! Because I know at one point, they talked about ten markers and things like that. I'm sure you've already done some interesting research in your labs where you're looking at effectively the degree of resistance in the high-risk subgroup and you're looking to find whether or not there are some potential targets that make these cells more resistant. I'm wondering if you've already found any or are you looking at any targets, and might we already have some drugs that have been developed to tackle these targets in other cancers?

Dr. Paiva: We have started this concept of MRD as a reservoir of chemo-resistant cells and therefore a clone for intensive research a couple of months, let's say one or two years ago. This has been presented in the American Society of Hematology meeting, I would say two years ago, last year also. And at the time, again limited by less high throughput techniques, we were able to characterize for the first time ever the MRD cells. We have detected that the phenotype is slightly different, as well as the genotype and the transcriptome, but then again, using intermediate, sensitive platforms. We are now also trying to validate this model and again the model for the identification of new drugs or new drug combinations. What we want to do now is really to synergize state-of-the-art technology to address this question. So only now with very sensitive flow will we be able to sort those cells with high accuracy. And also only now with the DNA and RNA sequencing tools, and that's for I would say almost single cells, we will appreciate all the genome and the transcriptome of those cells. Hopefully, this will give us information that so far has never been collected. We believe that in parallel to all the efforts that have been done so far to sequence, the myeloma cells of the patients with newly diagnosed myeloma, we believe that equally important will be to perform those sequencing efforts in clones that are typically not characterized, which is the circulating tumor cells as well as the minimal residual disease cells. We also believe that -- and this into some extent might even be rational -- for example, now they've been back into the newly diagnosed setting. It is common knowledge that for example, gene expression profile signatures are of great value to discriminate between standard versus high-risk patients, but they are different from scientific group to scientific group. Our personal belief in this regard is that those signatures are different because they were obtained with different treatment schemas. They worked in different trials, but they are obtained with different treatment schemas, which means that most likely, this strategy of trying to overcome the chemo resistance of MRD cells will be specific to the common backbone drugs beings used for each particular clinical trial because certainly, the nature of ultra chemo resistant cells will slightly differ according to the different drugs beings used.

Jack: Thank you. I have one other question and it requires you to think about a graph. Along the X-axis, you have standard risk and high-risk. Along the Y-axis, you have MRD negative, and above that, MRD positive. I think the genomic significance that you're looking at in your letter of intent focuses on the top part of that graph, in other words, the MRD positive for standard risk versus high-risk patients. I wonder if ultimately one will need to look at more. For example, are we sure it's better to be a high-risk MRD negative patient than it is to be a standard risk MRD positive patient?

Dr. Paiva: Those I believe, Jack, are two different questions. Regarding the first -- and again, looking into the outcomes we had with the initial follow-up, but of more recent clinical trials, it seems that the outcome of patients with high-risk disease reaching MRD negativity is indeed superior as compared to the outcome of patients with standard risk disease and not reaching MRD negativity, in other words, remaining MRD positive. That would be the answer for your last question. Regarding the first question, absolutely equally important is to characterize the biology. The genetic landscape of ultra chemo resistant cells from high-risk patients will be also to do the same approach in standard risk patients, also to understand the difference in the genetics of chemo resistance between the two patient cohorts, so this would be crucial. Also, and this should also be a research priority to all myeloma scientific groups, as you are probably aware that depth of a response is a key prognostic factor, but it does not afford the same answer to all the patients. In other words, there are patients that remain MRD positive and nevertheless experience very long-term outcomes, which means that we need to understand again the genetic profile of that resistant cell in order to identify clones that are left there after therapy, but that will behave like MGUS clones. These can be there for many years and the patients may even have persistent MRD, may even show the positive M spike in the serum, but the patient could be progression-free for many and many years, and really to dissect or to discriminate between an MRD positive patient that will unfortunately progress sooner from those that fortunately progress much later on in time, if they even progress at all, we believe will arise from two different research groups. Again, first -- and this is what we are discussing here -- to study the biology of those cells; secondly, to perform in-depth research on the immune system to understand how the patient's immune system are able or not to control residual disease levels after therapy.

