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Send us a textLet's talk about the BCR:ABL negative MPNs! Written by Dr. Sarah Ge (Internal Medicine Resident), reviewed by Dr. Christopher Hillis @HemeHillis (Hematology) and Dr. John Neary @jddneary (General Internal Medicine). Infographic by Dr. Caitlyn Vlasschaert @DrFlashHeart (Internal Medicine Resident).Support the show

Modern science, especially in the genetic and molecular biology spaces, generate vast amounts of data, and require vast amounts of data to be generated for thorough analysis. For example, finding a rare gene mutation such as BCR-ABL as a biomarker for chronic myeloid leukemia is like searching for a needle in a haystack. For a situation like this, dPCR is an ideal method, but high-throughput automation is also needed.Dr. Clarence Lee, Senior Product Manger at Thermo Fisher Scientific, tells how the QuantStudio™ Absolute Q™ AutoRun dPCR suite helps make the benefits of digital PCR available in an easy-to-use high-throughput system. The conversation covers how automation benefits are provided by MAP16 plates, system software, and the AutoRun plate hotel and loading robot. Clarence also talks about customer applications where he sees automation like this being applied to innovate and drive science forward. In the career corner portion, we learn about Clarence's journey from chemist and biophysicist, to roles in industry and his current role as a product manager. He shares what he loves most about his job and what he's most proud of over his career that has spanned several diverse roles. Visit the Absolute Gene-ius pageto learn more about the guests, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System. 

Drs. John Sweetenham and Marc Braunstein discuss practice-changing studies in hematologic malignancies that were featured at the 2024 ASCO Annual Meeting, including the ASC4FIRST trial in chronic myeloid leukemia and IMROZ and CARTITUDE-4 in multiple myeloma.  TRANSCRIPT Dr. John Sweetenham: Hello, I'm Dr. John Sweetenham from UT Southwestern's Harold C. Simmons Comprehensive Cancer Center and host of the ASCO Daily News Podcast. On today's episode, we'll be discussing practice-changing abstracts and other key advances in hematological malignancies that were featured at the 2024 ASCO Annual Meeting. Joining me for this discussion is an old friend, Dr. Marc Braunstein, a hematologist and oncologist from the NYU Langone Perlmutter Cancer Center.  Our full disclosures are available in the transcript of this episode. Marc, it's great to have you back on the podcast again. There were some important studies in the heme space at the Annual Meeting this year, and we're very pleased that you're able to share your takeaways.  Dr. Marc Braunstein: Thank you, John. It's great to be back again. Dr. John Sweetenham: Let's start out, Marc, with LBA6500. This abstract reports the primary results of the ASC4FIRST trial, and this was a trial comparing asciminib with investigator selected tyrosine kinase inhibitors in newly diagnosed patients with chronic myeloid leukemia. Could you tell us a little about the trial and how you think it's going to impact clinical practice? Dr. Marc Braunstein: Absolutely. So, asciminib is an oral tyrosine kinase of the ABL kinase domain. As we know in CML, the BCR-ABL translocation is characteristic of the disease, and asciminib is approved for chronic phase CML with a T315I resistance mutation or for patients who have received 2 or more prior lines of therapy. So the ASC4FIRST trial was a randomized trial of 405 patients with newly diagnosed chronic phase CML who are randomized one to one to receive either asciminib at 80 milligrams once daily, or investigator's choice of a first generation TKI imatinib or one of three second generation TKIs nilotinib, dasatinib, or bosutinib. The primary endpoint of the study was the major molecular response, or MMR, at 48 weeks. Pretty much, the study met its primary endpoint with a 67% rate of MMR at 48 weeks, with asciminib versus 49% in patients treated with the investigator's choice of TKI. And in addition, the major molecular remission or MMR of 4.5, which is a deep remission, those rates were higher as well, with asciminib versus investigator's choice at a rate of 39% versus 21% when comparing the groups. Furthermore, when we looked at toxicity, there were fewer grade 3 or higher adverse events, with the asciminib at 38% versus either 44% with the first generation, or 55% with the second generation TKI, and fewer discontinuations as well with asciminib.  So I think this abstract is practice-changing. I think it offers compelling data to use asciminib upfront for chronic phase CML. Those who don't agree with that sentiment might argue that we want to see longer term follow up. There's a planned follow-up at 96 weeks. We would want to see the rate of progression to acute myeloid leukemia and of course overall survival as well. But I think the abstract certainly shows an improvement in outcomes with asciminib versus our current array of TKIs. Dr. John Sweetenham: Yeah, I think it certainly is, at least at minimum, potentially practice changing. I agree with you. Just one question, and this may be a little bit speculative, but do you have any thoughts about treatment free survival with asciminib and how that might line up against some of the other TKIs? Dr. Marc Braunstein: Yeah, that's a great question. The abstract did not necessarily address that, patients were treated until progression, but we know that with the current landscape of TKIs, that in patients who have achieved a deep MR of 4 or 4.5 for at least 2 years who discontinue their TKI, the rate of relapse is about 50%. The current study, the ASC4FIRST, doesn't address that, but I think it's a really good question about whether, for those patients who have achieved a deep remission, whether they can eventually stop asciminib down the line. Dr. John Sweetenham: Yeah, I guess it's one of those ‘watch this space' things.  So we'll see how the data mature out. And let's move on to what I think is another potentially practice-changing study, at least in certain parts of the world. And that's [the] LBA7000 study in classical Hodgkin lymphoma. As you remember, this was a German Hodgkin lymphoma study group trial which looked at the tolerability and efficacy of a novel regimen, BrECADD versus eBEACOPP for patients with advanced stage classical Hodgkin lymphoma in their study, which is known as GHSG HD21. Can you give us your thoughts and take home messages from this trial? Dr. Marc Braunstein: Yeah, John, absolutely. So the German HD21 study is a phase 3 study of 1,500 patients with classical Hodgkin lymphoma. The majority were stage 3 or 4, 84%, that compared two regimens BEACOPP to BrECADD. The major difference between these 2 groups being that the newer BrECADD regimen swaps out bleomycin for brentuximab vedotin, which is an anti-CD30 antibody drug conjugate. Also, in the BrECADD regimen they eliminate vincristine that's incorporated into BEACOPP. Those are kind of the global differences between these 2 regimens. And when comparing these, they looked at the primary endpoint of progression-free survival. Of note, in this study there was a PET adjusted approach where if patients achieved interim PET negativity after 2 cycles, that was followed by an additional 2 cycles of their treatment as opposed to 4 cycles if they were PET positive after the initial 2 cycles of their respective treatment. And of note, there were similar rates of PET2 negativity between both arms, about 58% in both arms.  So at a median follow-up of 48 months, the 4-year progression-free survival was significantly better with the brentuximab containing BrECADD regimen at 94% versus 91% with a hazard ratio of 0.66. And the overall survival of the BrECADD arm was 98.6%, which is very high and impressive. The 4-year overall survival was similar between the arms at around 98%, but of note, there were fewer severe adverse events with BrECADD, the brentuximab containing arm versus BEACOPP at about 42% versus 59% and interestingly less peripheral neuropathy with the brentuximab containing BrECADD. So we're doing extremely well in treating advanced stage classical Hodgkin lymphoma. So the bar is set very high. But in this study, the rates of progression-free survival and overall survival are very impressive.  While these intensive regimens tend to be used outside of the U.S., there are several notable benefits of the study, including greater than 50% PET2 negativity and high rates of progression-free survival at 4 years. In discussing this abstract, it's worth noting that there are other competing regimens, if you want to call it that, that are more commonly used in the U.S. So the ECHELON-1 study looked at brentuximab AVD compared with ABVD with bleomycin and it was a 94% versus 89% 6-year overall survival rate favoring the brentuximab containing A+AVD regimen. And lastly, more recently, the SWOG S1826 study that hasn't been published but was presented in abstract form looked at nivolumab AVD versus brentuximab AVD at a median follow up of 12 months showed a progression-free survival of 94% versus 86%. And that study still has yet to be published and needs to mature. But both of those regimens are in the NCCN guidelines. So, we're definitely pushing the bar higher in terms of improving responses in treating advanced classical Hodgkin lymphoma. Dr. John Sweetenham: I think that there's no question that these results from BrECADD are very impressive. But I'm taken back to what I think has been a kind of philosophic discussion in Hodgkin lymphoma now for a number of years about balancing disease control and efficacy against the potential short-term and long-term toxicity of the regimens, particularly when you have very effective salvage therapies for those patients who may suffer a relapse. So I think that this is a discussion over whether you take a very intensive, upfront approach to Hodgkin lymphoma versus something that may be less and slightly less intensive. I suspect that's a discussion that's going to continue for a long time. I don't know what you feel, but my own feeling about this is that this study will likely have a major influence over treatment of Hodgkin lymphoma, particularly in western Europe. Less likely in the US.., I would think. I don't know what your thoughts about that are. Dr. Marc Braunstein: Well, it's a great question. In SWOG S1826, that study did include pediatric patients. In HD21, the median age was 31 and did not include pediatric patients. So I think we have to be selective in terms of fitness and which patients may be better suited for different regimens. But I think what all these studies show is certainly when we incorporate novel immunotherapies, whether it's brentuximab vedotin, nivolumab, we improve progression-free survival and even overall survival. Dr. John Sweetenham: Absolutely.  So let's shift gears now and take a look at Abstract 7500, the IMROZ study. This was the study of isatuximab, bortezomib, lenalidomide and dexamethasone versus VRD alone for transplant ineligible patients with newly diagnosed multiple myeloma. I know we discussed this in our preview podcast a few weeks back, Marc, but I just wonder now, having seen the data in more detail, what do you think of the important takeaways? And again, are we looking at a new standard of care? Dr. Marc Braunstein: You know, there are many standards of care in multiple myeloma, but we're always looking to make improvements on the regimens we have at our disposal. So, just to recap, IMROZ is a phase 3 randomized study of the anti-CD38 monoclonal antibody isatuximab with the backbone of bortezomib, lenalidomide, dexamethasone or VRD versus VRD alone, specifically, in transplant ineligible newly diagnosed multiple myeloma patients age less than 80. They studied 446 patients in this study, randomized 3 to 2 to Isa-VRD versus VRD alone, with the primary endpoint of progression free survival. Now, similar to other studies where they included a monoclonal antibody up front, the study met its primary endpoint of improving progression-free survival with the quad regimen containing the monoclonal antibody isatuximab versus VRD alone.  So what was interesting about the study, it's really the first of its kind to be presented that specifically looked at transplant ineligible patients, which is presumably a less fit or perhaps more frail population that wouldn't go on to consolidation with stem cell transplant. And in this study, the progression-free survival at 5 years was 63% versus 45%, clearly superior when you included isatuximab. And the rates of complete remission and MRD negativity were all significantly improved, too. However, that was also met with slightly more grade 3 or higher treatment emergent adverse events, 92% versus 84% in the control arm. There are also 11% grade 5 treatment emergent adverse events with the isatuximab group versus 5.5% with VRD alone. Although there was no major difference in treatment discontinuation. One small caveat worth noting, too, is that high-risk patients in this study, when presented at ASCO, did not necessarily show a difference in benefit, although there wasn't necessarily a detriment either.  So, John, I think that clearly quadruplet regimens are superior in outcomes of efficacy to triplets, even in transplant-ineligible patients. But I think we have to tailor these treatments to individual patients because I think when it comes to transplant-ineligible patients, it's a spectrum of patients who may be more or less fit for quad regimens versus triplet regimens. It's also worth noting, though, that in this study, the patients are really only getting a quad regimen for 4 cycles. They get their Isa-VRD, and then you drop the bortezomib.  So when we think about quads, it's not that they're getting the quad regimen indefinitely, it's really for the induction cycles. But still, I think we have to be aware of potential safety issues. Dr. John Sweetenham: Okay, great. And let's stay on the theme of multiple myeloma, Marc, and talk a little bit about Abstract 7504, which was a subgroup analysis of the CARTITUDE-4 study. This is a report on the use of ciltacabtagene autoleucel versus standard of care in patients with functional high risk multiple myeloma. Can you give us your thoughts on this and maybe put it into a bit of context for us?  Dr. Marc Braunstein: Absolutely, John. It's really a great time to be in the field of multiple myeloma. We're making tremendous progress, but when we think about one of the unmet needs, it's just consistently the high-risk patients who have shorter responses and are at higher risk for poorer outcomes. Just to review, cilta-cel is one of the 2 available anti-BCMA CAR T-cell products available for the treatment of relapsed or refractory multiple myeloma. Very recently, the FDA approved cilta-cel for lenalidomide refractory patients after 2 or more prior lines of therapy based on the CARTITUDE-4 study, which was published by San-Miguel and colleagues in New England Journal of Medicine in July 2023. And that study randomized 419 patients with multiple myeloma with 1 to 3 prior lines of therapy to receive either cilta-cel or physician's choice of standard of care, which was either 1 of 2 triplet regimens, a pomalidomide, bortezomib, dexamethasone or daratumumab, pomalidomide and dexamethasone. It's worth noting that about 25% of the patients in the CARTITUDE-4 study had prior anti-CD38 antibody treatment previously and the carfilzomib was not included in one of the standard-of-care arms, and we know that those regimens containing carfilzomib do increase survival in relapsed myeloma.  Nevertheless, the primary outcome of progression-free survival was not reached in the CAR T-cell arm versus 11.8 months in the standard-of-care arm, with a significant reduction in progression of 74%. So clearly a positive study and CAR T-cell therapy is included in the NCCN guidelines for patients who have an early relapse from their myeloma. The current abstract by Costa et al focused specifically on a subgroup of 79 patients from CARTITUDE-4 in second line of treatment and looked at what they called functional high-risk myeloma, defined as progression of disease within 18 months of initial treatment or after stem cell transplant. Again, the study showed a retained benefit of cilta-cel with significant improvement in progression-free survival either not reached or 12 months with the control standard of care arm, as well as complete remission rate and rates of MRD negativity of 65% versus 10% in the control.  The overall survival outcome was still immature and not presented. Nevertheless, cilta-cel is clearly superior to standard-of-care triplet regimens. I think that for patients with high risk, they clearly derive a benefit from CAR T-cell therapy if they have short progression-free survival after initial therapy.  Dr. John Sweetenham: Thanks, Marc. So let's round this out by talking about another area of unmet need, I guess in a way in a difficult to treat patient group. And that's Abstract 7007, the SYMPATICO study. This is a study which looks at the efficacy and safety of ibrutinib and venetoclax in patients with mantle cell lymphoma who had a mutated TP53. Can you just briefly review this for us and tell us what you think we should be taking away from this studys? Dr. Marc Braunstein: So, mantle cell lymphoma typically has an aggressive behavior, but the subgroup of patients with a P53 mutation tend to have the poorest outcomes and do represent an area of unmet need. Although BTK inhibitors are making important improvements in mantle cell lymphoma, they have yet to be approved in newly diagnosed mantle cell lymphoma. Acalibutinib and zanubrutinib are FDA-approved BTK inhibitors for previously treated mantle cell lymphoma. Ibrutinib was withdrawn from the market in the U.S. for mantle cell lymphoma. Dr. Michael Wang's group presented late-breaking data from the phase 3 SYMPATICO trial at ASH 2023, in which 267 patients with relapsed refractory mantle cell lymphoma were randomized to receive either ibrutinib plus the BCL2 inhibitor venetoclax or ibrutinib plus placebo after 1 to 5 prior lines of therapy. And that study showed a 32 versus 22 months progression-free survival at a median follow up of 51 months. The current abstract, also by Dr. Wang and colleagues, looked at the subgroup of patients who had a P53 mutation and included an open label cohort of 44 patients in the first line of treatment and a relapse refractory cohort of 75 patients, and compared this subgroup of patients with P53 mutation to those without. When we look at the outcomes, the patients who did not have a P53 mutation clearly did better in terms of progression-free survival being not reached in first-line treatment compared to 22 months progression-free survival in those patients with first-line [treatment] with a P53 mutation. As well as in the relapsed refractory setting, the PFS without the P53 mutation was 47 months versus 21 months with the mutation. However, when you look at these patients treated with ibrutinib and venetoclax comparing whether they got treated in first line or the relapse refractory setting, the overall response rates are very similar at about 80% to 90% and the CR rates were very similar at about 55% to 58%, which to me suggests that although patients with P53 mutation do worse than those without it, whether they're treated in the first-line or the relapse setting with this combination of venetoclax, they tend to do somewhat similar, suggesting that you can overcome resistance to prior therapy in the relapse setting. So I think further data are certainly warranted to explore the role of combination therapies that include novel agents such as BTK inhibitors in the first line setting.  It's worth noting that the TRIANGLE study was recently published, and this study looked at including ibrutinib at various phases, including at induction in combination with intensive chemotherapy and during the maintenance phase. And that study showed encouraging outcomes in patients who received ibrutinib even without stem cell transplant compared to those who received stem cell transplant. So the role of BTKIs in mantle cell lymphoma is certainly evolving, and I think it offers a very effective intervention without the same kind of toxicities we see with cytotoxic chemotherapy that's traditionally used in mantle cell lymphoma. But I think the subgroup of patients with P53 mutation in this disease still represent an area of unmet need that unfortunately have worse outcomes. But novel agents may be able to overcome some of those adverse outcomes. Dr. John Sweetenham: Yeah, I agree. I think these are intriguing data, and obviously it needs more follow-up and probably more prospective studies. But nevertheless, I think there are some signals there for sure that need to be followed up on.  Marc, as always, it's great to have your insights on key advances in the heme space from ASCO. An important year this year, and we really appreciate your time and effort in sharing with us your thoughts on what we've learned this year. So thank you as always. Dr. Marc Braunstein: My pleasure. Dr. John Sweetenham: And thank you to our listeners for joining us today. You'll find links to the abstract discussed today in the transcript of this episode. Finally, if you value the insights that you hear on the ASCO Daily News Podcast, please take a moment to rate, review and subscribe wherever you get your podcasts.   Disclaimer: The purpose of this podcast is to educate and to inform. This is not a substitute for professional medical care and is not intended for use in the diagnosis or treatment of individual conditions. Guests on this podcast express their own opinions, experience, and conclusions. Guest statements on the podcast do not express the opinions of ASCO. The mention of any product, service, organization, activity, or therapy should not be construed as an ASCO endorsement.   Find out more about today's guest:  Dr. Marc Braunstein  @docbraunstein    Follow ASCO on social media:   @ASCO on Twitter   ASCO on Facebook   ASCO on LinkedIn     Disclosures:  Dr. John Sweetenham:  Consulting or Advisory Role: EMA Wellness  Dr. Marc Braunstein:  Consulting or Advisory Role: Pfizer, Bristol-Myers Squibb/Celgene, Adaptive Biotechnologies, GlaxoSmithKline, ADC Therapeutics, Janssen Oncology, Abbvie, Guidepoint Global, Epizyme, Sanofi, CTI BioPharma Corp  Speakers' Bureau: Janssen Oncology  Research Funding (Institution): Janssen, Celgene/BMS   

