I'd like to thank you all for taking the time to attend this important session. My name is Jennifer Sipas. I am an endocrinologist at The Ohio State University. And I have the privilege of being up here with my good friend and colleague. Hi, I'm Eyal Robenstock. I'm an endocrinologist from Israel. And we have a fabulous team today with really interesting and novel ideas and research. So I encourage you to stay all the way to the end because it's really interesting. So we're going to go ahead and get started in the interest of time. I have the distinct privilege of introducing Dr. Mimi Hu. She is a graduate of the University of Texas Houston Health Sciences Center. And she completed her residency and endocrinology fellowship at Baylor College of Medicine. She completed an endocrine research fellowship at MD Anderson and joined the faculty in 2007. She is a professor in the department and has a clinical expertise in the evaluation and treatment of patients with thyroid carcinoma. She's going to talk to us today about re-differentiation strategies and neoadjuvant treatments. Good afternoon, everyone. Thank you so much for joining us for this late afternoon session. It's my honor and privilege to be here, especially in my second live conference since the whole pandemic. So I'm glad that you all made it here. It's a wonderful privilege to be here with my colleagues on this stage. And quite interesting how now on a topic of advanced thyroid cancer, four out of the five of us are endocrinologists. So, yes, we're taking over the world. All right. So I'm going to be speaking on re-differentiation strategy and neoadjuvant therapy. Here are my disclosures. We're going to just set the stage on what the current spectrum of approved systemic therapies are for advanced thyroid cancer. That's going to lead us into the outline of development of therapies targeting driver mutations. And that's really going to set the stage for this main talk on re-differentiation strategies for differentiated thyroid cancer and neoadjuvant treatments for locally advanced disease based on driver mutations. So you all know that since 2011, 11 years ago, we now have four multikinase inhibitors that were approved, vandetinib and cabozantinib for medullary thyroid carcinoma and serafinib and limbatinib for differentiated thyroid cancer. Well, recently, cabozantinib received FDA approval for second line therapy for patients with radioiodine refractory and progressive differentiated thyroid cancer that had already failed one line of systemic therapy. Now, these multikinase inhibitors have been used in clinical trials, and I list here all the various multikinase inhibitors that have been used, and the ones highlighted in green are the ones that are FDA approved. They're called multikinase inhibitors because they're pretty nonspecific in what they're targeting, but the most important target is VEGFR2, which is vascular endothelial growth factor receptor 2, which mediates angiogenesis, a very important oncogenic driver. So you see here the varying degrees of targeting VEGFR2 with cabozantinib being the most potent, but these are dirty inhibitors. They are targeting other kinases beyond VEGFR2, including RET, RET fusion, RAF, EGFR, fibroblast growth factor receptor. This is what's leading to some of the off-target side effects that we see in these patients, like hypertension, diarrhea, mucositis, weight loss, and hand-foot syndrome. And so although these drugs are effective and definitely have a role, they can have adverse effects that are dose-limiting and thus decrease some of their oncogenic efficacy. So instead of targeting oncogenic pathways in a more generalized fashion, is there a way of targeting specific driver mutations that can be even more effective in an oncologic perspective but also have less side effects because of less off-target effects? So the thyroid cancer genome atlas characterized the molecular mutations seen in papillary thyroid cancer. The most common and most important mutation that everyone should remember is the BRAFV600E, seen in almost 60% of patients. Amongst other mutations, today's talk we're talking about driver mutations that are targetable, we also see RET fusion, seen in about 6% in the TCGA, but we also would see ENTREC fusions and ALF fusions to a lesser degree. Now the group at Memorial Sloan-Kettering did analysis on poorly differentiated thyroid cancer and anaplastic thyroid cancer, and you see the poorly differentiated on the left, anaplastic on the right, and similar to papillary thyroid cancer, a high prevalence of BRAF mutations of 33% in poorly differentiated thyroid cancer, 45% in anaplastic thyroid cancer. Additionally, we see RET fusions with poorly differentiated thyroid cancer and also ALF fusions. Now ENTREC and RET fusions have also been seen in anaplastic thyroid cancer. So I'm going to start off with the most rare tumor, and of course Dr. Bible will be discussing this later on in further detail, but I really want to highlight anaplastic thyroid cancer because it's like the poster child of targeted therapy as a disorder. So given the prevalence of BRAF mutation in ATC, there was a phase one clinical trial that was enrolling patients with solid tumors and BRAF mutation, and we enrolled quite a few patients with anaplastic at MD Anderson, and you see here 16 patients who were enrolled into this trial, an objective response rate of 69%. That is amazing. Even one patient with a complete response. And this led to FDA approval for the dual therapy of Dibrafinib, the BRAF inhibitor, and Tremetinib MEK inhibitor combination therapy for anaplastic thyroid cancer. Since then, there's been updated data in 2022 where we now have 36 patients with anaplastic thyroid cancer, also very nice response rate. So in BRAF mutated papillary thyroid cancer, the combination of single agent Dibrafinib and the combination with Tremetinib was also used in a phase two trial. So this was for BRAF mutated PTC patients who had evidence of progressive disease in the 13 months prior to study entry. They were randomized one-to-one randomization schema to Dibrafinib, single agent twice daily, and the combination therapy with the MEK inhibitor Tremetinib. 53 patients were enrolled. The study was first presented by Manisha Shah at ASCO in 2017, and just recently it was published in Thyroid and is available by EPUB. So I apologize I didn't have time to put the graphics on because I had to upload my slides before this, so I just furiously updated some of the data. So in the single arm trial, the Dibrafinib only patients, objective response rate of 42%, and the combination therapy, Dibrafinib and Tremetinib was 48%. Now, Dibrafinib and Tremetinib combination therapy was not shown to be superior in efficacy versus Dibrafinib alone. Both arms were well tolerated. So we're able to sustain full dose strategy for a longer period of time. Now, before I go into the slide, when we think about Rett mutations, the first thing you think about is medullary thyroid cancer, obviously in all hereditary patients with a germline Rett mutation, but in the non-hereditary or sporadic cases, a significant portion of them will have a somatic Rett mutation. So in 2017, there were two highly specific and very potent Rett-specific inhibitors that were put in clinical trial in phase 1-2 trial design. So this is one of them, Selvacatinib, which used to be called LOXO-292. And these are the waterfall plots for the patients who had Rett-mutated medullary thyroid cancer. In the left graphic, you have the waterfall plot for the patients who had previous treatment with Vandetinib or Cabozantinib or both. The right waterfall plot are the patients who are naive to drug therapy. And look at these objective response rates of 69% in the previously treated group and 73% in the drug-naive group with some complete responses. Majority of them were partial responses. The table below just demonstrates that multiple Rett mutations were enrolled in these trials, predominantly 918, but there were a few V804 gatekeeper mutations. And for those who might not know, this is a mutation that actually leads to resistance against Cabozantinib and Vandetinib. So very notable that they had responses with this Rett inhibitor. So then the second Rett inhibitor is Pralsetinib, used to be called BLU-667. And it's really hard to show these data next to each other because they look so similar. They're great results. And so the Pralsetinib waterfall plot on the top are the previously treated group. The bottom plot are the drug-naive group and objective response rates of 60% and 71%. Also multiple Rett mutations including the gatekeeper 804 mutation included. So this trial enrolled patients not just with Rett mutations but also Rett fusions. Rarely seen in non-small cell lung cancer. Additionally, other solid tumors that had the Rett fusion, including our non-medullary thyroid cancer. So these are to show you the selprocatinib-treated patients with Rett fusion-positive thyroid cancer on the left, and on the right are the Pralsetinib-treated patients. Less patients because it's more rare to be found in papillary thyroid cancer. But you see objective response rates of 79% with selprocatinib, 89% with Pralsetinib. Really deep and long-enduring responses. So what's also remarkable, other than the responses, is the well-tolerability of these drugs. My patients come to me and say, Doc, I think I'm on the placebo because I feel great. I'm like, no placebo, you're getting the active drug. So it's really quite amazing. So these drugs, these two, selprocatinib and Pralsetinib, received FDA approval in 2020. So 2020 wasn't all the awfulness that it was. And it received approval for Rett-mutated medullary thyroid cancer and Rett fusion-positive non-medullary thyroid cancer, as well as non-small-cell lung cancer. So Ntrek fusions, even more rare, seen in 2% to 5% of patients with