So, let me introduce our first speaker, and Dr. Timo Rath, you're from University Elegon, Germany, world leading expert in the molecular imaging in IBD, and the professor of that university, and that now the platform is yours, Timo, Dr. Rath. And before we really start about talking to talk about molecular imaging and how we can guide medical and endoscopic therapy in inflammatory bowel disease. I would just like to set a common stage. That we define in one sentence what is molecular imaging. So one sentence definition of molecular imaging would be, it is the labeling of cellular or subcellular structures with fluorescent dyes, and the subsequent visualization with endoscopic or even shorter, a one word or three word definition would be it's in vivo, immunohistochemistry. And having said this, it's clear that we need different components to be successfully in using molecular imaging. And first we need a definite molecular targets target and in the majority of studies. These are membranous receptors. But clearly the target depends on the clinical scenario and the clinic question that we have. And secondly, and this is a very important if not the most important component of the fluorescent labeled probes. And finally, we need a molecular imaging devices that can adequately and specifically detect this fluorescent signal resulting from the fluorescent probes. So, what is an ideal molecular probe. In an ideal world, a molecular probe should have, first of all, high affinity for the target with at the same time, however, offer only low background signals. And then it is. It is desirable that molecular probe exhibits rapid binding kinetics to allow fast visualization visualization after administration, and also a fast clearance to avoid and limit side effects. And finally, an ideal probe should exhibit a deep tissue penetration to allow visualization of sub epithelial targets, and clearly immunogenic immunogenicity is a component that we rather don't want to see what label probes. And most importantly, when we try to broad scale molecular imaging, it's important that we can conveniently label our probes with fluorescent dyes or fluorophores, and also manufacture those probes at large scale volumes. Among the probes that have been utilized in the literature, or in the community are most commonly used peptides and antibodies peptides offer the advantage, they are very small molecules, thereby typically exhibit a very good tissue penetration and at the same time, have a very favorable toxicity profile. However, identification of specific peptides is rather labor intense, and sometimes requires sophisticated methods, such as phage display. Another commonly used molecular probe are antibodies, the beautiful thing about antibodies is there are largely available and offer high specific binding to what's defined molecular target. Whereas, a little bit with antibodies, they are large molecules so they only have a limited tissue penetration, and also a bear the risk of immunogenicity. And when we apply molecular probe, we need to decide whether this should be systemically systemic distribution usually offers sufficient distribution throughout the whole body, thereby enabling deep and almost complete tissue penetration. However, intravenous injection can clearly be associated with allergic reaction side effects topical and administration is typically done during endoscopy with the spraying catheter where we spray the molecular, molecular die onto the mucosa. However, it's clear that we only need very low amounts of a die to achieve high concentrations on the mucosa, and at the same time limit side effects. So from this point of view topical administration appears to be ideal for focally distributed diseases such So, if we then deep dive into the studies that are out there that show us how we can kind of not only great is intestinal inflammation and IBD but also can can influence our decision making with molecular imaging, it's important to note that one of the studies is already 15 years old. And interestingly enough, this is not a pure endoscopic study. It's rather in radiologic study, but still influenced and perpetuated the whole field. As you can see this was kind of a dual phase study in the first ex vivo set of experiments in, in, in, in a mouse in two different mouse models of colitis, the authors, the authors visualized apoptotic cells by annexing single photon emission tomography. And as the authors are observed by this annexing spec, the number of apoptotic cells was clearly increased upon anti TNF therapy. And with this knowledge, the authors, then went into a very first pilot study including 14 patients with Crohn's disease. And those patients were patients with clinical active disease as defined by a C die of 150 or higher. And those patients were scheduled for entity and after therapy. And what the authors did 24 hours after infusion of infliximab, the authors performed exactly this annexing spec tomography and assess clinical response 14 days later. And what the authors observed that the spec annexing spec segment was clearly increased in those patients that had subsequent response to what's biological therapy. So this was really a landmark study showing that we can only great intestinal inflammation