This presentation on cholangiocarcinoma covers epidemiology, including anatomical classification and risk factors; clinical management, including staging, treatment, and precision medicine; molecular pathogenesis, including the genomic landscape and the role of the demoplastic stroma. It also covers lab-to-clinic activities related to experimental disease models and identification of novel potential targets.
Chapters (Click to go to chapter start)
Outline Anatomical classification Epidemiology Management FGFR2 rearrangements and inhibitors IDH1 and IDH2 mutations Immunotherapy Genetic landscape Next-generation sequencing Signaling pathways Desmoplastic stroma Projects in the lab
Dr CIA. Um It's our pleasure to introduce dr daniel CIA. Today she obtained her PhD from the University of the studies of Milan and completed her postdoctoral studies at dr joseph Lovett's lab in Barcelona and then at the Icahn School of Medicine at Mount Sinai. She is currently an assistant professor in the division of liver diseases and an expert in the molecular biology and genomics of calandra carcinoma and a pedo cellular carcinoma. Her lab has left the field in translational research on hepatic malignancies with the generation of the first multi comics classification of intra Matic and extra dramatic calandra carcinoma. The first immune based classifications of HCC and the discovery of a target of all fusion protein involved in malignant pathways. A very warm welcome to doctors the next morning. Good morning number one. Thank you Dr Levin for the introduction and thank you dr kim as well. First of all, I would like to thank dr thomas and the whole department of medicine for the kind invitation and the opportunity to present at this meeting and to show you what we have been doing over the past year and a half since when I started my laboratory here at signing. Although as mentioned a joint lab johnson actually 2000 and 12 as a post doc. And then just last year I started to have independent visage lab focused on village tract cancer and as we heard in particular oncological cinema And in the next 15 minutes, I will try to give you examples. However, the years we've been trying to bridge the gap between the clinic and the laboratory. Mhm. I have no financial disclosure to this presentation and these are the strategy of this talk. By the end of this 50 minutes you will be able to describe the epidemiology including managing ecological cinema and also demonstrate awareness of future potential therapeutic approaches. This is the outline of my talk. We have four different key points. The first two will be strictly clinical. Well we'll try to provide you an accurate overview of the epidemiology, anatomical classification and this factor of the disease. In the second we will review currently current clinical management and how actually precision medicine. A few recent translational discoveries have improved the management occasion. We call in yoga cinema. What is in the last two points we will review will be able learned for pre clinical studies and translational studies and how this has impacted or not. They couldn't management of the disease. And more importantly in the last section, I will try to provide you with two examples of projects are being conducted in my lab in the past year and a house and how we try to improve the outcome of patients with client or customer For those of you who are not familiar with the disease. Clinical tsunamis. Overall considered a rare cancer. And indeed it accounts for only 3% of all gastrointestinal malignancies or whoever. It accounts for about 20% of all deaths calandra carcinoma is usually group would go blood there and and pull a cancer in the group. That is known as village cancer which is known to be highly heterogeneous. However, when it comes only to calandra carcinoma, as you can see, there are many different subtypes in this cartoon, you can see the contra customer can be mainly divided in intra particle and the custom of my next robotic according to the anatomical location. As the same name suggests anthropological Andrew carcinoma, which accounts for around one third of all cases, refers to those tumors arising from the small ball tax within deliver. On the other hand, extra pratica rises outside but can be far, they're subdivided in pretty high Lauren distal. The reality is the most common form of the virus is close to the bifurcation of the common bile duct. What is this that was on the opposite side away from deliver. It's interesting because over the years we thought that the anatomical location was the only difference across these subtypes. But actually we have learned that risk factors but also epidemiology and molly collaboration differ between these subtypes, suggesting they might represent different disease and by the end of the stock perhaps will convince Sydor. Indeed they should be considered completely different malignancies in terms of his factor. You can see that there are some common, mostly inflammation related like cholangitis and liver flukes. But over the years we have learned that intra political intrigue carcinoma can also be associated with risk factors typical of HCC peninsula carcinoma like viral hepatitis cirrhosis, obesity and diabetes. These have been really associated with extra Matic which is mostly associated with quality status lynch syndrome and political system. And this is just the tip of the iceberg. What the differences between intra Matic and extra batic as you can see, liver flukes are common to both in acrobatic and extra patty can actually represent the most common cause of cancer. Carcinoma particularly in some areas of the globe like Asia and Thailand. As you can see here china and Thailand where calandra comes to know, matt represent the most common form of liver cancer. Indeed here in these areas, indicated in this map in red, we have more than 80 cases per 100,000 people In the rest of the globe. As you can see in this map in the US and Canada and Europe landry carcinomas overall rare with less than six cases per 100,000 people. And this reflects the distribution of respecter since liver fluke infection and they make in china but they're not in there making other areas of the globe. However, what is important to understand is that as you can see in this box is the incidents of inter particle entre carcinoma has been steady increasing over the past 40 years. What is the incident extra patty has remained stable and indeed as you can see here this is the most recent report to the nation from the S. W. E. R. That the basis and you can see between 2000 and 9, 2000 and eight intraparty ecological system along with the potential carcinoma which always grouped together. You can see here is among the top malignancies with the in terms of new cases And this increase has been observed between males and females. And this does not impact over the past 10 years. But actually over the past 40 years there's been an increase of about 128 in cases of inter particle angelika Cinema. And the reason of these increases still not understood and probably also a better diagnosis contribute to these higher incidents. This disease is highly fatal. Indeed the number of deaths almost equal the number of new cases annually. Therefore with increasing incident has been increasing mortality. And again here in this map, you can appreciate the geographic distribution which reflect the distribution of these factors. But as you can