 All right. Well, thank you for getting that set up. So we got, I think, hopefully more of a Zoom crowd going on. So welcome to another Snowy Grand Rounds. I have the pleasure of introducing Dr. Wynne Chamberlain. He is coming to us from Portland, Oregon. And I know Wynne very well as we were co-fellows together at UC Irvine under the late great Roger Steinert. And I remember Wynne for being not only way smarter than I was, but also being one of the nicest guys and most humble guys I know. And I can't believe it's been like so long, almost 15 years since. Oh, no, you don't look any different either. So Wynne grew up in Southern California. He went to undergrad at Caltech with a original interest in aerospace engineering. It's wonderful what you see, what you find on Google. Yeah. So, but he started having an interest in the life sciences and majored in biology, minor in chemistry, went to medical school at the University of Colorado and also received his PhD in immunology also at University of Colorado. Then he went to UC Irvine for his ophthalmology residency, stayed on for his cornea fellowship, which is when I met him. And then after fellowship, he accepted a faculty position at OHSU in Portland, where he has stayed ever since and he's currently the head of the cornea division there. Wynne has several publications, talks and is very actively involved in the iBank Association of America, EBA. He's currently the chair of the medical advisory board there. So I'd like everyone to just join me in welcoming Dr. Wynne Chamberlain. And I'm going to try not to screw up this presentation for the virtual here. So I hope everyone can see the screen. Thank you for attending remotely and for the few that were able to make it in. I had to make it in because I was very eager to see this campus again. I was here in 2006 for a fellowship interview and it was a beautiful day. I think it was fall and so I remember seeing the sunshine. So today I brought you a little sunshine from Portland. And so this is, you'll see a little sunshine, sunrise, sunset theme in my talk, which is obviously intended to be a little bit symbolic. And so this is actually a picture I took 10 days ago driving to my Vancouver satellite and that's Mount Hood there in the distance. This is a sunrise. There's a little ambiguity between sunrise and sunset here. And there's no filters on this. I was driving across the I-205 into Washington to just snap this with my iPhone. So I have a few disclosures that are not really directly related to this talk. There is a product by Trefoil, which I'll mention. I've just been in some discussions with them on some basic scientific advisory stuff. And then I'm also a site PI for the COA phase two and phase three trials for DSO. And I'll talk about that briefly at the end. Just a reminder, I know probably a decent amount of the audience is not cornea. And so it's interesting that our field of transplanting tissue into humans has been around for a while. And so Edward Zerm, who is an Austrian ophthalmologist, gets the credit for doing the first transplant into humans. And he had a patient named Alois Glogar, who lived in this small town called Olmitz near Prague. And he's a farm worker who had a bilateral corneal burn from this unslaked lime, which is calcium oxide. And it can be converted to calcium hydroxide. It's very alkaline when it mixes with water. So he got this splashed in his face and ended up with bilateral blindness. And Zerm was aware of a young boy, an 11-year-old, who had unilateral blindness due to an intraocular foreign body. And so he actually enucleated his blind eye and harvested two small five millimeter graphs from that single eye. One was central and one was peripheral. And he used a tree find that was actually designed by Von Hippel to make these small graphs and actually transplanted both of Glogar's eyes. One of them failed. And it turns out the one that failed was actually the peripheral graft, the central graft that went to the left eye. And you can see in this picture in the lower right hand corner survived. And the patient was poured to have 2100 vision one year after surgery. And he actually held these graphs in place with congenital overlay flaps that get created with the way they put these in because they didn't have a kind of suture that we have available now. And Zerm died without reporting any more conial transplantations. And then Ramon Castro Viejo actually sort of advanced our field by developing techniques. And actually when Amy and I were fellows, I saw a Castro Viejo graft. Do you remember that? It was a square graft. So these these graphs had amazing longevity. And so there was a few remaining when I was training. And nowadays we even have femtosecond lasers to shape these grafts. So there's been quite a bit of advancement in kind of the way we do grafting. So just briefly, what is endothelial careplasty? Most of you know that this is a relatively modern addition, but it was thought of in 1956 by someone named Tillett, who published the first report on a posterior lamellar graft. It was a large incision and it was stitched into the eye. And then Jose Barracara is well known, came up with his own method of a stromal resection under a sort of a big fat lacy flap and did sewing anterior and posterior to hold posterior grafts in place. Garrett Mellis may be kind of the modern author of endothelial careplasty. He's thought up a lot of the modern ideas and described the posterior lamellar careplasty in 1999 with a corneal pocket. And then Mark Terry kind of advanced this by describing D-Lac, which was his version of that surgery with a modified technique in Portland, Oregon, starting about 2000. In 2004, Mellis said, let's not create the pocket. It's too much work. And so let's just peel off Decime's membrane and reattach a graft using air. And Frank Price reported the first large series of D-Sect in 2006. And then one of Frank Price's fellows, Mark Gorvoy, is still very active in the field, introduced the idea of using a microkeratone to actually make grafts. And that's where we get the A and D-Sect. The A stands for automated. It just kind of advanced the field by allowing us to cut these grafts with a microkeratone and get smoother interfaces. And that's kind of become the standard. And then in 2006, 2008, Mellis started reporting on DMAC, which is the most modern version of careplasty, other than some experimental stuff, which we'll discuss at the end. And this has kind of changed the way we do things. And this video at the top is showing a recent DMAC surgery that I did at the KCI Institute. And we use Interoperative OCT to help us a little bit with these cases. So this is just a reminder of what these are. Again, D-Sect is a piece of stroma, varying thickness that goes into the eye with endothelium and decimase intact. And DMAC is just decimase membrane and endothelium. So it's much thinner. And both these grafts can detach and develop little interface problems, which can create optical issues and postoperative issues. And they're both are kind of coaxed into the eye by some tapping and fluid techniques. Although it turns out the DMAC grafts require probably a little bit more manipulation in terms of tapping, whereas the D-Sect grafts kind of open readily, as you can just see there in that video. I tend to do both of these through an injector technique, but there's a lot of different techniques described now. So this is some statistical data from the EBAA. And I want to point out kind of two key things here. If you look at these top two rows, is that penetrating careplasty numbers have been going down since 2012. And endothelial careplasty numbers have been going up. And the most recent