So as all of us who are studying ketogenic diets and cancer do well, I’ll show you in a second. Our study from a few years ago is colloquially called the recharge trial. Because it was a very stupid acronym, but I won’t tell you what it was. So this is, you know, from recharge the novel biomarkers. And hope to just update you on some of the stuff we’re doing now, collaborators are many and I’m indebted to all of them wonderful people. Richard Feynman has been my colleague and collaborator for many years this year. So we all aim to target the Warburg effect and I can call and described it very well. And this is the glucose and the glycolytic dependent phenotype. It’s common to many aggressive cancers, and it’s evidenced by positive PET scan with 18 fluorine labeled FDG, FDG is fluorodeoxyglucose, you can hear the glucose is in the molecule, right? Do we have any pointers here? No, okay.Anyway, I think you can see what’s going on though, you can see the heart ’cause this is a rather fuzzy but still valuable PET scan that we obtained a long time ago, I still like it. And when a person is on a standard, rather high carbohydrate diet, well, insulin is secreted and drives glucose into the heart. So fluorodeoxyglucose is clearly visible in the heart. You can see the tip of the brain there, the liver is less well seen. Now, the lowest portion of the liver, and on the other side, you can see the kidney collecting systems because this is radioactive urine and thanks. All right, so there’s the collecting system of the urine and bladder. So the fluorodeoxyglucose is excreted into the urine. So you get that, but what you don’t really want to see, but you do, all these multiple sites within the liver, which are glucose added metastasis, and there’s also another one in the lungs. So this is a patient with the metastatic colon cancer. In 2012, somehow we’re all stuck in 2012 due to funding problems.But this was this was our study published at that time as again, as I say, called the recharge trial. And it was a pilot study in 10 patients, oops! You give me two clickers and I get confused. Alright, so the objectives were safety and feasibility of a 28 day ketogenic diet in 10 patients with advanced PET positive cancer. We use the entry and exit PET scan as a surrogate marker of efficacy. We’re looking at the change in the FDG uptake between the two. And the patients were monitored for compliance and the systemic metabolic effect by getting a weekly fasting beta-hydroxybutyrate level. I’m not gon na go over this in any detail, more information that I want. But you can see that well, first of all the patients were not selected as you can see by the tissue type, all right? Because there are a whole bunch of different, there’s breast cancer, ovarian, so forth, a lot of addition. The patients were selected by the metabolic phenotype by the fact that they had a positive PET scan using fluorodeoxyglucose. That was the selection of the patients.The circled patient here was excluded from analysis. We weren’t thinking, but you see that she had a biologically different tumor. She was surviving for 14 years, she had had no surgery on her breast cancer, she had no chemo, or no radiation therapy and was still alive. These other patients had multiple chemos failing. So she, we ended up having to exclude her from analysis. She was a biologically different patient. In any event, actually all the patients experienced a certain amount of calorie deficit. This was not by attention, they did not on average, they ate about 35% less calories. Five patients you can see in the blue had a stable disease or partial remission and forehead progressive disease, but there was no relationship between the calorie deficit that was the same in both groups. They also lost about 4% of weight, but again, it was about the same in both groups. What did matter was the level of ketosis between the groups, and you can see that the level of ketosis in the stable disease partial remission group is about three times higher than in progressive disease.These are all pretty consistent. I mean, even the lowest one is around 10 fold over baseline. These three of course, are below of, you know, four fold in lower baseline. This one, of course, looks like it, this patient should have responded but that’s actually part of the problem with cancer. Not everyone’s going to respond, then we’ll get to that in a little bit. Still, I thought was pretty promising overall. And the summary is that this was a prospectively designed and executed pilot study of ketogenic diet as a cancer therapy. 10 patients completed it without unsafe adverse effects. And ketosis was observed that correlated with the level of insulin inhibition. I didn’t show you that slide as you ‘d expect, and it also correlated with disease stability, which is consistent with the hypotheses affects on glucose dependent cancers.So new work, which is what I really came to talk about is we now are in the process of just starting a human trial of 65 patients with just diagnosed ER-positive breast cancer. Patients are randomized to two groups, one of ketogenic diet of 45 patients, and then versus a standard low fat diet of 20 patients total of 65. The ketogenic diet is supplied by Natural Ketosis, which is a company in UK these are vacuum sealed meals, we get them in boxes of 28 day supply, so they have long shelf life, and we can ship it out to the patient right after their diagnosis to start. The standard diet is high in carbohydrate foods, which is supplied from a list we called from a Fresh Direct menu and patients selected so they can come up to a high carbohydrate diet on that arm. And our question is do biomarkers of cell proliferation, this biomarker, KI 67 or Key 67, I guess, depending on where you’re