Dr. John Sweetenham and Dr. Marc Braunstein look ahead at key abstracts across the spectrum of hematologic malignancies that will be presented at the 2024 ASCO Annual Meeting, including the OPTIC trial in chronic myeloid leukemia, treatment options for transplant-ineligible patients with multiple myeloma, and the 7-year analysis of the ECHELON-1 trial in classical Hodgkin lymphoma. TRANSCRIPT Dr. John Sweetenham: Hello, I'm Dr. John Sweetenham from UT Southwestern's Harold C. Simmons Comprehensive Cancer Center and the host of the ASCO Daily News Podcast. I'm delighted to be joined again this year by Dr. Marc Braunstein, a hematologist and oncologist at the NYU Perlmutter Cancer Center in New York. We're going to be discussing some of the key abstracts in hematologic malignancies that will be featured at the 2024 ASCO Annual Meeting.  Our full disclosures are available in the transcript of this episode, and disclosures of all guests on the podcast are available at asco.org/DNpod.  Marc, it's great to have you back on the podcast. Dr. Marc Braunstein: It's a pleasure to be back, John.  Dr. John Sweetenham: There are some exciting abstracts to be presented at this year's meeting, and I would like to begin, if we can, with Abstract 6501. As you know, this reports the four-year results from the OPTIC trial of ponatinib in patients with chronic-phase CML and the T315I mutation. Can you tell us about the trial and about these latest follow-up results? Dr. Marc Braunstein: Sure. Well, we've made tremendous progress in managing patients with CML in the past two decades using these oral tyrosine kinase inhibitors such as ponatinib. Ponatinib is a third-generation TKI that has activity in both Philadelphia-positive ALL as well as CML, and can overcome the resistance mutation you mentioned, called the T315I mutation, which is sometimes found following prior TKI therapy. The OPTIC study is a multicenter phase 2 randomized study of various doses of ponatinib in 283 chronic phase CML patients who had received 2 or more prior lines of therapy or those who had the presence of a T315I mutation, with the current analysis examining the major remission at 48 months, PFS, as well as OS. Of note, in this study, after patients have achieved a major remission with a transcript level of 1% or less, the study allowed for dose reduction of ponatinib from the original dose of either 45 milligrams or 30 milligrams to a reduced dose of 15 milligrams.  So, when we look at the results, we find that the patients who had the highest overall response rates and higher rates of molecular remission were those who received the 45-milligram dose. And remember, these patients were allowed to be dose-reduced to the 15-milligram dose once they achieved a molecular remission of 1% or less. In addition, the rates of overall survival were highest in the 45-milligram dose as well. When looking at the T315I subgroup, the rates of molecular remission, the depth of remission, and the rates of progression-free survival, in general, were lower in that subgroup, but still higher in the 45-milligram dose than the 35- milligram dose.  Furthermore, when looking at the rates of treatment-emergent adverse events leading to discontinuation, they were 8% in the 45-milligram dose compared to 14% in the 30-milligram dose and 5% in the patients who only received the 15-milligram dose. The authors have concluded that the 45-milligram dose, with the potential to be reduced to 15 milligrams after achieving 1% or less of the BCR-ABL transcript level, seems to be the right balance between efficacy and safety.  Dr. John Sweetenham: Thanks, Marc. In the longer term, do you think that this study will, in any way, affect the position of ponatinib in the treatment algorithm for CML? Is it going to remain as a second or third-line option, or do you think there's any chance it will be moved up? Dr. Marc Braunstein: Well, that's a great question. There are other TKIs, such as asciminib, that also target the T315I mutation, and that mutation tends to develop after prior first-line or second-line TKI therapy. But given its activity in both ALL and CML, I think it's certainly reasonable to expect that ponatinib will be used in earlier lines of therapy given its efficacy in later lines. Dr. John Sweetenham: Let's change gears and move the focus to acute myeloid leukemia. There has been a lot of discussion around frailty in many different malignancies, but the impact of frailty on outcomes in AML is maybe something that hasn't been quite so well studied. In Abstract 6506, investigators did a population-based study in Ontario, Canada, that assessed the patient's frailty risk and the impact that might have on outcomes. What are your takeaways from this study, and how do you think these data will help optimize treatment decisions?  Dr. Marc Braunstein: Yeah, I'm glad we're talking about this abstract John, because frailty scores are increasingly being used in hematologic malignancies to help guide goals and intensity of care. And as opposed to using age or performance status alone, these composite frailty assessment tools, such as the MFI tool that they used in this particular study, take into account multiple variables that are both physiologic, such as the patient's comorbidities, as well as social, and what kind of support system do they have, and things of that nature. And that accounts for their overall fitness. So, in this retrospective cohort study that was a population-based study in Ontario between 2006 and 2021, they looked at 5,450 patients retrospectively with acute leukemia and grouped those patients into 3 categories based on this frailty index. Patients who are either fit, somewhere in the middle between fit or frail, which they call pre-frail, or frail. And they looked at outcomes such as overall survival, comparing patients who got intensive chemotherapy regimens for induction or those who got non-intensive therapy for induction. Patients in either group could have been assigned to either fit, pre-frail, or frail although there are much more fit patients than those who got intensive induction.  And so, looking at their findings, it was noted that patients who were in the frail category, not entirely unexpectedly, had lower overall survival when compared to those who were fit or pre-frail. I think the value of a study like this is not just to highlight the benefit of frailty scores to help predict which patients may ultimately have a shorter survival, but also to help potentially guide which patients may be more suitable for intensive versus less intensive induction. I will note that this study was conducted in an era where we didn't have the same sorts of less intensive induction that are very effective in less fit patients, such as the combination of azacytidine and venetoclax, which is commonly used in less fit patients nowadays. So, the study may encompass patients who didn't have access to that therapy because it wasn't available during that time. But I think it still, overall, does highlight the fact that assessing fitness or frailty in acute myeloid leukemia is important for predictive value. Dr. John Sweetenham: I agree. Marc, I don't know what your thoughts are on this, but it goes either way. I mean, I think that, if I remember the numbers correctly, 25% of fit patients received non-intensive therapy. So, is there a missed opportunity there for that group of patients who actually may have tolerated the intensive therapy but it was never offered? Dr. Marc Braunstein: That's an excellent point, John, and I think that highlights the importance of frailty indices because they take into account much more than one particular factor, or even just a subjective assessment of the patient in real time when they're first presenting. And they may have disease-specific features that are decreasing, say one element of their assessment such as their performance status. So, really taking these composite fitness scores into account may actually allow you to escalate therapy in a patient who may actually be fit but maybe perceived as less fit when they present. Dr. John Sweetenham: Yeah. So, I think, as you mentioned, there are better treatment options out there now maybe than there were at the time this study was conducted. Nevertheless, there may still be that opportunity for more intensive therapy for some of these patients when they are more holistically assessed.  Let's move on and switch gears once again and talk about a study in multiple myeloma, the so-called IMROZ study, which is Abstract 7500. So, this is a study looking at treatment options for transplant-ineligible patients with newly diagnosed multiple myeloma. Some of these patients may not have a chance for subsequent therapy if they are not eligible for transplant. What are your thoughts on this study? Do you think we're closer to a new standard of care for patients who are not going to proceed to an autologous stem cell transplant?  Dr. Marc Braunstein: It seems like every year there's a new standard of care for newly diagnosed multiple myeloma because there's so much data emerging, which is just wonderful. So, I think as background, at the 2023 ASH meeting, the IsKia study was presented, which is a randomized phase 3 study in newly diagnosed transplant-eligible patients. And that was using isatuximab with carfilzomib, lenalidomide, and dexamethasone upfront and that study did show a benefit in terms of reducing minimal residual disease compared to carfilzomib, lenalidomide, and dexamethasone alone. But that study was looking at fit newly diagnosed patients who were going on to stem cell transplant. Right now, the standard of care for patients who are not eligible for transplant is generally to use a 2 or 3-drug regimen, such as daratumumab, lenalidomide, and dexamethasone, based on the phase 3 MAYA study. But this study is really unique in that it looks at using a quadruplet regimen in patients who are transplant ineligible or not intended to go for transplant.  So, the phase 3 IMROZ study was a randomized study of 446 patients that compared isatuximab, bortezomib, lenalidomide, and dexamethasone to bortezomib, lenalidomide, and dexamethasone alone. So, a quad versus a triplet regimen. The primary endpoint in this study was progression-free survival, but they also looked at secondary endpoints, such as complete response rate and minimal residual disease negativity.   Just to quickly highlight the results and then discuss the standard of care, the median duration of treatment in this study was 53 months in the quad regimen and 31 months in the control arm. At a median follow-up of about 60 months, the progression-free survival was not reached with the quad regimen versus 54 months in the triplet, and that was a significant difference. In addition, the safety profile was pretty much consistent with the class, there were a bit more grade three or higher treatment-emergent adverse events with the ESA-containing regimen, 92% versus 84%, but no difference in adverse events leading to discontinuation in either arm.   So, this study is certainly compelling in terms of using quadruplet-based regimens that contain an anti-CD38 monoclonal antibody for newly diagnosed patients who are not intended to undergo transplant. I think at the meeting, I will be interested to see the patient population that was included. Patients who are over the age of 80, for example, are excluded. So, I would like to know more about their fitness level and performance status. But I think it's clear, John, that using quad regimens over triplet regimens is just consistently superior in terms of efficacy outcomes. Dr. John Sweetenham: Right. I guess that, even though maybe we can't focus on the specific agents right now, it looks as if quad regimens are going to be the standard of care regimens for the future in this group. Do you think that is fair?  Dr. Marc Braunstein: Very likely. Dr. John Sweetenham: Absolutely. Well, that's a pretty challenging group of patients.   And so to move on again, let's talk about another, perhaps equally challenging group - patients with mantle cell lymphoma, particularly those who carry certain mutations. The so-called SYMPATICO study, which is reported in Abstract 7007, presents data on the efficacy and safety of ibrutinib and venetoclax in patients with mantle cell lymphoma who carry a TP53 mutation. We know that this mutation confers a high risk of early progressive disease and poorer outcomes when these patients are treated with standard chemoimmunotherapy for mantle cell. Trials to date have been limited to small single-arm studies. Can you tell us a little bit about this study and the outcomes and what you think it means for the future?  Dr. Marc Braunstein: As a background, although BTK inhibitors such as ibrutinib have yet to be approved for newly diagnosed mantle cell lymphoma, acalabrutinib and zanubrutinib, which are second-generation BTK inhibitors, are FDA-approved for previously treated mantle cell lymphoma. Ibrutinib was withdrawn from the market. The lead author of this abstract, Dr. Michael Wang, had presented a late-breaking data from the phase 3 SYMPATICO trial at ASH last year, in which 267 patients with relapsed or refractory mantle cell lymphoma after one to five prior lines of therapy were randomized to receive the combination of ibrutinib plus the BCL-2 inhibitor venetoclax or ibrutinib plus placebo. That study showed there was a 32 versus 22-month progression-free survival with a hazard ratio of 0.65 at a median follow-up of 51 months, indicating the PFS benefit of the combination of ibrutinib and venetoclax compared to ibrutinib with placebo.   So that leads us to this subgroup analysis in the current study being presented at ASCO, in which they looked at a subgroup of patients with mantle cell lymphoma who are at very high risk for treatment failure and early relapse - those are patients who have a mutation in TP53, which again is high risk for treatment failure. This abstract examined an open-label cohort of 44 first-line patients, as well as 75 patients who were in the relapse/refractory cohort, and compared to patients who either did or did not have the P53 mutation. When we look at the progression-free survival outcomes, the median progression-free survival in the first-line cohort of patients who did not have a P53 mutation was not reached, whereas those with the P53 mutation had a median progression-free survival of 22 months, which is still meaningful but still less than those who did not have a P53 mutation. Which again is not entirely unexpected. But the overall response rate of the combination of ibrutinib and venetoclax was very high at 90%, and the median duration of response was about 21 months.  Now comparing this to the relapse/refractory cohort, in those without a P53 mutation, the progression-free survival of the combination of ibrutinib and venetoclax was about 47 months versus those who don't have the P53 mutation was about 21 months with an overall response rate of 80%. I think one takeaway looking at this comparison of the first-line and relapse/refractory setting is that patients seem to do very similar in terms of overall response rate and progression-free survival, whether they were in the first line or in the later lines of treatment if they had the P53 mutation, which says that the combination of ibrutinib and venetoclax is effective no matter which phase of the disease the patient might be in, indicating its overall activity and being strong.    Dr. John Sweetenham: Yeah, I thought that was an interesting observation, actually, how similar the outcomes were in those two groups.  Dr. Marc Braunstein: No, I agree. And I think although patients with TP53 mutations did comparatively worse than those without the mutation according to progression-free survival, overall response rate, or complete remission rates, they did seem to be similar whether a patient was in first-line or relapsed refractory if they were P53 mutant and were treated with this combination. So, I think we need further data in the first line, such as the data that's awaiting publication from the TRIANGLE study, which is examining upfront ibrutinib. But certainly, BTK inhibitors have significant activity in either the first line or the relapse setting of mantle cell lymphoma.  Dr. John Sweetenham: Great. Thanks, Marc.  Let's wind up with one more abstract, and this is Abstract 7053. It's a 7-year analysis of the so-called ECHELON-1 study. This was a study comparing the standard of care, ABVD, with the same regimen with bleomycin substituted by brentuximab vedotin for patients with previously untreated advanced-stage classical Hodgkin lymphoma. The study at the time it was originally reported, resulted in a significant practice change in the first-line therapy of Hodgkin's lymphoma. We now have mature follow-up. What are your take-homes from this study? Dr. Marc Braunstein: The ECHELON-1 study has certainly been a practice-changing clinical trial where, as you said, brentuximab with the backbone of AVD was compared to ABVD, which was the prior standard. And this was examined in newly diagnosed patients with classical Hodgkin lymphoma who were at advanced-stage, stage 3 or 4. The publication, first of the progression-free survival, and more recently, in the New England Journal of Medicine in 2022, where we saw the 6-year overall survival was 94% with the brentuximab-containing arm versus 89% in the control arm, established the brentuximab AVD, or otherwise called AAVD, as the standard of care in advanced stage newly diagnosed classical Hodgkin lymphoma. The current study is now reporting 7-year follow-up on about 1,300 randomized patients who were enrolled in this impressive study.   Though at a median follow-up of 89 months now, the 7-year overall survival was quite similar, 94% versus 89%, again favoring the brentuximab-containing arm. In particular, this was driven by patients who had stage 4 disease or those patients who were aged less than 60 in subgroup analyses. So, what I take away from this abstract in the 7-year follow-up of the ECHELON-1 is that brentuximab with AVD remains the standard of care for previously untreated advanced-stage classical Hodgkin lymphoma. It is worth noting that the SWOG S1826 study that was presented at ASCO last year compared nivolumab with AVD compared to brentuximab AVD and did show a slight PFS advantage of 94% versus 86% with nivolumab AVD. Obviously, these were different studies with different patient populations enrolled, so we're really just cross-comparing different studies. But I think brentuximab AVD, given the survival benefit that is retained now at seven years in the current abstract, still remains the standard of care for advanced-stage classical Hodgkin lymphoma. The role of immune checkpoint inhibitors like nivolumab is making headway in terms of treating newly diagnosed patients as well. Dr. John Sweetenham: Yeah, thanks, Marc. I mean, one of the observations that I thought was of interest in this study was the outcome for patients who were PET-2 positive, when you compare AAVD and ABVD. It does seem as if even in those patients who are PET-2 positive, having had AAVD, they still apparently have a better outcome than those who received ABVD in that situation who were PET-2 positive. So, I think that's another interesting observation. I'm not quite sure what it means, except speaking to the overall superior efficacy of that regimen. Dr. Marc Braunstein: You make a great point, John, because it's worth noting that in ECHELON-1, a PET scan was done after cycle 2, but the study was not PET-adapted. So even if you had a positive PET, you continued for the full six cycles of treatment. But PET-2 status is often used in various studies of Hodgkin lymphoma to guide whether to give additional cycles or escalate therapy. So, I think the benefit of presenting those subgroups is that even if you were PET-2 positive, you still did better by continuing on the brentuximab-containing regimen. Dr. John Sweetenham: Yeah, exactly. I mean, the other important takeaway message, I think, is that the outcome for patients with advanced Hodgkin lymphoma seems to continue to steadily improve, which is great news and also really remarkable. And I'm excited to see there may be some additional data presented at one of the late-breaking abstracts in this year's meeting, so it will truly be interesting to see what that shows us as well.  Dr. Marc Braunstein: Incredible. Dr. John Sweetenham: Well, Marc, as always, thank you for sharing your insights with us today on the ASCO Daily News Podcast. We look forward very much to hearing the updated data from these abstracts at the meeting.  Dr. Marc Braunstein: As do I and thank you so much for inviting me again.  Dr. John Sweetenham: And thank you to our listeners for joining us today. You'll find links to the abstracts discussed today in the transcript of this episode. Finally, if you value the insights that you hear on the ASCO Daily News Podcast, please take a moment to rate, review, and subscribe wherever you get your podcasts. Disclaimer: The purpose of this podcast is to educate and to inform. This is not a substitute for professional medical care and is not intended for use in the diagnosis or treatment of individual conditions. Guests on this podcast express their own opinions, experience, and conclusions. Guest statements on the podcast do not express the opinions of ASCO. The mention of any product, service, organization, activity, or therapy should not be construed as an ASCO endorsement.   Find out more about today's guest: Dr. Marc Braunstein @docbraunstein   Follow ASCO on social media:  @ASCO on Twitter  ASCO on Facebook  ASCO on LinkedIn    Disclosures:  Dr. John Sweetenham: Consulting or Advisory Role: EMA Wellness   Dr. Marc Braunstein: Consulting or Advisory Role: Pfizer, Bristol-Myers Squibb/Celgene, Adaptive Biotechnologies, GlaxoSmithKline, ADC Therapeutics, Janssen Oncology, Abbvie, Guidepoint Global, Epizyme, Sanofi, CTI BioPharma Corp Speakers' Bureau: Janssen Oncology Research Funding (Institution): Janssen, Celgene/BMS