data suggests that endothelial careplasty exceeds penetrating careplasty by about double the number. And so this is about two thirds of the careplasties done in the U.S. now are endothelial careplasty. And in 2011 was kind of the turning point where endothelial careplasty started to exceed the number of PKPs done. And now we have a new turning point we're coming to. You can see on the left hand side here that DMAC is actually catching up with DSEC. And it's been a little bit slow because the surgery is a little bit more difficult. It's been a little bit slower to adopt. And there's higher complication rates that have been reported with DMAC. But it looks like in 2022 or 2023 DMAC may actually start to surpass DSEC as the most common form of careplasty in the U.S. At least it'll be equal and it'll be equal to the numbers of PKs. So PK is that blue line on the right. They'll all kind of enter a point of equality in the next year or so, which is kind of an interesting turning point for careplasty. So this is another graph in that same statistical report. And what it shows is that, so I've just told you that endothelial careplasty is the most common careplasty in the U.S. And more than 50% of those are done because of endothelial dystrophy in the U.S. If we look at the foreign data from the EVAA, it's probably skewed because the graphs that get sent out from the U.S. actually probably get used for things that don't reflect the true distribution in places like Europe and New Zealand and Australia. So we send graphs to other parts of the world where endothelial careplasty is not quite as common. So I would say in modernized countries, endothelial careplasty is probably the most common surgery as well as in the U.S. And DMAC numbers and DSEC numbers are probably very similar to what we see in the U.S. There's a meta-analysis done in 2017 pointing out that we didn't really have good randomized trials to compare DMAC to DSEC. This is important because a lot of people are arguing if we should go to the learning curve of DMAC was a hard surgery. There's more complication rates and we still see those. And then there was this emerging data that thinner DSEC graphs may behave as well. And so when Amy and I were fellows, Christiana Neff, who was Ed Holland's fellow that year, published a study showing that DSEC graphs under 140 microns actually saw better than DSEC graphs over 140 microns. So people started describing thin DSEC and then ultra-thin DSEC. And these numbers actually don't have clear definitions, but nowadays in the literature, it seems like most people find ultra-thin DSEC is under 100 microns. So that would be stroma, decimase, and endothelium. So in about 2015, I started designing the decimase and endothelial thickness comparison trial with Jennifer Rose, news bomber, after going down to UCSF to give a talk. And we started arguing about the merits of these two surgeries. And one of our conversations was based on my early experience, my DMEC learning curve kind of looked like the graft on the left. You can see in that blue line that at six months, my DMEC patients were seeing better, except if I included my failed DMECs. If I included my failed DMECs and looked at the visual acuity of all the patient population, they're actually doing significantly worse than my DSEC patients. And because those failed graphs didn't see as well even after I regrafted them. This was obviously kind of a steep learning curve and a lot of people were struggling with the surgery. And then also on the right, I looked at endothelial cell counts in my first 40 cases and the endothelial cell counts were doing poor in the DMECs. So Jennifer Rose said, you're going to do better with ultimate DSEC. Let's do a trial and confirm it. And so that was the purpose of the decimase endothelial thickness comparison trial, which we're now calling pilot for reasons I'll make clear in a few moments. But this was a multicenter double mass randomized controlled clinical trial. So couldn't mask the surgeon, but we could mask the evaluator after the procedure. So anyone who's doing refraction or scans was masked to certain components, so they weren't aware and the patient was masked throughout. Primary outcome was six month ETDRS best corrected visual acuity. And we looked at a number of secondary outcomes, which I'll discuss. We screened a lot of patients. It was hard to enroll because in Portland, we have a lot of patients traveling long distances and they couldn't make it back for all the follow up visits. But we randomized 50Is to either DMEC or ultrathin DSEC. We had a pre specified analysis that had a power and 80% power to detect about a little over a one line difference in snow and acuity between ultrathin DSEC and DMEC. And that was our, again, our primary outcome. And in this study, we thought it was important to keep all patients in the study. So even patients that had failed graphs during the study, we kept them in the study. We had to intervene and re graft a few of them, but we still follow their visual acuity because we thought that was a fair real world assessment of how we were doing these types of surgeries. So I'm going to go through a number of slides and I'm going to kind of give you a take home point from each slide because there's a lot of data here. The first one is just the baseline characteristics of the patients. And the take home point here is because of randomization, and this is powerful when you randomize patients, you really get a fair distribution between so you don't introduce biases of the surgeon or the patient. So in this study, because of randomization, there was no difference in age, gender, diagnosis. Most of these were folks, there was a couple of PBKs, preoperative, best spec corrected, ETDRS, visual acuity, corneal thickness, manifest spherical equivalent, or whether they needed cataract surgery at the same time as they needed endothelial care to plastic. So all these were the same between patients who received all within the second to Mac. We also randomized the graphs. And in the graphs, there was no difference between the donor age, sex, depth of preservation, depth of surgery, cell counts before or after processing is a firm out of processing that goes on with endothelial care to plastic in the eye banks these days. And then the donor graph thickness was obviously different because desex are thicker by definition, but these were truly ultra thin graphs averages about 73 microns. And there was none that were greater than 90 microns in thickness. So our primary outcome again is our six month ETDRS visual acuity. But even at three months, we were seeing significantly better vision in our DMAC group. And at six months, it had improved the scatter plot on the right shows that kind of clustering of the DMAC and the better visual acuity range based on the log Mars scale in six months compared to baseline. And we looked at adverse events, and it's important to note that because of the size of the study, we weren't necessarily powered to detect differences in adverse events. And we didn't, even when we compared the most common one, which is re bubbling, which was, as you can see from this table was more common in the DMAC group, not surprisingly so. So one of the questions we started asking is why are we seeing better, especially since some people didn't feel that that would necessarily be the case at six months. And so we kind of asked some theoretical questions about the shape and can imagine if you add a chunk of tissue to the back of the cornea versus a very thin layer, you might alter the optics of the cornea here. And so one of the things that can be altered is these higher order aberrations, which are probably created