Win-Win -The Development of Novel Immunotherapies for Brain Tumors Using Pet Dogs

On Thursday June 10^th  at 3pm EST/12pm PST, we were delighted to welcome Dr. Liz Pluhar to our webinar series.

Dr. Pluhar is a full professor and small animal surgeon in the Department of Veterinary Clinical Sciences at the University of Minnesota and leads the Canine Brain Tumor Clinical Trials Program.

Dr. Pluhar shared insights on developing novel immunotherapies for brain tumors using pet dogs.

We'll also discussed the benefit of pursuing clinical trials and the important research her team has uncovered on the development of bone graft substitutes and animal models of disease.

Christina: Topic du jour is brain cancer. And specifically, how this is such a big unmet need, frankly, on both sides of the leash. So we, little known fact, is that we're very actually inspired by the brain cancer journey, specifically, frankly, of Joe Biden's son, who we got to know a bit, the Biden Foundation, and especially the cancer moonshot that he really led, and folks in the cancer moonshot, and the journey of one just really, really not having access to therapeutics.

So that's one. Two, in learning the data in the journey, the cancer journey for his son's glioblastoma, having to take medical records from one hospital in Pennsylvania to another in Texas on Air Force Two, taking from floppy disks, taking photos with an iPhone. And this inspired a lot of what we designed here in FidoCure, firsthand.

We actually were honored by Joe Biden for our work at South by Southwest a few years ago. And his folks are fans of also, given the intimate learning, very sadly, that the Biden family had with brain cancer. They're really aware of the translational, the animal model limitations, right? And are very aware, which we'll hear a little bit more today about the opportunity, in a way, to solve these two unmet needs really creatively, right? For canine brain cancer and human brain cancer. And how can we bring these sides of the leash closer so we can really unlock both sides. As many of you probably know from the animal model side of really unlocking therapeutics for brain cancer, currently the state of affairs is they induce brain cancer in a mouse by literally putting it in the mouse's butt, in a way such that it's almost like a spoon that you scoop out. This has like, is it fair to say, Dr. Pluhar, like zero translation to humans?

Dr. Liz Pluhar: Well, some of them very little, yes. I would say that. I think they're a necessary evil, they're necessary, but they're not the most informative.

Christina: So it's a starting point, absolutely. However, in the dog, location matters, brain matters. So this is a fantastic opportunity to help both sides. And this is going to be what we're going to be spending our time here today. And I'm so excited you're all with us because this is fascinating and so important to the very DNA of what we do and our thinking and elaboration.

And for anyone that has had a family member or a friend touched by brain cancer, you know, or a dog, right, on the other side of the leash, you know this is a very, very, very challenging tumor type. So without further ado, I'm going to quickly pass over to our chief medical officer, Dr. Jerry Post, who's going to do a more, formally introduce Dr. Pluhar for all her fantastic accolades. And then we'll jump right in. So start thinking of your questions and we'll be taking them in the second half. Over to you, Dr. Post.

Dr. Post:  Thanks so much, Christina. It is again my pleasure and honor to introduce Dr. Pluhar, who is a colleague and friend of mine from many, many, many years ago. Dr. Pluhar is a full professor and small animal surgeon in the department of veterinary clinical sciences at the University of Minnesota, my alma mater. Following her undergraduate education, she received an MS degree from Northern Illinois, DVM from Oregon State, a second master's degree from Washington State, and then a PhD in comparative orthopedics from the University of Wisconsin, Madison.

She completed advanced clinical training with her internship at the animal medical center in New York, which is where I first met Liz. And then she did a surgical residency at Washington State. She is a diplomat of ACVS. She also had postdoctoral training under the tutelage of ACVS diplomats. She has received numerous awards, William Harris award from the Orthopedic Research Society, excellence award for veterinary research excellence from the University of Minnesota.

And she's involved with numerous professional organizations, including the Society for Neuro-Oncology, the Orthopedic Research Society, American College of Veterinary Surgeons. And she even served on an advisory committee for the FDA. Her research interests include immunotherapy for brain tumor, and we'll talk about how a surgeon gets interested in immunotherapy, just in a bit. The development of bone graft substitutes in animal models of disease. She's supported by funding from a variety of sources, including the National Institutes of Health, American Cancer Society, the Department of Defense, the American Brain Tumor Association, and several foundations.

She's published over 50 peer reviewed papers and has given over 150 scientific presentations and lectures. And she leaves their canine brain tumor clinical trials program at the university of Minnesota. So we're so thrilled that Dr. Pluhar has given of her time and expertise to be with us today.

Dr. Gerry Post: Why did you get involved in brain tumors first? That's the first question.