Jack: Thank you very much, Dr. Paiva. I wish you the very best of success for all myeloma patients.

Dr. Paiva: Thank you.

Jenny: Great, thank you, Jack. Pat, did you have any questions before -- I have a couple of things I would like to ask.  

Jenny: Go ahead, Pat.

Pat: Oh, sorry. See, I'm not familiar with the mute button, so I thought it was off; it was on. I was going, "Can you hear me?" I just have one question and it's related, but it's not. I think in a basic myeloma patient's mind, it's probably related, but they are two different things. Is any of the work you're doing, doctor, helping doctors monitor non-secretors so that we can more closely follow the progress of their myeloma?

Dr. Paiva: Absolutely. Again, I'm really appreciating this now already one hour of debate --

Jenny: It goes by quite fast.

Dr. Paiva: This has been tremendously stimulating hopefully for all of us that are online. This is a very good question. You're probably aware that just recently in 2014, the diagnostic criteria for myeloma was updated by the International Myeloma Working Group. For example, one of the updates was to really remove the need of an M protein to diagnose a patient with multiple myeloma, realizing the existence of the non-secretory patient. Now, for the non-secretory patient, there was always and always from the last five, six, ten years the possibility, as you are probably aware, of using the free light chains that are more sensitive as compared to standard electrophoresis or immunofixation methods to detect the M components. So the free light chains are for information on how to measure the M protein that's around 50% of the non-secretory patients, but certainly to measure the clonal cells in the bone marrow for information in virtually all the patients. It should be emphasized that from a therapeutic point of view, from a cellular point of view, so far we are not seeing differences between the phenotype that is the way we see the cells between secretory versus non-secretory myeloma patients, which means that the technique or the methods we developed, the method we used can be commonly applied, equally applied to both secretory versus non-secretory myeloma patients.

Pat: Thank you. That helps. Jenny, I want to commend you. I know a lot about multiple myeloma, but I'll tell you, this is pretty complicated stuff and you do a great job helping to simplify things. The panel has done a wonderful job I think helping patients understand what's going on because this is pretty technical stuff.

Jenny: Yes, it is, and we appreciate all the good questions -- they're excellent -- and your great responses, Dr. Paiva. What I hear you saying overall is that you want to both capture the baseline through some standard genetic tests and then you want to use this flow cytometry, the next generation, to compare against that baseline to see what genes are regulated, how does myeloma change and morph so you can create a plan of attack to manipulate the genes that are causing those problems. I think one thing that you said that really stood out to me is that you want to test a higher amount of cells so you can look at single cells. I would say to patients who are -- and this is a little complicated, but to patients that need to understand this better, that is so important because that single cell can become the problem over time. It might not be a problem when you first get hit with your induction therapy or even your transplant, but after that's finished, if you have that cell left over, that new cell that maybe wasn't very aggressive before can become the single problem. In my mind, that's why this work is so important.

Dr. Paiva: Well, thank you. For us, it has become I'd say a very passionate research area of our group. Again, we are really committed to study two very rare cells, the first being the circulating tumor cell, which is not contemplated in this research problem, but we are deeply committed to study that cell because we believe that the circulating tumor cell is the one responsible for the disease dissemination. Secondly, to the MRD cell, and for a second now, we close our eyes and we try to not think on the prognostic significance, on the potential role for treatment decisions tailoring therapy, and we only think on biology. If we close our eyes, we can imagine or we can envision that these cells are very few. Only a few have the true potential to resist such effective treatment strategies. These cells are likely quiescent after therapy. They are not producing, in the majority of cases, the M protein, the monoclonal protein. These cells can be dormant for many years, but then again, these cells are the ones responsible for the patient's relapse. These three descriptions or these three features that I just mentioned have been let's say the three hallmarks of the worst cancer cell that has ever been postulated, which are the cancer stem cells. So we truly believe that this MRD cell could be enriched on cancer stem cells. If we are able to really reduce their levels through treatment, if we are able to monitor their sensitivity, and now if we are able to study or to unravel the biology of these cells, hopefully these could be of great value to this disease as well as to other diseases in hematology, so we are truly looking forward to this research problem in our laboratory.  