by little micro distortions in tissue. So we used the penicam, which is a shime flu image, or to actually assess anterior and posterior or higher order aberrations between ultra thin DSEC and DMAC patients. And the left graph shows that basically there was no difference in the anterior surface between ultra thin DSEC or DMAC between the groups and also between comparing them to any point post stop and points from baseline. But we looked at the posterior surface and you can imagine this is where the action might be because that's where the surgery is. We saw a significant difference in posterior surface higher order aberrations. So in DMAC, the aberrations got better immediately after surgery and they continued to improve. And in a year, they were significantly better and they were baseline. So we're actually improving the backside of the surface by doing DMAC. But the opposite was true for DSEC. The aberrations got worse and stayed worse for up to a year after surgery in this graph. And these aberrations included things like coma and trefoil and higher order of stigmatism. And then the total higher order aberrations were significantly higher in the ultra thin DSEC group. And these aberrations did correlate with visual acuity, which we did on a correlation analysis. It wasn't a fantastic correlation, but it was definitely strongly suggested that they were correlating with visual acuity outcomes we're observing. Recently, we've looked at the 24 month data on these higher order aberrations and done a slightly different statistical analysis showing what's called mediation, which means that certain variables will mediate better visual acuity. And this just shows the data again on the left that really the entire higher order aberrations are no different between the two study groups with DSEC and blue and DMAC in sort of orange. But in the posterior side, DSEC is persistently higher in all of these time points after surgery, although it is getting better. And at 24 months, it's still higher significantly higher than DMAC, but it is getting better over time. We also wondered about light scatter because back to the time when I was even a fellow, I remember people saying, well, the DSEC is causing light scatter because you had extra interfaces. You've got this slab of tissue and so you might have light passing off the back of the host cornea as well as off the back of the donor cornea. Whereas in DMAC, because the layer is so thin, we may not see that effect. And we'll actually measure this with shine fluid photography again. As we asked the question, is there a difference in light scatter between DSEC and DMAC? And we actually looked at different layers of the cornea. So we looked at the anterior cornea, the middle cornea and the posterior cornea, which you can see on the table below. And from the p-values, you can really see that there's no difference between DMAC and DSEC for light scatter at any time point after surgery. The values did significantly improve from baseline. So light scatter got better after surgery, but it wasn't better between the groups. So obviously visual acuity got better in both patients. And 24 month data, very similar analysis. So even up to 24 months later, there's no difference between light scatter between DMAC and DSEC. So image on the right is a sunrise again coming up over Mount Hood, driving to Vancouver as seen through an aberrated cornea. Actually it's through an aberrated windshield, but I also thought it looked a little bit like some of the multifocal IOL pictures that we see in the literature. But in this mediation analysis, we noted that the posterior corneal aberrations had this significant effect on mediating visual acuity, whereas the anterior aberrations in the dense telemetry basically had no effect. So we do think these higher aberrations are a big component of why DMAC sees better. What if we ask the patients what they think? So in the detect trial, we also had patients do standardized visual quality functioning surveys. And this was the NIH standardized validated surveys. And what we learned was is that ultrathin DSEC patients saw as well as DMAC patients, at least based on their vision related quality of life surveys. So there's no difference in terms of what the patients felt they were seeing. And we look at some predictors at what gave them better scores on that survey. And the predictors were PBK, although very poorly powered because we had so few PBK cases in this randomized controlled trial. But the posterior densitometry seemed to be a predictor. So if they had more light scatter on the posterior cornea preoperatively, they actually felt like they saw better postoperatively after surgery. And also they had two eyes and roll they saw better, which kind of makes sense. So those were kind of predictors. What about endothelial cell counts? So three, six and 12 months, we didn't see any statistical difference in endothelial cell loss between our ultrathin DSEC group and our DMAC group. But if you look down on that lower right column, you'll see that that DMAC group is actually dropping off at what appears to be a faster rate. And by 12 months, it looks like it's getting close to visual or to statistical significance. Central cornea thickness obviously improved in both groups of patients as well. And this is 24 month data. 24 month data shows that visual acuity is still better in DMAC than it is ultrathin DSEC. So ultrathin DSEC may be catching up, but at 24 months, DMAC still better. We're actually starting to collect five year data on these patients. And so we will be looking at that to see if there's actually a catch up at five years. So based on the data, we wondered if it was time to put DSEC to better to the grave, because the visual acuity at least on the chart was better. And the endothelial cell counts at least statistically didn't seem to be different. But we were forced in a more recent publication reporting our 24 month data by an ophthalmology reviewer to do a secondary analysis on our endothelial cell counts, which was not a pre specified analysis, but a statistician from UCSF helped with this. And basically, we use the repeat measures method from baseline to 24 months with clustering by patient. And when we did this non pre specified sensitivity analysis, actually DMAC had a significantly lower cell count or significantly higher endothelial cell loss rate at 24 months than DSEC. And so this kind of brings DSEC back into the picture is maybe potentially a type of graph that will last longer than DMAC. So we're still asking the question, should we stick with with DSEC as our as our mainstay? Good question. And so the answer to that is no. But the numbers were low enough that it probably wasn't sensitive to pick that up. And there were higher rebubble rates for sure in the DMAC. So there was only one rebubble in the DSEC group. So it would have been a kind of a shoddy analysis because the low numbers. Yeah, I'm sorry. So Jeff is asking me to repeat the question. Did the end cell loss correlate with graph rebubbling, which I think is a very good question and one that we hope to go answer. And there are studies out there now, I believe they're all retrospective showing that there is a definitive correlation between rebubbling. In our study, it wasn't powered to detect it and we didn't detect the small numbers we had. Griffin Jardine, who I think is on faculty here now and I haven't seen in a while, helped us do an analysis of end-of-the-fill cell loss and graph preparation several years ago, which we published in current eye research. And what was noted was that there was a significant amount of cell loss in DMAC graphs, much more than DSEC graphs, just in the preparation. And so this may explain to some extent why we're seeing this cell drop off, although you wouldn't expect it to be progressive as the study is suggesting. But there was a fair amount of baseline loss of cells as a result of processing. And then we went on to publish a few more studies showing that there's various things that contribute to this from our markers or S-stamps. We now use F-stamps, our treflination points, and even sliding the graphs through different types of injectors can induce linear damage on them, which you can see in that left-sided photo next to the S. And these are all things that contribute to maybe more cell loss in the manipulation of DMAC graphs. I'm going to change topics here for a minute and I'll come back to the endothelial cell counts. But we were interested in understanding another component of how the cornea changes. And that was the change in corneal power. And this was motivated by the fact that we've known for a while and we teach our fellows that if you're going to do cataract surgery combined with ultrathin DSEC, that you have to sort of plan ahead that you're going to have a hyperopic shift. And when you put an IOL in, you've got to kind of do this sort of ad hoc adjustment to your IOL and shoot a little more myopic, because the graphs probably going to cause the eye to have a hyperopic shift after surgery and want to avoid that. And so you can see here on the left the anterior corneal curvature and posterior corneal curvature values between ultrathin DSEC and DMAC. And the anterior corneal curvature doesn't change much. It changes a little bit at three months. But beyond that, it's pretty much nonchanged from baseline. The posterior corneal curvature stays significantly changed, and it actually steepens. So this is kind of confusing because it's a negative K that I'm showing on this scale. So the drop in that line is not showing a flattening of the posterior corneal curvature. It's actually showing a steepening because the posterior cornea contributes a negative power to the surface. And it turns out that negative hour or steepening causes a hyperopic shift in the cornea, which may be a contributor to what we're seeing. And we looked at the patients in the study that had cataract surgery, and we basically wanted to know if we calculated something and using the Barrett II formula, if we calculated where they should end up with an IOL master, where did they actually end up compared to what we expected them to? So remember, we weren't shooting for planar. We were shooting for myopia, because we expected them to end up a little bit hyperopic. But the zero here reflects what our target was. And so the bottom scale is the delta or the change of what we expected versus what we actually saw. And you can still see that a majority of the patients had hyperopic shifts, many more than we wanted to. But there was also about 38% of the patients that have myopic shift for reasons which were unclear. And there's been several reports on why this might be what popular theory early on with DSEC was the DSEC graph actually creates a negative lens on the back of the cornea. It's like a meniscus lens. And so it actually subtracts power from the cornea with this meniscus effect and causes a hyperopic shift. But DMACC does it too. And DMACC doesn't really have that effect. So there's a chance and this is kind of a still a theory, but it kind of makes sense to me that when we strip decimates an endothelium off the center of a thick cornea, and then we add back a new graft, the healthiest cells are probably in the center of the graft, not at the periphery. And I showed you some pictures a minute ago of how trefination damages those cells. And so they're getting a healthy load of cells centrally, which causes central thinning faster than peripheral thinning of the host cornea. But then over time cells sort of redistribute. And so the peripheral thickening, which you see initially after a lot of DMACC surgeries, at least my DMACC surgeries actually starts to improve. So by three to six months, that periphery starting to thin out more too. And so that thinning out of the periphery then causes a little bit of a back drift or a myopic shift back. And you can see that in the graph here and see where it's actually drifting back a little bit. And so the important thing to learn from this is that this actually isn't stable for maybe up to 12 months after surgery. And so when we think about guessing on an IOL or if we're going to do an IOL after DMACC or DSEC surgery, when's the right time to do it? And this may be relevant nowadays even with a light adjustable lens is when will be the right time to just unlock in that lens based on changes in the cornea after care to plastic. So the American Academy of Ophthalmology, Technology Assessment Group, published right before we started publishing detect data that they felt DMACC to be safe and effective as a for endothelial failure, superior to DSEC in terms of visual recovery, visual outcomes and rejection rates. And this backed up a lot of the randomized control data that we were starting to present induced less refractive error, which actually we didn't see in the detect trial. They felt the surgical risk were equivalent to DSEC. And I think that's still a point of debate. And then the rate of air injection with repeat care to plastic were similar and DMACC versus DSEC. And I would say that's still a point of debate too and in a lot of the retrospective literature and in some of the randomized control trials. There's two other randomized control trials that look very much like detect, which came out shortly after ours. One's from Holland. And interestingly, they didn't see a difference between DMACC and DSEC in postoperative visual acuity. They saw no difference in endothelial cell loss similar to us using standardized statistical techniques. And they did see a hyperopic shift. And then another group in the UK noted early DMACC superiority in terms of visual acuity, but by 24 months, ultra thin DSEC had caught up. So these randomized control trials gave different results than our trial gave. And they had sort of similar patient satisfaction between groups in that last trial. This is a recent meta analysis kind of looking at those trials and a few retrospective trials. And in terms of the endothelial cell counts, which I think is critical to this, is that there's a little bit of a distribution here, but if they take all the data together and analyze it, they don't really see a difference in endothelial cell loss between any of these randomized control trials or the one retrospective trial that they included in this graph on the forest plot. So one thing we know is that, and this is relevant to endothelial cell loss is that DMACC is a harder surgery to do in difficult eyes. So post-retracted my eyes, AFAKA guys, large iris defects, eyes with compromise or no capsule, eyes with glaucoma shut devices, and trabeculectomies. And so we need more literature to guide us because these are the ones that are starting to challenge us now. And I think these are the eyes that most corny surgeons still feel are best to approach with DSEC because it's a little bit more predictable. We don't know what those long-term visual acuity outcomes are going to be. We don't know what the endothelial cell loss is going to look like in those eyes or late graph failures. And there's a lot of changing trends. We're starting to see a lot more reports of people tackling these difficult eyes with DMACC. I wouldn't be giving a good talk here if I didn't mention a few things that are changing in the field. And so this is a landmark paper in the New England Journal of Medicine that was published