Patients with BCR-ABL negative myeloproliferative neoplasms (MPNs) are at risk of progressing to accelerated-phase or blast-phase disease (MPN-AP/BP). Several mutations, including... The post Accelerated-phase MPNs: mutational landscape, challenges, and novel treatment strategies being explored appeared first on VJHemOnc.

This month on Episode 45 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the February 3rd and February 17th issues of Circulation Research. This episode also features an interview with Dr Hind Lal and Dr Tousif Sultan from the University of Alabama at Birmingham about their study Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation.   Article highlights:   Pi, et al. Metabolomic Signatures in PAH   Carnevale, et al. Thrombosis TLR4-Mediated in SARS-CoV-2 Infection   Cai, et al. Macrophage ADAR1 in AAA   Koide, et al. sEVs Accelerate Vascular Calcification in CKD   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cynthia St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting the articles from our February 3rd and 17th issues of Circulation Research. I'm also going to have a chat with Dr Hind Lal and Dr Tousif Sultan from the University of Alabama at Birmingham about their study, Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. But before I get to the interviews, here are a few article highlights.   Cindy St. Hilaire:        The first article I want to highlight comes from the laboratory of Dr Peter Leary at the University of Washington, and the title is Metabolomic Signatures Associated With Pulmonary Arterial Hypertension Outcomes. Pulmonary Arterial Hypertension or PAH is a rare but life-threatening disease in which progressive thickening of the walls of the lung's blood vessels causes increased blood pressure and that increased blood pressure ultimately damages the heart's right ventricle.   Interestingly, progression to heart failure varies considerably among patients, but the reasons why there is variability are not well understood. To find out, this group turned their attention to patient metabolomes, which differ significantly from those of healthy people and thus may also change with severity. Blood samples from 117 PAH patients were analyzed for more than a thousand metabolites by mass spectrometry and the patient's progress was followed for the next three years. 22 patients died within a three-year period and 27 developed significant right ventricle dilation. Other measures of severity included pulmonary vascular resistance, exercise capacity and levels of BNP, which is a metric of heart health. Two metabolic pathways, those relating to polyamine and histidine metabolism, were found to be linked with all measures of severity suggesting a key role for them in disease pathology. While determining how these pathways influence disease as a subject for further study, the current findings may nevertheless lead to new prognostic indicators to inform patient care.   Cindy St. Hilaire:        The next article I want to discuss is coming from our February 3rd issue of Circulation Research and this is coming from the laboratory of Dr Francisco Violi at the University of Rome and the title is Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection. Thrombosis can be a complication of COVID-19 and it is associated with poor outcomes, including death. However, the exact mechanism by which the virus activates platelets, which are the cells that drive thrombosis, is not clear. For one thing, platelets do not appear to express the receptor for SARS-CoV-2. They do however, express the TLR4 receptor and that's a receptor that mediates entry of other viruses as part of the immune response. And TLR4 is ramped up in COVID-19 patient platelets. This group now confirms that, indeed, SARS-CoV-2 interacts with TLR4, which in turn triggers thrombosis.   The team analyzed platelets from 25 patients and 10 healthy controls and they found that the platelet activation and thrombic activity were both boosted in the patient samples and could not be blocked using a TLR4 inhibitor. Additionally, immunoprecipitation and immunofluorescent experiments further revealed colocalization between the virus protein and the TLR4 receptor on patient platelets. The team went on to show that the signaling pathway involved reactive oxygen species producing factors p47phox and Nox2, and that inhibition of phox 47, like that of the TLR4 receptor itsel,f could prevent platelet activation. As such, this study suggests that inhibiting either of these proteins may form the basis of an antithrombotic treatment for COVID-19.   Cindy St. Hilaire:        The third article I want to highlight is coming from the lab of Shi-You Chen at University of Missouri and the title of this article is ADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic Aneurysm. Macrophage activation plays a critical role in abdominal aortic aneurysm development, or AAA development. Inflammation is a component of this pathology; however, the mechanisms controlling macrophage activation and vascular inflammation in AAA are largely unknown. The ADAR1 enzyme catalyzes the conversion of adenosine to inosine in RNA molecules and thus this conversion can serve as a rheostat to regulate RNA structure or the gene coding sequence of proteins. Several studies have explored the role of ADAR1 in inflammation, but its precise contribution is not fully understood, so the objective of this group was to study the role of ADAR1 in macrophage activation and AAA formation.   Aortic transplantation was conducted to determine the importance of nonvascular ADAR1 in AAA development and dissection and angiotensin II infusion of ApoE knockout mice combined with a macrophage specific knockout of ADAR1 was used to study the role of ADAR1 macrophage specific contributions to AAA formation and dissection. Allograft transplantation of wild type abdominal aortas to ADAR1 haploinsufficient recipient mice significantly attenuated AAA formation. ADAR1 deficiency in hematopoietic stem cells also decreased the prevalence and the severity of AAA and it also inhibited macrophage infiltration into the aortic wall. ADAR1 deletion blocked the classic macrophage activation pathway. It diminished NF-κB signaling and it enhanced the expression of a number of anti-inflammatory microRNAs. Reconstitution of ADAR1 deficient but not wild type human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries. Together these results suggest that macrophage ADAR1 promotes aneurysm formation in both mouse and human arteries through a novel mechanism of editing the microRNAs that target NF-κB signaling, which ultimately promotes vascular inflammation in AAA.     Cindy St. Hilaire:        The last article I want to highlight is also from our February 17th issue of Circulation Research and it is coming from the lab of Shintaro Mandai at Tokyo Medical and Dental University and the title of the article is Circulating Extracellular Vesicle Propagated MicroRNA signatures as a Vascular Calcification Factor in Chronic Kidney Disease. Chronic Kidney Disease or CKD accelerates vascular calcification in part by promoting the phenotypic switching of vascular smooth muscle cells to osteoblast like cells. This study investigated the role of circulating small extracellular vesicles or SUVs from the kidneys in promoting this osteogenic switch. CKD was induced in rats and in mice by an adenine induced tubular interstitial fibrosis and serum from these animals induced calcification in in vitro cultures of A-10 embryonic rat smooth muscle cells. Intraperitoneal administration of a compound that prevents SEV biosynthesis and release inhibited thoracic aortic calcification in CKD mice under a high phosphorus diet. In Chronic Kidney Disease, the microRNA transcriptome of SUVs revealed a depletion of four microRNAs and the expression of the microRNAs inversely correlated with kidney function in CKD patients.   In vitro studies found that transected microRNA mimics prevented smooth muscle cell calcification in vitro. In silico analyses revealed that VEGF-A was a convergent target of all four microRNAs and leveraging this, the group used in vitro and in vivo models of calcification to show the inhibition of the VEGF-A, VEGFR-2 signaling pathway mitigated calcification. So in addition to identifying a new potential therapeutic target, these SUV propagated microRNAs are a potential biomarker that can be used for screening patients to determine the severity of CKD and possibly even vascular calcification.   Cindy St. Hilaire:        Today I have with me Dr Hind Lal who's an associate professor of medicine at the University of Alabama Birmingham and his post-doctoral fellow and the lead author of the study Dr Tousif Sultan. And their manuscript is titled Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. And this article is in our February 3rd issue of Circulation Research. So thank you both so much for joining me today.   Tousif Sultan:              Thank you.   Hind Lal:                     Thank you for taking time.   Cindy St. Hilaire:        So ponatinib, it's a tyrosine kinase inhibitor and from my understanding it's the only treatment option for a specific group of patients who have chronic myelogenous leukemia and they have to harbor a specific mutation. And while this drug helps to keep these patients alive essentially, it's extremely cardiotoxic. So cardiotoxicity is somewhat of a new field. So Dr Lal, I was wondering how did you get into this line of research?    Hind Lal:                    So I was fortunate enough to be in the lab of Dr Tom Force and he was kind of father of this new area, now is very developed, it's called cardio-oncology. On those days there were basically everything started in cardio-oncology. So I just recall the first tyrosine kinase approved by FDA was in 2000 and that was... Imagine and our paper came in Nature Medicine 2005 and discovering there is... so to elaborate it a little bit, the cancer therapy broadly divided in two parts. One is called non-targeted therapy like chemotherapy, radiations, et cetera, and then there are cytotoxic drugs. So those cytotoxic drugs because they do not have any targeted name on it so they are, cardiotoxic are toxic to any organ was very obvious and understanding. When these targeted therapy came, which is mainly kinase inhibitor are monoclonal antibodies. So these are targeted to a specific pathway that is activated only in the cancer cells but not in any other cells in the body so they were proposed as like magic bullets that can take off the cancer without any cardiotoxity or minimal side effects. But even in the early phase like 2005 to 2010, these came out, these so-called targeted, they are not very targeted and they are not also the magic bullets and they have serious cardiotoxicity.   Cindy St. Hilaire:        And so what's the mechanism of action of ponatinib in the leukemia and how does that intersect with the cardiovascular system?   Hind Lal:                     Yeah, so this is very good question I must say. So what we believe at this point because, so leukemia if you know is driven by the famous Philadelphia chromosome, which is a translicational gene, one part of human chromosome nine and one part of human chromosome 22 and they translocate make a new gene which is BCR-ABL gene. And because it was discovered in Philadelphia UPENN, is named that Philadelphia chromosome, which is very established mechanism, that's how CML is driven. But what we have discovered that the cardiotoxicity driven by totally, totally different from the ponatinib is one of the inflammatory So it's kind of goodening. So this question is so good. One kind of toxicity is called on-target, when toxicity is mediated by the same mechanism, what is the mechanism of the drug to cure the cancer? So in that case your absolute is minimal because if you manipulate that, the drug's ability to cure the cancer will be affected but if the toxicity and the efficacy is driven by two different mechanism, then as in case of ponatinib seems like it's NLRP3 and inflammasome related mechanism. So this can be managed by manipulating this pathway without hampering the drug efficacy on the cancer.   Cindy St. Hilaire:        So what exactly is cardiotoxicity and how does it present itself in these patients?   Hind Lal:                     So these drugs like ponatinib, they call broader CVD effects. So it's not just cardiac, so they also in hypertensives and atherosclerosis and thrombosis, those kind of thing. But our lab is primarily focused on the heart. So that's why in this paper we have given impresses on the heart. So what we believe at this point that ponatinib lead to this proinflammatory pathway described in this paper, which is just 108A9-NLRP3-IL-1β and this inflammatory pathway lead to a cytokine storm very much like in the COVID-19 and these cytokine storms lead to excessive myocarditis and then finally cardiac dysfunction.   Cindy St. Hilaire:        Is the cytokine storm just local in the cardiac tissue or is it also systemic in the patients? Is cardiotoxicity localized only or is it a more systemic problem?   Tousif Sultan:              I would like to add in this paper we have included that we look this cytokine things and explain blood circulation, bone marrow. So the effect is everywhere, it's not local. So we didn't check other organs, maybe other organs also being affected with the ponatinib treatment.   Cindy St. Hilaire:        And what's the initial phenotype of a patient has when they start to get cardiotoxicity, what's kind of like a telltale symptom?   Hind Lal:                     So good thing that in recent years cardio-oncology developed. So initially the patient that were going for cancer treatment, they were not monitored very closely. So they only end up in cardiology clinic when they are having some cardiac events already. So thanks to the lot of development and growth in the cardio-oncology field, now most patients who going for a long-term cancer treatment, they are closely monitored by cardiology clinics.   Cindy St. Hilaire:        Got it. So they can often catch it before a symptom or an event. That's wonderful.   Hind Lal:                     Yeah, so there's a lot of development in monitoring.   Cindy St. Hilaire:        Wonderful. So you were really interested in figuring out why ponatinib induces cardiotoxicity and you mentioned that really up until now it's been very difficult to study and that's because of the limitation of available murine models. If you just inject a wild type mouse with ponatinib, nothing happens really. So what was your approach to finding relatively good murine models? How did you go about that?   Hind Lal:                     So this is the top scientific question you can ask. So like science, the field is try and try again. So initially this is the first paper with the ponatinib toxicity using the real in vivo models. Any paper before this including ours studies published, they were done on the cellular model in hiPSC, that isolated cardiomyocytes. So you directly putting the ponatinib directly the isolated cells. So this is first case when we were trying to do in vivo, maybe other attempt in vivo but at least not published. So first we also treated the animals with ponatinib and that failed, we don't see any cardiotoxic effect. And then when we going back to the literature, the clinical data is very, very clear from pharmacovigilance that ponatinib is cardiotoxic in humans. So when we're not able to see any phenotype in mouse, we realize that we are not mimicking what's happening in the humans.   So we certainly missing something. Now once again I quote this COVID-19, so many people get infected with COVID-19 but people are having preexisting conditions are on high risk to developing CVD. So there was some literature on that line. So we use this very, very same concept that if there is preexisting conditions, so likely who'd have developing future cardiac event will be more. So we use two model in this paper one atherosclerosis model which is APoE null mice mice, another is tag branding which is pressure overload model for the heart and as soon as we start using what we call comorbidity model like patient is having some preexisting conditions and we very clearly see the robust defect of ponatinib on cardiac dysfunction.   Cindy St. Hilaire:        Yeah, it's really, really well done and I really like that you use kind of two different models of this. Do you think it's also going to be operative in maybe like the diabetic mirroring models? Do you think if we expand to other comorbidities, you might also recapitulate the cardiotoxicity?   Hind Lal:                     So you got all the best questions.   Cindy St. Hilaire:        Thank you. I try.   Hind Lal:                     So because this is CML drug and lot of the risk factor for cardiovascular and cancer are common and even metabolic disease. So most of the time these patients are elderly patients and they're having metabolic conditions and most of the time they have blood pressure or something CVD risk factors. So I agree with you, it'll be very relevant to expand this to the diabetes or metabolic models, but these were the first study, we put all our focus to get this one out so news is there then we can expand the field adding additional models et cetera. But I agree with you that will be very logical next step to do.   Cindy St. Hilaire:        Yeah. And so I guess going back to what you know from the human study or the clinical trials or the human observations, are different populations of patients with CML more predisposed to cardio toxicity than others or is that not known yet?   Hind Lal:                     So one other area called pharmacovigilance. So what pharmacovigilance does patient all over the world taking these drugs. So WHO have their own vigilance system and FDA have their own, so it's called BG-Base for the WHO and it's called the FAERS for the FDA. So one can go back in those data sets and see if X patient taking this Y drug and what kind of symptoms or adverse effect they are seeing and if these symptoms are associated with something else. So there is data that if patients having CVD risk factor, they are more prone to develop ponatinib induced cardiac events. But it needs more polish like you asked the just previous question, diabetes versus maybe blood pressure means hypertension, atherosclerosis, or thrombosis. So it has not been delineated further but in a one big bucket if patients are having CVD risk factor before they are more prone and more likely to develop the cardiac events.   Cindy St. Hilaire:        So after you established that these two murine models could pretty robustly recapitulate the human phenotype, what did you do next? How did you come upon the S100A8/A9-NLRP3-IL-1β signaling circuit? How did you get to that?   Hind Lal:                     So in basic science work, whenever we do mouse is called until we get there is cardiac dysfunction, it's called phenotype, right? So mouse had a cardiac phenotype. So next step is, "Why? What is leading to that phenotype?" That's what we call mechanism. So there the best idea to fit the mechanism is using one of the unbiased approaches like you do unbiased proteomics, unbiased RNC analysis, something like this that will analyze the entire transcript like RNC and say, "Okay, these pathway are," then you can do further analysis that will indicate these pathway are different, are altered. So in this case we used RNC analysis and it came out that this yes A8 and yes A9, 100A8 and nine, they were the most upregulated in this whole set. And thereafter we were very lucky. So we started this study at Vanderbilt, where my lab was and thereafter we very lucky to move here and found Sultan who had a lot of experience with this inflammation and immune system and then Sultan may add something on this so he'll be the better person to say something on this.   Tousif Sultan:              So after our RNC analysis, so we got this S100A8 and nine as top hit with the ponatinib treatment. So then we validated this finding with our flow cytometric, qRT PCR aand then we started which pathway is going to release cytokine and all that. So we found that is NLRP3 inflammasome.   Cindy St. Hilaire:        Yeah and well and I guess maybe step back, what is S100A8/A9? What are those? Tousif Sultan:              Yeah, S10A8/A9 is a calcium binding protein. So that's also called alarmin and they basically binds with the pathogen associated pattern and other TLR2 like receptors and then start inflammatory pathway to release cytokine and all that and it's stable in heterodimer form. So S100A8 heterodimer with A9 and then bind with TLR and a start in this inflammatory pathway.   Cindy St. Hilaire:        And what type of cell is that happening in? Is that happening in the immune cells only or is it also in the cardiomyocyte, or...?   Tousif Sultan:              Yeah, we have included all this data. So from where this alarmin is coming with ponatinib treatment, so literature also suggested that neutrophils and monocytes, those cells are the potential to release the alarmin. So here we also found these two type of cells, neutrophils and monocytes. They release huge alarmin with the treatment of ponatinib.   Cindy St. Hilaire:        And so really taking this really neat mechanism to the next level, you then tried attenuating it by using broad anti-inflammatory steroid dexamethasone but also by targeting these specific components, the NLRP and the S100A specific inhibitors and they worked well. It worked really nicely. Does your data show that any of these therapies work better than the other and then are these viable options to use in humans?   Hind Lal:                     Yeah, we have some data in the paper. Are very broad which help a lot in COVID patients, far very acute infections. So in this case, situation is very different cause most of CML patients will going to take ponatinib for lifelong, there is no remission, right? So in those case, its certainly not a very attractive option. We have shown data in the paper that dexamethasone help with the heart but lead to some metabolic changes. So we have compared those with the NLRP3 inhibitors, those metabolic alterations, dexa versus the NLRP3 inhibitors, CY-09. And we demonstrated that targeting is specifically with paquinimod, our NLRP3 inhibitor CY-09, feel better. It can still rescue the cardiac phenotype without having those adverse effect on metabolic parameters.   Cindy St. Hilaire:        That's wonderful. Do you think though that because you have to take ponatinib for life, that long-term NLRP inhibition would also cause problems or...?   Hind Lal:                     So because not every patient who taking ponatinib would develop the cardiac phenotype, right? Which is like a 10%, 12%, patient developing cardiac dysfunction. So I think someone like I strongly believe paquinimod, which is inhibitor of S100A9, will be really good option or at least we have enough data that make us nail for at least a small clinical trial. And we quickly moving on that. At UAB we have our clinical cardio-oncology program and we are already in touch with the director for the clinical cardio-oncology program. So what we trying to do in that small trial is if one of the standard therapy for heart like beta blocker or ARBs inhibitor, is there any preference like one work better than the other in the standard care? So first we doing that project, then we obviously looking forward if one small clinical trial can be done with paquinimod. I strongly believe it should be helpful.   Cindy St. Hilaire:        That is wonderful. And so do you think... There's other chemotherapeutic agents or probably even other non-cancer drugs that cause cardiotoxicity, do you think this mechanism, this pathway, this S100A-NLRP-IL-1β axis is operative in all cardiotoxicities or do you think it's going to be very specific to the ponatinib?   Hind Lal:                     So it's certainly not all, but it'll be certainly more than ponatinib. So in our lab we are using another kinase inhibitor, which is osimertinib and it's not published yet, but now we know that it's also cardiotoxic because it's taking metabolic root or energetics disruption but not this pro-inflammatory part, but we're doing another project which is strep pneumonia induced cardiac dysfunction, which is called pneumonia. So strep pneumoniae, which leads to the pneumonia ,and lot patient die because of the failing heart we see here in the hospitals and we see these pathways operational over there and we gearing up to do clinical trial on that aspect as well, but it's not generalized like all kind of heart will have the same mechanism.   Cindy St. Hilaire:        It's wonderful to see you're already taking those next steps towards really kind of bringing this to a translational/clinical study. So what was the most challenging aspect of this study?   Tousif Sultan:              The challenging aspect, ponatinib is a kinase inhibitor and that was surprising for us how it's activating immune cells. Generally kinase inhibitors, inhibits all the cells like that. So that was challenging. So we repeated it many times did in vitro experiment to confirm that. So we just added, just treated in vitro immune cells with the ponatinib and confirmed it. So that was little challenging.   Cindy St. Hilaire:        So what's next? You mentioned you're going to try some clinical trials, early stage clinical trials. What's next mechanistically, what do you want to go after?   Hind Lal:                     So what we are doing next and we are very, very eagerly trying to do that. So what it was done, we used the cardiac comorbidity models, but as you know, anybody who will take ponatinib will have cancer, right? So we strongly believe that we miss one factor. There was no cancer on these. So that is very logical next step. What that will allow us to do, what rescue experiment we'll have done in this paper. So we saw, "Okay, this rescue the cardiac phenotype, which is taken care of now," but very same time, we not able to demonstrate that this is happening without hurting the cancer efficacy. So if we have the dual comorbid mouse, which have CML a real thing and we have cardiac thing, then that will allow us to demonstrate, "Okay, we got something that can take care of the cardiac problem without hurting the efficacy on the cancer." And it will be best if you also help little bit to more potentiate the cancer efficacy.   Cindy St. Hilaire:        Yes. Excellent. Well, congratulations on a beautiful study, really exciting findings. Dr Lal and Dr Sultan, thank you so much for taking the time to talk with me today.   Tousif Sultan:              Thank you so much.   Hind Lal:                     Well thank you, Cynthia. We really appreciate your time. Thank you for having us.   Cindy St. Hilaire:        Yeah, it was great.   Cindy St. Hilaire:        That's it for our highlights from the February 3rd and February 17th issues of Circulation Research. Thank you so much for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Hind Lal and Dr Tousif Sultan. This podcast is produced by Ishara Ratnayake, edited by Melissa Stoner and supported by the editorial team at Circulation Research. Some of the copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover CircRes, you're on-the-go source for most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2023. And the opinions expressed by the speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.  