papillary thyroid cancer. Some rarely in anaplastic thyroid cancer. And so larotrectinib is a Ntrek inhibitor, and it actually has FDA approval for a tumor agnostic. So it doesn't matter what tumor you have, as long as you have an Ntrek fusion. And so these data are the updated data recently published with lead author, one of my colleagues, Dr. Steven Wagaspak, in European Journal of Endocrinology, where 29 patients with Ntrek fusion thyroid cancer were enrolled, predominantly papillary thyroid cancer. There were a couple follicular and actually a few anaplastic thyroid cancer patients. So you see on the left side the waterfall plot where the well-differentiated cancers in the blue on the left and the purple represented the more de-differentiated anaplastic thyroid cancer. And with the papillary thyroid cancer, follicular thyroid cancer group, the objective response rate was 86%. I know it's kind of small on your screen, but that is remarkable. And with anaplastic thyroid cancer, 29%. So certainly less than the papillary thyroid cancer cohort, but still that's pretty good for anaplastic thyroid cancer. So then you see the swim lane plot on the right side. Those little hash marks are three-month increments. So you see the long duration of effect that this drug has in these patients. So 24-month duration response, 81%, 24-month progression-free survival, 69%, and overall survival, 76%. So beyond the multikinase inhibitors now, we have these very specific inhibitors of driver mutations of BRAF, Ntrek, and RET. So now six additional drugs approved for differentiated thyroid cancer, ATC, and MTC, which harbor these alterations. But with this group, of course, how can we forget radioactive iodine, our original targeted therapy, right? So multikinase inhibitors and even these very highly potent kinase inhibitors, they're effective. But with long-term use, there can be significant accumulation or even just a fatigue that our patients have with dealing with these adverse effects associated with these drugs. But also they could potentially lead to resistance mutations. And also there's financial burden. These patients have to come often to their visits, get imaging studies, and also just the drug therapy. So the idea of maybe going back to radioactive iodine and re-differentiating these patients so that radioactive iodine can be given after a short course of a kinase inhibitor is very attractive to limit the amount of adverse effects and potentially lead to longer-term remission in these patients. So why does de-differentiation occur in our follicular-based thyroid cancer? So I'll actually defer to at Memorial Sloan Kettering, Dr. Fagan's lab that did wonderful work in this. But essentially mutations that lead to a gain of function of map kinase pathways such as BRAFV600E or fusions of RET and NTREC or RAS mutations is going to lead to increased expression of ERK. But additionally, it's going to down-regulate thyroid differentiation genes. And by down-regulating those thyroid differentiation genes, it's going to reduce NIS or sodium iodine symporter expression and also decrease the ability to localize the NIS to the cellular membrane to allow for iodine uptake. Now in mouse models of thyroid cancer, Dr. Fagan's lab showed very elegantly that selected map kinase pathway inhibition will increase NIS expression and also iodine uptake. So what about in humans? So this led to Alan Ho's group at Memorial Sloan Kettering where they did a pilot clinical study of 20 patients with radioiodine refractory papillary thyroid cancer and poorly differentiated thyroid cancer. They used the MEK inhibitor selumetinib. And what they did was they evaluated the I124 PET CT, not a study that's widely available at many institutions. But they did that. And then after five weeks of selumetinib, they re-imaged with the I124 PET. And what you see here are various patients in A, B, C, and D panels where the before selumetinib and then the after where there was increased iodine adiabatity in the metastatic lesions. Now the patients enrolled had variable mutations. There were BRAF, RAS, RETPTC, fusion, wild type. And out of the 20 patients, 12 of them had increased tumor uptake. Of those 12, eight met the dosimetry threshold and they received a treatment dose of I131. And five of them had a partial response on continued follow-up six months after radioiodine. Three of them had stable disease. Notably, all five of the RAS mutated patients had iodine uptake and received radioactive iodine. And thus, the RAS mutation predicted a better response. Now, Mike Rothenberg at Mass General at the time followed this up with, well, could maybe targeting BRAF specifically with a BRAF inhibitor be even better at leading to resensitization to radioactive iodine? So this small cohort of patients of 10 with radioiodine refractory PTC with the BRAF mutation, they were enrolled and they received a BRAF in it for four weeks. And they used the traditional diagnostic whole body scan. And you see the baseline where this is a representative patient that did not have significant uptake in metastatic lesions. After four weeks of the BRAF in it, if there was increased radioiodine avidity, then they gave a treatment dose empirically of 150 millicuries. And you'll see here in that panel some additional lesions took up iodine on the post-treatment scan. Just a couple of patients showing representation of tumor regression after both the BRAF in it and iodine. That top panel is a patient with a mediastinal lymph node that regressed after both treatments. And then the bottom is the metastatic lung lesions that had regressed with treatment. And they followed these patients up to six months after radioiodine. This is just a waterfall plot showing those ten patients where out of the ten, six had new findings of radioiodine avid distant metastases. On follow-up at six months, two patients had a partial response and four had stable disease. Now our group at MD Anderson also evaluated our patients where we used this re-differentiation strategy. So patients were either treated with a BRAF inhibitor, if they had a BRAF mutation, or a MEK inhibitor, if they had a RAS mutation. And this was published by Tanya Jaber, who was one of our oncologic endocrine fellows who has since graduated. And showing our 13 patients who were treated with one of these agents. And just a few representative patients where they had their pre-inhibitor diagnostical body scan. And after being treated with BRAF or MEK, the significant uptake, look at that lung uptake in patient number four. I mean, it's just very remarkable. And so this waterfall plot, let me walk you through it. The blue represents what the patient's tumor lesions did in response to the kinase inhibitor. So modest results, most of them kind of stable. But after radioiodine was given, you see the orange bars, significant regression of disease afterwards. So resensitizing them to radioiodine and treating them with a dose of radioiodine was very beneficial. And on follow-up of these patients with a median of 14.3 months, a significant amount of them still had stable disease off of their BRAF and MEK inhibitor. Well, what about the Ntrek and RET fusions? So there have been a few case reports, and I'm going to show them to you all here, that with these TREK fusion and RET fusion patients that they're treated with a specific inhibitor, there might be also a resensitization to radioiodine. So this was the first report by Dr. Grossin in New England Journal of Medicine two years ago, where there was an Ntrek fusion PTC patient who was on lumbatinum for metastatic disease. And you see that top left panel where the whole body scan was negative in the distant metastases. The patient was having progression, so found to have the Ntrek fusion, went on to larotrectinib. Three weeks later, you see all of that uptake in the lungs. And you have the SPECT CT imaging overlying and seeing those areas of uptake in the lung disease. They followed up with another very interesting report with a RET fusion papillary thyroid cancer patient who had no evidence of radioiodine at baseline due to widely metastatic disease, was treated with selprocatinib for three weeks. And you see that middle panel of reuptake of radioactive iodine metastatic lesions. They gave the patient radioactive iodine, and two weeks later, you see that increased areas of uptake. Now, this patient was maintained on selprocatinib due to the burden of distant metastatic disease. These panels are very busy, but I just wanted to highlight that there have been pediatric patients with Ntrek and also RET fusion that have been described in the literature. The top C panel was described by the Korean group where they had two young female patients who had either an Ntrek fusion or a RET fusion. And the top patient is the RET fusion patient who, after being treated with selprocatinib, significant uptake of radioiodine in the lungs, actually received two doses of radioactive iodine while she was maintained on selprocatinib and had a regression of disease. Whether this was just the selprocatinib or the combination with the radioiodine is hard to tell because the selprocatinib was maintained long-term after the radioiodine. But the bottom panel is a patient of my colleagues, Dr. Steven Waguespack, who is a dual-boarded adult amputee endocrinologist. And so this was a young gal of his who had an Ntrek fusion. And after being on larotrectinib for about six months, he did the iodine scan, and you see all of that lung uptake. So he did treat her with radioactive iodine, I131, and stopped the larotrectinib. And one year on follow-up, the patient's disease is still regressing, where some lesions are no longer seen in the lungs. So I've just kind of shown you a few representative case series of re-differentiation strategy with MEK and BRAF inhibitors. This is just a listing of all the published case