by a Keneshina's group in Japan. This was back in 2018. And so what do they do? They took donor endothelial cells. These are allogeneic cells. So they're cells that are matched to the host. They pulled them out, and they have figured out ways to grow these in culture. And there's a long body of literature now demonstrating that molecules known as rock inhibitors, which I'll discuss a little bit more in a minute, can actually drive proliferation of endothelial cells in vitro. The benefit of driving proliferation in vivo is a little less clear. But in vitro, if you take these cells out and you grow them in a tissue culture plate, you can actually get fairly significant proliferation of endothelial cells. And Noriko Koizumi, who developed a lot of this technology, told me that she can get about 30 graphs worth of cells out of a single donor. So it's pretty significant if you think about tissue shortages in certain parts of the world. Of course, it's not easy to grow cells. And it's not cheap to grow cells in culture. But they did a kind of proof of principle study where they looked at patients with bolus keratopathy in Japan. And the picture on the right shows four of these patients that had pretty impressive corneal clearing two years after injection of endothelial cells. So all they did is they denuded off endothelium centrally. They injected endothelial cells with a needle into the anterior chamber and had the patient's slave face down for three hours after surgery. And so it wasn't even a significant amount of laying down time. And remarkably, these cells found where they were supposed to go, landed on the backside of the cornea and repopulated and cleared these corneas. And more recently, the same group published a follow-up at five years. And this was a subset of their original number of patients. And what they noted was normal corneal endothelial function in 10 of the 11 eyes that they assessed. And the endothelial cell counts in those eyes were about 12 to 1300. Now, this to me is significant because these are actually numbers similar to what we might be seeing in a DMET graft at two years out. And so the best corrected visual acuity improved significantly in 10 of the treated eyes. Obviously, there was one failed eye in this cohort and there was no major adverse events from injecting these cells. So the cells didn't end up in the wrong location and cause glaucoma or other types of abnormal tissue proliferation. So studies definitely confirmed safety and efficacy of this technique. And as a result of this, and because of innovation going on around the corneal world, these are three companies that are based in the U.S., MSL, Arcuscel and Orion, which are now setting up or running clinical trials, looking at endothelial cell cultures to use as a method to repopulate corneas that have sick or dysfunctional endothelium. MSL is planning a phase two trial. MSL is a company that's developed a magnetic bead technology to tag their endothelial cells, and they're using magnets to actually draw these cells to the backside of the cornea for location. And it'll be interesting to see how that pans out. Arcuscel is a little newer to the game, but they're also developing limbalsome stem cell cultures, and they'll be entering into early safety trials, phase one trials in the near future. And Orion's probably the furthest out the gate. Orion has had kinesheed as an advisor, and they've already run about 70 to 80 patients in Central America, and I believe they're going to present their first data at ASCRS in about a month, a month and a half in San Diego. So it's interesting to see how the results look, but this could be a big game changer for those of us doing endothelial caretoplasty. Let's talk just briefly about row-associated kinase inhibitors or rock inhibitors. Picture of it on the right is rapazodil. Rapazodil is the rock inhibitor that was found by a drug screening protocol and was used in Japan, and this drug is approved in Japan to treat glaucoma, but it was noted that had activity on corneal endothelial cells. So endothelial cells have limited proliferative capacity, as we know, and the mechanisms that may play a role in healing endothelium that are related to rock inhibitors are that rock inhibitors may promote proliferation, they definitely promote proliferation in a tissue culture plate by regulating cell division. So they actually push cells out of the G1 phase and into the DNA synthesis phase of the cell cycle, because this can promote proliferation. So that's one potential benefit, and this may be happening at a low level inside the eye as well, inside a living eye, so in vivo. They also enhance some components of actin myosin contraction, and this is important because endothelial cells have to migrate to heal and have to migrate into areas of damage, and this is a current model, as you damage, you take off dysfunctional endothelium, and then the other endothelial cells are kind of sitting there static, have to be stimulated to migrate in. The injury itself probably promotes migration, but it seems that rock inhibitors may actually promote faster migration to promote intracellular healing, and they also rock inhibitors probably also block apoptosis, and they do this through a slightly subtle mechanism of acting on the actin contractile forces within the cell that keep them from actually breaking down the nucleus, nuclear disintegration is actually an essential component of cell apoptosis, and so they actually keep cells alive, and they also cut down on membrane bleb formation, so when cells go into apoptosis they start letting off all these little bubbles of cells and break up into little tiny individual bubbles of cell components, and that's how the cells die off in apoptosis, and rock inhibitor blocks this process, so it may prolong cell survival, and I think this is interesting because this may play a role in grafting as well, we may be able to keep stressed endothelial cells from dying by giving them rock inhibitor during endothelial keratoplasty, so decimates stripping only or also known as decimates stripping without endothelial keratoplasty or DEWEC is a newer surgery that's been developed which doesn't involve any transplantation, basically this is one of my cases, and I've not done a lot of these cases for reasons which I'm happy to talk about later, but basically we make about a four millimeter zone centrally over the pupil, I like to dilate because I can see the endothelium better, we just reach in with a pair of forceps, these are Gore-Voy forceps developed by Mark Gore-Voy, and they're nice because they allow you to peel off decimates which is actually usually quite easy in Fuchs patients without beating up the underlying or the underlying stroma, you don't want to beat up that stroma too much because if you create an evenness in it the endothelial cells have a hard time migrating over it to come back to the center, so it's not a very it's not a very stimulating surgery from a surgeon who's gone through the DMEC learning curve, but it is quite easy, so you could probably teach this comprehensive ophthalmologist and it basically gets rid of all the dense goutte in the center, and then postoperatively you let those cells migrate back in with or without the help of a drop of a rock inhibitor, and so this patient, I'll show you some data on them, but actually let me let me give you a little bit of background on DSOD WEC, there's a couple key studies, but if you look at the column three in this table, and this was out of a recent review and in seminars on ophthalmology, you can see the numbers are pretty small, we don't have a lot of data on this surgery, and so I think that the two most interesting trials