Dr. Liz Pluhar: Well, it was a very interesting journey. I was doing my surgery residency at Washington State University. And during the second year of my residency, Washington State University hired Rod Bagley, who is a neurologist and a neurosurgeon, and he didn't have a resident at the time. And he had a really interesting project and asked me if I'd be interested in doing the project with him on looking at the effect of acute occlusion of one of the transverse venous sinuses and to see what would happen with intracranial pressure.

And so, that was my master's project during my residency. So I did that with Rod and I ended up scrubbing in on a lot of brain surgeries with him as well, doing corpus callosotomies and tumor removals and things like that. And so that was my initial introduction to brain surgery.

And Rod was a great teacher. I learned a lot from him and he had an obvious love for that topic. And that rubbed off on me. Then my career kind of led me in a little bit of a different direction once I finished my residency, because I actually was training primarily in orthopedics and my PhD at Wisconsin was in orthopedics, and I did help out people at Wisconsin if they had a tumor that was at the cerebella modularity pontine angle, that's a tough, that was what the approach that we developed was for. And so I had to assist with that, but that was about the extent of my brain tumor experience during that time. And then my first position was at Colorado State as an orthopedic surgeon. And so I did nothing but orthopedics there.

And then Minnesota hired me away from Colorado State. And about two years after I started here at Minnesota, I was approached by a guy, named John Olfest, who was at that time, a post-doc and he had a very small goal of trying to cure brain cancer. And he heard one of my colleagues give a talk at the medical school about dogs and cancer, or veterinary cancer.

And it was Elizabeth Neal. And she didn't mention anything about brain cancer. So John approached Elizabeth after the meeting and asked if dog's got brain cancer. And she said, yes, they do. And John got very excited and started talking to her about this project that he had in mind and how we wanted to do brain surgery and immunotherapy. When he got excited, there was no stopping him.

And Elizabeth was just like, wait, wait, wait, wait. She said, I'm a neurologist. She said, I'm not a brain surgeon. I do in radiation oncology. But she said, I don't do that kind of stuff, but I know somebody who's had that experience. And so she introduced me to John.

And when we first got together, we started doing, we got some research beagles and we did some injections to see whether or not we could do some, could infect with, we tried infecting with antivirus, wanted to see how well that worked in dogs and whether or not there were any adverse effects.

And I really thought that that was kind of going to be the extent of my work. And it has completely taken over my life. We've been very blessed in the number of grants that we've been able to get. It was right early on when people started recognizing that dogs with spontaneous disease are a great model for human disease. And so we were kind of caught up very early in that process and have been very, very successful in our granting. So that's kind of how I got started in it. And now it's basically my life, it's dog breaking and brain tumors.

Dr. Gerry Post: Fantastic. So for the other folks in the audience, I think your story brings up really two important points, at least for me. One is, the value of saying yes and the value of collaboration. Often we think of our, we work in our silos and that peripheral vision, that breaking down of silos, for me, and certainly based upon your story, has really tremendous advantages. And so I'm wondering, do you counsel, mentor, your residents, your students in this, and the value of saying yes, and the value of collaborating?

Dr. Liz Pluhar:  Yeah, absolutely. I do think it's important. And one of the great advantages of being here at the University of Minnesota is that we do have the medical school that is here in the same area. We've got that, you know that we have all these different campuses, but they're all interconnected. And there's buses that go around them all the time. So it's very easy for me. I can be over to the medical school in five minutes to talk to the people that I'm working with at the medical school, or in the school of dentistry or in the genomics area.

So there is a lot of interaction and it has been, I mean, the administration has really promoted this and people are talking about one medicine and translational medicine and the former dean, the dean that just stepped down, he actually moved on to a higher position in the academic health center. He told me at one point, he said, I'm getting so tired of telling people about the canine brain tumor program. And I was like, what?

What do you mean? That's appalling.

And he said, no, no. He said that, "When people ask me what one medicine or translational medicine is, the best example I have is the work that you do with the canine brain tumors that's being translated for people with brain tumors." He said, "But that's the one, that's kind of my go-to." And he said, "I've talked about it so much. I'm kind of getting tired of, I want something new to talk about."

But that's been fantastic. And yes, absolutely, I think that that's something that we have to tell the people that are up and coming in the different areas of medicine about, that we need collaborations. I don't think you can work as a silo anymore, the way things are. And there's, we have areas of expertise, we kind of go, we have immunologists, we've got surgeons, we've got pathologists, we've got internal medicine people, we've got a variety of people that are all working together.