see in all these countries globally, particularly in the us, there has been a state increased mortality between 24 and 2013 and the projection are even more the small. In fact here, this reflection of the US living in dramatic indicator here in green, you can appreciate the front 2020 it is expected. It will keep increasing in terms of a number of deaths and actually the W. H. O. Estimates in their annual projection, but by 2000 and 40 more than one million people will die globally of living in traumatic. By that, to summarize this first introduction calandra carcinoma is a rare, the quite fatal cancer of the bile ducts. There is different in terms of subtypes, we have the different subtypes present this anatomical location but also these factors. And we will see how also piece. We have different management and different molecular alteration in particular incidents and mortality of intra Matic larger customers being still increasing what wine indicated. This represented global health challenge, increase in incidence and mortality. Particular inter particle Angelica's norma over the years has prompt clinicians and scientists to focus on this disease. And for the first time in 2010 intraparty ecological cinema received its own staging system up to the date. Indeed, anthropological Angelika cinema was merged with the Paterson local tsunami in terms of staging system. Although the two diseases are very different Again, clinicians were prompt to focus on dramatic, logical cinema and in 2014 and the international liver Cancer Association for the first time published guidelines of management for the disease. These were published in general pathology and the most important figure that management guidelines can be here are summarized in these slides. Indeed, as you can see anthropological angiograms, cinema can be classified in four different stages. The first two are early stage and here the disease is considered acceptable although as you can see from the numbers, this is just only one third of patients, Even when resection is visible. Unfortunately recurrence remains high with up to 70% of five years. Therefore, in case of a curative resection, you can see here below the five year survival rate of 40 that it drops to 20% in case of a non creative wrist action, even more problematic. The majority of patients are diagnosed when resection is not an option and in this case we can only offer palliative chemotherapy region side of being classes platinum With the median survival of 12 months, Johnson had been pluses platinum became the standard of care in 2010 following the space free clinical trial published in New England Journal of Medicine. In this case, we had 400 patients enrolled in the abc go to trial. As you can see considering calandra has enormous group we go bladder and pull account so you can see that the primary tumors that were quite different. We had intra particle angelika, cinema, extra patty, Kyler gold bladder and so on. And you can see that while the total was quite significant. We only had 80 patients with traumatic logic. A Snowman 73 with extra patrick in the whole court. The combination was significantly better than the inside have been alone. But the indication for intra particle entre carcinoma is basically based on this subgroup analysis showing that the combination was better than the inside of being alone. This is the most recent treatment algorithm for calendrical cinema particular. Intra pathetic. Published last year in nature reviews gastroenterology and hepatology and unfortunately although the structure is different you will appreciate that there are many very few differences compared to the previous guidelines. Indeed as you can see the majority of patients are diagnosed when all the political equipment can be offered in James's is still the standard of care. Although there is one novelty compared to the previous guidelines that is all those up to is represented by this box here. Basically what we're saying is the molecular profiling of the tumor is highly relevant for patient. We contract a cinema because it can unveil opportunities like every year for an I. D. H. Inhibitors and we will review these novel opportunities in the next slides. Unfortunately still under development is the role of immunotherapy. Move on to review this in the next slides the most promise promising targets for conjugal cinema have emerged in the past five years and our group was happy to contribute to these discoveries and culminated in two important clinical trials that were published last year in Lancet Oncology. This is the first started, it is the phase three clinical trial demonstrating for the first time that I was seeing. It is an I. D. H. One inhibitor was able to provide clinical benefit in advanced patients. We Klinger carcinoma are very 90 H one mutation and the other novelty came from this space to study. And this study always published by abu off any steam as low and capturing the demonstrating the pima. Got any and every year for two inhibitor was able to provide clinical benefit in patients with colon carcinoma harboring energy effort to fusion and this is perhaps the most promising therapy. And indeed last year in 2020 april 2020 the FDA, based on the results of this phase two study, were granted accelerated approval for the first time to a targeted therapy in patients with colon carcinoma. This action has been followed this year a few months ago, but the second accelerated approval to invigorating it Also known as B. J. J. Free 98. There is another F G F R 20 better for patients with calandra carcinoma are very F G F R to fusion as you can imagine everything for the future represent probably the greatest breakthrough in the field of contra carcinoma. FGFR two fusion were discovered a few years back by our group and others by applying irony sequencing technology to more patients with landry carcinoma. The first report ever came from a basket trial publishing cancer discovered 2013 including only two cases of colon carcinoma. In both cases were found positive for this. FGF are too big. There is now we know to be the most common. How does you can see in this cartoon? It these are different fusions they've been reported. This is the one that we found in our lab G F R. Two people and one But there are many others. And actually over the past few years, we have discovered there are more than 60 partners that always fused to GFP to to create the civil judgments As you can imagine. And as you can appreciate from these figures, the Fusion's have always the same structure. Regardless of the second partner. The FGFR two is almost intact. They contained the 1st 19 extend. It only loses the last one. But what is important is the terrace in Canada's domain are here are maintained and that will confirm the activity to this future. Indeed, as we know if Jeff are twisted, known oncogene, but it requires usually the presence of the lions to be activated and to demand rise. In this case with fusion's the second partner was responsible of maintaining the FDA far too, except during a constitutive states even in absence of the lycans. Therefore, you can imagine that when a tumor is expressed in F g r two, D f G F R two, signaling is always activating and signaling within the self. In our case, we were curious to understand the mechanisms behind the generation of these fusions. We performed whole genome sequencing and we validated the results by fluorescent in situ hybridization. This is a to mark, this is a patient with FGF two PP Children. One. This is the healthy liver and this is the tumor. In our case, F G F R two is located on chromosome 10 and p pHO one is located on chromosome 12. Therefore we use two probes mapping FGF 20 P 21. And here you can see the healthy liver, you can identify the two sets of chromosomes, the wild type. In the case of the two more, you can see that we have one probe recognizing after 4-1 recognizing people and one and one where you actually can see the fusion where the two genes are fused together to generate this protein. In our case, we also were interested in understanding if this FGFR two fugitives unhygienic and if only depends on the FGFR two but not on the second partner. So what we did we use NIH 53 which are normal Miriam fibroblast cell line we ever expressed. If Jeffrey took the picture one and all of a sudden we observed these cells are able to grow as a colony. The normal salon is not able to form colonies in vitro because again, it's not a transforms the line. But when we over expressed, jennifer took a picture line. The cells were beautifully beautifully forming these colonies and this growth was completely dependent on FGF two. Indeed when we added B J J free 98 now known as integrating it, we completely suppressed this proliferation. Therefore, at the time we already understood F g f R two was like a great promise for patients with calandra carcinoma. Since it was highly frequent in patients, we will perform a screening in 119 patients and we upset the 25% of all cases were positive for at least one F g F R two fusion and of course was a psychogenic And considering the presence of agenda two inhibitors with a potential target. And indeed several clinical trials have followed the discovery of average of two fusion and this is the most important as mentioned before. This is the face to study the light to FDA accelerated approval of Penny Got any. This was published last year and in this study we had 145 patients here. The majority of patients with our brain FGF two fusion. We had one of seven, 20 had other actual fate alteration in 18 hand, none as you can appreciate from the stable objective responses rate with all the observed in the group would have G fr to fusion to be got enemy was also able to improve progression free survival although as you can appreciate The survival was modest. We had a million PFS of 6.9 Here in the patients with FGFR two Fusion vs two months and 1.7. So although significant, although promising is certainly is concerning that this progression free survival is quite short. Regardless, the FDA granted accelerated approval. This represented the first great promise for patients with calandra carcinoma and accordingly we're waiting for the results of the phase three. Again, I want to focus your attention the short PFS and actually two studies following these discoveries have tried to understand the reason and the mechanism behind this quick progression free survival. And there are two studies that have been published political google and the team of Donna Farber in cancer discovery. The first was published in 2017 and the second one was published two years later when they actually clarified the mechanism of acquired resistance that was responsible for this quick progression free survival. Here. In this study, they analyzed two more and the circulating tumor DNA from patients enrolled in clinical trials with F G F R inhibitors here. Just for you to understand, I'm showing you two patients, these two patients before the start of treatment. Of course we checked and there was screened for the presence of a G F R two fusion. As you can see here. The first patients were found positive for N F G F R two, that the M Y M four and the second was positive for N F G F R two, R P T N. They didn't have any other alteration in terms of FGFR. These patients were enrolled in clinical trials with FGF two inhibitors. As expected, there was a response actually good response in this case and then it was quickly followed by progression at the time of progression this time. The others analyzed again, the tumor and the circulating tumor DNA. And interesting what they observed there on top of the fusion that that was was identified before this time. They were able to identify mutations Activating mutations in the FGFR two receptor. More importantly, what this mutation were doing is they were changing the confirmation of the FGFR two and in particular the majority of these mutations were cut in the pocket that was binding the drug. Therefore, by changing the confirmation the cell was becoming resistant to the average. In fact to accept her because the drug was not able to buy the defacto anymore. And more importantly, this was definitely another breakthrough which clarified the mechanism of acquired resistance. This is the first mechanism acquired resistance in these patients but I also found the ex solution or at least a partial solution. Indeed in all these clinical trials and the Pima got even into integrating the whole f G F. R to accept of second generation. It means that there are 80 P competitive inhibitors Right now we have a third generation inhibitors like test 1 20 which is an irreversible inhibitor. And in the second study they were able to demonstrate the third generation of Jeff are inhibitors are actually effective against these mutations. Therefore here once again and showing the example of two patients, the baseline there were positive for fusion. They were enrolled in this clinical trial with a T. P. I believe that this was J. J. 3 98 or W. 347 at the time of progression. The authors checked once again identify specific mutation and enroll the patient in a second clinical trial where they're receiving tasks 1 20. That's 1 20. This time was able to overcome this mechanism of acquired resistance and inducing a longer program, a longer benefit clinical benefit. Although once again uh they observed that there was progression and further investigation is currently ongoing it now and of course there is a better I need is to better understand how these mutations can be further blocked. Like what is important is that this led to the facebook like outside address currently ongoing. And with this irreversible F. G. F. R. Inhibitor interface between taliban insurgencies and were anxiously waiting for these results. Another important promising for patients with colon carcinoma is represented by mutation in 98 1 and two. For us. For those of you wanna familiar I. D. H. One to our metabolic enzymes located either in the sight of us of the south where we have A. D. H. One or in the mitochondria, they both have the same function of converting. As aside trading to help but get a good rate as you can see here when these genes are mutated, submitted the form is responsible for converting the house to get obliterate into a drugs obliterate. This has been reported to be toxic for the south. They can even alter the mutilation profile. But what is most important how described in this paper where we were lucky enough to collaborate, collaborate published in nature doesn't 14 win and the group of dana Farber were able to demonstrate the two hydroxy glitter. It is particularly relevant for Colangelo as normal because this a christian is to a drugs to go to a block, H and M for alpha H and F four outfits of transcription factor that is responsible for the expression of liver specific