were prospective trials by Marion Moxai who did 18 patients, 10 of which just got stripped and didn't get any drug, and then eight other patients that got stripped and got rapazodil, and she was able to get an IND from the FDA to run this trial, and the patients that got treated with rapazodil, they all cleared and they cleared faster, and they had higher endothelial cell counts centrally after surgery, but actually most of her patients that didn't get rapazodil cleared as well, she had one fail in this trial. Maloney did a prospective study treating all of his patients with rapazodil, 22 out of 23 cleared, one of them failed and had to have a DMAC rescue, and he also reported there was a most common side effect of rapazodil, which was GI upset and ocular irritation, and this has since been reported in a couple of case reports as well. There's some other retrospective studies, including one by Catherine Colby that had seen that the size of the amount of decimates that you strip is very important. If you go too big, you'll fail, and it's probably because you're taking off too many endothelial cells, and there may be other reasons for that as well, such as the cornea has a harder time clearing and normalizing, and then the lower study by Wong and colleagues actually noted something similar and also made a point to point out the DMAC patients actually get much faster visual recovery than DSO patients, and there may be a problem with personalities in terms of waiting that long. The COA trial, which has finished its phase two enrollment of about 60 patients, is now sort of getting ready to publish this data, although we haven't seen any of the results yet, so this will be the biggest study to date. This is an industry-sponsored trial by the company that makes Repazodil, and we're kind of interested to see how they do, and they're currently setting up to do a phase three trial, which is a much larger group of patients to look at the efficacy in that group. This is a hard trial to enroll patients in. We actually screened out 22 patients and only got one end because they were so fussy about the kinds of patients that we could enroll, so this is the one patient I enrolled in that trial, and I don't know if they got Repazodil or not because I was masked to that, but this is two weeks after DSO and the surgery I just showed you, eight weeks after DSO in the middle and 12 weeks after DSO, and what's interesting here is this 12-week, you can actually still see the desmetorexus, but the cornea is clear in the center now, so you can see the edge of it with a red arrow through indirect illumination, through direct illumination you can actually see the edges of it on that slit, and if you look carefully at the slit there's a little bit of haze in the posterior cornea there, so it's still existing, but in eight weeks this patient was seeing 20-25, their baseline was 20-40, so that's some improvement, and by 12 weeks they're seeing 20-15, so there's no endothelial cell transplantation here, and these are specular images of that particular patient at that time, and you see the grossly abnormal endothelial cells at eight weeks, and normalizing more at 12 weeks, but still low cell counts around 850. I suspect this patient got repasital just because of the way they behaved, but still don't know the answer to that. One of the things that's been brought up is like, yeah the cornea is clear, but they're abnormal, they're irregular still because they've cleared, but they don't have enough endothelial cell function in the middle to kind of normalize the surface, and so in this patient, this is just one example, so it's an end of one, you can't draw a conclusion from this, the patient had zero change in their penicam axial maps between baseline when they were 20-40 and 12 weeks out when they were 20-15, the penicam looks essentially identical in terms of the corneal powers and the distribution of the astigmatism in this eye, so but there have been many reports that suggest that patients have irregular astigmatism, which worsens after doing DSO, so that may be problematic. So as a result of this data that's out there, the old detect gang got back together and by this point we enlarged a little bit, Charles Lin from Stanford had joined our group and we had Benny Yang who's now at Penn who was kind of interested in the study, we still had the Francis Proctor Group which does outstanding data management and Tom Lippman is kind of the king of randomized control trials at UCSF and then Jen Lee joined us from UC Davis and then Jonathan Lass agreed to do the endothelial cell analysis because he's got an outstanding center that allows us to do standardization of our endothelial cell counts and he'd helped us with our 12 and 24-month endothelial analysis in the first trial, so we designed a new trial and this one got funded by the NIH and most of the credit year goes to Jennifer Rose Neusbaum who's an outstanding grant writer, but we decided to target patients with endothelial cell dysfunction, but these patients in the first arm of this trial known as detect one are going to still be DMAC versus ultra then DSEC, but they're going to be in complex eyes, so eyes that have previous retractomies, very advanced fuchs with lots of edema, history of retinal surgery, glaucoma, and even failed grafts, so grafts that we're trying to rehabilitate with endothelial keratoplasty. And the primary outcome is not going to be vision in this arm, it's going to be central endothelial cell counts at six months and we're going to look at those counts obviously at 12 and 24 months as well. We're interested in knowing if we continue to follow our trend that DSEC outperforms DMAC in terms of cell counts, but the other thing we're doing with this detect one group is we're randomizing them in a rapazodil, so a quarter of the patients or one half of the DMAC patients and one half of the DSEC patients are going to get rapazodil and the other half are going to get placebo and they're going to be massed to it and we're interested in knowing if rapazodil is actually going to speed up the rate of recovery and also help us to see less graft failure and endothelial cell attrition over time. So this is a question that hasn't been asked yet in a randomized perspective fashion and actually very little data in the retrospective literature on this as well. And then we're also doing a second arm and the second arm is also asking a question which hasn't been looked at in a perspective fashion is does DMAC and DSO do as well? And so we're going to have an arm of milder fuchs where they either get randomized to DMAC or DSO with rapazodil so the DMAC group won't get rapazodil and we're going to look at the primary outcome, their best spectral corrective visual acuity and we're actually currently enrolling patients so so far the trials enrolled about 25 patients and our goal was about 200 patients for arm one and about 50 patients for arm two. A couple more points and I'm going to stop. This is a totally different topic but I think it's very interesting. This is I mentioned at the beginning there's a company out there called Trefoil which through the work of David Avelith has developed fibroblast growth factor in a recombinant form and they modified this protein because it has a very short half-life in the anterior chamber of animals and they have done animal in vivo and in situ human studies on this modified growth fibroblast growth factor. And so Pizzuto and colleagues have published two papers on this now looking at the effects of endothelial healing in cultured human corneal scleral rims looking specifically the endothelial layers and what they see is that patients that get the fibroblast growth