series here. Now, just so you know, there is a Phase II ongoing trial in Europe looking at Tremetinib followed by radioactive iodine for patients with RAS mutations. This is led by Dr. Sophie Leveleau. So what about the other targeted therapy, surgery? So can we be more elegant than what this cartoon showed? This is an actual cartoon that was published in 1793 that I found. But anyway, could some of our patients who have really, really advanced disease in the neck, could we potentially improve or maximize the surgical effectiveness in resecting as much disease as possible and to maybe get them down to an R0 level with potentially less comorbid side effects such as recurrent laryngeal nerve damage or brachial plexus injury and all the others? So the idea of neoadjuvant therapy prior to surgery is not unusual. This has been seen in other solid tumors like breast cancer and such. So this is a patient of mine with medullary thyroid cancer, really locally advanced disease. You can see the compression of the tracheal lumen and deviating the trachea off to the right. You may not be able to appreciate this, but there are significant laryngeal wall and also esophageal wall involvement. This patient actually did not have distant metastases. Unfortunately, he did not have a targetable RET mutation. But can trace that to this anaplastic thyroid cancer patient of my colleague, Dr. Maria Cabanillas, where this ATC is so aggressive, that's tumor coming out of the neck. And the trach is in place for airway protection. Luckily, she had a BRAF mutation treated with dabravin and trimetinovim four weeks later. This is just the regression. The surgeon did not take it out yet. So this is just regression with treatment. So our group had published our experience with using BRAF with MEK inhibitor therapy in our BRAF mutated ATC patients. So you see the PET CTs for a few of these patients, the before and one to five months after using dual therapy, significant regression disease on the PET CT. And then on the swim lane plots, what you see are the yellow lightning bolts. Those are when the patients received surgical resection, and then they continued with therapy. And you see that some of these patients have had durable responses even beyond 20 months for anaplastic thyroid cancer. That is amazing. So we have a active and rolling phase two trial using neoadjuvant dabravinib, trimetinib, and pembrolizumab combined with immunotherapy for BRAF mutated anaplastic thyroid cancer. Now, Dr. Cavania has also looked at a trial using Vemurafinib in a neoadjuvant setting for BRAF mutated papillary thyroid cancer. So 17 patients were enrolled. 14 of them completed two months of Vemurafinib. And what you see in the waterfall plot is of those 14 patients who received Vemurafinib, three had a partial response to the BRAF inhibitor, nine had stable disease, but you saw regression of their disease. So 11 out of the 14 went on to surgery. One just refused the surgery. He didn't mind the chemotherapy, wanted to stay on it. The other two had progressive disease. So of the 11 who went to surgery, eight had an R0 pathologic response, which means negative surgical margins, and or an R1 path response, microscopic positive margins, and three had an R2 response. These are patients with medullary thyroid cancer with a RET mutation. The panel on the left labeled as one was a patient with a RET deletion. And this patient's tumor, if you can appreciate, was more than 180 wrapped around the subclavian artery and also the aorta. And the patient underwent subacademy therapy, significant regression disease that you see on the right side of those blue arrows, and went to surgery. The patient continued on self-recatenib due to distant metastases. So one of our head and neck surgical fellows just recently presented that case in addition to two other MTC patients of mine who had RET mutations at the AHNS annual meeting, showing the amount of regression of disease after self-recatenib and able to go for surgery. None of these patients developed recurrent laryngeal nerve damage or even hypoparathyroidism. We also have a phase two trial with neoadjuvant self-recatenib for RET-altered thyroid cancer. We're trying to open it up at a few other sites around the country. So we are eagerly awaiting results on this trial. Now finally, neoadjuvant limbatinib is being evaluated for locally advanced thyroid cancer. This is a phase two open-label multicenter trial led by Dr. Greg Randolph at Mass General. So limbatinib is gonna be given to patients, adult patients with advanced thyroid cancer with extra-thyroidal extension and or locally invasive disease. They're excluding ATC and MTC. Also patients with intraluminal airway tumor, complete carotid encasement or infiltration, prior radiotherapy to the neck anticoagulation or antiplatelet therapy, mainly because limbatinib does have very significant anti-angiogenic effects and there is risk for fistula as well as bleeding. And also they're excluding patients who've had prior anti-VEGF therapy. Primary outcome is looking at R0, R1 resection rate. So in summary, I hope I've impressed upon you that the management of locally aggressive and advanced thyroid cancer is rapidly evolving and it's very exciting. For all of the fellows who might be in the room, this is an area to get into because it's rapidly growing and we need more fresh faces to come into this field. Personalized care involves knowing the molecular drivers of disease to effectively guide treatment. At our tumor board, we present our cases as some such year old male with papillary thyroid cancer with a BRAF mutation. It comes in that first sentence now. New strategies with systemic therapies are being investigated to optimize hallmarks of treatment that have traditionally been used, radioactive iodine surgery. But further investigation is needed to understand the variable responses that we see with re-differentiation strategy and neoadjuvant strategy and how long are these responses gonna go on and what are the long-term consequences of these treatments. So thank you very much for your attention. I'll take your questions. Thank you. Can I take one? Yeah. Thank you for that excellent talk. We have just one question from the audience here and it's when do you consider the benefits of these targeted therapies versus the risks of side effects and how do you use that to sort of gauge when is it appropriate to start thinking about treating a patient? When am I not thinking about this? So honestly, a lot of us see the run-of-the-mill papillary thyroid cancer not exceedingly advanced and no evidence of distant metastasis surgery. Radioiodine is still the most standard therapy. But when patients have advanced disease, recurrent disease or fibroglobulin rising and you're looking and you're hunting, you're finding lesions or you know that they have a more aggressive subtype. These are the patients where in whom I'm checking for molecular mutations. I need to understand what is the biology that's driving their disease. And if they have a targetal mutation like a BRAF mutation or rarely even those Ntrek fusions or RET fusions, those drugs are highly effective and significantly better tolerated. Now I do give kinase inhibitors, multi-kinase inhibitors on Vatnib, Cavo, Vandy. I give it and under skilled hands, you can control those adverse effects and patients can learn to live with them. But why do that if I have drugs that are maybe as effective, not more, with less side effects? So if they have a targetal mutation, we're cheering. Yeah, yeah. So another question, you showed promising results with re-differentiation therapies, really attractive approach. So you have a patient, the disease is advancing. When would you consider targeted therapies and when would you consider trying re-differentiation? Oh, thank you. Yeah, this is the question that we always have is, are we just gonna give a short-term kinase inhibitor for re-differentiation strategy or sometimes long-term? So in my experience, it feels like the patients who have, even if they have widely metastatic, but lower volume disease, like lesions that are less than two sonometer, those are the ones that tend to have a little better response because you're not starting off with a big bulky lesion that you're trying to shrink. And then radioiodine can be potentially more effective in getting into those lesions. So I find that that's helpful. Also metastatic bone disease. So our multi-kinase inhibitors don't have great penetration to the bone environment. And so radioiodine re-sensitization strategy in that group is very promising because if we can get radioiodine to the bone, we could potentially lead to long-term remission there. Great. Thank you so much. One last question. No, I'm asking. Yeah. Is mutation analysis widely available? So how do you do molecular testing? Right, so sometimes if you're at a, especially in an academic institution, you might already have a CLIA-certified molecular panel. So usually we want to do next-generation sequencing to get really good testing. And for the fusions, you really need good RNA-based sequencing. So you do need to talk with your institution to see if they have a good panel and what mutations they find, okay? Or they can be able to pick up. If you don't have that at your institution or you're in private practice, you can actually go online and request these molecular testing from various platforms. And so there are various platforms that are out there and I've used many of them. And just order online and those platforms will actually go to the pathology lab, pull the blocks that they need and analyze it. Yeah. And we could talk about those if you want to afterward. Okay, thank you. Okay, thank you. Thank you. ok so that was breathtaking all the options that we have and now for the next speaker professor Keith Bible he's a well-renowned oncologist specializing in