And we do have a brain tumor program here that is a mix of people from several of the different colleges and different areas within those colleges that all work together as a group. And as far as saying, yes, I think it's important to say yes, but I will tell you that I am trying to tell my new faculty to learn to say no as well.

Dr. Gerry Post: Absolutely. Both are important. Well, congratulations, that's wonderful that the shining example of that one health concept  because I agree, it's incredibly important. And one health concept, as you know, FidoCure is a brand of the One Health company. And so we fully support that.

In terms of the brain cancer, I get how a surgeon can go from cutting, with a neurologist to brain cancer research. Now talk to me about how a surgeon gets involved with the immune system. Because that's just fascinating to me.

And I think, you're certainly not the only one, but I think it's just a fascinating story of how a surgeon can get involved with immunotherapy. And I think it really illustrates the value of the veterinary education as a whole is that, I remember in Minnesota, one of my professors, years ago, was talking about, we know multiple species, multiple systems better than anybody. It's lovely to see that our core education can be utilized across such a breadth of fields, and so I'd love to hear your story of how a surgeon gets involved with immunotherapy.

Dr. Liz Pluhar:  Yeah, so I mean I am by no means an expert immunologist but it is interesting because when I was doing my PhD at Wisconsin, I did take some advanced immunology courses, but that was a few years ago, to say the least. And I actually TA'd in some of those courses but we've learned so much and there are so many new immune cell types that we know about. I mean, we didn't even know about suppressor cells then, so the depth of knowledge and the breadth of knowledge within immunology has grown so much since I did my PhD that I had to do a lot of reading and talk with immunologists about what's going on. And it's been a lot of work to try to keep up and understand what's going on in the area of immunology.

But certainly a lot of it is working with these people that I've been working with. Initially, it was John Ohlfest who was my first research partner, and unfortunately he passed away from metastatic melanoma a little over eight and a half years ago now. And one of his post docs named Mike Olin has kind of stepped into John's place and has taken over and he's the person that I work with the most now. He actually has an immunology background from the University of Minnesota from the vet school. So he and I work closely together and he's very patient with me, and when I forget something he'll explain things to me, but it's been... John's dream was to try to find an alternative therapy to the current standard of care for brain tumor patients. And so the standard of care is surgical removal as much as possible, followed by a combination of chemotherapy with temozolomide and radiation therapy followed by Cipla temozolomide therapy. And there are obvious toxicities when you're dealing with radiation and chemotherapy, and when you put them together it can be pretty devastating.

One of my very best friends was diagnosed with a glioblastoma a couple of years ago and hers recurred within four months of the initial surgery, and she had a second surgery and was never quite the same after that. But John was trying to figure a way out to avoid having to use these, what he called atom bombs, something that attacks the entire body, chemotherapy and radiation therapy. And he really felt that the way to go was to try to stimulate our body's immune system to recognize these tumors and then kill any residual tumor cells to try to prolong the disease progression-free time and the overall survival time in these patients.

And so everything that we did together was trying to avoid using radiation and chemotherapy and strictly using some sort of an immunotherapy approach. And he was very invested in using autologous tumor lysate state vaccines, and so that was kind of our initial foray into immunotherapy, and it started with some murine work that he was doing that showed, like it does in murine models, really great success. And one of the things that he did was combine vaccine immunotherapy, along with adenoviral-mediated interferon-gamma gene therapy, and in the murine model that combination of therapy was extraordinary. It was so much better than the vaccines by themselves.

So we did a canine study that was funded by the American Cancer Society where we looked at dogs treated with the vaccines alone or with our [inaudible] cerebral interferon-gamma gene therapy. And the interesting thing was that we did not see any effect of the gene therapy in the dogs. So what was seen in the mice did not translate to the dogs and I think that's such a great example of why I really feel pretty strongly that these dogs with spontaneous brain tumors are a much more translational model for people with spontaneous brain tumors.

And I think that the numbers... It's like there's 11% of the new therapies that are developed that can cure a mouse of a brain tumor and prevent recurrence, ever work in people. So that's pretty low numbers and considering the expense that it takes to get through a Phase I/II clinical trial, we definitely needed a better model. So the fact that we have these dogs that get these spontaneous tumors and they appear to be a better model, I think, is fantastic. I think that... I kind of just lost my train of thought there a little bit but so, yeah.

Dr. Gerry Post: I think you're certainly preaching to the choir, but absolutely I think all of us, I think most veterinarian oncologists, really are comparative oncologists and really do believe incredibly strongly in the power of veterinarian oncology and translational medicine. And again, super impressed and I think you're such a great shining example to all of the people in our audience, whether they're in academia or in private practice, again, about how we can collaborate, how we can learn new things while still utilizing the basic knowledge that we have and how valuable that knowledge of being a veterinarian is. So seeing what you have accomplished, what a great thing to aspire for the young veterinarians in our audience.