factor as you can see in this picture to the left heal almost completely when they're treated with it but the patients that are the control that get a or the tissues that are controlled that get just kind of a placebo drop that endothelial defect remains constant at 14 days and the immunofluorescence staining on the right shows the incorporation of a fluorescent deoxyuridine component which correlates with DNA proliferation and what they're showing here is different zones in the control group and in the group that's treated with the fibroblast growth factor and there is more cellular proliferation in the group that's treated with fibroblast growth factor. And I think this is interesting because that means that this protein which we all make may be able to stimulate endothelial cell proliferation in vivo in the human eye and if that's the case then this may actually be a solution for bolus caretopathy where there's a global shortage of endothelial cells. It also play a role in doing DSO and scraping off Fuchs Gute and allowing those cells to proliferate and come back in but it might be very important in bolus caretopathy which is a bigger problem in many parts of the world. So I guess this makes me a little bit nervous as a corneal surgeon because I wonder if we are going to become surgeons that are going to be injecting like our retina colleagues or just putting drops in the eye with rokinase inhibitors like our glaucoma doctors to allow the cornea to heal and specifically that injection for the fibroblast growth factor would be into the anterior chamber not in the vitreous space at this point but it does seem like that's a possibility and the trefoil study is going into phase two trials. We haven't seen a lot of data published from their safety trial as of yet. One last slide this is a completely different approach to endothelial cell dysfunction which I would have said would never work but seems to be catching a little bit of storm and it's like kind of a stormy sunset there off the Cincinnati sort of Kentucky airport which I took a few months ago but this is a company that I believe is based in Israel called Ion and they have developed a posterior I guess you could call it an underlay it's a synthetic artificial endothelial layer which is basically made of a hydrophilic acrylic so what we use for our IOLs and they do a small sort of stripping of decimates in the center of the cornea and place this small diameter underlay against the cornea and they published two patients that they followed for 17 months after surgery these underlays or artificial endothelial layers were difficult to get to stick to the back of the cornea they had to re-bubble them a few times but they managed to clear the cornea and the thought behind this is that they are preventing fluid from flowing passively into the cornea by preventing this inhibition pressure which is kind of the difference between the IOP in the eye and how much fluid is drawn in by all the extracellular matrix proteins that are in the cornea and so these two forces are at play and by putting this barrier in there you actually keep fluid from entering the center of the cornea the interesting question is is wouldn't you keep nutrients rendering the center of the cornea as well and their theory is that the nutrients is getting in peripherally but they still have endothelium intact so obviously a long way to go with this but they're also in a larger scale trial now to look at the long-term outcome it's very appealing because there's no cells here you can touch this thing you can push it around and if you get clearance with significant visual acuity improvement we may not need cells anymore there are a few future things being discussed this is sun rise over mount hood there's synthetic bioengineered stroma it's being generated by a company called cornea bioscience which they may be able to repopulate with endothelial cells seems like cells like to grow on this spun collagen matrix that they can generate this goes back to May Griffiths original research that was done in Ottawa there is some talk about RNA antisense therapy where you deliver antisense all the nucleotides to basically shut down bad transcriptions of TCF gene that plays a big role in Fuchs and then there's gene editing which is actually a real thing now the KCI Institute is doing gene editing trials in the retina for leavers congenital amaurosis and this may become a thing in the cornea in the near future as well we actually just snip out the defective components of the Fuchs gene and give them a good copy in which case we wouldn't need anything related to implants or tissue culture in the future so the residents are listening they may be thinking about going into retina glaucoma if you're worried about the future of cornea surgery so I'm going to stop at this point and just to give a little bit of a couple minutes to ask questions but I guess just philosophically I kind of did the sunrise sunset midday thing because I've been thinking like how much longer are we going to be doing what we've been doing for the last five to ten years and for a surgeon at my sort of level who's spent a lot of time and frustration trying to get these techniques to work well and watching patients recover and seeing good things but also seeing some bad things happen I have mixed feelings but obviously the the technology is marching forward and there's a lot of promising avenues I do ask the question you know how much will this apply to other parts of the world where first of all it's very expensive to adopt this technology and secondly the distribution of endothelial dysfunction in many parts of the world looks so different than it does in the U.S. and so we really have to think about tools that will apply to cornea blindness which is much more predominant in other parts of the world and much more debilitating than it is in the U.S. and in Europe and Australia and New Zealand so I'll stop at that point I'm happy to take questions I think Jeff's going to help me with probably the majority of questions coming from online and thank you for anyone who tuned in and thanks for the the core that came in this morning thank you there aren't there aren't any online questions right now I'll kind of read them as they come just just following up in your comment about you know distribution of corneal blindness different parts of the world you know do you envision a scenario where there's a DSO you know with or without medication or injection of cells for severe pbk which is such an issue in other parts of the world yeah so before I answer that I will say that it's interesting so I Jeff we're talking about this last night I got to spend a couple years in Myanmar helping set up a training program there and helping kind of promote the i-bank that they had there and I was shocked to see how much fuchs dystrophy they had Myanmar which may actually genetically be different than what we see but they do have fuchs in other parts of the world I've done some mission trips to Guatemala and I've seen quite a bit of fuchs down there as well but I do think that definitely a drug approach would be much cheaper than cells easier to get there so rock inhibitors may play a role for fuchs for pbk I think maybe the most promising thing is end of art if it takes off because it's just got a really long shelf life and we might be able to improve those patients with a very simple procedure which we could teach anyone that can do cataract surgery can do this and then also this trefoil medication may be applicable although again it's it's biologic it's a it's a protein it has to be stored appropriately and because it's a biologic it's going to be really expensive we struggle