genes. Therefore when a to a drug secretary accumulate in the liver, this is blocked and the party opportunity result cannot differentiate into a battle site to actually maintain a novel cell states and in presence of additional alteration like for example mutations in chaos. The unusable cell expansion information of contra carcinoma. The most important is that I. D. H. One intimidation is they are quite common in conjugal cinema particular. I. D. H. Has been identified around 15% of all cases. But as you can imagine also we have the first specific I. D. H. One and I. D. H. Two individuals. And here you have uh at least a few mostly from address to company address And over the past few years and several clinical trials have been conducted with these compounds and unfortunately the ones without the H2 inhibitors for unknown reasons, completely understood have not been successful. But the 13 day th-1 inevitably being quite successful. And actually as mentioned before published last year, the results of the pastry clinical child would ever see the name This clinical trial include 185 patients that were randomized to 21 to receive I received in the versus placebo. There were no many difference between the two groups, but what was interesting to observe is that most of patients where patients with intra particle angelika cinema, whereas the ones with extra padding but almost quite rare are completely absent. And there was one particularly hot spots that was identified. That is this hot spot 90 H1 I wanted to see In terms of progression free survival of the city was able to prolong progression free survival and the significant this was quite significant, although once again, similar to actual for two inhibitors. As you can appreciate, the benefit is modest. We have a median PFS of 2.7 anniversaries, 1.4. Therefore, these represent a step forward. But as you can imagine, the marginal benefit suggests that we need to design combination strategies and actually the FDA is currently deciding on the approval of these drugs in the case of I. D. H. Just mention is um different from F G F. R. And it's probably the short progression free survival is probably due to the mechanism of action of the drug which is a sight a static doesn't kill the cell but blocks the proliferation in this context, we can now talk about immunotherapy. Indeed, you're probably familiar with checkpoint inhibitors that have been quite successful in a subset of patients across several histology is unfortunately when it comes to plant because you know, my first clinical clients have been disappointed and we will review this in the next slide. Before I get into the details. I would like just to provide an overview how checkpoint inhibitors work in this cartoon, you can see the track to activate the T cell. We need two signals. The first one required the TC are to recognize the new anti gin express an MHC complex and a second signal that is called the costume military signal. And here we have the example of City 28 in case of chronic antigen exposure, there are system in place when excessive immune response, particularly physiological condition, which consists in the expression of immune checkpoint proteins. You're probably familiar with PD one PD L. One City lay forth some to Marcel take advantage of the system to evade the immune response before the design of monoclonal antibody targeting description checkpoints have been quite successful because basically the way the work is that they re activated t cells and unleashing immune response against the tumor. As mentioned, checkpoint inhibitors are quite successful but in a subset of patients, whereas the majority are resistant. Therefore, over the years, it has been interesting identifying biomarkers of response in particular. The FDA has approved the one in dread PD L one expression to mean additional burden, a mismatch repair deficiency. For some indications, but not others when it comes to killing dragons. No MA PD L one expression has not been find associated with response interim additional burden unfortunately is overall law. So it doesn't seem to be a good indicator. The only by a market that stands for contractors norma is represented by mismatch repair deficiency. To most. With this efficiency basically have a deficiency in the machinery responsible for decoration of mutations. Therefore, the tur as accumulates a large number of mutations which increase the likelihood of this mutation to be recognized by the immune system. There are, there have been several clinical trials that are demonstrated a high response rate in patients we mismatch repair. One of the most famous was published in New England 2015 were actually up to 40% of patients we mismatch repair deficiency were responded to checkpoint inhibitors and as a consequence, the FDA approved for the first time in this cancer treatment for any solid tumors with specific genetic futures. Regardless of the histology, including Kalenjin carcinoma. Unfortunately, these marginal impact patients with colon carcinoma because mismatch repair deficiency is quite a rare event with less than 3% of calandra carcinoma, heart brain this alteration when tested in all commerce Without any selections of fire. We're still in early face. But there are two keynote studies completed to date. The first is a key no 158 and the second is the keynote 28. As you can appreciate from this table, the keynote 150 is the largest conductors of firing patients will be like the track cancer. And unfortunately the objective response rate has been quite discouraging with only 6% in the keynote 158. This of course suggests that contracts numbers are factory to therapy including checkpoint inhibitors and therefore there is a need to to design combination therapist, increase their feet as it went to PT one. And in this regard, the first technical studies have emerged. I'm showing here to one published last year and one published this year. This was published last year by our collaborators at the medical clinic where they provided the rational to combine anti CSF one R. To deplete macrophages with Auntie LI six G to deplete G. M. D. M. C. And only this triple therapy in their experimental lawn that is able to increase the response to anti PD one. The rationale behind it and that they observed the PD L. One in Colombia to cinemas mostly expressed from tomorrow. So City macrophages but the depletion of macrophages induces a compensatory mechanism which leads to the accumulation of the immuno suppressive G. M. D. C. Therefore only by eliminating jim DNC and macrophages that were able to improve response 20 P one and actually the first clinical trial particularly minor cleaning based on these studies are currently are emerging. The second study is much simpler. It's just a combo therapy with CD 40 agonist uh and anti PD one therapy city 40 belongs to the TNF family and it binds the receptor CD 40 receptor which sally frequenting calandra carcinoma. And the combination of the 40 with entropy one was able to completely suppress to more broad this experimental model. Therefore, as you can see there is a dire need to design a combination therapies and the first preclinical studies are emerging and an even higher number of clinical trials is currently ongoing and we will see what we can learn from these studies. To conclude the second part, surgery remains the only curative option for patients with calandra casino and early stage J. M. C. S. Is