treatment of advanced endocrine cancers he does also basic transit translational research is the co-chair of the ATA anaplastic thyroid cancer guidelines task force is an emeritus professor of oncology at the Mayo Clinic in Rochester and he is a member of the ATA endocrine society and I talk so the topic of the the talk would be therapies that impact survival in ATC please Keith no I control thanks much it's good to have someone else in control once in a while so thanks everyone for attending and I appreciate the opportunity to speak today so today we were to talk about in part therapies that impact or have potential to impact survival in anaplastic thyroid cancer which is actually somewhat of an early topic this photograph here actually Mimi asked me where it was taken but she didn't ask me why I was showing it and there this was actually taken in April the Milky Way above arches but there's some analogy to anaplastic thyroid cancer so first of course historically the outcomes for anaplastic thyroid cancer have been dismal they've been very dark just like the night sky and I'm hoping that we can make things a little brighter as seen in this photograph second thing is we can only see part of the Milky Way here you can see part of it peeking through the one of the windows in the arches area and so we're getting a glimpse of what's to come but there's still a lot hidden and we also want to present a very broad array of information about anaplastic thyroid cancer not just specific to cure provided by endocrinologists but also by radiologists radiation oncologists medical oncologists surgeons they all have an important role they all have important roles to play in therapy and impacting survival positively so I have no financial disclosures and you've heard some of my unpaid conflicts and I will state that there will be off-label therapies discussed just like in Mimi's talk disclaimer that is really very important here is that there have been no fully accrued adequately powered randomized therapeutic clinical trials in anaplastic thyroid cancer and this is impediment truly making firm pronouncements over survival advantages now I have the QR code up here if you want to send us questions electronically if you don't have that already so learning objectives gain an understanding of the factors that are currently known or implicated as positively impacting survival in ATC and then to start to get you to think practically about how to integrate these findings into your own practice when you see patients with ATC so the first thing that I must say is that it is no longer appropriate in our view to consider ATC as a death sentence necessarily there are therapies that have evolved as Mimi alluded to and we'll talk about more today that are impacting survival and many of these therapies are available fairly broadly now not all of them but many are available broadly and you'll have potential access to further progress in anaplastic thyroid cancer is going to require more work and very rigorous randomized trials so historically before the more modern era when I entered oncology almost everyone died of anaplastic thyroid cancer and if you did not refer them for aggressive treatment most everyone would die quickly in often a matter of weeks and from disease in the neck from asphyxiation so that's illustrated by the study referred to in British Columbia which showed that 85% of anaplastic patients died within one month of diagnosis unless they were referred on to get treatment and so when we look at impacts on survival we have to start historically and here what we found is in earlier studies survival was longer amongst the patients who were referred on to treatment and who received surgery radiation or a combination of the above and also associated with chemo radiation is mentioned in this study from Thailand so in all parts of the world including areas which are very distant from the US and may have more limitations in terms of access ready access financially to kinase inhibitors you always will have access to surgery radiation and cytotoxic chemotherapy and those alone were the first treatments which started to impact survival in ATC so to illustrate this just with a Mayo series but this is not unique to Mayo Clinic we found when I started trying to move the need a little bit on anaplastic thyroid cancer that in the year 2020 our median survival was three months for patients with anaplastic thyroid cancer and it was so miserable as we're working on with others developing other treatments to try and improve outcomes we wanted to implement a treatment quickly that might have some potential to help our patients so at that time the two class of agents which were known to have some efficacy in a plastic thyroid cancer we have recyclings and taxing's and because there is synergy between doxorubicin and dosetaxel a representative of each of those two groups and that had been used in breast cancer we combine them with IMRT as early treatment to see if we might get some impact to make a head roads in roads into improving outcomes with anaplastic patients and we treated 10 consecutive patients these were our patients who were 4a and 4b excluded 4c and in the first 10 patients our median survival was about 60 months and this led us to be stunned and to expand the cohort if you look at all of our patients in historical cohort from before the year 2000 with a median one year survival 10% this practice change shifted the median survival this is all comers this is a being for a for a B and for C combined what you found is that the one year survival went from 42% now this is not unique to the Mayo Clinic you can all do this in your practices you don't need fancy drugs you need ancient drugs like cytotoxic chemotherapy and IMRT radiation therapy and surgery so this slide illustrates that in patients with 4b there can be a very great impact these were patients in the upper curve who elected the multimodal therapy and in the lower curve palliative treatment they requested no aggressive therapy you can see that the patients who were for even 4b who elected only palliative care no one survived a year whereas at five years we had in the neighborhood of 30% patients surviving so this is really started to move the needle even before we come to our more modern era of drug development so the take-home points are ATC is rapidly and uniformly fatal disease if untreated time is of the essence in treating ATC so you can't delay you have to move forward quickly you have to establish a diagnosis as fast as you can and treatment as fast as you can and combined modality treatment without newer drugs can impact survival the application of these commonly available approaches is a important tool that you all have regardless of availability of other drugs so how about newer drugs let's not talk about dinosaur drugs that I've used in medical oncology the cytotoxics what about multi kinase inhibitors checkpoint inhibitors mutationally guided therapeutics and combinatorial approaches and as Mimi pointed out to guide some of this care it is critical to do early somatic genomic interrogation in ATC so that we know what targeted mutations might exist because in ATC generally you're going to need a next treatment and a next treatment and a next treatment so you want to queue up the next treatment while the patient is still responding to the treatment you've just started so we know that there has been great success from differentiated therapy cancer treatment with multi-kinase inhibitors and in Japan ATC has an approval for latvatin in for anaplastic not just differentiated and that was based upon a study done there which demonstrated a partial response rate of almost 40 percent thirty point seven percent interestingly in an italk study in another smaller series that have been done outside of Asia there has been a much lower partial response rate more like five percent and I summarize these in this slide but curiously the overall survival is very similar between the studies so there are a couple of take-home points here one is this may these data may help to reconcile in your minds why there was approval in Japan and not the u.s. and really in terms of overall survival the studies really were not much different it was a response rate difference but not overall survival and it also points out that in anaplastic thyroid cancer unlike many other cancers we really cannot use response rate as a surrogate for overall survival we really have to use a harder endpoint and overall survival is going to be an important primary endpoint in trials going forward so what do we know from this well there is a transient effect of some multikinase inhibitors in anaplastic thyroid cancer and some patients will respond brilliantly but usually not for very long so the question is what other approaches can we use checkpoint inhibitor have really been game-changing in a number of cancers and in this first really stunning result in anaplastic thyroid cancer using this fertilism ab which is a PDL one antibody directed antibody what was reported here is in study of 42 anaplastic patients a partial response rate of almost 20% and there was an association of response with PDL one staining greater than 1% but critically the median survival was not three months like you saw with van van it was 12 months so this is really a little more encouraging data so what else have we learned from other immunotherapeutic trials so there's a recently published online trial from as I remember memorial which looked at a combination of a PD anti PDL one therapy and CTLA for treatment hoping to do better than even the prior prior spartalism and trial but curiously they had no responses and their overall survival was only four months so the question is why you have different PDL one inhibitors what's going on what what accounts for the differences and I don't have an answer for that yet but I