Speaking about accomplishments, in 2015 you were one of the organizers and participants, and really created the NCI Comparative Brain Tumor Consortium. Can you tell us why this group was formed and what has resulted from this group that you helped bring together?

Dr. Liz Pluhar: Yeah, so that group was formed to try to bring together people from both sides of the leash, so from the human medicine and veterinary medicine and bring them together to try to figure out ways of making dogs more translational and trying to figure out the similarities and the differences between canine and human brain tumors. And the first big push with that was to get a group of veterinary pathologists and human neuropathologists together to look at canine brain tumors and try to figure out how we were going to classify the different tumors that we were seeing in dogs, and see how comparative they were based on histology alone to the human tumors.

So that was the first big publication that came out was in that realm of the pathology of the canine brain tumors. There's an immunotherapy group, there's a surgery group, so there's all these different groups within that canine brain tumor consortium that are working together towards trying to promote dogs with brain tumors to represent and to help to find new cures that are going to help both our canine patients and human patients as well.

Dr. Gerry Post: Fantastic. And getting into the specifics of that, you've done some really groundbreaking work with vaccine technologies and and brain tumors. Can you tell us about some of that research?

Dr. Liz Pluhar: Sure, sure. So, as I said, that that was John Ohlfest's baby, trying to utilize the body's own immune system, stimulate the body's own immune system. And it was kind of funny because I think he really thought that this was going to work really, really, really well and it worked okay. I mean, definitely I think that if you do surgery alone or palliative care, the prognosis for dogs with high grade gliomas is about the same. It's about two months. And we were getting about a seven or eight month survival times in dogs with high grade gliomas.

And one of the things that I think has been really important for us is that we get a definitive diagnosis for every dog we treat. We know what we're treating. And that was one of the things I found interesting when I read through the veterinary literature on canine brain tumors is that it was pretty unusual to have a diagnosis prior to treating with radiation therapy, which was the primary modality that was being used. And so I really feel it's important to make sure that we always know what we're treating. And we started out doing frozen sections but that was kind of very time-consuming and we didn't always get a good answer because we didn't have the logistics down really well.

But we still do get a diagnosis and if a diagnosis comes back with something other than a high grade glioma, we still will treat those dogs with whatever's the most appropriate treatment. If there's a low grade glioma, then we will continue with the vaccine therapy or whatever therapy we're doing at that time. And if it's something else, because we do see dogs with something like GME, the pre-op MRI looks very much like a glioma but it'll come back as GME. We've had infarcts, we've had a variety of different diagnoses and so we'll treat whatever is most appropriate for that final diagnosis.

So we didn't hit a home run and we were kind of disappointed about that, so we started looking at different things to add in along with the vaccine immunotherapy to improve our immune response. But one of the interesting things we learned in that first study from the American Cancer Society with the interferon-gamma was that dogs... We did have a couple of dogs that had low grade gliomas that were included in that trial because it didn't specify that it had to be high grade, whereas some of the other trials did. and it turns out that dogs with low grade gliomas do really well with vaccine immunotherapy. I think the median survival time for those dogs is well over 900 days, so it's quite amazing.

And then the other serendipitous thing that happened was we had a couple of dogs very early on that the biopsy came back as anaplastic meningioma.

Dr. Gerry Post:  Oh wow.

Dr. Liz Pluhar:  And those dogs lived their lives without having any recurrence after a series of six vaccines starting two weeks after surgery. So three months after surgery, we stopped the vaccines and these two dogs with anaplastic meningiomas didn't have a recurrence. So we said, "Huh, maybe we should try using this surgery and immunotherapy instead of radiation for meningiomas," so we did do a trial with that. And so that worked so for low grade gliomas and meningiomas, even anaplastic meningiomas, grade three meningiomas, the vaccines worked really, really well along with surgery. And of course the extent of resection makes a difference so we want to try to get as much out, because I think that there's a limit to how much tumor the immune system is capable of controlling and so we want to try to minimize disease and that's true for any of the tumor types.

But we tried a lot of different things, different types of immunostimulants. We used OX40, we tried adding in a little bit of chemotherapy with temozolomide. We did the interferon-gamma. We did a combination gene therapy with thymidine kinase and Flt3 ligand, and nothing really helped. I mean, we just seemed to get stuck at that seven to eight, maybe nine month median survival time.