but also make predictions about therapeutic success success by visualizing apoptotic cells. So with this knowledge, in the back of our heads, Roger Atreya from our department went into a phase two trial in Crohn's disease to show that kind of the actual molecular target of entity and F therapy therapy, which is membranous TNF can be used as a companion diagnostic to predict therapeutic response to its subsequent biological therapy with anti TNF. And this was a kind of a study that that had three different steps. The first step was of course to generate a molecular probe and for this purpose. I used commercially available few mirror and labeled that with with fits, and all this labeling was done under GMP confirm condition conditions to ensure that really this die can be used in patients in vivo. The second step then was that during endoscopy 25 patients with Crohn's disease, prior to the initiation of entity and F therapy. This die was topically applied to the inflamed parts of the mucosa. And the fluorescent signal, resulting from labeling of the mucosa with fits labeled Humira was then visualized with confocal laser and the microscopy. So those gray dots here, those are single cells expressing membranous TNF, and as you can already see from this there were kind of two cohorts of patients. One cohort of patient had a very low amount of membranous TNF expressing cells, whereas other patients at high amounts of membranous TNF expressing cells. And this really turned into prediction of therapeutic response. So after this molecular endoscopy patients had a second appointment in our outpatient department, and receive the first dose of Humira. And as we can see here, clinical response assessed at week 14 was way higher in those patients with high baseline empty and F expressing cells, as compared to those patients with low amount of empty and F expressing cells at baseline during molecular endoscopy. And if you calculate diagnostic performances. For this approach to use empty and F expressing cells as a companion diagnostic. We observed a positive predictive value of more than 90% to predict response towards subsequent biological therapy. So here in naive patients we can kind of stratify the cohort into those that have a high likelihood of responding to therapy compared to those with only a low likelihood of responding to therapy. This approach was limited to entity TNF therapy as shown in a very small case series that we subsequently performed in Crohn's disease here similar approach. We used commercially available video leads him up so anti alpha four beta seven antibody therapy label this antibody with fits on the bench, and then in kind of on the bench experiment showed that we still have specific binding towards alpha four beta seven fluorescently labeled antibody, and then also went into an ex vivo molecular imaging study. And the result of this pilot study okay serious wasn't it was kind of similar in in the key message, those patients with high amounts of alpha four beta seven expressing cells in the mucosa or in the lamina proper. They are the candidates that respond to subsequent video leads map therapy, as compared to no response in those patients that that had zero or very low amounts of alpha four beta seven expressing cells. So here, the study show us that in proof of principle concept, really can use molecular imaging to predict response to subsequent therapy. And with that, I would like to change gears a little bit. As I said initially molecular probes are kind of one of the key components, if not the key components in molecular imaging and a probe that has two very unique features, as shown on this slide and this probe is called gamma glutamyl hydroxymethyl rhodamine green, and the beautiful thing about this probe is the fluorescent signal from rhodamine green is in the unclean state of this of this molecular die is kind of encaged or hidden, and only after activation or cleavage of this die by gamma peptidase an enzyme that is highly upregulated in cancer, the fluorescent die becomes liberated is transferred to the lysosomes, and thereby enables specific visualization of cancer cells, as shown in this initial study in a mouse model of peritoneal cancer here. Even after topical spraying terminals were specifically identified. With this in mind, in 2013 in a rodent model of colitis associated cancer it was shown that we really can precisely identify colitis associated cancer or dysplasia. So here the authors use a well established model of colitis associated cancer which is called a OEM DSS model so the mice received one injection of a carcinogen which is a OEM, followed by three courses of colitis as conferred by dextrane sodium and it is well described in the literature that these, this resembles collided associated cancer, and as shown in the study. When this molecular probe gamma glutamate hydroxyl methyl rhodamine green was administered topically spray to the colon of these mice. It was possible even after five minutes to precisely identify colitis associated cancer or dysplasia, even a lesion that are one were one millimeters or even below. The second beautiful thing about this study, it was, it was able to show that we can not only visualize single tumor bearing areas, but also