think we can learn a little bit more so in many cancers there has been a rational development of the combination of immunotherapy in particular pamphlet is a man with multi kinase inhibitors and in most probably with lenvatinib, based upon a synergy, immunologic synergy, between these two drugs in treating a variety of cancers. And Jenna French and Brian Haugen have developed rationale mainly in differentiated thyroid cancer, which you're probably familiar with. But this has been borne out in many different cancers. In a few smaller trials, this combination has led to both impressive response rates, here 42% and 66%, and with overall survivals, which are much better than the three months we otherwise saw. So there is this signal from this combination of pembrolizumab and lenvatinib, which has been worthy of pursuing. So a German consortium is studying this in a trial which is not yet published, but was presented at the more recent ITOG meeting, which confirmed that the response rate is very high from this combination in ATC, 50% or 60%. And their overall survival thus far was about 11 months, suggesting that this combination may be the way forward using immunotherapeutics as a next step. So we'll here watch this space. The problem with this combination is in our patients who have been treated with prior neck irradiation to a high dose, there are many complications, infectious complications. And we noticed this at Mayo, and this was noted in Germany. In the German study so far, about half of the patients actually died from therapeutic complications, not from anaplastic thyroid cancer, and they seemed to be largely infectious. And this has not been noted with this combination in lung cancer, non-small lung cancer, for instance. And the question is why. I think this is because of the higher dosage of radiation that we generally give in radiation with regard to ATC, with regard to radiation in lung cancer. But we still have a lot to learn. So the way forward here is thought to be to administer, co-administer antimicrobial agents, including antifungal agents, to see if we can tamp down this collateral damage which is leading to the deaths of a large portion of our patients who are not dying from anaplastic thyroid cancer. So we already heard from Mimi about the new adjuvant approach to ATC in BRAF V600E mutated ATC, which can significantly downstage cancer. But there is a comment here. This is specific to the BRAF V600E mutation, not to other BRAF mutations. So it's important to remember V600E is the important mutation to consider. I think most of us were very strikingly affected by these data. Now, this combination of Dubrafinib and Tremetinib has also been used in metastatic anaplastic thyroid cancer and also been very effective, again, with high response rates, but importantly with high, longer overall survival of a year or greater. And so this is starting to look much more favorable than a pankinase inhibitor or a multikinase inhibitor like Lenvatinib. So the difference here isn't really the response rate so much, but the durability response and overall survival. So this really, as Mimi pointed out, is an important therapy in anaplastic thyroid cancer, hence need for early screening. So how about other treatments? You also heard about all of these agents or classes of agents from Mimi. So RET, Fusion ATC, ELK Altered ATC, TREK Fusion ATC. There are patients who can respond to each of these targeted treatments, but you need to know the alteration. Not all genomic interrogation of tumors, however, will demonstrate fusions, and so it's important to make sure that the platform you use is going to detect the targetable fusions. I think these are creeping into most of the platforms now, but historically they've not been included in all interrogation platforms. So how about the dinosaur approach of cytotoxic treatment? Well, the cytotoxic treatments have mainly found a place in conjunction with early upfront initial treatment of anaplastic as a multimodal treatment with IMRT, often combined with surgery. But there are circumstances in which you can get very good in vitro and in vivo synergy of agents combining targeted or multi-targeted small molecule kinase inhibitors with chemotherapy, in particular with taxanes. This seems to be mediated by a death driven by what's called mitotic catastrophe, whereby you take a delay in cell cycle traverse from the antimicrotubule inhibitors, such as taxanes, and you prolong that with a kinase inhibitor, which also targets cell cycle, to delay traverse through the cell cycle and trigger this cell death called mitotic catastrophe. So in a large but not large enough trial that was done through a consortium, initially a radiation oncology treatment group, combining Paflitaxel with Pazopanib or placebo, you can see that there were numerical advantages to this combination of humans. But the trial was underpowered, and we need to learn more about this. So this is not statistically significant, but numerically significant. We really need to do more randomized trials, and we need to power them and to invest in the trials so that we really know what's impacting survival. But this trial illustrates that, despite having 20 patients per arm, roughly, in non-metastatic patients, it still was not sufficiently powered to show a difference one way or the other with regard to the two treatment arms. Here you see response, which is six months into treatment of a patient with lung metastases from anablastic thyroid cancer. So how about other treatments? Combinatorial debrafinib, trabetinib, and intensity modulated radiation therapy is being looked at through an ITOG trial, which is now moved from, as the epicenter was, Ohio State. Now it's the city of Hope. And so this is ongoing, and this is an early up-front trial that you can refer patients to. And it has multiple sites, and the number of sites will probably increase further. But it's something that is an initial treatment trial that is open now and is available and something that merits further consideration for your patients. So we talked about the need for randomized therapeutic trials and using overall survival as an endpoint. I want to highlight that the ETA guidelines, though published sometime previously, most all of the data and the recommendations contained in those guidelines are absolutely relevant today. There is a pocket card, which can be very helpful to you and may give you some initial guidance with the decision trees, first with regard to approaching anablastic thyroid cancer, the need for early assessment of tumor mutations. And I want to also highlight that patient preference is critical because there are some patients who will like to decline treatment because of the rigors of treatment, the potential collateral damage, the requirement for travel to be at a center that can offer such treatment. So this sometimes will affect patients' decision-making with regard to whether to do treatment or not do treatment. So always with a very lethal disease like anablastic thyroid cancer, consideration of hospice and palliative care are all ways of consideration. In terms of the guidelines, the ETA guidelines for anablastic thyroid cancer, this highlights also, as we did today, the need for consideration of targeted therapy, the importance of consideration of debrafinib-trametinib, and they also stress this option for early treatment with surgery, radiation, chemotherapy, with definitive intention. 4C is very similar, but there there is not expected any curative approach, and so there's more emphasis on targeted therapies and systemic approaches early on. Key points. So anablastic thyroid cancer, as we already discussed, is rapidly and uniformly fatal if untreated. When I started treating anablastic patients, the majority died from disease in the neck. Now, in our experience, we seldom have recurrences in the neck which are threatening. It's less than 1 in 10 of our anablastic patients. So we've shifted away from disease which is lethal local regionally to a disease which is more threatening with regard to systemic disease. Time is of the essence, and MD Anderson Group has published on this. If we hear about a patient with anablastic thyroid cancer, we're generally offering to see the patient within a day or two and to start treatment very quickly. Most often we can see the patients faster than they can get to us, and this is true with most centers of excellence in anablastic thyroid cancer. It truly does make a difference. Stage is a critical determinant of survival. For A patients treated with a combined modality approach, we've remarkably had no deaths with this approach. For Bs, you saw the data, it points out that it is critically important to treat patients when they're early on with regard particularly to this multimodal initial treatment. In stage 4C, we have difficulty demonstrating a survival advantage from even the most aggressive initial approaches, so we really need to see the 4A and the 4B patients. Early mutational interrogation is key, and so as soon as you suspect the patient may have anablastic thyroid cancer, you want to start genomic interrogation. Alternatively, if you have a time delay in your lab, you can do immunochemistry for BRAF, which at least gives you a starting point while your other genomic data are maturing. Checkpoint inhibitor immunotherapy, particularly in this combination of multi-kinase inhibitors like lenvadinib and PD-L1 antibodies, seem to have promise, and there seems to be a signal not just with regard to response rate but also with regard to a apparent improvement in overall survival, and this is going to be something that we're going to learn more about in the near future. Combinatorial therapeutics combining other agents, including cytotoxics and targeted agents, may also find application, but we really have a lot more work to do. So just like in the photograph