And then Mike Olin, who took over John's position kind of after John passed away, was looking at different immunosuppressors that were in the human patients and he found this one called CD200. It's a immunosuppressive protein called CD200 and it's basically a checkpoint inhibitor or an immune checkpoint. And he's done a lot of research on that and what he found was that there are certain peptides of that native protein that either act as an agonist or an antagonist. And we have found an antagonist peptide that we produced and we're using that with our vaccine therapy, and we doubled our median survival time just by adding in this peptide, this checkpoint inhibitor. And so that's been the biggest advance that we've seen, and we have been able to translate both the vaccines alone, and now the vaccines with the checkpoint inhibitor, a human specific checkpoint inhibitor, into human clinical trials and have seen some success.

It's kind of like the dogs where some people respond really well, some people not so well, but then there's kind of that middle group. But we started this new clinical trial in January with our peptide, the CD200 peptide, and the human patients are all responding. We're actually seeing an immune response in their brains and in their blood in response to the vaccines plus the peptide that is much more robust than the vaccines alone.

Dr. Gerry Post: That is just a fantastic story. How wonderful to be able to see your work. not only work in dogs, whether it be meningiomas gliomas, but then see that work also work in people. How incredibly right.

Dr. Liz Pluhar: Yeah. It's been pretty amazing for me because I've met some of the human patients that have been treated, and one person stands out in particular for me, and this was a man who had had his third recurrence. Because for the human clinical trials, they don't treat [inaudible] patients usually when they're trying out these new therapies. It's really hard to get people to move beyond the current standard of care.

So we're treating these patients that have recurrent glioblastoma and sometimes it's the second or third or fourth recurrence. So this man was a third time loser, unfortunately, and he was enrolled in this clinical trial with just the vaccines alone, and he was one of our best responders. When I met him, he said, "I can't tell you how much I appreciate you and what you're doing in the dogs," because he said, "I haven't felt this good since before I was actually diagnosed with the tumor in the first place."

So it had been years, and he said, "I felt so terrible with the chemo and the radiation and I've had absolutely no side effects with any of the vaccines." And the wonderful, unfortunately he passed away, but the nice end of that story is that he and his wife were able to take a trip around the world while he was getting the immunotherapy, which was on his bucket list. He was just so thankful for all that time that he got, and the time was, it was really good quality of life for him as well.

And that's something that most of the owners of my canine patients tell me as well, that their quality of life is really good while they're getting the same immunotherapy because there's not some of those adverse effects we see with other therapies.

Dr. Gerry Post: How wonderful. I can tell you, you have changed my mind in terms of how strongly I would recommend brain biopsy. I think knowing, because again, I think a lot of people are very reluctant to recommend brain biopsy, but when you're talking about the difference of a couple of months versus over 900 days, if it's a certain type of cancer treated in a certain way, that definitely changes the equation for me.

Dr. Liz Pluhar: It does.

Dr. Gerry Post: And likely for our clients. So thank you. That's a really powerful statement.

Dr. Liz Pluhar: Yeah. I think it's been actually been published that if a third of the tumors that look like a glioma on an MRI turn out to be something other than that, and really that's what I have seen. It's almost one third of the cases. So if you're treating an infarct or a GME with radiation therapy, you might get a really good response, and when you talk about the responsive gliomas to radiation therapy, you're going to include that group, which is can be a third-year cases. So I really do think it's quite important to know, absolutely to know what you're treating, because the prognosis is quite different.

Dr. Gerry Post: So what are you most excited about? I mean, I'm not sure it gets any more exciting than what you just described, but looking to the future, what are you most excited about as it relates to comparative oncology?

Dr. Liz Pluhar: I think what I'm really excited about right now is to really see how this clinical trial in our human patients pans out, because adding temozolomide in with radiation therapy was the biggest advance in treating glioblastomas in history. It literally added two months to the median survival time, adding temozolomide in, and people were really, they're really excited when you can increase the median survival time by two or three months.

And if what we're seeing in the dogs translates well, if we're going from the vaccines themselves to vaccines, plus our peptide, we're doubling the survival time. We could be talking about years for these patients even with recurrent glioblastoma. So that's really exciting for me, I think.

And the other thing that we're doing is we're starting to look at combining different modalities as well. I don't think that any type of monotherapy is going to be what's going to ultimately cure any type of brain cancer. But I think that we're starting to look at combining certain types of radiation therapy with immunotherapy, trying to figure out timing to get these things to work together rather than against each other.

So we're working on writing a grant now to look at that. We're really exploring the CD200 peptide, because this checkpoint inhibitor, not only does it affect the CD200 checkpoint, but it also down-regulates CTLA four and the PDL1 access as well. It affects all of those checkpoint inhibitors. So that instead of having to use checkpoint inhibitors and getting that additive toxicity, we can just maybe use this one that will end up having an effect on all those different checkpoints. So that's kind of what is really exciting me for the future right now.