Jenny: Okay. Perfect! My final question is -- you've talked about your research aims, what you're hoping to accomplish. Could you outline for us the specific milestones in this research proposal and then just give us an idea of the general amount of budget that you would need to accomplish those specific milestones?

Dr. Paiva: The milestones, give me five seconds to give three milestones for this research program. 1. The first would really be to establish ultra sensitive MRD negativity as a surrogate for long-term disease control with high-risk myeloma as well as the biomarkers for every drug approval. This would be the first. 2. The second will be to define for the first time ever the genetic signature of MRD cells in both high-risk as well as standard-risk patients. 3. The third milestone would be to establish the MRD cell as a new model for identification of novel targets, as well as drug development. I think this would be the key three milestones of the research program.

Jenny: And then do you have an idea of what you would need budget-wise to accomplish those three milestones?

Dr. Paiva: Again, being very reasonable and trying to be cost-effective in any way we can. In fact, the way we developed this flow assay was to -- one of the persons that is online asked about the number of colors. I don't remember now his name unfortunately and I'm sorry for that. He asked for the number of colors, eight or ten or more. Certainly, the more, the better, like always, but we need to commit and we need it to be cost-effective so it can be available to all the patients and we need these techniques to be available for all the centers, those that are more specialized, perhaps with also more resources, as well as other centers that don't have the resources of these groups, which means that all the biomarkers that we try to develop here are really with that purpose of being tremendously cost-effective. So if we want to study the therapeutic characteristics of the myeloma cell diagnosis as well as the MRD cell that is there after therapy, if our goal is to perform these longitudinal comparison, these would be two time points in a series of 100 patients, for example, comparing 50 high-risk patients versus 50 standard-risk patients, which means again that a cytogenetic characterization must also be performed in the baseline to identify the two-patient cohorts, then RNA or DNA sequencing studies to be performed both at the baseline, as well as the MRD level, then also for in vitro studies to develop the model of MRD and the transformative new drugs or new drug combinations. I would say that these would be more or less in the range of $1500 to $2000 per patient. Jenny: And you're trying to study 100 patients?

Dr. Paiva: Yes.

Jenny: Perfect. Well, that gives us an idea of scope, I guess, so it's very helpful to have that. Well, I think that one of the most exciting things about your project is that for the first time, this flow test is weighing in on the biology of the disease, which has never really happened before, and the biology of myeloma is so complex. Even within a single myeloma patient, they can have several different kinds of myeloma cells within their own bodies, so the more testing and the more detailed and the more biology you're studying, I think we are far better off having that.

Dr. Paiva: Can you restate the question?

Jenny: I was just saying how significant I think it is that for the first time, you are studying the flow cytometry test to study the biology.

Dr. Paiva: Again, we are extremely hopeful that this new approach may really afford information that was never there before. And then again, I think that really the laboratory as well as the biology should keep the same pace as the improvement in treatment options. It is our opinion that again -- and perhaps coming back from where we started with this radio show, I think the time has come to really improve the response criteria and the biology regarding response criteria in the same level as the essential diagnoses, risk stratification, and treatment options that have been improved in the last five to ten years.

Jenny: Absolutely. Well, we agree and we're so thankful for the work you're doing, the study that's been done by you and your group. We're just very grateful because it's helping discover many new approaches and targets for myeloma. Dr. Paiva, we've kept you way over time today, but we think it's worth it, so we're so thankful for your participation today. Thank you for all you're doing.

Dr. Paiva: It was my great pleasure. It has been one of the most exciting and stimulating hour of my research life. Again, I can assure you that all of us here in our institution work hard every single day to really improve the lives of myeloma patients who have hematological malignancies. It is our really great pleasure to participate in this event.

Jenny: Well, thank you so much. We've very fortunate that there are people like you working so hard for us, so thank you. Now, we'd like to thank you for listening to Myeloma Crowd Radio and the new MCRI series. We know that patients can help support the discovery of a cure and we encourage you to become more involved.