Featuring perspectives from Dr Daniel DeAngelo, including the following topics: Introduction: Perspectives on chronic myeloid leukemia (CML) (0:00) Asciminib — ASC4FIRST study (8:33) Case: A woman in her early 60s with a PMH of Stage IA hormone receptor-positive breast cancer on adjuvant tamoxifen with newly diagnosed CML — Michael R Grunwald, MD (22:38) Case: A man in his mid 40s with low-risk CML who achieves MR4 on imatinib but loses molecular response 3 months after discontinuing therapy — Shams Bufalino, MD (27:23) Case: A man in his early 40s with CML on dasatinib who does not achieve treatment milestone (MR3) at 18 months but does at 24 months — Dr Grunwald (33:19) Case: A man in his early 80s with CML who initially receives imatinib followed by nilotinib develops CHF and has a BCR-ABL of 0.8% (IS) — Namrata I Peswani, MD (37:33) Case: A woman in her late 20s with CML that is resistant to multiple TKIs and therapy compliance concerns — Minesh Dinubhai Patel, MD (40:44) Case: A woman in her early 40s who initially receives dasatinib for chronic-phase CML with a good response but then develops lymphoid blast crisis — Dr Grunwald (52:17) Case: A woman in her mid 70s with CML who achieves MMR on nilotinib but then develops peripheral arterial occlusion after 9 years on therapy — Dr Grunwald (58:10) CME information and select publications

Featuring perspectives from Dr Jorge Cortes, including the following topics: Introduction: Journal Club with Dr Cortes (0:00) Case: A man in his early 40s with chronic myeloid leukemia (CML) and osteopenia after long-term imatinib therapy — Rajalaxmi McKenna, MD (15:00) Case: A man in his early 60s with chronic phase CML (CP-CML) and continued detectable BCR-ABL transcript on bosutinib — Bruce Bank, MD (23:55) Case: A man in his early 70s with CP-CML and notable toxicities from several BCR-ABL tyrosine kinase inhibitors (TKIs) — Gigi Chen, MD (27:37) Case: A woman in her late 40s with CML in complete molecular response for more than 3 years with nilotinib — Michael R Grunwald, MD (36:05) Case: A woman in her mid 60s with CP-CML and negative BCR-ABL mutational analyses who experiences lack of disease response to several BCR-ABL TKIs — Shams Bufalino, MD (43:27) Case: A man in his early 40s with CP-CML who is found to have a T315I mutation — Dr Grunwald (44:45) Appendix of Key Recent Data Sets (53:26) CME information and select publications