Dr. Gerry Post:  Fantastic. And you and I spoke yet yesterday or the day before about not only the value of CD200 in terms of utilizing it as a checkpoint inhibitor, but also aiding in diagnosis. So I'm wondering if you could just tell the audience how CD200 can be used in potentially diagnosis of recurrence.

Dr. Liz Pluhar: One of the things that we've realized is that the CD200 protein is actually shed by the tumors. And so it goes into, it drains out of the brain, and it gets into the systemic circulation. And so we can measure CD200 levels in the blood of our patients. And what we have found is that when a person is responding to the therapy, their CD200 levels will decrease, CRMCD200 levels. They tend to start going up again when there's a recurrence.

So what we've seen is they're seeing a recurrence on the MRI correlates to there's the increase in the CD200 levels. And interestingly, we've been doing the same thing in dogs, and one of my really long-term survivor cases, dogs in the gene therapy, or no, sorry, the vaccine plus a CD200 peptide pilot study we did. Was a little dog, a little Boston terrier from Florida. And we collected Marley's blood samples, but we hadn't run the CD200 levels on them. And we were also monitoring Marley, and he had no recurrence after a year of his tumor.

So we told the owner we're going to continue to give him treatment, but he's no longer in, this trial ended at a year, but we want to follow him and we're going to continue to treat. And about five or six months later, he started to have some clinical signs that were consistent with maybe starting to have a recurrence. So we repeated the MRI, sure enough he had a recurrence, and so we actually did a second surgery and then treated him again with the vaccines plus the checkpoint inhibitor.

But when we finally ran the CD200 levels on Marley's blood, what we found was that at that one-year timeframe, when the MRI was completely negative, and I went back out like, there's got to be some tumor on here that I'm missing. His CD200 level went from almost zero to, it was one of the highest levels I'd seen. Then it stayed up. It actually went up a little bit more at that six months later when we did note that recurrence.

So we really feel that that CD200 may be a biomarker that we can follow in these dogs. So if their CD200 level really climbs, we should maybe start doing repeat MRIs a little more frequently to try to get that recurrence as soon as we can, because definitely a prognosis, it's much better if you can get the tumor when it's small and, and get a better excision.

Dr. Gerry Post:     Fantastic, your story about utilizing multiple therapies in combination to me is truly one of the values and strengths of comparative oncology. Utilizing veterinary oncology, spontaneous cancer models, because we do have the ability to utilize combination therapy as frontline. Utilizing these combination therapies when they're most biologically appropriate, rather than waiting for all of the quote unquote standard of cares to fail.

I love being a veterinary oncologist, but understanding why, and the value that we can bring not only to our patients, but to the wider biomedical community, I think it's just awesome. And I think your stories and your journey have really displayed and shown the value of comparative oncology, and also the value of working with not only oncologists, but surgeons and neurologists as well. How fantastic.

At this point, I'd like to encourage any of our participants to ask any questions if they have any. My last question to you is, is there anything on the      immunoncology front that you are, in the human realm, that you're excited to bring back to the dog?

Dr. Liz Pluhar:  One of the exciting things that's going on in the human realm is the use of 5-ALA along with photodynamic therapy. So using that and then using light to stimulate the 5-ALA to kill cancer cells that preferentially take up that compound. The problem with brain tumors is that the light doesn't have a very, it doesn't penetrate through the tissue very deeply, but ultrasound does.

So one of the things that's pretty exciting that somebody, a group that I'm working with has developed a ultrasound device that they place on the skull, on the brain, on the head, that delivers Sono therapy, so ultrasound therapy that can penetrate centimeters. Since measuring from one side of the dog's brain to the other is five centimeters, we can treat the entire brain with this therapy. So we're getting ready to start a new clinical trial where we're going to be treating with 5-ALA and this Sono dynamic therapy by putting this little helmet on the dogs and treating them, adjusting the amount of ultrasound based on some work that they've done previously, and then seeing how well that works.

I find that to be very exciting, and I think that, we could do that. We can add an immuno, maybe we can add in vaccines with that, and just kind of hit those tumors from as many directions as we can. Radiation therapy may play a role in there as well. So I think that trying to be able to combine a lot of different therapies that are working in different ways is probably going to be the way to go in the future.

Dr. Gerry Post: Fantastic. When you told me about sono-dynamic therapy, that is just fantastic.

Dr. Liz Pluhar:  Yeah, it's exciting.

Dr. Gerry Post:  So you talked about diagnosis and treatment. Is there any insight relating to prevention or brain cancer in dogs and people?