still the current standard of care for patients with acceptable conduct a cinema. Although we are anxiously waiting for the results of the first uh clinic faced technical trust with Jeff I inhibitors and definitely what we have learned is the molecular profile is essential for this disease. Since it can provide access to personalized therapies and again as a mention of face technical class will actually find any business are currently ongoing. Whereas for I. D. H. Maybe this is clearly we need to design a combination therapies and the same applies to immunotherapy since a single agent has been quite disappointing so far. Moving on to the next section has we have learned by the application of RNA SEq technology. We have learned a great deal in the past few years about the genetic landscape of Kwanzaa carcinoma. And going back to the cartoon that I showed you before, I try to highlight how intra Matic and extra batic are different. And the application of a holic some sequencing and RNA seq further clarify these discrepancies. Indeed here I'm showing the molecular alteration. There are more commonly found in tropical America cinema. And in particular I want to focus your attention on the target of adulteration their I. D. H. Next year part And as you can see here what they are quite frequent in intra particle and carcinoma around 2025% depending on the cohort and the studies these are actually rare, completely absent. An extra battery which further highlights the molecular pathogenesis of these. Uh to canter's is distant and therefore perhaps we should really consider them as distant disease not only in research studies but also in clinical trials. Since of course marginal this patient together with a different molecular profile. My actually confound the results of this study. The application of next generation sequencing to large Quartz has been conducted over the past five years And this is one of the first published in 2015 in nature genetics. You can see a large number of realtytrac cancer was analyzed Around 239 and all the studies in neuropathic is always the most common subtype. But we have also extra patty can go bladder. Your brother is significantly less explored compared to the others. But the first studies are currently emerging and actually my group is currently collecting tissue from patients we call go bother to apply single cell based technologies. These studies have been quite helpful because not only a further highlighted the differences of the subtypes have identified additional targeted by alteration here. You have some unfortunately all these uh less frequent than 90 H and R. Jafar in patients with ecological cinema. Once they found positive for mutations in BRAC idea far or be wrapped, they usually enrolled in basket trials. More important thing is here indicated clearly in this figure once again into a particle and a custom um an extra batic have a very different like europe lo fi. And here you can see all the different alteration of intra Matic which present really little overlap with those of extra batic. And even if we go bladder, which of course is outside of the liver is even a different organism for the difference are even higher than within the subtypes of cancer carcinoma, regardless of the musicians. Over the years we have also learned a great deal in terms of signaling pathways and the ones that are more commonly dis regulated in clans. Because you know my regardless of the subtype and in particular there's been a lot of interest in targeting china using chinese inhibitors. These are the most common. And over the past years, several clinic culturalism being conducted here in this box, you can see the most important and unfortunately none of them has elicited positive results. The only one here that want to mention is the raw material that is still ongoing and we will see what happens with this one. I call antibody Directed against video for two. Unfortunately all the others has mentioned a B negative and have been quite discouraging further, suggesting that particular, we need to move away from the old commerce approach and apply a more personalized therapy for country customs, other important pathways they're relevant and potentially targeted ballin contra carcinoma include a notch. Singularly then here, yup, signally, unfortunately over the years, several notch inhibitors have been designed targeting either the like guns with the receptor or the gamma secretaries and it's a kind of pressure from the box. Unfortunately the first bullet poets already spoiled the problem with these clinical trials. All of them have been halted because of toxicity. And right now we only have this clinical trial. It would not need bitters. This is a novel notch. Neither it seems to work downstream without not at the level of the party seems to work downstream, although the mechanism not clear and hopefully there will be less successively with this drug. 11 message that I want to convey is that of course hold this negative clinical trial suggests that we need to better understand the disease and we need to gain a more personalized approach. So those so we need to understand better the disease. And over the years I think that what has been neglected from the visit standpoint is to focus on the macro environment. Indeed from other cancer pancreatic cancer we have learned a great deal in about the role of the micro environment and this seems to be quite relevant to us in the case of Qalandiya carcinoma in particular. Here you can appreciate the complexity of the macro environment. The strawman is quite dense in these tumors consist of cancer associated material cells, cancer society. Fabulous. Which represent the majority of cells but also, you know suppressive cells. And over the years particularly with the advance of a single cell based technology that has been a great interest in part of dissecting the micro environmental Qalandiya custom in particular consider that these two most highly dismal plastic cuff cancer is to study the bible but seems to play an important role the role in driving the growth but also in time in the response to therapy and cancer associated fibroblasts are heterogeneous group of cells. There are many different subtypes with different functions that can promote cancer progression and the way they work is that by can directly or indirectly interact with tumor cells rather cells to the macro environment in particularly by secreting specific factors that can only induce an immune suppressive mac environment and also influence to more growth. This year. We were lucky to be involved this beautiful study published in cancer cells and by applying single cell technologies to build muting and human cells. We were able to identify different subtypes. This is mostly based on muting models of conduct a cinema but similar results were observed in a cross position analysis with human patients was performed in particular. We have identified three different subtypes of cancer society fiberglass. The first is called maya fibroblast decaf and is responsible for the deposition of access a lot of metrics. This was called inflammatory caffe here in red because it was released in an over expressing inflammation related cytokines and other factors and this is the third missing kind of cap with less clear role importantly what was identified as that my calf and I caF can secrete specific factors and here we are highlighting H. A. Which yellow