Dr. John Sweetenham, of the UT Southwestern's Harold C. Simmons Comprehensive Cancer Center, and Dr. Marc Braunstein, of NYU Langone Health, discuss key data from the CAPTIVATE and GRIFFIN trials and other compelling studies in hematologic malignancies featured at the 2022 ASCO Annual Meeting.  Dr. John Sweetenham: Hello. I'm John Sweetenham, the associate director for Clinical Affairs at UT Southwestern Harold C. Simmons Comprehensive Cancer Center and host of the ASCO Daily News podcast.  For my guest today, I'm pleased to introduce Dr. Marc Braunstein, a hematologist, and oncologist at NYU Perlmutter Cancer Center. We'll be discussing key posters on advances in hematologic malignancies that will be featured at the 2022 ASCO Annual Meeting.  Our full disclosures are available in the show notes and disclosures of all guests on the podcast can be found on our transcripts at asco.org/podcasts.  Marc, it's great to have you on the podcast today.  Dr. Marc Braunstein: Thank you, John. It's a pleasure to be here.  Dr. John Sweetenham: So, Marc, there are going to be some very interesting abstracts with some provocative results presented at the ASCO Annual Meeting this year. I know we've selected a number of these to speak about today, beginning with Abstract 8027 on the subject of multiple myeloma. And this I think is a long-term follow-up study for long-term survivors of multiple myeloma more than 12 years out now. Can you comment on this and let us know what you believe the key takehomes from this study are?  Dr. Marc Braunstein: Sure. Absolutely, John. So, this was a prospective registry that has been in place since 2009, and it's composed of various practice settings, but primarily community practices where most patients get their myeloma care. And what they did was they looked at about 1,400 patients with newly diagnosed multiple myeloma across 250 sites in the U.S. between 2009 and 2011 who were included in the registry. The participants also filled out the quality-of-life surveys, and they compared a group of long-term survivors who had more than 8 years of follow-up to patients who were not long-term survivors below that 8-year threshold.  So, about 20% were in the long-term survival group and 80% in the non-long term survival group. And they basically characterized those 2 groups. What they found was that the individuals who did have long-term survival were generally younger—median age of 62 versus 68, and had better performance status, were more likely to receive stem cell transplants, about 66 versus 60%.  And therefore, the implication of this is that patients who fit those criteria may have a better prognosis in addition to the other cytogenetics and other factors we use as prognostic indicators. And what was also interesting was that the 8-year overall survival of the overall group was about 36%.  So, we still have room to go in terms of bringing new therapies to extend survival in this condition. And only 20% of the total population were long-term survivors at that 8-year threshold. So, those were the general findings of the abstract.  Dr. John Sweetenham: Do you think it gives us new information on patient selection for more intensive therapies upfront?  Dr. Marc Braunstein: Well, I think it certainly tells us which patients are more likely to have longer-term survival. I think we know in multiple myeloma that it's essential to really use the patient's presenting features, their disease features, their comorbidities, and their degree of fitness or frailty to guide how intensive a therapy or regimen we can devise for that individual patient. But I think it certainly says that if you have a patient who is on the younger side of the spectrum, who is eligible for stem cell transplant, who has a better performance status, those are the patients that are more likely to have the long-term survival. It doesn't necessarily say that if you're not in that category, you won't have long-term survival, but on average, those were the patients who fared better in the long term.  Dr. John Sweetenham: Okay. So, staying for a little while on the subject of multiple myeloma, Abstract 8037 is really addressing a very different question. It's the application of circulating tumor DNA analysis and its association with relapse in patients with refractory myeloma. Would you comment a little on this and maybe let us know what you think the significance of this will be for the future?  Dr. Marc Braunstein: Sure. My colleagues in the solid tumor space are using circulating tumor DNA regularly and in the myeloma field, we're a little bit jealous of them.  So, it's helpful to have a study like this that's looking at circulating peripheral blood markers, in this case circulating tumor DNA, to help guide various prognostic or predictive indices that will help us guide therapeutic decisions.  So, this was a study where they looked at patients who were enrolled in a phase 2 study of a free-drug regimen of carfilzomib-thalidomide-dexamethasone the MM17 study, and they took 50 transplant eligible multiple myeloma patients who were refractory to their first line of therapy, and they collected bone marrow samples and peripheral blood at 3 time points at the third cycle of treatment and at the end of the study or at the point of refractoriness to that regimen.  They collected about 187 samples in total. They used a sequencing technique to determine the variance of 22 gene signatures known to be mutated in multiple myeloma. And what they found was a particular gene signature that was associated with shorter progression-free and overall survival in that phase 2 study. And those genes included known oncogenic drivers, including BRAF genes, ATM, and P53.  What was particularly interesting among the circulating tumor DNA mutations was that they were found in about 88% of patients at the start of the study. So, what that tells us is, number 1, circulating tumor DNA offers a wealth of information that can be highly valuable in multiple myeloma, which is a disease where we typically rely on the bone marrow to assess the status of the plasma cells and status of the mutation profile.  And number 2, that many of these mutations may be present earlier on in a disease that we know evolves in a clonal way that leads to disease progression. So, I think there's still a lot of information we have to learn about the utility of circulating tumor DNA in myeloma, but this study certainly shows that there's a lot to be explored in terms of the mutational profile and peripheral blood in myeloma.  Dr. John Sweetenham: A couple of questions that arise for me out of this study. First of all, do you think this is going to have any implications for future study design and patient selection?  Dr. Marc Braunstein: Definitely. I think the whole field in multiple myeloma is progressing quickly in terms of how we assess response, how we use minimal residual disease, and moving more towards using novel markers in peripheral blood, including mass spectrometry, and now perhaps circulating tumor DNA to look at surrogate markers for survival.  And so, what this abstract is showing is that we could potentially use circulating tumor DNA both as prognostic markers, potentially as disease response markers, and prognostic markers to guide which patients may be more likely to have shorter survival. So, I think this has a lot of implications for how we design future studies.  Dr. John Sweetenham: Yeah. And the second question, do you think this is the beginning of the end of bone marrow analysis in multiple myeloma?  Dr. Marc Braunstein: So, I can tell you if it is, patients I think will be very happy and so will clinicians because we really want to know at the core what the degree of residual disease is in a patient. And right now, the only way to do that is through a bone marrow biopsy.  And so, I think that this is the beginning of the use of peripheral blood studies with higher resolution to allow us to gain more information on patients that hopefully will allow us to obviate the need for more invasive testing like bone marrow biopsies.  Dr. John Sweetenham: Yeah, absolutely. Thanks. Just changing gears now, moving on to Abstract 7050. This is an abstract that addresses what I think we'd all agree is becoming an increasingly important question in the management of chronic myeloid leukemia (CML), and that is number 1, is it safe to discontinue therapy in responding patients? And number 2, when is it safe to discontinue that therapy?  Dr. Marc Braunstein: So, this is an abstract that is looking primarily at CML. You know that we're making a lot of progress when we can begin to talk about discontinuation and de-escalation of therapy.  And so, in the field of CML, the use of tyrosine kinase inhibitors (TKIs) and the targeting of the BCR-ABL mutation has brought about tremendous progress in patients in the chronic phase.  So, there have been several retrospective studies that have looked at the role of discontinuing one of the TKIs. Most of the studies have focused on imatinib since that was the first one that was discovered, but they've looked at others in the class as well.  What struck me the most is that there's a remarkable consistency between these studies. So, when you discontinue one of these TKIs, the percentage of patients who remain in remission is somewhere between 40 to 50%. And what this abstract looked at was a single institution retrospective assessment of 284 patients with CML, between 1999 and 2017, who were treated with a TKI for their CML and then subsequently discontinued the therapy.  Now, what's worth noting in the various studies that have looked at discontinuation therapy is that patients who were taken off of the TKI generally were in a good molecular remission, MR 4 or 4.5, for at least 2 or 3 years. And in this study, about 70% of patients had electively discontinued and 24% of patients stopped due to adverse events.  So, it wasn't necessarily guided by their response to treatment at the time of discontinuation. What they found actually was fairly consistent with the literature that at a median follow-up of 36 months after TKI discontinuation, about 19% lost their molecular remission and 88% had achieved a molecular remission after resuming therapy. And that is consistent with the literature that fortunately, even if a patient loses their molecular remission off of the TKI therapy, the majority of patients will go back into molecular remission when you re-challenge them.  Dr. John Sweetenham: Important data, indeed. And you know, on something of a similar theme, the next abstract that we're going to look at is the Abstract 7519. In this case, in chronic lymphocytic leukemia (CLL), and certainly, those of us who remember when ibrutinib was initially introduced into the second-line treatment of CLL, didn't really know whether discontinuation or fixed duration treatment with agents like this was going to be something that we could pursue or whether treatment with these drugs was going to be indefinite. This abstract certainly addresses that specific question, and again, I'm interested in your insights into this.  Dr. Marc Braunstein: Sure. So, this is an abstract looking at CLL, where we've really begun to move away from chemotherapy, and we have a variety of targeted oral therapies that target the underlying pathology of this leukemia.  And so, as you mentioned, ibrutinib is approved both in the relapsed and more recently in the frontline setting, wherein the RESONATE-2 study that was published in the New England Journal of Medicine in 2015, there was actually an overall survival benefit of ibrutinib even in higher-risk patients.  So, the CAPTIVATE study is an ongoing phase 2 study that is looking at whether we can improve the efficacy of single-agent ibrutinib in the first-line setting when combined with venetoclax.  Ibrutinib targets protein tyrosine kinase and venetoclax targets Bcl-2, and that combination is hypothesized to further weaken the resistance of CLL and lead to better outcomes.  So, this was a multicenter phase 2 study. And in this abstract, they looked at the 3-year follow-up of patients who were actually able to discontinue therapy on this regimen. So, just as a bit of background, ibrutinib is typically continued until progression, and venetoclax as it's been studied in the first-line setting with obinutuzumab is given for about 12 months.  So, in this study, at 3-year follow-up, they looked at the patients in the cohort who were off therapy and looked at the percentage of patients who maintained a complete remission at 3 years. And that complete remission rate was about 57%.  The majority of patients, greater than 95% of patients, were alive at 3 years even in the high-risk cohort. So, I think the implications of the study is that upfront or oral targeted therapies when you combine ibrutinib and venetoclax really produce tremendous responses that are durable, and it's found even in the high-risk patients who are expected not to do quite as well at 3 years.  Dr. John Sweetenham: Yeah, I agree. I think it's very reassuring actually to see these durable responses with this fixed duration regimen. And to conclude, Abstract 8011 was an abstract which addressed treatment in the first-line setting for multiple myeloma. And again, I wonder if you could comment on this study.  Dr. Marc Braunstein: Sure! So, this is a study looking at the GRIFFIN regimen, which was a phase 2 randomized study of daratumumab (DARA), plus lenalidomide, bortezomib, and dexamethasone.  So, DARA RVd versus RVd alone. In that study, the primary endpoint was stringent, complete remission, and it has been previously presented and published that the stringent complete remission (CR) rate was significantly improved, 42% versus 32%, when you include daratumumab upfront.  In this abstract, they looked at the sustained rate of minimal residual disease negativity, which is basically the deepest possible remission you can achieve in upfront therapy and in myeloma.  What they found was that, again, when you looked at the quadruplet regimen versus the triplet regimen, the rates of minimum residual disease (MRD) negativity were just improved with the quad regimen.  So, at a median follow-up of 38.6 months, there were about 54 versus 20% of patients who were MRD negative at 12 months amongst the patients who had achieved a CR, and 59 versus 17% MRD negative among the patients who achieved a stringent CR favoring the daratumumab arm.  So, I think this abstract shows the benefit of including a monoclonal antibody upfront in newly diagnosed patients with myeloma combined with stem cell transplant and maintenance, allowing for sustained MRD negativity.  Dr. John Sweetenham: Do you think this represents a new standard of care?  Dr. Marc Braunstein: I do. At our institution, we've adopted this regimen for most newly diagnosed transplant-eligible patients. I think the data clearly show an improved depth of response and MRD negativity rates, and I think that there are a number of ongoing studies looking at the role of monoclonal antibodies in the maintenance phase as well.  I'm especially excited this year, at ASCO Annual Meeting there's a plenary session involving myeloma looking at patients who received RVd upfront and then went for transplant. But I think we can improve on that regimen by including monoclonal antibodies and immunotherapies upfront, and I do think it represents a new era of immunotherapies in multiple myeloma.  Dr. John Sweetenham: Well, thanks, Marc. I mean, to your last point, it sounds as if there is a lot, including these abstracts, to look forward to at the upcoming ASCO meeting. So, we really appreciate you sharing your insights into these abstracts with us today.  Dr. Marc Braunstein: Sure. My pleasure. Thank you for having me, John.  Dr. John Sweetenham: And thank you to our listeners for joining us today. You'll find links to the abstract discussed today on the transcript of this episode. Finally, if you value the insights that you hear on the ASCO Daily News podcast, please take a moment to rate, review and subscribe wherever you get your podcasts.      Disclosures:  Dr. John Sweetenham:  Consulting or Advisory Role: EMA Wellness  Dr. Marc Braunstein:  Consulting or Advisory Role: Celgene, Janssen, AstraZeneca, Amgen, Takeda, Verastem, Celgene, Janssen, Karyopharm Therapeutics, Epizyme, Morphosys, Takeda, Pfizer  Research Funding (Inst): Janssen, Celgene/BMS  Travel, Accommodations, Expenses: Takeda  Disclaimer:   The purpose of this podcast is to educate and to inform. This is not a substitute for professional medical care and is not intended for use in the diagnosis or treatment of individual conditions.  Guests on this podcast express their own opinions, experience, and conclusions. Guest statements on the podcast do not express the opinions of ASCO. The mention of any product, service, organization, activity, or therapy should not be construed as an ASCO endorsement. 

「慢性骨髄性白血病のBCR-ABL-IS」（解説）国立がん研究センター中央病院 血液腫瘍科長　　伊豆津宏二氏（ききて）順天堂大学教授　　池田志斈氏

MECP2, RAS, and TP53. JAK2, RET, and BCR-ABL. Genetics is full of abbreviated names, pedigrees, and surprisingly, math. Not to worry, though - genetics TA Aaron D'Souza shares best practices to succeed in this class.

生物科技驱动制药创新的历程来自青侨阳光主播：小陆https://xueqiu.com/3770558188/150465923 医药行业里的新机会，是供给还是需求在驱动呢？ 其实，供给驱动是在寻找【改写游戏规则】的技术，而需求驱动呢，则是在寻找【远未满足的迫切需求】。最好的状况，是用技术去满足需求。我们更关注供给驱动，因为，如果仅仅有需求，不一定找得到技术和供给。但只要技术够颠覆，创新供给够颠覆，迟早能找到释放价值的地方、合适的使用场景。 今天，我们就来简单梳理一下，生物科技是如何驱动生物药和化学药的创新发展的。 首先先说生物药。在过去几十年，生物药技术驱动的核心是什么？当然就是——生物科技。在几十年的时间里，生物科技在医药行业有了层层突破。 简单回顾一下历史—— 50年代的时候，DNA双螺旋模型被提出，分子生物学诞生。DNA双螺旋模型简洁地解释了众多复杂现象，震撼学界，由此也获得资源和市场的青睐，加速了学科发展。 70年代，内切酶、连接酶、质粒、PCR都出来了，微生物研究也越来越清晰。产业化尝试也从这个阶段开始。 80年代，基因泰克生长激素、胰岛素相继获批，多肽药物进入爆发期。多肽药物最先爆发，它没有人源化免疫原性的问题，用大肠杆菌就可以。1975年，抗体开始尝试产业化，1983年，第一个抗体药物上市了。80年代就有了抗体，但2000年左右才真正爆发，正是因为免疫原性。 90年代后，人源化逐渐被突破，单抗开始爆发，因为最初的限制瓶颈被解决了。单抗行业刚打开的时候，大家尽情的去筛临床价值好的靶点，比如现在的药王阿达木单抗，未来的药王pd1单抗，一个是下调免疫系统治疗自身免疫性疾病，一个是上调免疫系统治疗肿瘤。但是，胞外靶点毕竟是有限的，筛到一定程度，好靶点就筛的差不多了。 抗体要进一步创新，要提高临床效果，更多是在现有靶点上的精耕细作。所以工程抗体是一个必然趋势，双抗、ADC、合成生物学都是其中的重要细分。 抗体之后，接下来一波比较大的浪潮，我们称之为入胞时代。抗体比多肽难，时间轴上对应的爆发时间点也晚些，但入胞的技术比抗体更难。多肽、抗体针对胞外的蛋白，难度小很多。但90%以上的生命过程在细胞内，而且蛋白只是下游功能元件，胞外远比不上核酸调控的效果。 令人振奋的是，入胞相关的前沿性基础研究在过去几年，随着技术的突破也有了新发展。2010、2015年后，入胞技术逐渐成熟，第一个真正有显著临床优势的基因疗法、第一款RNAi疗法药物相继获批。NGB-也就是下一代生物技术成为一个很大的浪潮。 接下来，我们看下：过去几十年，化学药技术驱动的核心是什么？答案同样是生物科技。为什么这么说呢？大家都熟悉青霉素、二甲双胍，但很难说清它们对应的靶点。因为以前化药的发现和研发，都是基于某种现象来反向筛选。比如，发现菌落周围不长菌，根据这个现象反向推导回溯，找到青霉素，更像盲筛的过程。 但70到80年代间，随着分子生物学的渗透，化药的研发过程被极大的颠覆。生物学带来“靶点”的概念，照着靶点来开发药物，极大提升了小分子化药的成功率。 很多创新药出现在八、九十年代之后，一方面是因为这个时期有一些关键的政治决策颁布出台，比如1984年颁布的“Hatch-Waxman法案“在美国医药产业百年发展历程中具有里程碑意义。但更重要的一方面是生物科技的进步。生物科技改写了小分子化药的研发格局，也促进了生物药的发展。 从80到90年代开始，HMG-CoA的靶点带来了一批的他汀；ACEI和ARB的靶点带来了普利类、沙坦类。再到2000年后，肿瘤驱动基因的概念被验证，开山之作就是诺华的格列卫，也就是伊马替尼，靶向BCR-ABL融合蛋白。大家发现某些肿瘤的发生也是受核心基因所驱动，抑制了融合蛋白BCR-ABL，急淋白血病就得到了极好的控制。 之后，一批小分子抗肿瘤药物奔涌而出。很多小分子靶向药物其实就是酪氨酸激酶抑制剂也就是我们常听说的TKI。比如典型的在膜上的EGFR，贝达的埃克替尼；在胞内的BTK抑制剂，诺诚健华的奥布替尼；JAK抑制剂，辉瑞托法替尼等。还有一类较大的多靶点激酶抑制剂，比如恒瑞的阿帕替尼。这也是一个比较大的浪潮，相关的上市公司非常多。 回顾整个发展历程，分子生物学对整个生物制药有着重大影响。 化药的发现是基于生物的靶点，生物药更是基于生物科技。过去生物制药领域出现众多机会，生物科技功不可没。很多时候，表面看上去是公司个体的成就，实际上都是技术与时代发展的烙印。对于医药行业的投资者来说，值得庆幸的是：往前看，生物技术的大潮仍将汹涌，技术创新与突破仍将不断。

生物科技驱动制药创新的历程来自青侨阳光主播：小陆https://xueqiu.com/3770558188/150465923 医药行业里的新机会，是供给还是需求在驱动呢？ 其实，供给驱动是在寻找【改写游戏规则】的技术，而需求驱动呢，则是在寻找【远未满足的迫切需求】。最好的状况，是用技术去满足需求。我们更关注供给驱动，因为，如果仅仅有需求，不一定找得到技术和供给。但只要技术够颠覆，创新供给够颠覆，迟早能找到释放价值的地方、合适的使用场景。 今天，我们就来简单梳理一下，生物科技是如何驱动生物药和化学药的创新发展的。 首先先说生物药。在过去几十年，生物药技术驱动的核心是什么？当然就是——生物科技。在几十年的时间里，生物科技在医药行业有了层层突破。 简单回顾一下历史—— 50年代的时候，DNA双螺旋模型被提出，分子生物学诞生。DNA双螺旋模型简洁地解释了众多复杂现象，震撼学界，由此也获得资源和市场的青睐，加速了学科发展。 70年代，内切酶、连接酶、质粒、PCR都出来了，微生物研究也越来越清晰。产业化尝试也从这个阶段开始。 80年代，基因泰克生长激素、胰岛素相继获批，多肽药物进入爆发期。多肽药物最先爆发，它没有人源化免疫原性的问题，用大肠杆菌就可以。1975年，抗体开始尝试产业化，1983年，第一个抗体药物上市了。80年代就有了抗体，但2000年左右才真正爆发，正是因为免疫原性。 90年代后，人源化逐渐被突破，单抗开始爆发，因为最初的限制瓶颈被解决了。单抗行业刚打开的时候，大家尽情的去筛临床价值好的靶点，比如现在的药王阿达木单抗，未来的药王pd1单抗，一个是下调免疫系统治疗自身免疫性疾病，一个是上调免疫系统治疗肿瘤。但是，胞外靶点毕竟是有限的，筛到一定程度，好靶点就筛的差不多了。 抗体要进一步创新，要提高临床效果，更多是在现有靶点上的精耕细作。所以工程抗体是一个必然趋势，双抗、ADC、合成生物学都是其中的重要细分。 抗体之后，接下来一波比较大的浪潮，我们称之为入胞时代。抗体比多肽难，时间轴上对应的爆发时间点也晚些，但入胞的技术比抗体更难。多肽、抗体针对胞外的蛋白，难度小很多。但90%以上的生命过程在细胞内，而且蛋白只是下游功能元件，胞外远比不上核酸调控的效果。 令人振奋的是，入胞相关的前沿性基础研究在过去几年，随着技术的突破也有了新发展。2010、2015年后，入胞技术逐渐成熟，第一个真正有显著临床优势的基因疗法、第一款RNAi疗法药物相继获批。NGB-也就是下一代生物技术成为一个很大的浪潮。 接下来，我们看下：过去几十年，化学药技术驱动的核心是什么？答案同样是生物科技。为什么这么说呢？大家都熟悉青霉素、二甲双胍，但很难说清它们对应的靶点。因为以前化药的发现和研发，都是基于某种现象来反向筛选。比如，发现菌落周围不长菌，根据这个现象反向推导回溯，找到青霉素，更像盲筛的过程。 但70到80年代间，随着分子生物学的渗透，化药的研发过程被极大的颠覆。生物学带来“靶点”的概念，照着靶点来开发药物，极大提升了小分子化药的成功率。 很多创新药出现在八、九十年代之后，一方面是因为这个时期有一些关键的政治决策颁布出台，比如1984年颁布的“Hatch-Waxman法案“在美国医药产业百年发展历程中具有里程碑意义。但更重要的一方面是生物科技的进步。生物科技改写了小分子化药的研发格局，也促进了生物药的发展。 从80到90年代开始，HMG-CoA的靶点带来了一批的他汀；ACEI和ARB的靶点带来了普利类、沙坦类。再到2000年后，肿瘤驱动基因的概念被验证，开山之作就是诺华的格列卫，也就是伊马替尼，靶向BCR-ABL融合蛋白。大家发现某些肿瘤的发生也是受核心基因所驱动，抑制了融合蛋白BCR-ABL，急淋白血病就得到了极好的控制。 之后，一批小分子抗肿瘤药物奔涌而出。很多小分子靶向药物其实就是酪氨酸激酶抑制剂也就是我们常听说的TKI。比如典型的在膜上的EGFR，贝达的埃克替尼；在胞内的BTK抑制剂，诺诚健华的奥布替尼；JAK抑制剂，辉瑞托法替尼等。还有一类较大的多靶点激酶抑制剂，比如恒瑞的阿帕替尼。这也是一个比较大的浪潮，相关的上市公司非常多。 回顾整个发展历程，分子生物学对整个生物制药有着重大影响。 化药的发现是基于生物的靶点，生物药更是基于生物科技。过去生物制药领域出现众多机会，生物科技功不可没。很多时候，表面看上去是公司个体的成就，实际上都是技术与时代发展的烙印。对于医药行业的投资者来说，值得庆幸的是：往前看，生物技术的大潮仍将汹涌，技术创新与突破仍将不断。