Dr. Liz Pluhar:  Oh, it's a great question. I think that if we knew what caused brain cancer, we might win the Nobel Prize. But I actually had, I worked with Matthew Brene. We were trying to get funding to look at early MRIs in dogs, in those breeds that are prone to developing gliomas and starting to do early MRIs to see whether or not that would be helpful in trying to diagnose the tumors when they're small. The primary presenting sign with dogs with brain tumors are seizures. The tumors have to get pretty large before they start causing seizures, and only about 20% of human patients with brain tumors have seizures, with gliomas, have seizures on diagnosis because they're able to tell ... people can explain that they've got these subtle signs that the dogs can't.

We did a little study looking at the volume of brain tumors in dogs versus the total brain volume versus people, the volume of their brain tumors versus total brain volume, and the dog's tumors were, volume-wise, much, much larger per volume of brain. So I think these tumors, we're getting these tumors when they're really big and really advanced, and although sometimes they don't look invasive on the MRIs, we know that these are very invasive tumors. And even if we remote, get what we consider patting our backs because we've got a gross total resection on our post-op MRI, we know we've left something behind, and that's why the dogs that had surgery alone had recurrence in two months.

But yeah, so I think that's the only thing I can think of for prevention, is maybe early surveillance. If the CD200 biomarker works out, then maybe setting up a biomarker test with just looking at serum levels of CD200. And I think we definitely would want to look at a normal cohort to see what that would be in normal dogs to make sure that that is something that is definitely associated with the development of glioma rather than some other disease process, because it is kind of a ubiquitous protein.

Dr. Gerry Post: Absolutely. And given that brain tumors do have a breed specificity, has anybody or do you of anybody that's looking at the genetic predisposition that may play a part in brain tumor formation and thereby ultimately may play a part in cancer prevention?

Dr. Liz Pluhar:  Yes, I mean, there have been some studies that have tried to look at that and they haven't really found anything specific, but with the advances in genomics that are going on right now, I think that there's still that possibility that there may be something that's more subtle, that there may be some differences within those specific breeds that we just haven't recognized thus far that may help with that. Certainly, if we can figure out why, then we're going to be able to be better at preventing. But one of the things that I've recognized in my research is that, we believe that French bulldogs may be a little bit different than the other brachycephalic breeds.

Dr. Gerry Post:  Millie's listening very carefully right now.

Dr. Liz Pluhar:  When we first started the CD200 peptide plus vaccine pilot study, the first four dogs that we operated on had a very short survival time. I went, called Michael and I said, "Mike, we got to stop this trial because this is making the dogs die faster, not ... I mean, they're doing worse than with the vaccines alone." And he said, "How can that be? How can that be?" And I said, "I don't know, but it is." And then we actually, we kept going. He said, "Let's keep going for a while." And it turned out that three of the first four dogs were French bulldogs. So we're like, "Oh," and then I looked at other French bulldogs that I treated in previous clinical trials, and their median survival time was dismal compared to any, to boxers or Boston terriers or any of the other types of bulldogs.

Now, we have that as an exclusion criteria. We are not enrolling French bulldogs in the immunotherapy trials. For the 5-ALA trial, when that starts, we will take Frenchies, but when we're specifically stimulating the immune system ... we don't know why, and I actually have a PhD student that that is her thesis, to try to figure out if and why French bulldogs are different.

When we're talking about numbers for anything that we do in veterinary medicine, we say an N of 10 and we get really excited. We're talking about hundreds or thousands of human patients. It's only been, I think, 13 Frenchies in total, but the median survival time is 48 days for those guys versus months for everybody else, so I really believe that it's real and we're excited about looking into that to try to see if and why Frenchies are different. I know there's some information that they may function under more anoxic conditions than some of the other brachycephalic breeds, and definitely with HIF-1 and that's really important, I think. So we may be on to something there, but I think that's kind of exciting.

Dr. Gerry Post: Well, fantastic. As much as I adore you and your work, I hope myself and Millie never have to come see you.

Dr. Liz Pluhar:  I hope so too. You don't want to see me. Not as a patient, anyway.

Dr. Gerry Post:  Absolutely. Socially, absolutely. One additional question. Sounds like many exciting new developments and treatments. What are the prospects of commercializing the treatments that you work on, but will it have to be proven to work for humans first for anybody to take it to market?