moronic acid Potosi growth factor and this had a direct impact on the growth of the um are indeed When these two factors were suppressed particularly specifically in counselors to 35 of us. You can see here at the bottom this was a significant decrease in proliferation. Our contribution to this paper really rely mostly on the design of specific signatures for my calf by calf and all caf and we observed that the presence of country associate the fiber bus was associated with poor prognosis and in particular that might have signatures. Those patients would hire my cup signature. We're the ones with the shortest survival before this study along with few others emerging right now are highlighting the importance of the macro environment and how the different components of the macro environment in particular the immune and this trauma should be included and considered especially at the time of patient stratification before we need to better understand the elements of the macro environment. Since they have an important role in determining response to therapies summarizing this part. Next generation sequencing technology have elucidated the genetic landscape of calandra carcinoma have contributed to further clarify that I. C. C. A. The inter pathetic and extra batic represent this disease with different molecular aberrations and profile and the role of several targeted path to risk has been elucidated. But unfortunately once again in clinical trials in all commerce have been negative suggesting once again that personalized purchase are needed for patients we call Angelika cinema particular in the view of such a complex and heterogeneous macro environment. A better patient stratification is needed which in this ideally this stratification will take into account not only elements of the tumor but all elements of the immune system, A macro environment. In the last 10 15 minutes I would just like to provide your review what we've been doing in the lab. The main projects and go and how we're trying to reach our long term goal which is to identify more effective therapies for patients we call angie carcinoma to improve the outcome of these patients. My lab focuses not only an intra patted but also go bladder and extra batic. And over the past year in the house we have been collecting tumor samples from patients undergoing resection for all these these different indications. Unfortunately in the past and half, especially because of the covid pandemic with hit a couple of months after actually started my lab we focus most on intra particle and focus on um in terms of experimental models since for I. C. C. A. Over the years we'll be able to collect large number of clinical and genomic 11 patients and also we have a higher number of experimental models although starting in the next few months we want to also start exploring experimental models of this disease. These are the disease. Over the past year the house we have mostly focused on four different aspect. The first one is that clearly mentioned before is that we need a better patient stratification and consider the role of the micro environment. You really believe that the perfect patient stratification needs to take into account all elements not only the tumor cells but also the stromal cells and immune cells. And I will show you the results of the first study that are currently under review. A second important focus on my lab is identifying targets. For example the discovery general confusion was definitely fortunate event but it really changed the management of the disease and disease. Why we really want to focus identification other potential targets to mimic what happened, the success with FGF I inhibitors of course to do so we need pre clinical models. Therefore we have established several million models were working unclear and more also for what concerns go bladder and extra batic. Since the experimental models for the extra batic disease are really, really few and more recently in the past few months we also started focusing mechanism of metastases. Since metastases the first cause of death for patients with calandria cinema. And then we have actually started to perform single cell based technologies uh in much primary and metastatic tumors. Focus on the first example in this first study as I mentioned and stress several molecular stratification of being proposed and several from our group. Unfortunately one of the caveats with this classification was at the time we were not looking at the macro environment. Uh in my case it was 2000 and 18 when I joined yourself lab in Barcelona and we designed the first molecular classification of intra particle Angelika cinema. This was published in 2013. We had collected a large court, 150 patients. We call in yoga cinema and by focusing on the tumor, we identify that there are two main classes. The two classes were called proliferation and inflammation Proliferation class was the largest around 60% of the disease and was quite complex and actually quite a very genius. They were definitely aggressive tumors. We prove survival and early recurrence, but also where the tumors that were enriched in several angiogenic pathways. Most of them target double also several alterations, particular mutations and chaos give up an epidemic growth factor receptor. But also this class included a small subset that is what is called virginity like a C. C. A. These are two more that expressed temperature in their cells in this subgroup within which 98 1 and two. The second group was the inflammation class, which was enriched interleukin six and start face signaling. And this were indolent us really with well differentiation and good prognosis as mentioned this we have learned a lot in terms of like about the joints for this classification, but at the time and it was almost 10 years ago, we really did not take into account the limits of the macro environment and I think it's one of the caveats of this classification. Many other studies have been published and there is general consensus on the existence of these two classes, although once again, this classification did not take into account the tumor micro environment, which for me, is the greatest limitation of this classification and in in this classification has not been implemented in clinical practice there for what we did over the past year and a half because of the covid pandemic, we were mostly forced to work remotely in the first few months. What we did was to generate a new algorithm this time, we prioritize elements of the immunity from a macro environment. And we generated another classification what we call the steam classification. This is trauma tumor immune classification. And in reality we actually focus on elements of this trauma and elements of the immune micro environment to design this novel stratification This time instead of two classes within 255 indicated here by different colour boxes at the top. The most important message is that two classes present a really high immune infiltration. These are called inflamed. Whereas the majority of cases were not inflamed had really scared immune infiltration and more importantly by adding other levels of characterization. And we found out not only the immune composition was different but also the Strama composition was different. In particular between these two inflame classes with both high immune infiltration. We always said that this one in yellow that was called inflammatory Strama. I