of the Milky Way, partially obscured and hidden behind arches of the windows area, we still have a lot of hidden areas of therapeutics with regard to anablastic thyroid cancer that we need to explore further. Acknowledgements. You know, there are many people, including those in this room, that have contributed to develop therapies and pioneered new novel therapies in anablastic thyroid cancer, but importantly, most importantly from my standpoint, is the relentless commitment of our patients and their families to moving the needle forward and to investing in very laborious, time-intensive, often toxic treatments to try and not only gain benefit for themselves but also to provide evidence to help the next generation of patients. I also list the members of the ATA, ATC Guidelines Committee below, and I also want to thank the Endocrine Society for inviting us to present here today and you to attending. Also acknowledge the American Thyroid Association and the International Thyroid Oncology Group, which I mentioned with regard to several of the trials and maybe did also. And with that, we'll stop and take questions. Thank you. Thank you very much for an excellent presentation. Thank you very much for an excellent presentation. In patients with R1 or R2 disease locally, and they have BRAF positive disease, which modality would you prefer, tramatinib, deprafinib, or radiational chemo-radiation, chemo-sensitization? So that answer may vary depending upon whether you ask Mimi or I, but I think the evidence, at least the most persuasive evidence, would be that we have a potential curative intent approach if we use multimodal chemo-radiation therapy after surgery in that circumstance. So that would generally be the approach that I would favor, but there are exceptions. There are patients who will have rapidly recurrent disease and by the time you're setting up for radiation therapy, they're already progressing and they may need systemic therapy. So the particulars of the case and how it evolves over time as you're doing treatment planning really matters a lot. So I think the answer could vary in a very case-dependent manner, but my general preference is if they truly have no visible disease but only microscopic disease, say R1, I would be inclined myself to offer curative intention chemo-radiation in that setting. Can I have another question? Another situation, I have a patient now with BRAF-positive disease, but the BRAF fraction is minimal. I mean, we had difficulty actually detecting the BRAF, and then when we did whole exome sequencing, we found that the fraction of BRAF is only around 12%. So is the depravenib-tramatinib effective in this situation? I mean, the dose of the mutant, basically. So Mimi may want to weigh in on this, but I'll tell you my take on the situation. So there's another question, and that is, is this a heterogeneous tumor where you have an anaplastic component and also a poorly differentiated component or different fractions of the tumor? I think the answer is right now I would be inclined to use depravenib-tramatinib even if the fraction is relatively low. Would you agree, Mimi and Matt? I think we're all in consensus. We don't go so much by the fraction. And, in fact, you'll notice in the PD-L1 antibody trials, they use a positive rate for PD-L1 of 1% positive. And so you don't need a large fraction necessarily. So I would start with depravenib-tramatinib. Thank you. Good. Thank you. Yeah, thanks very much. Okay, and our third and final speaker is Dr. Matthew Ringel. He is the Ralph W. Kurtz Professor of Medicine and Director of the Division of Endocrinology and Metabolism at The Ohio State University. He's also the co-director of the Center for Cancer Engineering and co-leader of the Cancer Biology Program at Ohio State Cancer Center. He has a very active lab, and his group's work is focused on PI3 kinase slash AKT and PNPAC signaling, metastasis suppressing pathways, as well as transcriptional regulation of oncogenes and thyroid and other cancer types. He is currently the co-chair of the American Thyroid Association Thyroid Cancer Guidelines Task Force and is editor-in-chief of Endocrine-Related Cancers. So he's going to talk to us about advances in transcriptional targeted therapies in thyroid cancer. Well, thank you, everyone. I'm actually thrilled to see so many of you staying. It's always a bad spot between being the last speaker of the day before dinner and drink somewhere else, so I really appreciate that you're here. We are going to really move to the future now a little bit in this talk, and I'm going to start a little bit with some stuff we've heard about just so you can see where we are. My disclosures, I really have none relevant for this. Most of them you've heard about. Funding is all from the NIH and Department of Defense, so no conflicts with any companies as well, and you heard about the rest. So we talked about this in the context of a question earlier, and I want to come back to this. I think in the end it's really important that you understand this aspect. So the first thing that we're going to always give a talk on this of going beyond typical thyroid cancer therapies is who should we be thinking about this in. So this really is for patients with progressive, distant, metastatic, or high-volume disease that have symptoms due to metastasis in bad locations. They typically are growing. Typically they could be either distant or locally invasive, but progressive, as we've heard about earlier, and they usually are patients who have failed standard therapy, whatever that standard therapy is, including XRT in many cases for IMRT-based therapies. And, of course, as you heard about, patients with anaplastic thyroid cancer almost straight up because of their prognosis. This is not for patients with stable or very slow-growing asymptomatic metastatic thyroid cancer, with the possible exception of some of the re-differentiation therapy you have. Now, it may become that in the future. We just don't have the data because we haven't selected our clinical trials that way, and I'll explain why I think that's an important concept as we go through today's talk. So this is from a recent review article that we published in JCNM just a few months ago, and this is to kind of put together what you've heard about a little bit in the context of cancer, and hopefully you guys can see this. Can you see that okay? So we heard a little bit about immune checkpoint inhibitors and other forms of immunotherapy. Those are driven in part by the tumor cell and in part by the immune response to that tumor cell. The second are other extracellular targets, such as mutated or rearranged, mostly mutated genes that would still be in this extracellular area of RET, and these other targets that you heard about, VEGFR2, for example, CMET would be hit, for example, by cabizantinib uniquely, and there may be some specific reasons to use that in the context of second-line therapy that we talked about. And then you can move inside the cell. These are still largely tyrosine kinases. These are some of the fusions that we heard about that are signaling from inside the cell directly rather than outside. And then you've got other kinases, BRAF being a serine 3ne kinase, MEK, PAK that I'll not talk about today, PI3 kinase, all of which can drive tumors and all of which are being studied in clinical trials, if not in thyroid cancer, in other forms of cancer. And this, again, you heard about. This is just in one table form from a paper that Jen and I just published last week, I think, right? Just looking at a similar table to what you heard about, and just to stress a couple of things. One thing is that we have use of therapies that are not directed toward thyroid cancer but are driven by having a biological marker, meaning these are things that are approved off of what we used to call basket trials. So if you have a marker, any solid tumor type, you could get into the study. And some drugs are FDA approved in that way, immune checkpoint inhibitors for patients with tumors that have high tumor mutation burden or a mismatch repair deficient and have microsatellite instability, both uncommon in differentiated thyroid cancer but common in anaplastic thyroid cancer. Okay? We heard also about PD-L1, not on that FDA approval list, but clearly studied in thyroid cancer and something you can get as a companion test for use, say, example of some of the PD-L1 inhibitors. Then you've got things that are approved specifically for thyroid cancer. We heard about dibraphinib-tremetinib, selprocatinib, pralsetinib, can be used in tumors with RET mutations, RET fusions, but very specifically have approvals that include medullary thyroid cancer. Entrec fusions, again, can be used in any progressive solid tumor that has the biomarker. And then you've heard about the multikinase inhibitors that are really nonspecific. You don't require a specific biomarker to use these, and in fact, most of the data would suggest that no particular mutation predicts response other than in some secondary sub-analyses of these studies. But what are the challenges? Well, we heard about side effects, but let's talk a little bit more specifically about resistance. One thing that is true that we've not talked about really entirely is that these drugs do not cure cancer. These drugs all will knock down cancer. We even have a couple of complete responses, but they are not durable. There invariably is going to be resistance to these drugs. So these are great. I mean, I have the patients right now who have terrible stage IV-C medullary thyroid cancer, 10-centimeter masses that have melted away for four years. So you can see remarkable, remarkable responses that really impact on their lives. But they will eventually get resistant through a variety of mechanisms. Most of them are not 100% inhibitory. So they will knock down signaling even at the