to get tissue to many parts of the world because it's too costly for them even though it's heavily discounted so I think coming up with non tissue solutions are definitely going to be a game changer if we can find things that work long term but but what I think we're seeing is we're seeing that a lot of the surgeons even in countries that don't have access to tissue are selling us we're not going to settle for less than what you settle for in the U.S. and so we say well you just take our bad tissue and use it it's like that's actually not respectful to them and they don't want to use it they want to get the highest standards as well and so a lot of conversations with international cornea surgeons have kind of driven that home to me. Thank you Griffin Jardine has a comment you're gonna I think Ethan is unmuting you Griffin I know you just want to take credit for your paper but go ahead. Thank you Jeff but Dr. Chamberlain Boy what a treat to hear from you this morning you really were one of my most influential mentors and heroes in residency so it's just been a treat to hear from you I'm so sorry I have to be that person I met a satellite office this morning and barely made it here alive but the I was going to say amazing presentation so interesting you talked about some of the you know this question of endothelial cell loss you're kind of later in DMACC your DMACC's been around long enough that maybe we don't have the you know the the good trials and you know around my good objective data on late graft failure with DMACC but what what have you seen anecdotally and in terms of just the survival time of that that graft? Yeah well so there is some there is definitely retrospective date on this and a lot of it comes out of Mela's group and long-term survival looks outstanding the cell counts do drop and it looks like they continue to drop at a faster rate than they would in the native cornea but they look really good and I can tell you I told the story a lot my first DMACC case at the KCI Institute was an absolute nightmare it was it was so bad that the resident who was with me in that case along with the fellow looked at me afterwards actually he went to glaucoma for understandable reasons and he probably didn't spoke to him now Rory Aller and he said Dr. Chamberlain will you ever do one of these cases again after my first DMACC? It was three hours patient had sneakier and people that was actually never quite normal again but you know that patient has about 800 cell counts he's about let's see he's about 12 years out now is that right maybe maybe 11 years out and he's 2015 in that eye with 800 cell counts and so even though that graft was just completely beat up by a surgeon didn't know what he was doing I think it's a real testimony how sturdy these are over time and he hasn't redeveloped Gute so I don't think his own endothelial cells have grown back in totally to cover that graft that I put in there so I suspect that we're going to see a lot of graft survival low cell counts which was actually a result of the cornea donor study as well they saw that grafts that even had cell counts as low as 500 lasted a long time maybe because some of those cells were actually host cells but probably some of them are continued donor cells as well so anyway long long answer to a good question and Griffin it's privileged to talk to you over the microphone I'm glad thank you for those kind words. Such great memories working with you the last question to follow up to that so if the endothelial cell count maybe doesn't have a perfect correlation or maybe isn't directly in causation of graft failure maybe why use that as your primary metric in this new study do you think do you think there's maybe I guess I'm just unclear if that if that is really what is going to determine graft survival and visual acuity what are your thoughts on that? Yeah that's a very fair question one of the advantages we have is is the double randomization in this trial because we actually predict that we'll see a difference between patients that get rock inhibitors and not and you might still say well it still doesn't matter because even if their counts are low without rock inhibitor and they're higher with rock inhibitor which is kind of what we anticipate although we may not see that it may not matter long term in terms of visual acuity but it might matter more again for this third world argument in patients that have bullish keratopathy because they have a global decrease in their endothelial reservoir which creates a lot of problems with the peripheral cornea as well and so having more cell counts long term and sicker eyes and having healthier cells and sicker eyes is probably going to allow us to tackle some of the tougher cases so that's a really good question. Well great answers great great to hear from you again thanks again for coming to visit with us with Moran and thanks again for your just your incredible mentorship to me over during those critical years so take care. Thanks Griffin. So just kind of it's going to be interesting to see how iBanking will change a lot with the new technology with it seems like it's iBanks are now competing to get like intellectual be in on these new technologies and i feel like iBanks will need to really evolve to keep up otherwise they're going to be left in the dark so it'll be interesting in the next five to ten years. Yeah it's a great comment Amy and there's so much discussion on that going out of the EVAA and it'd be interesting to have a side conversation about even what that implies for iBanks. Amy and I both work in the EVAA and this is a great community and they've done a lot of research to advance criminal transplantation and I still feel like in many parts of the world criminal transplantation is going to be the mainstay because I think it's going to be too expensive to do this stuff first off but I do think we're going to see a significant consolidation of iBanks which may not be a good thing if they have to compete with other technologies so we may see our approximately 50 iBanks to windle down to a handful like maybe five or six if these other technologies take off because there won't be reasons to have so many iBanks and that could impact the international group because a lot of these corneas that they're getting come from the US the US has the highest corneas supply of any other of any country in the world so I think it's a really good point. Any other questions? Or ostensibly normal glaucoma patients with endothelial with no obvious endothelial problems? So the question is are we seeing a honeycomb dystrophy when you put patients on rock inhibitors you see kind of this unusual almost macrocystic edema in the epithelium of corneas that are treated with the rock inhibitors and I think this is a little bit more common with metarsidil or rupressa than it is with rapazidil but it's been reported in both. So the short answer to the question is we haven't done a ton of these cases yet. I anticipate we will see it. My understanding is that it's short-lived from the standpoint of unmasking patients in a randomized controlled mass trial it might be interesting if somehow that becomes symptomatic to them and it could unmask it's not going to unmask the surgeon because the surgeon is already unmasked but it could unmask the other evaluators as well. It seems to be a short-lived phenomenon and it's because row kinases, row associated kinases are ubiquitous and I'm sure the expression in the epithelium is impacted by this drug as well and it's changing their behavior. Thank you very much for your attention. Thank you Win for that wonderful, wonderful talk and yeah we look forward to maybe some future collaborations.