had a really dance. This more plastic reaction was enriched in maya fiber plastic cap. That is the same that I mentioned before, which are responsible for high stiffness and hide the position of access alarm matrix in which also contributes significantly to the growth of the tumor. On the other hand, the non inflamed anthropological and your question on my head really scares him an infiltration and each one of them and specific tumor alteration. For example I. D. H. One interpretation of the effort to fusion were enriched in this group along with abundant micro Fiji's type two. On the other hand, this blue for example was enriched in regulatory T cells. All this to say that we have identified five different subtypes and each one of them is not only unique in terms of two more aberrations but also in terms of immune it's trauma and this provides a better platform for us to design a combination strategies and we'll give an example in the next slide just to mention that in addition to the different molecular and micro environment differences were also observed difference in clinical futures and in particular disc two more this group of tumors in red and the yellow with the one with the worst survival. Also stage was different in particular this group in green. E. Was the one with the earlier stage tumors. Why? Why I really highlights this classification is that by providing a better understanding of how tumor genotype correlate with immuno phenotype and Strama. We can now design more rational combination strategies. For example based on this, we are speculating that those tumors with an inflammatory strawman activation of T. J. B. To need the dual innovation of this mule surprise developments like T. J. B. To but also now with the novel curiosity because perhaps we can block the androgenic chaos to elicit a better response to pD one. Therefore we need to eliminate the brakes and what is inducing an immune suppressant micro environments to make these tumors more sensitive to checkpoint inhibitors and for example here where we have I. D. H. One and two but also not collapsing money we need to combine this targeted therapies with immunotherapy to get a better response. And although of course this is only based on in silica data, what we've been doing in the past few months is that using your model is able to replicate the different subtypes. Were actually testing this combination to demonstrate and prove uh have the proof of concept that uh this molecular stratification is valid and then of course specific combination therapy can improve the outcome of patients with calandria cinema. Moving on to the last example that I want to provide today is as I mentioned we are really interested identifiable targets for effective therapies. In an attempt to identify these novel targets we performed in immuno genomic analysis in particular. We looked at the expression of checkpoint molecules here are the most common. You're probably familiar with most of them across the five classes that I described uh just a few seconds ago. And as you can appreciate as also we have seen in a potential carcinoma usually the expression of checkpoint molecules especially at the messenger level really correlate to the presence of immune cells. Therefore it was not surprising that the majority of this checkpoint were highly expressed in the studio every time an infiltration but actually one was different and cauterize you can see here. B seven H four is an immune checkpoint But for whatever reason we found that there was really highly expressed in this group of tumors, it was almost specific to this group of humor but also we know that this group of tumors percent really low immune infiltration. This was quite really intriguing for us and therefore we looked at the T. C. G. A. We wanted to see really if this image for bozo so highly expressed in other data sets and interesting when we look contractor cinema was ranked at the top. It means there really isn't much for is highly expressing contractor cinema but seems to be also quite relevant in this disease compared to other tumors. And this was an effort to prompt as to further investigate before going and further, I just want to mention the B seven H four is an immune checkpoint protein. It belongs to the B seven family along with PD one and PD L. One and as you know, they all have the same functional blocking the activity of T cells. In the case of the so much four here it binds an unknown receptor and is able to exert a profound individual activity on T cells. As a result of the first model client bodies have emerged and actually the first one called up 150 is currently being tested in a phase one clinical trial in breast and ovarian cancer. Of course we're not immunologist. So we were intrigued but what actually called more our attention was that there are few reports suggesting that regardless of the activity of this image for on the immune cell, it seems that the syringe for one is over expressed only two more. It can promote the growth of the tumor to an unknown cell autonomous mechanism. This was really interesting for us and prompt that's really in front of investigating this. Uh and we actually have a project of the ongoing about this image for just to show, we were interested in understanding the expression of percentage for on the tumor before we did um, you know, standing and compared to the classical Pd L one and Pd one as you can appreciate here, less than 10% of contractors. Cinema patients are positive for one or the other, which is quite scarred presence. On the other hand, The Syringe four, we were right, really surprised to see the 42% of the tumors really present high expression, really highly positive for B seven H 4 26. Short low expression between zero and 50% of to themselves and only 32 are completely negative. So this part was suggested to be 74 can really represent a potential target and more importantly, percentage for was completely absent on the health deliver. I really just really little expression with absurd of countryside suggested it could be really safe target, intrigued by the suggestion that is NH four can promote tumor growth. We use the neurons align. This is called Nac which has high expression of the seven H four and using the CRISPR CAS nine system we knocked down this NH four you can see that we different physical guides. We really achieve the good suppression of dissent H four. More importantly disease the BRD you which measure proliferation of the south. You can see that compared to the control there was last proliferation and those cells lacking B. Seven H four. And even more obvious here, you can see when we block B seven H four with these three single guides. The cells are not able to form colonies, suggesting that indeed B seven H four. Besides inhibiting T cells also has a cell autonomous role in promoting tumor growth in Colombia carcinoma. Which is quite appealing because it means that we can block B seven H four and blocked due to an activity not only in the tumor but also on the micro environment. This was a mirren's align. So we were interested in understanding if the same happens in human cancer carcinomas alliance and in this time with in collaboration with the next question is lab we have designed antisense illegal nucleotide. These are really short sequence 2025 nucleotides that can bind specific messenger RNA. And the gradient. Use either the degradation of the production of a non functional