Guest Name and Bio: Suneer “Sunny” Jain, M.S., Chief Executive Officer, Sun Genomics. Mr. Jain earned his Bachelor of Science (B.S.) degree from the University of Iowa in 1999. He went on to graduate with a Masters in Anatomy and Cell Biology from the Department of Anatomy and Cell Biology at the University of Iowa in 2004, where he conducted research in the field of Microbiology and Gene Therapy. Following graduation, Mr. Jain began his career as a Research and Development Supervisor for Pathway Diagnostics. Here he developed new molecular biomarker assays for clinical trials and out-licensing opportunities, planned and performed studies for the development and optimization of molecular assay(s) such as RNA/DNA extractions, (q)RT-PCR, PCR, SNP genotyping, Indel detection, gene rearrangement, and mutation status. Mr. Jain focused his career in 2007 towards clinical patient testing while working for LabCorp in the molecular oncology laboratory. During his time at LabCorp, Sunny participated in the development and launch of new lymphoma and leukemia molecular assays such as T-Cell GR, BCR/ABL, improved and troubleshooted existing assays such as Jak2, FLT3, IgVh somatic hypermutation, B-Cell GR, and MSI; he also reviewed and oversaw the scheduling and completion of Proficiency testing for CAP and NYS, and submitted validations and received approval from the New York State DOH on multiple assays. In 2009, Mr. Jain began working as a Lab Manager and Technical Supervisor in a Clinical Services Laboratory for industry leader, Illumina. At Illumina, Sunny managed the world’s first CLIA Whole Genome Sequencing Lab at Illumina for seven (7) years and was a part of the team that established the Illumina Clinical Services Laboratory and the first Clinical Laboratory to sequence the whole human genome in a CLIA-certified, CAP-accredited laboratory introducing NGS technology into a clinical setting. Additionally, he led a zero deficiency CAP inspection as acting quality manager/general supervisor/technical supervisor from 2011-2015, authored state of the art assay validation studies for Whole Genome Sequencing as an LDT, managed site relocation, re-verification studies, and HiSeq 2000 validations for Whole Genome Sequencing, and restructured payment processing workflow in a SAP for a laboratory generating over $5 Million per year. What you will learn from this episode: 1) What is a precision probiotic? 2) Can probiotics help and how? 3) What is the best way to sequence the gut microbiome 4) How to take a highly personalized approach to gut health 5) Understanding how to optimize gut health How to learn more about our guest: https://sungenomics.com/ https://www.instagram.com/sungenomics/ https://www.facebook.com/sungenomics/ https://www.linkedin.com/company/sungenomics/ https://twitter.com/sungenomics?lang=en Please enjoy, share, rate and review our podcast and help us bring the message about precision health care to the world!

Dobrý deň, počúvate podcast ONKLÓGIA, ktorý je súčasťou skupiny podcastov ZDRAVIE. V dnešnom diely sa budeme venovať otázke „Dá sa chronická myelocytová leukémia (CML) vyliečiť”? Odpoveď sa dozviete v dnešnom podcaste, ktorý pre Vás pripravil Dr. Chudej, číta Marián Mitaš.Dá sa chronická myelocytová leukémia (CML) vyliečiť?, MUDr. Juraj Chudej, PhD., Klinika hematológie a transfuziológie, Univerzitná nemocnica Martin, Jesseniova lekárska fakulta v Martine, Univerzita Komenského v Bratislave.Chronická myelocytová leukémia je typom rakoviny krvných buniek. Bola prvým opísaným typom leukémie vôbec. Čo je dôležité si zapamätať je skutočnosť, že pod hrôzostrašne znejúcim označením leukémia sa neskrýva len jedno, ale celý rad ochorení.A tieto jednotlivé podkategórie leukémií sa líšia v prognóze, ale aj v spôsobe liečby.Leukémie teda delíme podľa:1.ich agresivity a2.typu postihnutej bunky.Podľa agresivity leukémie rozdeľujeme na akútne a chronické. Termín “chronická” poukazuje na skutočnosť, že ochorenie má pozvoľný priebeh. Pacient nemusí mať žiadne príznaky aj niekoľko rokov. Druhé delenie leukémií vychádza z typu postihnutých bielych krviniek.Dá sa povedať, že v princípe rozlišujeme štyri typy leukémií, a to:1.akútnu myeloblastovú leukémiu2.akútnu lymfoblastovú leukémiu3.chronickú myelocytovú leukémiu4.chronickú lymfocytovú leukémiu.V tomto podcaste sa bližšie budeme venovať len chronickej myelocytovej leukémii, ktorú budeme nazývať skratkou CML. Prvé pôvodné odborné články o prípadoch pacientov s CML z roku 1845 pochádzajú z Edinburghu vo Veľkej Británii a mali názvy: ,,Prípad zväčšenia sleziny a pečene, kde smrť bola spôsobená hnisaním v krvi” a ,,Prípad ochorenia sleziny, kde k smrti došlo v dôsledku prítomnosti hnisavej hmoty v krvi”. Zhruba v rovnakom čase v Berlíne publikoval známy vedec Virchow články s názvom ,,Biela krv” a ,,Biela krv a nádory sleziny”, v ktorých uznal, že ,,biela krv” a zväčšenie sleziny neboli spôsobené hnisavým procesom, ale predstavovali samostatnú chorobnú jednotku, ktorú dnes voláme leukémia. Veľký prelom v pochopení mechanizmu vzniku CML nastal až v roku 1960. Teda viac ako 100 rokov po prvom opísaní tohto ochorenia. Peter Nowell a David Hungerford z Philadelphie objavili v jadrách nádorových buniek malý neznámy chromozóm. Chromozómy sú de fakto nositeľmi genetickej informácie. Ľudia disponujú celkovo 23 pármi chromozómov. Ale u pacientov s CML sa objavil nový, doposiaľ nepoznaný chromozóm. Neskôr sa zistilo, že spomínaný malý chromozóm je v skutočnosti chromozóm 22, ktorého veľká časť (nazývaná BCR) je premiestnená na chromozóm 9 v oblasti (nazývanej ABL) a naopak. Hovoríme o vzájomnej výmene časti dvoch chromozómov za vzniku Philadelphia chromozómu. Chromozóm Philadelphia je nositeľom génu, označovaného ako BCR-ABL gén, ktorý produkuje proteín (tzv. Bcr-Abl onkoproteín) s enzymatickou (tyrozínkinázovou) aktivitou (TK). Tento proteín spôsobuje, že kostná dreň produkuje príliš veľa nezrelých bielych krviniek, nazývaných leukemické bunky. CML sa tak stala prvým nádorovým ochorením, ktoré je spájané s genetickou abnormalitou. Keď je existencia tohto chromozómu teda priamou príčinou CML, mala by jeho úplná eliminácia viesť k vyliečeniu pacienta. CML je častou leukémiou v dospelom veku, tvorí 15-20% prípadov všetkých leukémií, s výskytom 1-1,5 prípadu na 100 000 obyvateľov a rok. Ročne na Slovensku pribudne 60 nových pacientov s CML, v susednej Českej republike je to 150 nových prípadov. Ochorenie postihuje o niečo častejšie mužov, v pomere 2:1. Rodinný výskyt CML ochorenia je zriedkavý. Rasové alebo geografické predispozície pre CML neboli doteraz opísané. Výskyt ochorenia narastá s vekom. To znamená, že choroba je vzácna v detstve. Najčastejšie sa CML vyskytuje v druhej polovici šiestej dekády života.Ochorenie je charakteristické svojim dvoj- alebo trojfázovým priebehom. Rozlišujeme chronickú, zrýchlenú (akcelerovanú) a akútnu, resp. blastovú fázu. Približne v dvoch tretinách prípadov, ak už dôjde k progresii, tak progreduje chronická fáza do akcelerovanej fázy, po ktorej nasleduje blastový zvrat. U jednej tretiny prípadov sa blastový zvrat objavuje náhle, bez evidentnej zrýchlenej fázy. Zhruba 90% prípadov ochorenia sa zachytí v chronickej fáze.Ako sa vlastne toto ochorenie prejavuje? Približne 70% pacientov sa najčastejšie sťažuje na únavu, zníženú toleranciu k záťaži, nechutenstvo, bolesti brucha, skorý pocit sýtosti, úbytok na váhe, nadmerné potenie. S postupujúcou progresiou ochorenia sa príznaky zhoršujú. U viac ako 30% pacientov sa ochorenie zistí náhodne pri bežnom vyšetrení krvného obrazu. Vyšetrenie u lekára môže odhaliť splenomegáliu, t.j. nadmerné zväčšenie sleziny. Medzi menej časté príznaky patria príznaky napodobňujúce ochorenie štítnej žľazy (nočné potenie, neznášanlivosť tepla a váhový úbytok), hučanie v ušiach, bolesť v ľavom ramene ako dôsledok infarktu sleziny, cukrovka a akné. Spontánne prasknutie sleziny je vzácna komplikácia. V zrýchlenej fáze sa intenzita príznakov stupňuje. Výrazná splenomegália spôsobuje útlak okolitých orgánov, objavujú sa tráviace ťažkosti a silné bolesti v ľavej polovici brucha. Pacienti sa často sťažujú na bolesti v kostiach. Blastová fáza pripomína príznakmi už akútnu leukémiu.Diagnostika ochorenia zahŕňa vyšetrenie u lekára špecialistu a laboratórne vyšetrenia. K laboratórnym vyšetreniam patrí vyšetrenie krvného obrazu, vyšetrenie kostnej drene a vyšetrenie biochemických parametrov z krvi.Na rozdiel od iných druhov rakoviny, ktoré vzniknú na jednom mieste (ako napríklad rakovina prsníka je v prsníku, alebo rakovina pľúc v pľúcach) a potom sa šíria (metastázujú), rakovinové bunky u pacientov s leukémiou sú prítomné v celom tele už na začiatku diagnózy z dôvodu prirodzenej cirkulácie v krvnom obehu. Z týchto dôvodov sa prognóza neurčuje podľa rozsahu ochorenia, ale podľa už vyššie spomenutých fáz, a to chronická, zrýchlená a akútna.Pacienti, ktorým pred 20 rokmi diagnostikovali CML, nemali pred sebou veľmi priaznivú budúcnosť. Ochorenie obyčajne sprevádzalo vyradenie z pracovného a spoločenského života. Podporná liečba zahŕňala dlhodobú liečbu antibiotikami, ako aj častú transfúznu terapiu červenými krvinkami. Často podstupovali aj náročnú chemoterapiu a transplantáciu kostnej drene, obe s neistým výsledkom. Len necelá tretina pacientov prežívala viac ako päť rokov. V roku 1996 profesor Brian Druker začal experimentovať s látkou so zvláštnym esoterickým názvom CGP57. Táto látka dokázala zastaviť činnosť už spomenutého Philadelphia chromozómu. Nakoľko užívanie látky CGP57 malo zázračný účinok u pacientov s CML s malým počtom nežiaducich účinkov, jeho implementácia do klinickej praxe trvala neobvykle krátko. Už v roku 1998 sa dostal k prvým pacientov. Látka CGP57 patrí medzi tyrozínkinázové inhibítory. Tyrozínkinázové inhibítory prvej generácie znamenali zásadný obrat v liečbe CML. Pacientom zrazu stačila jedna tabletka denne, ktorá im umožnila žiť mnohé roky plnohodnotného života. Mohli ďalej pracovať, venovať sa rodine a svojim záujmom. Časom okrem tyrozínkinázových inhibítorov prvej generácie prišli aj ďalšie podobné lieky vyššej generácie. Tieto inhibítory fungujú tak, že sa naviažu na Bcr-Abl onkoproteín a blokujú jeho aktivitu, čo vedie k zníženiu tvorby leukemických buniek. Veľkou výhodou je, že sa jedná o cielenú liečbu, keďže Bcr-Abl onkoproteín sa nachádza len v leukemických bunkách, a tak nedochádza k ovplyvňovaniu zdravých normálnych buniek.Približne u 75% pacientov nie je Philadelphia chromozóm nájdený po 1. roku liečby - hovoríme, že pacienti dosiahli tzv. cytogenetickú odpoveď (0% Philadelphia chromozóm pozitívnych buniek). Jej dosiahnutie je dôležitým cieľom liečby, pretože je spojená s dlhším prežívaním. Ďalším cieľom v liečbe je dosiahnutie tzv. molekulovej odpovede. To znamená, že nie je možné už detegovať ani produkt Philadelphia chromozómu (0% buniek s BCR-ABL génom). Úplne nedetegovateľné ochorenie by malo znamenať vyliečenie. Túto otázku si položili aj vedci. V roku 2008 profesor Mohone z Francúzska prezentoval na najväčšej hematologickej konferencii na svete výsledky svojej štúdie. U 15 pacientov s CML, ktorí dosiahli tzv. veľkú molekulovú odpoveď, sa rozhodol zastaviť podávanie tyrozínkinázového inhibítoru. Ukázalo sa, že 7 pacientom sa vrátilo ochorenie do 6 mesiacov späť. Avšak čo bolo zaujímavé je fakt, že u 8 pacientov, napriek prerušeniu liečby, vyliečenie pretrvávalo. Na základe tejto pilotnej štúdie boli uskutočnené podobné rozsiahlejšie klinické skúšania, a to napr.: ENESTfreedom a ENESTop. Obe štúdie ukázali, že prerušenie liečby tyrozínkinázových inhibítorov viedlo u väčšiny pacientov k udržaniu veľkej molekulovej odpovede po dobu najmenej troch rokov. Navyše prerušenie liečby viedlo k zníženiu počtu nežiaducich účinkov, ktoré sa spájajú s dlhodobou liečbou tyrozínkinázových inhibítorov. Druhou dobrou správou je, že väčšina pacientov, u ktorej sa ochorenie opäť prejavilo, dosiahlo po opätovnom zahájení liečby rýchlo späť pôvodnú molekulovú odpoveď. V súčasnosti ešte neexistujú presné odporúčania na to, u koho sa môže liečba tyrozínkinázových inhibítorov prerušiť. Nevyhnutnou podmienkou takéhoto prerušenia je však určite ustálená dlhodobá hlboká molekulová odpoveď na liečbe tyrozínkinázovými inhibítormi. Podmienkou jej dosiahnutia je kľúčové dodržiavanie liečby. Pacientom sa to však často nedarí. CML je jedným z mála druhov nádorového ochorenia, kde existuje účinná, život zachraňujúca liečba a pacienti môžu viesť prakticky plnohodnotný život...Kompletný prepis podcastu nájdete na webstránke www.webakademia.sk v časti laická verejnosť pod podcastom OnkológiaDnešnú tému podporila spoločnosť Novartis Slovakia s.r.o.SK1911772335