Dr. Liz Pluhar: We're actually in the process of trying to do that right now. We're working with some people on trying to do that. And so I think that the peptide, we're really excited about the peptide because what we've found is that the peptide sequences for the peptides are a little bit different. The homology is not, it's not 100% homology between most human or canine. So we're doing really specific testing within each species to make sure that we're using the peptide that has the optimal activity. And so the peptide that OX2 Therapeutics, which is the company that Dr. Olin started to market the human peptide, checkpoint inhibitor peptide, that's a little bit different than the peptide that we're using in dogs. And so there are no canine specific checkpoint inhibitors that are available right now.

And this is kind of unique, because it is a peptide, it's not a monoclonal antibody, which all the other checkpoint inhibitors are. So this is able to cross the blood brain barrier, and we're not sure that it's necessary, because we're actually injecting it peripherally right now. And it still seems to be ... it's working quite well.

We are going to be looking at doing interest cerebral injections of the peptide as well down the road here to see whether or not that helps to stimulate, to suppress the immunosuppression or reverse the immunosuppression that's there in the brain in the tumor micro environment. But yeah, we are starting to look at getting this marketed, because I think that would be a tool that would be useful in several different types, or any type of cancer, just like they're using the checkpoint inhibitors in human patients for almost any type of cancer. And there are some types of cancer where it's being very effective, and others where it's not, and unfortunately, brain tumors, they don't work very well against any of the brain tumors.

Dr. Gerry Post: Fantastic. Just looking at the time, we have I think time for one last question. And this question I would say is really interesting. So with the new administration, there seems to be more likely that they're interested in cancer. Is there an expectation for more government funding for this research in dogs and comparative oncology in general? And are there any indications of that happening already?

Dr. Liz Pluhar: Absolutely. Yes. I agree. I mean, I have an NIH grant through the National Cancer Institute to do the immunotherapy trial that we're doing. And I was one of five researchers that received this funding, and it's millions of dollars that was being given to people that were specifically looking at canine immunotherapy for cancer, for canine cancer. And it had to have a translational aspect to it, but still, I mean, millions of dollars for a canine study is amazing. I mean, just amazing to have. And I think that that's being recognized more and more and for more and more types of cancer, if there's that translational part to it. And there were so many of the cancers that dogs get are similar or identical to what people get, or there may be variations like angio sarcomas and hemangiosarcomas in dogs.

So yes, and absolutely. I think that my funding is through the Cancer Moonshot that came to into play when Joe Biden was Vice President. And so now as President, I think that he is still very invested in promoting this. And I'm hopeful that they will continue to provide funding for these types of studies that have this translational potential.

And the great thing is that even though we may be looking to try to find something that is going to help people, human patients, our canine patients definitely are benefiting from this as well. And it gives people hope to be able to have something on the horizon for their own pets. And I found it pretty amazing how many people that I talked to or whose pets I enroll, dogs I enroll in the clinical trial either have a friend or a family member that has been diagnosed with the same type of cancer. And so they're not just invested because they want to help their dog. They really want that translational part as well. They want to be a part of that, and it's been amazing to see that, how people are invested in that.

Dr. Gerry Post: Yeah, I think over the last year and a half with unfortunately the COVID situation, it really has highlighted how important pets are to people. I think now over 90% of people really believe and think are their pets as members of their family. And so what you're seeing in terms of the research helps not only the veterinary market or the human market, but really increases the understanding of that interrelationship, that familial connection that you have with your pet. Absolutely. It's a really lovely circle.

We'll be sharing some similar information about the mutational landscape in a paper that we'll be publishing shortly about really the incredible similarity between the mutational hotspot landscapes in dogs across many tumor types and the mutational landscapes in people. So I think really validation of just what we've been talking about for the last hour, how applicable translational oncology is between dog and people. [crosstalk] time?

Christina Lopes:  It was so wonderful to spend this time with you, Dr. Pluhar. I hope we get to collaborate as we are working also in our pipeline to have more sophisticated diagnostics and therapeutics in the immuno-oncology space. We recognize how important that is, and already have mindfulness and some thinking there.

And it's a real honor to be able to keep the dialogue going and the connection going, even hopefully at the tail end of COVID in some places, but still connecting here together as a community. Echoing also what Gerry said, which is we have a manuscript coming out. It's the largest study in the world, the deepest study in the world, looking at the genomic landscape in cancer, comparing dogs and humans.

And I'll tell you, there are some proteins that are 100% homologous, 100%. I mean, this is wild. And what it unlocks is there's so much information from the human world and a positive data in dogs. However, if there's a similarity, then this just becomes, right, there was like a little crack of the door, the door just swung open. The possibilities are immense, and the dog as patient, I think unlocks a lot also, right? So not as model, but as really a patient, just there's so much for us to do.

So, thank you for your amazing contribution and so exciting. And I cannot wait to see how this goes and hear the updates.