recommended doses, 70, 60, 70, 80, 90%. And in some cases, that may not be enough within therapeutic windows. There are side effects, which we talked about, and some tumors lack the biomarkers, okay, that predict it. And that's where the multikinase inhibitors are in there, but we don't really have much beyond some of those things for those patients. So what are the alternatives that are being looked at in the future? Where are we going? And for the fellows in the room, you need to listen really closely to this because we need people doing these in thyroid cancer. So one is that there's a lot of work, and you guys know this, using alternative antitumor approaches, whether that's intrinsic, through tumor vaccines, chimeric agents, CAR-T therapies, NKT therapies, neutrophil, whatever, they're being studied in thyroid cancer, okay? Peptide PRRT radionuclide therapies, there's quite a bit of literature on these, including in neuroendocrine thyroid cancers specifically. How can, there are also methods to improve that target inhibition so that a 60, 70, 80%, how can we do better than that? So one really popular thing that there's a lot of work going on right now are called degrons or PROTACs that degrade protein targets. So some of them might even use a drug like Pralsatinib or Selprocatinib, link it to something that leads to the degradation of the protein that it's bound to. So they can not just block the kinase to get in there, they can get rid of the protein. So the target is no longer there, okay? A lot of work on that, and it's been looked at in a number of thyroid cancer relevant targets in vitro, not yet in clinical trials for thyroid cancer. Covalent inhibitor inhibiting targets, and I mention this because I actually just have a patient who's on this now. This is an uncommon mutation in thyroid cancer, but there's a specific FDA approval for covalent inhibitor of this RAS mutant, which has been remarkably effective in lung cancer mostly, but also in pancreatic cancer, and rarely in thyroid cancers, okay? This is a common, not common, but occasionally seen, Keith's group, we have, and MD Anderson have all shown this to occur in some resistant tumors to BRAF inhibitors, okay? So this can happen. And lastly, what we're going to talk about are ways that we can maybe stop the gene, the offending genes from being expressed at all, right? So one is you can degrade it after it's expressed, and other approach is to block the ability of a cell to even make it. And that's what we're going to talk about a little bit now, because there's data from a couple of drugs, or pro drugs of drugs that are currently in phase two clinical trials right now, that I think is an approach worthwhile talking about. And that is something we've studied a little bit, which is super enhancer targeting of specific oncogenes, and I'll explain what I mean in English in a moment. Okay, so what is a super enhanced gene, what's a super enhancer? So these are clusters of transcriptional regulators that can get looped in to a gene, and they're marked by these things called histone acetylations, I'll show you what that is in a moment. And this is something that is part of normal human development, it's how organs have specific overexpression that cause, say, a thyroid to become a thyroid, or a lung to become a lung, and they can go up and down in development, okay? So that's what this is, and cancers can sort of hijack that, right? And use that to overexpress at very high levels certain cancer causing genes, so you can get not only mutant genes, but you can get high expression of those mutant genes. So you have two reasons now why they're activated, one is a mutation, the other is that they're expressed at a very high level. That make sense to everyone? Okay, so two components. The normal genome, and this is different than we learned, doesn't always get transcribed across in a linear way, there are these clusters and regions within chromosomes that get pulled together, these are very sharply defined, you'll see them written as tads, but the current literature, again I'll show you what I mean in a moment, are called condensates, okay, that are very sharply defined in the normal genome. So I'm going to give you some pictures of what I actually mean. This is again from this article that's recent, and this is a normal gene, this is kind of the way we learned how they're transcribed, you've got this area of intergenic DNA, you've got an enhancer in what's called the promoter of the gene, it binds to this whole group of regulators that are required to get DNA to become RNA for a gene to be transcribed, and you transcribe one gene copy off of this promoter, right, that's what we were all taught in medical school. But if you have a super enhanced gene, you have something that's way, way away from the gene, sometimes 10 kb is away, it's got all these histone acetylation marks that I mentioned, all of these transcription factors that get pulled in by this thing called the cohesin and mediator complex, so you get all these positive activators, all get pulled into this promoter and instead of getting one copy of a gene through activation, you can get hundreds of copies of a gene from that activation. And that's a normal thing to occur, and it occurs within the nucleus. So what happens in cancer? Well cancers can take advantage of this, as I mentioned, they can hijack it, they can do that in a number of ways, they can get amplification of super enhancers, and we have a paper that just came out with Marie-Claude Hoffman about amplification of chromosome 7 regions in BRAF resistant thyroid cancer. You can get mutations that lead to changes, you can get insertions that can cause something to get pulled in, or you can have what would be depicted here, which would be a fusion, where you have now the activator of a gene instead of being regulated by its own promoter is now regulated by a different promoter, which might be a super enhanced promoter. So that's how cancers hijack this. The other way to hijack it is this thing called condensate, so this is a normal compartmentalization of a chromosome, kind of seen on end in this thing called a condensate, and they get dysregulated in cancer through this process, so you bring all kinds of regulators in that can then get looped in to lead to transcription of a gene. So when you think then about targeting genes, you want to be able to target genes that are super enhanced with some level of specificity, right? Because otherwise, you could kill off a patient, right? You don't want to block all RNA from being made. If you're going to find a therapeutic window, you've got to have a marker to be able to address this, just like if it's a mutation, okay? So the key regulators of this that have been looked at in thyroid cancer are some kinases. So this is actually using kinase inhibitors to block transcription, and this is the most commonly looked at one is called CDK7, cyclodependent kinase 7, cyclodependent kinase 9, 12, and 13, and then this other regulator called the bet-bromo domain proteins, and this is the most commonly targeted one, BRD4. Now you say, well, what is—are we looking at cyclodependent kinases in this context? It turns out that there's a whole family of cyclodependent kinases that don't really function to regulate cell cycle. They function to regulate gene transcription. They just happen to be in the same overall-looking gene family. So what I'm going to show you now are data using a CDK7 inhibitor and a bet-bromo domain inhibitor in thyroid cancer all the way through mouse models, because I think we're gearing up now to be considering this for clinical trials in medullary thyroid cancer as well as differentiated progressive thyroid cancer. So this is just going to show you just a schematic to sort of orient you. So the way we started this a number of years ago in medullary thyroid cancer is work that we and others had done as part of our SPOR, looking at the fact that patients with medullary—patients that have retinoblastoma abnormalities have an increased incidence of thyroid cancer. Mice where you lose retinoblastoma all get medullary thyroid cancer, of all things. And we had previously published that if you have loss of retinoblastoma activity, you have a worse prognosis for medullary thyroid cancer, whether or not you have a Rett mutation. Okay, so this is independent of Rett. So we were very interested in this. So we started this work using a drug called dynacycline, which inhibits like all of these cycle-independent kinases, including the ones that regulate RNA. And we showed that this effect that we were seeing with dynacycline was not due to the cell cycle effect, unexpectedly, but was due to the RNA transcriptional activity. So this was a not predicted finding. So our null hypothesis failed, but we picked something else up, okay? And what we picked up was it led us to hypothesize that CDK7 might be an important target. And this is the probe, the drug probe that we use called THZ1, which was developed as a covalent inhibitor of CDK7. So here's some data. I'm not going to make you memorize all this. But what... I'm going to go backwards here. What we showed, and I just want to point out these doses, we're talking, you know, 50 nanomolar, 2550 nanomolar doses, IC50s, in TT cells and MZCRC1, two models of medullary thyroid cancer with RET mutations where it kind of kills bugs dead, okay? Really kills off the cells. We show that it blocks growth inhibition at 5 and 10 nanomolar doses, really impressive for these cells in particular that are slow growing. And it induces apoptosis, as you can see here, with a cleave cast-based assay. So this drug is very effective in these medullary cancer cells. And here's just demonstrating the activity of this, showing that it blocks the CDK7 activity. This is phosphorylation of RNA polymerase II. And this is the cyclin inhibition, which