Dobrý deň, počúvate podcast ONKLÓGIA, ktorý je súčasťou skupiny podcastov ZDRAVIE. V dnešnom diely sa budeme venovať otázke „Dá sa chronická myelocytová leukémia (CML) vyliečiť”? Odpoveď sa dozviete v dnešnom podcaste, ktorý pre Vás pripravil Dr. Chudej, číta Marián Mitaš.Dá sa chronická myelocytová leukémia (CML) vyliečiť?, MUDr. Juraj Chudej, PhD., Klinika hematológie a transfuziológie, Univerzitná nemocnica Martin, Jesseniova lekárska fakulta v Martine, Univerzita Komenského v Bratislave.Chronická myelocytová leukémia je typom rakoviny krvných buniek. Bola prvým opísaným typom leukémie vôbec. Čo je dôležité si zapamätať je skutočnosť, že pod hrôzostrašne znejúcim označením leukémia sa neskrýva len jedno, ale celý rad ochorení.A tieto jednotlivé podkategórie leukémií sa líšia v prognóze, ale aj v spôsobe liečby.Leukémie teda delíme podľa:1.ich agresivity a2.typu postihnutej bunky.Podľa agresivity leukémie rozdeľujeme na akútne a chronické. Termín “chronická” poukazuje na skutočnosť, že ochorenie má pozvoľný priebeh. Pacient nemusí mať žiadne príznaky aj niekoľko rokov. Druhé delenie leukémií vychádza z typu postihnutých bielych krviniek.Dá sa povedať, že v princípe rozlišujeme štyri typy leukémií, a to:1.akútnu myeloblastovú leukémiu2.akútnu lymfoblastovú leukémiu3.chronickú myelocytovú leukémiu4.chronickú lymfocytovú leukémiu.V tomto podcaste sa bližšie budeme venovať len chronickej myelocytovej leukémii, ktorú budeme nazývať skratkou CML. Prvé pôvodné odborné články o prípadoch pacientov s CML z roku 1845 pochádzajú z Edinburghu vo Veľkej Británii a mali názvy: ,,Prípad zväčšenia sleziny a pečene, kde smrť bola spôsobená hnisaním v krvi” a ,,Prípad ochorenia sleziny, kde k smrti došlo v dôsledku prítomnosti hnisavej hmoty v krvi”. Zhruba v rovnakom čase v Berlíne publikoval známy vedec Virchow články s názvom ,,Biela krv” a ,,Biela krv a nádory sleziny”, v ktorých uznal, že ,,biela krv” a zväčšenie sleziny neboli spôsobené hnisavým procesom, ale predstavovali samostatnú chorobnú jednotku, ktorú dnes voláme leukémia. Veľký prelom v pochopení mechanizmu vzniku CML nastal až v roku 1960. Teda viac ako 100 rokov po prvom opísaní tohto ochorenia. Peter Nowell a David Hungerford z Philadelphie objavili v jadrách nádorových buniek malý neznámy chromozóm. Chromozómy sú de fakto nositeľmi genetickej informácie. Ľudia disponujú celkovo 23 pármi chromozómov. Ale u pacientov s CML sa objavil nový, doposiaľ nepoznaný chromozóm. Neskôr sa zistilo, že spomínaný malý chromozóm je v skutočnosti chromozóm 22, ktorého veľká časť (nazývaná BCR) je premiestnená na chromozóm 9 v oblasti (nazývanej ABL) a naopak. Hovoríme o vzájomnej výmene časti dvoch chromozómov za vzniku Philadelphia chromozómu. Chromozóm Philadelphia je nositeľom génu, označovaného ako BCR-ABL gén, ktorý produkuje proteín (tzv. Bcr-Abl onkoproteín) s enzymatickou (tyrozínkinázovou) aktivitou (TK). Tento proteín spôsobuje, že kostná dreň produkuje príliš veľa nezrelých bielych krviniek, nazývaných leukemické bunky. CML sa tak stala prvým nádorovým ochorením, ktoré je spájané s genetickou abnormalitou. Keď je existencia tohto chromozómu teda priamou príčinou CML, mala by jeho úplná eliminácia viesť k vyliečeniu pacienta. CML je častou leukémiou v dospelom veku, tvorí 15-20% prípadov všetkých leukémií, s výskytom 1-1,5 prípadu na 100 000 obyvateľov a rok. Ročne na Slovensku pribudne 60 nových pacientov s CML, v susednej Českej republike je to 150 nových prípadov. Ochorenie postihuje o niečo častejšie mužov, v pomere 2:1. Rodinný výskyt CML ochorenia je zriedkavý. Rasové alebo geografické predispozície pre CML neboli doteraz opísané. Výskyt ochorenia narastá s vekom. To znamená, že choroba je vzácna v detstve. Najčastejšie sa CML vyskytuje v druhej polovici šiestej dekády života.Ochorenie je charakteristické svojim dvoj- alebo trojfázovým priebehom. Rozlišujeme chronickú, zrýchlenú (akcelerovanú) a akútnu, resp. blastovú fázu. Približne v dvoch tretinách prípadov, ak už dôjde k progresii, tak progreduje chronická fáza do akcelerovanej fázy, po ktorej nasleduje blastový zvrat. U jednej tretiny prípadov sa blastový zvrat objavuje náhle, bez evidentnej zrýchlenej fázy. Zhruba 90% prípadov ochorenia sa zachytí v chronickej fáze.Ako sa vlastne toto ochorenie prejavuje? Približne 70% pacientov sa najčastejšie sťažuje na únavu, zníženú toleranciu k záťaži, nechutenstvo, bolesti brucha, skorý pocit sýtosti, úbytok na váhe, nadmerné potenie. S postupujúcou progresiou ochorenia sa príznaky zhoršujú. U viac ako 30% pacientov sa ochorenie zistí náhodne pri bežnom vyšetrení krvného obrazu. Vyšetrenie u lekára môže odhaliť splenomegáliu, t.j. nadmerné zväčšenie sleziny. Medzi menej časté príznaky patria príznaky napodobňujúce ochorenie štítnej žľazy (nočné potenie, neznášanlivosť tepla a váhový úbytok), hučanie v ušiach, bolesť v ľavom ramene ako dôsledok infarktu sleziny, cukrovka a akné. Spontánne prasknutie sleziny je vzácna komplikácia. V zrýchlenej fáze sa intenzita príznakov stupňuje. Výrazná splenomegália spôsobuje útlak okolitých orgánov, objavujú sa tráviace ťažkosti a silné bolesti v ľavej polovici brucha. Pacienti sa často sťažujú na bolesti v kostiach. Blastová fáza pripomína príznakmi už akútnu leukémiu.Diagnostika ochorenia zahŕňa vyšetrenie u lekára špecialistu a laboratórne vyšetrenia. K laboratórnym vyšetreniam patrí vyšetrenie krvného obrazu, vyšetrenie kostnej drene a vyšetrenie biochemických parametrov z krvi.Na rozdiel od iných druhov rakoviny, ktoré vzniknú na jednom mieste (ako napríklad rakovina prsníka je v prsníku, alebo rakovina pľúc v pľúcach) a potom sa šíria (metastázujú), rakovinové bunky u pacientov s leukémiou sú prítomné v celom tele už na začiatku diagnózy z dôvodu prirodzenej cirkulácie v krvnom obehu. Z týchto dôvodov sa prognóza neurčuje podľa rozsahu ochorenia, ale podľa už vyššie spomenutých fáz, a to chronická, zrýchlená a akútna.Pacienti, ktorým pred 20 rokmi diagnostikovali CML, nemali pred sebou veľmi priaznivú budúcnosť. Ochorenie obyčajne sprevádzalo vyradenie z pracovného a spoločenského života. Podporná liečba zahŕňala dlhodobú liečbu antibiotikami, ako aj častú transfúznu terapiu červenými krvinkami. Často podstupovali aj náročnú chemoterapiu a transplantáciu kostnej drene, obe s neistým výsledkom. Len necelá tretina pacientov prežívala viac ako päť rokov. V roku 1996 profesor Brian Druker začal experimentovať s látkou so zvláštnym esoterickým názvom CGP57. Táto látka dokázala zastaviť činnosť už spomenutého Philadelphia chromozómu. Nakoľko užívanie látky CGP57 malo zázračný účinok u pacientov s CML s malým počtom nežiaducich účinkov, jeho implementácia do klinickej praxe trvala neobvykle krátko. Už v roku 1998 sa dostal k prvým pacientov. Látka CGP57 patrí medzi tyrozínkinázové inhibítory. Tyrozínkinázové inhibítory prvej generácie znamenali zásadný obrat v liečbe CML. Pacientom zrazu stačila jedna tabletka denne, ktorá im umožnila žiť mnohé roky plnohodnotného života. Mohli ďalej pracovať, venovať sa rodine a svojim záujmom. Časom okrem tyrozínkinázových inhibítorov prvej generácie prišli aj ďalšie podobné lieky vyššej generácie. Tieto inhibítory fungujú tak, že sa naviažu na Bcr-Abl onkoproteín a blokujú jeho aktivitu, čo vedie k zníženiu tvorby leukemických buniek. Veľkou výhodou je, že sa jedná o cielenú liečbu, keďže Bcr-Abl onkoproteín sa nachádza len v leukemických bunkách, a tak nedochádza k ovplyvňovaniu zdravých normálnych buniek.Približne u 75% pacientov nie je Philadelphia chromozóm nájdený po 1. roku liečby - hovoríme, že pacienti dosiahli tzv. cytogenetickú odpoveď (0% Philadelphia chromozóm pozitívnych buniek). Jej dosiahnutie je dôležitým cieľom liečby, pretože je spojená s dlhším prežívaním. Ďalším cieľom v liečbe je dosiahnutie tzv. molekulovej odpovede. To znamená, že nie je možné už detegovať ani produkt Philadelphia chromozómu (0% buniek s BCR-ABL génom). Úplne nedetegovateľné ochorenie by malo znamenať vyliečenie. Túto otázku si položili aj vedci. V roku 2008 profesor Mohone z Francúzska prezentoval na najväčšej hematologickej konferencii na svete výsledky svojej štúdie. U 15 pacientov s CML, ktorí dosiahli tzv. veľkú molekulovú odpoveď, sa rozhodol zastaviť podávanie tyrozínkinázového inhibítoru. Ukázalo sa, že 7 pacientom sa vrátilo ochorenie do 6 mesiacov späť. Avšak čo bolo zaujímavé je fakt, že u 8 pacientov, napriek prerušeniu liečby, vyliečenie pretrvávalo. Na základe tejto pilotnej štúdie boli uskutočnené podobné rozsiahlejšie klinické skúšania, a to napr.: ENESTfreedom a ENESTop. Obe štúdie ukázali, že prerušenie liečby tyrozínkinázových inhibítorov viedlo u väčšiny pacientov k udržaniu veľkej molekulovej odpovede po dobu najmenej troch rokov. Navyše prerušenie liečby viedlo k zníženiu počtu nežiaducich účinkov, ktoré sa spájajú s dlhodobou liečbou tyrozínkinázových inhibítorov. Druhou dobrou správou je, že väčšina pacientov, u ktorej sa ochorenie opäť prejavilo, dosiahlo po opätovnom zahájení liečby rýchlo späť pôvodnú molekulovú odpoveď. V súčasnosti ešte neexistujú presné odporúčania na to, u koho sa môže liečba tyrozínkinázových inhibítorov prerušiť. Nevyhnutnou podmienkou takéhoto prerušenia je však určite ustálená dlhodobá hlboká molekulová odpoveď na liečbe tyrozínkinázovými inhibítormi. Podmienkou jej dosiahnutia je kľúčové dodržiavanie liečby. Pacientom sa to však často nedarí. CML je jedným z mála druhov nádorového ochorenia, kde existuje účinná, život zachraňujúca liečba a pacienti môžu viesť prakticky plnohodnotný život...Kompletný prepis podcastu nájdete na webstránke www.webakademia.sk v časti laická verejnosť pod podcastom OnkológiaDnešnú tému podporila spoločnosť Novartis Slovakia s.r.o.SK1911772335

In this episode, Keith Flaherty, director of clinical research and targeted cancer therapy at Massachusetts General Hospital, shares his vast wealth of knowledge in cancer starting with the history of treatment from chemotherapy to radiation to surgical therapy and where those methodologies seemed to have leveled off. He also walks us through the timeline of advancements (and lack there of) from when the War on Cancer was declared in the 1970s, through the sequencing of the entire human genome, and all the way to today. Keith dives into the topic of immunotherapy, probably the most exciting recent development in cancer therapy, and also provides us a rundown of his notion of a different approach to cancer that attacks all the essential pillars of cancer growth and survival. Finally, we talk a little bit about liquid biopsies, we discuss the roles of CRISPR and other potentially over-hyped therapies with respect to cancer. We also touch on stem cell therapy a bit, as well as some other common cancer-related questions such as the role of vitamin D and sun exposure in melanoma, and much more. We discuss: Growing up around medicine, and finding a career that you love [7:30]; Medicine as a career, limitations of the med school teaching approach, and the dynamic and accelerating field of medicine and technology [16:30]; Explaining chemotherapy, radiation, and how a cancer develops [23:45]; Surgical oncology, cure rate of solid tumors, and survival rate after tumor removal  [33:15]; 25 years after the War on Cancer is declared, gene sequencing, and why Keith’s was fascinated by the HIV case study [37:15]; Cancer immunotherapy: History, how it works, and why some cancers respond and others don’t [46:00]; MHC complexes, and cancer cloaking mechanisms [56:00]; Comparative biology of cancer: Why some cancer can evade immune detection better than others [1:03:00]; What we learned from the Cancer Genome Atlas Project [1:07:00]; Defining targeted therapy, HER2 breast cancer, chronic leukemia, and the translocation of chromosomes [1:12:00]; Tumor protein P53, the most famous tumor suppressor gene and its ubiquity in cancer [1:17:45]; Activated oncogenes, the RAS pathway, PI3 kinase, RAF gene, and Keith’s “aha moment” [1:24:15]; Advice for starting your career as a scientist/clinician [1:37:00]; Fusion-driven cancers, targeted therapy, and the Bcr-Abl/chronic myelogenous leukemia case study [1:39:45]; Targeted therapy for fusion-driven solid tumors, adjuvant systemic therapy, and the HER2 breast cancer example [1:53:00]; Advancing melanoma treatment, survival, and cure rates with BRAF-MEK combo therapy [1:59:15]; The fundamental pillars of cancer growth and survival, and the toolkit we need to attack cancer from all angles [2:02:40]; Peter’s clinical framework for thinking about cancer and how Keith might improve it, and how the biotech environment is hampering our ability to put together novel cancer treatments [2:05:00]; How useful is CRISPR in terms of tumor suppressing? [2:16:15]; Liquid biopsies as a therapeutic monitoring tool [2:18:00]; Stem cell therapy: The efficacy and potential risks [2:25:15]; Aging and cancer: Is cancer inevitable? [2:28:45]; Vitamin D supplements, sun exposure, melanoma, and exercise [2:32:30]; How and why Keith has straddled the line between science/research and industry/drug companies, and the importance of getting more voices of practitioners at the table [2:42:00]; and More. Learn more at www.PeterAttiaMD.com Connect with Peter on Facebook | Twitter | Instagram.

Dr Boer speaks with ecancer about the genetic subtypes of ALL, and how to best treat BCR-ABL-like patients. She outlines the network of mutations that can drive leukaemia through JAK, ABL and RAS pathways, and describes Philidelphia-like genetic signatures. Dr Boer also considers how druggable these pathways may be, including the use of tyrosine kinases.

Sat, 6 Feb 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19273/ https://edoc.ub.uni-muenchen.de/19273/1/Grziwok_Sandra.pdf Grziwok, Sandra ddc:590, ddc:50

We present a case of a 42-year old female with the rare diagnosis of a myeloproliferative syndrome harboring both a BCR-ABL transclocation and a JAK2V617F mutation.Initially diagnosed with a CML, the patient underwent treatment with imatinib followed by dasatinib. Despite a major molecular response, the patient developed a thrombocytosis. Molecular analyses revealed a heterozygous JAK2V617F mutation, which was detected retrospectively in the bone marrow at the time of CML diagnosis.This case underlines the complexity of MPS pathogenesis. For the clinician, a JAK2 mutational screening should be performed in CML patients without hematological response in the absence of BCR-ABL.

Dr Mullighan talks to ecancer at the European Hematology Association congress 2012. Whole genome sequence analysis of MLL-rearranged infant ALL has revealed remarkably few mutations in a study from the St Jude Children's Research Hospital and Washington University Pediatric Cancer Genome Project. (MLL = Myeloid/lymphoid or mixed-lineage leukemia gene) A small number of genetic changes are needed to drive this disease, meaning epigenetic approaches may be therapeutically beneficial. BCR-ABL-like leukaemia is also discussed, and better screening for patients at diagnosis. Whole genome sequencing for ALL is still in the early stages, but in the next few years enough should be understood to put new therapies in the clinic.

The study by Marin et al is another milestone in our understanding how to manage CML patients on tyrosine kinase inhibitors.

Negative feedback mechanisms fail to limit Src family kinase activity in leukemias driven by the oncoprotein Bcr-Abl.