protein unless to design three different A. O. N. Here. I'm showing just two of them 1935 and 1936. These were able to block the protein. You can see there is a significant reduction in the protein. What is more important compared to the control. Once again we were able to reduce the proliferation of the cells And actually it's quite significant with 1936. This is quite important because of course once again we demonstrate and we confront the results of the CRISPR CAs nine we demonstrated the B seven H four is an important target by blocking this image for we have the potential of blocking tumor growth but also interfering with the micro environment. And more importantly, oh and actually being used in clinical trials, the first against TJ Evita are being tested in clinical trials, suggestion the RIO and represent even better strategy or potential strategies for blocking the seven inch foreign calandra carcinoma. Just to conclude this presentation, calandra customer represented health challenge with increasing the incidence and mortality rates. Genomic profiling has helped us to elucidate the molecular nitrogen native disease. Has identified the molecular classes the computation and genomic rearrangements. Unfortunately immunotherapy despite being promising, many other cancer has been quite disappointed in contact a cinema. Whereas on the other hand, position medicine with FGFR. IDH two inhibitor seems to be quite promising other treatments again, when performed in all commerce have been negative in particular decay and notch inhibitors have failed to provide encouraging results suggesting once again that we need a better patient stratification and ideally uh I hope that I've showed you some results uh to convince you that we will be achieved a better patient certifications with our steam classification. But of course validation is needed and actually we're currently providing this additional classification and many models of the disease. Finally we're really excited about the discovery and how this image four seems to be highly expressed in contact a cinema. It's an immune regulatory protein but seems to play an important role in inducing a cell autonomous growth of the tumor. Before we hope that by using a a an antisense oligarchical today we can interfere with this duel functional design french foreign provide another potential targets Which will benefit a large number of contractors cinema. Since the 50% of them have really high expression of this molecule on this under us. Last but not least I would like to thank my lab our internal and external collaborators, the department of medicine from the invitation and the support the division liver disease and teach cancer institutes and all our current and past funding and of course all of you for your attention. Thank you very much hope to see you. Thank you so very much for that terrific literature. I know that a lot of us were texting and just um admiring your body of work. Um your ability to go between the clinical and the and the science in the lab And so really congratulations on what has been uh really a terrific presentation as well as a wonderful body of work. There are a few questions in the chat but I actually have a question for you to um my question to you is this um you had mentioned in the beginning that uh there were changes over time in terms of the clinical epidemiology. Certain increases over time. I wonder if you can comment on whether there are changes like this also in the molecular uh in the molecular space. So in other words do you I think that there are changes in time perhaps as patients are treated that there are different mutations, different pathways therefore that you can leverage and and so I wonder if you can comment a little bit on that. Yeah, this is actually a very good question. Especially because most of our knowledge that is from resected patients especially for the application. Next generation sequencing. I mean we need a large at least so far. We have needed a large number or amount about 10 a.m. D. N. A which is impossible to obtain from biopsies. Especially because biopsy is not required For the diagnosis of conduct a cinema. I mean not necessarily uh therefore imaging sometimes is enough there for most of the knowledge derives from resected patients. So your question is actually quite valid to know if advanced cases have a different molecular profile and there are only few studies actually one I showed it was the clinical cancer visions 2018 where they analyze red sector cases and biopsy. It doesn't seem that there are additional targets but I think we need larger cords to understand if advanced stage there are additional multi collaboration. There are not present at the early stage. It's quite difficult to obtain biopsies unfortunately. Yes. Yes. Especially it's not required. Yeah. Right right now I imagine so. Okay so there are a few questions in the chat. Uh There's one question regarding as chinese immigrants to the U. S. Are here uh sort of in the 2nd and 3rd generations. Do they maintain this increased fraction with a number of generations or do they uh start do you start to see a decrease in the incidence of the legacy? Uh No I think I will say that also for example in in our study we collected a lower large number of samples from the U. S. And there were a lot of asian patients and I know that the incidence is maintained Hi even when they migrate to the US probably there are some risk factor that some genetic risk factors in addition thinks they've been exposed. So even when they moved to another country some of those are retained in the DNA. Like exposure to risk factor. As I say the liver fluke infection. For example if that has happened before it has already had an impact. Especially the exposure to liver fluke infection that's not cause immediately the answer to cancer. What happens up to 50 years later, before we still see a high increase in those populations. Wonderful. Also another question on whether obesity increases the incidence of the inter paddock collage carcinoma. Yes, it does. That are yes. Has been reported. Okay. All right. Uh and any unique features of intra paddock, of clash of carcinoma in the setting of inflammatory bowel disease. Not to my knowledge is something interesting. Unfortunately, as I said, the majority of cases arises in a healthy liver. So, we don't have enough uh large number enough of patients like PSC or inflammatory bowel disease to do these correlations. But uh, a doc studies are required to address these questions. They're all valid questions. Yeah. Do we have any additional questions for dr CIA? Feel feel please feel free to type them into the chat. This has really been a superb presentation. Um As I said, I can tell just by the degree of engagement that we've had on this call that uh that there are many people who really felt this is a terrific body of work. So, thank you so very much for your time and your efforts and yes, I can see you clapping. So wish we could thank you. It was an honor to present at this meeting. Thank you for inviting me. All right, thank you so much. But there's actually one last question here about role of cholesterol esters and metastases. Perhaps you'd like to just come? I don't think I'm able to address this, but I will. Yes, I can definitely follow up. There is not much known in terms of metastases. Okay. All right, well, thank you George. All right. Bye bye. Everyone have a great day.