is a target of RNA polymerase II. And this is MCL1, which is actually a target of CDK9, which can be regulated by CDK7. And this shows that CDK9 activity goes down. So bottom line, drug is effective in medullary thyroid cancer cells. What we did not expect was this, okay? This shows, and this is looking, these are cells that are driven by RET mutations, was that the RET protein was gone, okay? This was not expected. The RET protein was gone at 50 nanomolar concentrations. This is actually well below the tolerable dose of this drug, okay? So the question then was, was it happening at the RNA level or was it happening at the protein level? And we show that the RNA levels are also reduced. So it suggested that there might be something special about the effect of this drug on RET. So there's a couple of different ways you can look to identify a super enhancer. I'm just showing you this schematic because I'm going to show you the data in a moment. This is, again, showing a super enhancer. You do something, in this case, we did something called ChIP-seq, where we immunoprecipitate using an antibody against this particular mark that marks a super enhancer. And then you sequence through it to identify all the genes that seem to have a lot of these acetylation marks on it. And you get a plot like this, where something that has a ton of these will show up in this vertical side, because this is the number of histone acetylation marks on it. So your super enhancers are all going to be sitting here in this area. And sometimes you'll see where they flip the other way, but it's the vertical part that's got the super enhancers. So what do these thyroid cancer cell lines look like? So this is looking now at a ChIP-seq using acetylation, and this is using BRD4. These are now the super enhancers, just to give you a sense, there are 1,000 of them. This happens to be in the TT cell line, it's about the same for the MZCRC. And these are the gene types, this is what would be predicted. What I want to point out is RET is a super enhancer. It was the 43rd pick out of this 1,000, so it had hundreds of acetylation marks. And here is the visual of those acetylation marks in intron 1 of RET. So not in the promoter, in the first intron, and about 50% of super enhancers are in intron 1. So we have definitely shown that there's a super enhancer in RET. We've got lots of other data I could share with you, but I want to move on away from medullary cancer and show you some of the data from some of the other groups that have shown this also in differentiated cancer and anaplastic models very quickly. So this is from Gallaudet, published around the same time as our first paper. This is using a BET inhibitor called JQ1, showing that it is inhibiting in multiple thyroid cancer cell lines growth. It's functioning properly, and in vivo in mice, it's blocking growth. Not entirely, but pretty reasonably in these very rapidly growing thyroid cancer cell lines. So it's effective in vitro, and it's working in vivo, and it's working, they think, in collaboration or likely due to overexpression of MYC, and MYC has a known super enhancer, so a predictor here could be MYC overexpression. Using this drug again, Mio et al. did an array study and identified MCM5 as a potential target using an unbiased approach and then looking at a number of these genes in two different cell lines. So we've got a couple of targets that might be predicting the activity in this case now in anaplastic thyroid cancer. So this is from Xu et al., which is Xi and Cheng's lab at NCI, demonstrating something kind of similar again, but extending this to say that the BET inhibitors not only are targeting MYC, but that they're synergistic with MYC inhibitors. So again, looking now beyond, now looking at drugs that are resistant to some of these things, and again, not showing you every piece of data, but showing you that it's blocking cell growth, ERK activity is gone, MYC expression is gone at the same time, and these are from xenograft tumors, so these are in mice showing loss of MYC, loss of RB, as well as inhibition of growth. But perhaps most impressively, Xu Yan's group then has looked at this in an endogenous model of anaplastic thyroid cancer. So this is a mouse model that is a combination of a mutation, which is a thyroid hormone resistance mutation, and a KRAS mutation, which you can see in anaplastic thyroid cancer, that gets aggressive disease, and this is, you can see the cancers that develop. Now, what I want to show you here is these cancers are characterized by a very high level of MYC, and that you can, as well as of cell division, mitotic figures in KI67, for example, and that treating with the BET inhibitor targets MYC and reduces the size of the tumors in this endogenous anaplastic thyroid cancer model. And this is now showing that MYC is the likely target in this particular model, and you can see the ratio of positive MYC cells goes down. So all of this seems to correlate really nicely and sets up a model where the drug in anaplastic thyroid cancer that is high MYC levels might be particularly responsive to either this drug, JQ1, or perhaps also a CVK7 inhibitor alone or in combination with some of the other drugs. Interestingly, just published about two years ago in Thyroid, Cayetal did a drug screen with roughly a couple thousand compounds at the beginning to identify drugs that might be cytotoxic in anaplastic thyroid cancer cells, and you can see they whittled it down and the one cell line is their screening cell line. I can't see the number here, I think it's 117 or so, and then when they extended it to about six thyroid cancer cell lines, they came with one target. That one target, totally objectively, THZ1, which is the same drug that we were looking at in medullary thyroid cancer, and sort of a cousin of the BET inhibitors in here, number three or four, is the CVK12-13 inhibitor, which would react similarly. So they went ahead and did these cell models showing that it was killing the cells and also did in vivo xenograft models with THZ1 in a much larger panel now, anaplastic thyroid cancer, showing pretty impressive results as well. So objective, not a targeted look, but taken from a screening look of chemicals, the same pathway, potentially really important. So how might these activities against these be identified? I mentioned MYC, here's anaplastic thyroid cancer looking at CDK7 overexpression as a biomarker model for use of not only a CDK7 inhibitor, but this is actually an experiment using CRISPR-mediated loss of CDK7, so much more specific, and then using a compound showing very similar activities, and now looking at human tumors and looking at the frequency of CDK7 overexpression, and showing that those patients with high CDK7 levels did much more poorly than those with low CDK7 levels in both papillary thyroid cancer and anaplastic thyroid cancer. And I won't go through this in detail, this is now looking at cell lines and looking at the markers that they think might be important in correlating with this effect. Finally, and I'll just put a plug in for those people who are looking for interesting things in this area, we had a poster rapid fire presentation from my lab by Neil Rajan, who really did a wonderful job demonstrating not only are these drugs inhibiting CDK and CDK9, but part of their on-target effect is actually to degrade RNA polymerase II, this is all new, and I think really exciting, so functioning as an inhibitor of transcription, but also degrading the target, which is probably why we're seeing such a remarkable activity. So challenges for this, nothing's perfect, we need to know which drivers are super enhanced, we need to know their expression levels, so we need to identify biomarkers of targets for selection, we've got some, but we'll need more, and the therapeutic margin I think is going to be really important and we're going to keep a close eye on these now phase two studies using oral versions of these combinations as well as combinatorial strategies. So just to finish up, we think it's really important to move forward away from the standard kinase inhibitors, okay, we know we're going to do this, we know we've got these combinations and we're going to continue our work in those areas, but we need to think beyond that a little bit because we know we're going to get resistance, and we have opportunities with degraders, with covalent inhibitors, with super enhancer inhibitors, and with other types of immunotherapy like CAR T and others approaching thyroid cancer. We need the right biomarkers, and importantly, we need the right patients, and we need to make sure as these therapies are getting better and better targeted, we might need to think about opportunities to potentially treat a little bit earlier if that's going to impact on their outcomes. So I want to thank my lab. I mentioned Neil, who did a lot of work on this particular project, Tila Canal as well in my lab, Ani Valenciaga, who did some of that initial work in Motosagi, our funding sources as well as the James for supporting our work, and that's what I have for you, so I'm open for questions. I have a question. Do we know how specific, like CDK7 and BET, what's the distribution in the body, so how specific can we be? So these are all expressed in the body. They're part of the normal cellular context. What makes a therapeutic margin is by having a target use the super enhancer or not. So if you just go high dose on this, it's going to be very toxic. And this is known already from the phase one, two studies, but there is a therapeutic margin. Yeah. Okay. Thank you, and thank all the speakers for a fabulous session, and all the best.

therapies

survival

anaplastic thyroid cancer

aggressive treatment

surgeries

radiation

cytotoxic chemotherapy

immunotherapy

PD-1 antibodies

somatic genomic interrogation

pembrolizumab

lenvatinib

BRAF V600E