This thesis applies quantitative mass spectrometry to research topics in relation to cancer. Proteome-wide quantification at the protein expression level and phosphorylation level were achieved. The technologies developed and used here cover the latest improvements in instrumentation in mass spectrometry, strategies in phosphopeptide enrichment in large scale, algorithms in data analysis and their streamlined implementation, and data mining in downstream bioinformatics. For each of the projects described in this thesis, proteome mapping routinely resulted in identification and quantitation of around 4,000 proteins and phosphoproteome mapping often lead to quantitation of more than 5,000 phosphorylation sites. This ‘systems-wide’ quantitation of the proteome and phosphoproteome is a completely novel development, which has not been used in cancer related topics before. Three major biology topics are studied in this thesis. In the first project, the phosphoproteome of a mouse liver cancer cell line Hepa1-6 was analyzed in-depth, by using phosphatase inhibitors (calyculin A, deltamethrin, and Na-pervanadate) to boost phosphorylation. The characterization of the phosphoproteome revealed a broad spectrum of cellular compartmentalization and biological functions. Quantitation of phosphatase inhibitor treatment using the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) method revealed the quantitative effects of these inhibitor compounds on the whole phosphoproteome. To our surprise, these three broadband phosphatase inhibitors displayed very different efficiency, with tyrosine phosphorylation significantly boosted but serine/threonine phosphorylation much less affected. Additionally, a method to estimate an upper bound of the stoichiometry of phosphorylation was introduced by comparing phosphorylation in three SILAC conditions: non-treated cells, stimulated cells (e.g. with insulin), and only phosphatase inhibitor treated cells. The methods developed here can be used directly in development of drugs directed against kinases and phosphatases, key regulators in cancer and other diseases. The second project continues with the application of phosphoproteomics techniques. Kinase inhibitors influence cellular signal transduction processes and therefore are of great potential in rescuing aberrant cellular signaling in tumors. In fact they constitute a significant portion of drug developing programs in pharmaceutical industry. With the aim of quantifying the effect of kinase inhibitors over the entire signaling network, the second project first set out to study two very commonly used kinase inhibitor compounds for MAPKs: U0126 and SB202190. Their effect on epidermal growth factor (EGF) signal transduction was quantified and compared using the HeLa cell system. The study confirmed that the MAPK cascades are the predominant signaling branches for propagating the EGF signaling at early time points of stimulation. These large scale examinations also suggest that U0126 and SB202190 are quite specific inhibitors for MAPKs as the majority of regulated phosphopeptides appears to belong to the MAPK pathways. In the second part of the project, the effect on phosphoproteome changes of the chemical compound dasatinib, which was demonstrated to effectively inhibit the constitutively activated fusion protein BCR-ABL and was recently approved for chronic myelogenous leukemia (CML) therapy, was quantified in the human CML cell line K562. Bioinformatic analysis revealed that the most influenced signal transduction branch was the Erk1/2 cascade. Overall more than 500 phosphorylation sites were found to be regulated by dasatinib, the vast majority not described in the literature yet. The third project compared the proteomes of mouse hepatoma cell line Hepa1-6 with the non-transformed mouse primary hepatocytes. This was performed by combining the SILAC heavy labeled form of Hepa1-6 with the primary hepatocytes. To characterize the features of these two proteomes, quantitation information (i.e. protein ratios between the two cell types) was used to divide all proteins into five quantiles. Each quantile was clustered according to the Gene Ontology and KEGG pathway databases to assess their enriched functional groups and signaling pathways. To integrate this information at a higher level, hierarchical clustering based on the p-value from the first Gene Ontology and KEGG clustering was performed. Using this improved bioinformatic algorithm for data mining, the proteomic phenotypes of the primary cells and transformed cells are immediately apparent. Primary hepatocytes are enriched in mitochondrial functions such as metabolic regulation and detoxification, as well as liver functions with tissue context such as secretion of plasma and low-density lipoprotein (LDL). In contrast, the transformed cancer cell line Hepa1-6 is enriched in cell cycle and growth functions. Interestingly, several aspects of the molecular basis of the “Warburg effect” described in many cancer cells became apparent in Hepa1-6, such as increased expression of glycolysis markers and decreased expression of markers for tricarboxylic acid (TCA) cycle. Studies in this thesis only provide examples of the application of mass spectrometry-based quantitative proteomics and phosphoproteomics in cancer research. The connection to clinical research, especially the assessment of drug effects on a proteome wide scale, is a specific feature of this thesis. Although this development is only in its infancy, it reflects a trend in the quantitative mass spectrometry field. We believe that more and more clinical related topics can and will be studied by these powerful methods.

The BCR/ABL1 fusion protein is found in virtually all cases chronic myeloid leukemia (CML) and a large proportion of acute lymphoblastic leukemia (ALL). The fact that the BCR/ABL1 fusion protein is crucial for the development of leukemia makes this fusion protein an attractive target for therapy development. We have developed a strategy for the in vivo detection of the BCR/ABL1 fusion protein, in which the presence of the BCR/ABL1 fusion protein is detected intracellularly and if the fusion protein is present an arbitrary action is initiated in the cell (e.g. mark the cells or selectively kill the cells). Our BCR/ABL1 detection strategy is based on protein-protein interactions. Two detection proteins are expressed in the cells: 1) protein A, a GAL4-DNA binding domain/BCR interacting protein fusion protein (GAL4DBD-BAP-1) and 2) protein B, a GAL4-activation domain/ABL interacting protein fusion protein (GAL4AD-CRKL). Only when BCR/ABL1 is present in the cell, do protein A, protein B, and BCR/ABL1 form a trimeric complex which activates the transcription of reporter genes under the control of GAL4-upstream activating sequence (UAS). A proof of principle for the strategy was implemented in the yeast system. We did not use full length BAP-1 or CRKL but only those portions of the proteins that directly interacted with BCR or ABL, respectively. We showed in the yeast two hybrid system, that the C-terminus of BAP-1(amino acids 617-879) binds to full length BCR. The site of interaction of CRKL and ABL was confirmed to be the N-terminal SH3 domain (SH3n) of CRKL as described in the literature. Yeast cells (strain CG1945) transformed with a protein A expressing plasmid (pGBT9-BAP), a protein B expressing plasmid (pGAD424-CRKLSH3n), and a BCR/ABL expressing plasmid (pES1/BCR-ABL) showed expression of the reporter genes HIS3 and LACZ. The expression of the HIS3 reporter gene was assayed by growth of the yeast cells on medium lacking histidine. The expression of the LACZ gene was verified by a beta-galactosidase filter assay. Yeast cells that were transformed with the pES1 plasmid without the BCR/ABL1 coding region did not show activation of the reporter genes. Several other negative controls demonstrated the specificity of the assay. Thus the method was able to clearly distinguish between BCR/ABL expressing cells and cells did not express BCR/ABL1. We then adapted this system for use in mammalian cells. The open-reading frames encoding the proteins A and B were recloned into mammalian expression vectors. The human embryonal kidney cell line HEK293 and the murine myeloid progenitor cell line 32D which had been stably transfected with a BCR/ABL expressing plasmid were tested. The firefly luciferase gene and the yellow fluorescent protein (eYFP) were used to evaluate the whole cell population and single cell, respectively. Unfortunately, the system failed to work in the mammalian cell lines tested. Even though the detection system did not work in mammalian cells, most likely due to the cytoplasmic localization of the BCR/ABL1 fusion protein, it should still be a viable strategy for the detection of leukemia-associated fusion protein, which localize to the nucleus (i.e AML-ETO). This strategy could be adapted for purging the bone marrow of leukemia patients using therapeutically more useful effector genes like suicide genes, which encode pro-drug converting enzymes (e.g. HSV thymidine kinase), or markers that can easily be assayed (e.g. YFP).

The application of global gene expression profiling allows to obtain detailed molecular fingerprints of underlying gene expression in any cell of interest. In this work gene expression profiles were generated from a comprehensive cohort of leukemia patients and healthy donors referred to and diagnosed in the Laboratory for Leukemia Diagnostics, Munich, Germany, which is a nation-wide reference center for the diagnosis of hematologic malignancies. Thoroughly characterized clinical samples were analyzed by high-density microarrays interrogating the expression status of more than 33,000 transcripts. In one specific aspect of this work the potential application of gene expression signatures for the prediction and classification of specific leukemia subtypes was assessed. Today the diagnosis and subclassification of leukemias is based on a controlled application of various techniques including cytomorphology, cytogenetics, fluorescence in situ hybridization, multiparameter flow cytometry, and PCR-based methods. The diagnostic procedure is performed according to a specific algorithm, but is time-consuming, cost-intensive, and requires expert knowledge. Based on a very low number of candidate genes it is demonstrated in this work that prognostically relevant acute leukemia subtypes can be classified using microarray technology. Moreover, in an expanded analysis including 937 patient samples representing 12 distinct clinically relevant acute and chronic leukemia subtypes and healthy, non-leukemia bone marrow specimens a diagnostic prediction accuracy of ~95% was achieved. Thus, given these results it can be postulated that the occurring patterns in gene expression would be so robust that they would allow to predict the leukemia subtype using global gene expression profiling technology. This finding is further substantiated through the demonstration that reported differentially expressed genes from the literature, namely pediatric gene expression signatures representing various acute lymphoblastic leukemia (ALL) subtypes, can be used to independently predict the corresponding adult ALL subtypes. Furthermore, it could be demonstrated that microarrays both confirm and reproduce data from standard diagnostic procedures, but also provide very robust results. Parameters such as partial RNA degradation, shipment time of the samples, varying periods of storage of the samples, or target preparations at different time points from either bone marrow or peripheral blood specimens by different operators did not dramatically influence the diagnostic gene expression signatures. In another major aspect of this work gene expression signatures were examined in detail to obtain new insights into the underlying biology of acute promyelocytic leukemia (APL) and t(11q23)/MLL leukemias. In APL, microarrays led to a deeper understanding of morphological and clinical characteristics. Firstly, genes which have a functional relevance in blood coagulation were found to be differentially expressed when APL was compared to other acute myeloid leukemia (AML) subtypes. Secondly, a supervised pairwise comparison between the two different APL phenotypes, M3 and its variant M3v, for the first time revealed differentially expressed genes encoding for biological functions and pathways such as granulation and maturation. With respect to 11q23 leukemias it could be demonstrated that leukemias with rearrangements of the MLL gene are characterized by a common specific gene expression signature. Additionally, in unsupervised and supervised data analysis algorithms ALL and AML cases with t(11q23)/MLL segregated according to the lineage, i.e., myeloid or lymphoid, respectively. This segregation could be explained by a highly differing transcriptional program. Through the use of biological network analyses essential regulators of early B cell development, PAX5 and EBF, were shown to be associated with a clear B-lineage commitment in lymphoblastic t(11q23)/MLL leukemias. Also, the influence of the different MLL translocation partners on the transcriptional program was directly assessed. But interestingly, gene expression profiles did not reveal a clear distinct pattern associated with one of the analyzed partner genes. Taken together, the identified molecular expression pattern of MLL fusion gene samples and biological networks revealed new insights into the aberrant transcriptional program in t(11q23)/MLL leukemias. In addition, a series of analyses was targeted to obtain new insights into the underlying biology in heterogeneous B-lineage leukemias not positive for BCR/ABL or MLL gene rearrangements. It could be demonstrated that the genetically more heterogeneous precursor B-ALL samples intercalate with BCR/ABL-positive cases, but their profiles were clearly distinct from T-ALL and t(11q23)/MLL cases. In conclusion, various unsupervised and supervised data analysis strategies demonstrated that defined leukemia subtypes can be characterized on the basis of distinct gene expression signatures. Specific gene expression patterns reproduced the taxonomy of this hematologic malignancy, provided new insights into different disease subtypes, and identified critical pathway components that might be considered for future therapeutic intervention. Based on these results it is now further possible to develop a one-step diagnostic approach for the diagnosis of leukemias using a customized microarray.

Die vorliegende Arbeit beschreibt die durchgeführten Experimente zur Charakterisierung der molekularen Assoziation der Rezeptor-Tyrosinkinase c-Kit mit dem Onkoprotein Bcr-Abl. Es wird in der zeitlichen Abfolge der chronisch myeloischen Leukämie (CML) besonders für die chronische Phase eine Beteiligung des Stammzellfaktors (SCF), dem natürlichen Liganden von c-Kit, an der Proliferation des malignen Zellklons vermutet. Ob hierbei eine Stimulierung durch SCF wesentlich ist, oder ob der Rezeptor von intrazellulärer Seite durch Bcr-Abl stimuliert wird ist eine wichtige Frage für das Verständnis der Progression der CML. Um diese Frage zu beantworten, wurden Expressionssysteme in Säugetierzellen und Insektenzellen optimiert, um eine hohe Produktion von c-Kit zu gewährleisten. Es wurde eine Koexpression von c-Kit und Bcr-Abl hergestellt um gute Bedingungen in den nachfolgenden Immunpräzipitationen (IP) zu schaffen. In diesen Studien konnte eine Kopräzipitation von c-Kit und Bcr-Abl erstmals nachgewiesen werden. Die Bindungspartner waren jeweils im Western-Blot der IP nachweisbar, sowohl nach Präzipitation von Bcr-Abl, als auch von c-Kit. Im Western-Blots konnte der Status der Tyrosinphosphorylierung von c-Kit detektiert werden. Eindeutige Hinweise auf die Aktivierung von c-Kit durch Bcr-Abl konnten durch diese Experimente zum ersten Mal nachgewiesen werden. Punktmutationen an funktionell relevanten Positionen in Bcr-Abl änderten nichts an der gezeigten Interaktion, bis auf eine Kinase-inaktive Mutante, die keine Phosphorylierung von c-Kit mehr bewirkte. Um eine Bindungsstelle in c-Kit zu charakterisieren, wurden dann Trunkationsmutanten von c-Kit hergestellt. Dabei wurde der extrazelluläre Rezeptoranteil entfernt und immer kürzere Mutanten, durch Entfernung einzelner struktureller Domänen von N-terminal, hergestellt. Diese wurden in IPs mit Bcr-Abl eingesetzt. Es konnte für sämtliche Mutanten eine Kopräzipitation gezeigt werden, so dass eine Assoziationsstelle von c-Kit nicht klar ermittelt werden konnte.

Bcr-Abl, eine onkogene Tyrosinkinase, ist maßgeblich an der Entstehung der chronischen myeloischen Leukämie (CML) sowie einem Teil der Fälle von akuter B-lymphoblastischer Leukämie (B-ALL) beteiligt. Die essentielle Rolle der Kinase-Aktivität von Bcr-Abl für dessen leukämogenes Potential konnte in zahlreichen Arbeiten demonstriert werden. Entsprechend ist Bcr-Abl Zielstruktur des ersten klinisch geprüften Tyrosinkinase-Inhibitors, STI571. STI571 führt bei Anwendung während der chronischen Phase der CML zu einer anhaltenden hämatologischen Remission, die nicht selten begleitet wird von einem kompletten Verschwinden Ph-positiver Zellen aus dem Knochenmark (zytogenetische Remission). In dieser Arbeit konnten zusätzlich zu Bcr-Abl weitere, therapeutisch relevante Zielstrukturen für die Behandlung Bcr-Abl-positiver Leukämien identifiziert werden. Es handelt sich hierbei um die Mitglieder der Familie der Src-Kinasen, die über einen komplexen Mechanismus, letztlich aber unabhängig von der Aktivität der Abl-Kinase, aktiviert werden (siehe Abschnitt 3.1 und 3.2). Tierexperimente zeigten, dass diese Kinasen zumindest im Rahmen der Bcr-Abl-positiven B-ALL von essentieller Bedeutung sind (siehe Abschnitt 3.3). Diese Beobachtung war vor allem deswegen interessant, weil gerade die Bcr-Abl-positive B-ALL nur ein sehr schlechtes Ansprechen auf STI571 als Monotherapie zeigt und weil STI571 Src-Kinasen weder direkt noch indirekt über Bcr-Abl inhibierte. Durch Exploration des Bindungsmodus von STI571 konnte eine Bindetasche in Abl identifiziert werden, die Grundlage für die hohe Spezifität von STI571 ist (Abschnitt 3.4). Die dabei gewonnenen Daten wurden zur Etablierung eines Zellkultursystems genutzt, das die Selektion und Validierung einer neuen Substanzklasse, der dualspezifischen Src-/Abl-Inhibitoren, ermöglichte. PP1 und CGP76030 waren Prototypen derartiger Inhibitoren. Sie blockierten die Aktivität von Src-Kinasen und Abl über einen identischen Bindungsmechanismus. Experimente mit Bcr-Abl-positiven Zellinien deuteten darauf hin, dass die alleinige oder additive Inhibition von Src-Kinasen durch diese Inhibitoren eine zusätzliche Option für die Therapie Bcr-Abl-positiver Leukämien darstellt (Abschnitt 3.5). Dies gilt insbesondere für Patienten mit fortgeschrittener Leukämie oder dann, wenn sich aufgrund spezifischer Punktmutationen eine Resistenz gegenüber STI571 ausgebildet hat. Derartige Substanzen sollten daher in naher Zukunft in Tiermodellen getestet werden.