monitor to tumors over time as you can see on the right hand side of this slide for 150 days it was possible. to monitor exactly the same colitis associated cancer regions, and also precisely discriminate those regions against background inflammation that those DSS mice typically have. The proof of principle was corroborated in another study a couple of years later here endothelial receptor, a was chosen as a molecular target and in three different mouse models, a UMD SS and acute on chronic model, as well as a model of sporadic colorectal cancer which are APC minimizing was shown that with molecular endoscopy, we can identify colorectal colorectal cancer and colitis associated cancer and with the same results we can discriminate colitis associated cancer or dysplasia from background inflammation and this is, as you all know, one of the major changes in the surveillance of IBD patient especially colitis patient that we have a hard time even with chromo endoscopy of precisely delineating only inflammatory pure inflammatory errors from dysplastic and another probe I would like to introduce to you is is shown here because this recently gained traction here in the field this is called SGM 101. This is a monoclonal antibody, a chimeric monoclonal antibody occur against casino embryonic antigen. And this is linked to a molecular diet that has fluorescent in the near infrared range. And as you can see here, it is fairly enriched in tumorous tissues. And as you can see here in those radiologic images in a mouse model of peritoneal cancer. This probe. When administered intravenously allows to delineate tumor bots and single tumor cells in the peritoneal cavity in these rodents. And if you look at the tiny images here. It's, it's very fascinating to see how bright this fluorescent signal is. So, in the middle panel those are mice with the were spontaneous colorectal cancer is simulated. And as you can see, even in the closed mouse so peritoneal cavity is intact, you can see how bright, the, the molecular diet fluorescent fluorophores through the through the And the reason why I mentioned this probe is because just recently in a major publication and Lancet gastroenterology hepatology from the Dutch group. It was shown, not only that we can use it safely in humans, after intravenous injection, but also that we can unmask tumor lesions that were not suspected before. So this was a study that included 26 patients with colorectal cancer. And the focus of the study was a dose escalation cohort so different doses of this, this molecular probe were used five to 10 milligram. And even in the 10 milligram intravenous dose, there were no serious side effects. And most importantly, what was observed in this study so all these patients had elevated serum CAA levels, and known or recurrent peritoneal metastasis, and then 40% of the patients. were found that were not suspected before. And in 35% of the patients, this led to a change in the treatment plan and as you can see here in the right panel, this is very fascinating panel A and B, these are lesions, kind of buried in the momentum that are only visualized with fluorescence, fluorescence endoscopy or molecular imaging and panel C, you see the right ovary, where the fluorescent signals very strong indicative of peritoneal metastasis, and here in panel D you even see retroperitoneal lymph node that only are detected with fluorescence endoscopy or molecular imaging. Now, what is the future. Well, one thing I would like to highlight is, although I've mentioned a couple of times that molecular probes are the key components. Do we at all need molecular problems, maybe not. What I would like to introduce on the last two slides is the context of multi photon microscopy or multi photon micro and endo microscopy so what is multi photon microscopy. Well, this is based on the nonlinear excitation of autofluorophores such as NADH, FAD or collagen, and when we then use in our endoscopic devices as you can see here in a custom built mini endoscope, we use suitable filters, the spectral signature of NADH, FAD, and collagen can be separately visualized, meaning that in parallel can, without the need of exogenous dyes visualize stroma epithelial cells and cells in the lamina propria, all in the same device without additional dyes and Sebastian, Christ from our department just showed in gastroenterology, at least in rodents that in different models of colitis, we can precisely great intestinal inflammation and various models of mirroring colitis. So, ladies and gentlemen, I would like to summarize. So, molecular imaging can be used not only to assess therapeutic success on single cell or molecular level, but also be used to predict therapeutic efficacy prior to treatment initiation. And we already have in vivo in human proof of concept. And first evidence also suggests that molecular imaging can be used to precisely delineate and unmask colorectal and colitis associated cancer and as we all know, we have a very high need for this in our IBD patients and future developments might be able to facilitate molecular imaging and enable label free molecular imaging. So with this, thank you very much.

molecular imaging

inflammatory bowel disease

medical therapy

endoscopic therapy

molecular probe