from, versus cell death, which is a tunnel acid whether these biomarkers change between biopsy and surgery, which is a period of usually around three weeks between two and four depending on how long it takes to get the patient schedule.Now, biomarker study isn’t exactly a therapy trial, but it’s clearly related to one because it provides information about the likely behavior of a therapy trial, right? And question you might reasonably ask is, is three weeks or two to four weeks enough time to see a difference in a biomarker? And Goodwin and Dowling, I mentioned Goodwin before from Canada studied Metformin in a very comparable study. They did it a few times until they got up to about 500 subjects and during the same time interval. And what they found was that there was a significant drop in KI 67 in the Metformin treated patients, but not in the control group.And I think everyone in this room will agree that Metformin, you know, while we don’t really know quite how it works, we know it drops blood glucose and thereby drops insulin. But we also know that a ketogenic diet is quite a bit better at doing that. And so if this work for Metformin, we at least have some reason to believe that we may be successful in showing a change in a three week trial using our diet. So, you know, I came to show you a study of 65 patients and so far, we’ve managed to complete one patient. So, I don’t have a lot of data, but, it’s illustrative. So this is KI 67. This is the biomarker of proliferation. And you can see these brown stains in a lot of the cells, you can see a lot of other cells which don’t have that stain, they’re sort of grayish. And Susan Feinberg, a pathologist has the laborious task of counting the numbers of cells and determining the percentage, and you have to do this in four high powered fields. So it’s very laborious, but and then you get an average. So she found one of these high powered fields at 44%, another 72, 73 and 79%. That stain for KI 67, and this is on the biopsy. Now, the tumor excision specimen, this is, you know, three weeks later, you can see, it looks like it’s more, but I’m just, you know, I deliberately picked that ’cause I wanted you to see that it got worse, but it’s still she did all the counting. And it was, you know, a little bit more hard to say if this is statistically significant from four high powered fields slides, but it was 51%, 79, 80 and 81 that stained in this follow up, you know, biopsy, you know, from the from the actual tumor excision, so it certainly was no better. Now, when I actually try to press Susan for what’s the normal value of KI 67 and breast et cetera she waffled you know, but I’ll just say that by large, the value is probably less than 10%. So these are both very abnormal values.This is a patient who didn’t start off well and maybe got worse but certainly didn’t get better. This is the standard human toxin and a nuisance name the HNA and the central area is the tumor with normal tissue around it mostly, and this is three weeks later. So higher power field, but the important thing here is here you can see my mitotic figures, this is a nuclear materials separating and a mitosis and it’s only one slide, but it looks worse. And basically, there’s a lot of mitosis if you look at a lot of other fields. So in three weeks, the tumor appears to have gotten worse. Displaying mitotis figures, certainly not a good possibly higher KI 67 and also apoptosis relating actually to tumor growth, not to an improvement.And yet patient one was randomized to the ketogenic diet. So this isn’t very good, right? This is not what we were expecting anyway, but you know, we have to take the data as it is, however, it turns out, the patient was not in ketosis. The baseline shows ketone body, sort of at the upper limits of normal first week, if you want to call that marginal, you know, borderline physiologic ketosis you can but you can see that really nothing happened. And the problem was our fault it wasn’t hers, she was supposed to be on a ketogenic diet, but turns out I don’t know what happened. But when we gave her the list from Fresh Direct, we included some foods which were not actually ketotic, and she jumped at them, and then she ate them. And so entirely our fault. I forget, someone once told me that whenever you do a clinical trial, you have to expect that you’re gon na screw up the first two patients. Well, we’ve done it on the first one.But anyway, we’re still on the second we’re about, so far, we have patients, the second patient is two thirds of the way through the diet. So we’re pretty close. But in any event, the other thing, so I just wanted to give you a flavor of what we’re doing now, in terms of the human trial, we’re excited to be doing it. We’re hoping to get more patients and to start doing it correctly, and that’ll be good. And the other things we’re doing is we’re trying to develop some novel biomarkers. KI 67 is not a novel biomarker, but it’s a cell proliferation.It’s been around, but it’s not used routinely in clinical practice, it’s too cumbersome to use and the standardization is difficult. So it’s not really truly standardized, as I say, not part of routine evaluation. And our team is working on developing a standardized method from large statistical samples, basically, through automatic cell recognition, or cell properties, counting these stains in a computerized fashion, and being able to get a large representative number. So we’re very excited about possibly bringing this to the clinic. Probably the most exciting thing that we’re also doing is another novel biomarker. And Stephanie Mattingly is a colleague of ours who’s working at University of Alberta in Canada and she’s after three years of very hard work she managed to actually label ketone body with 18 Fluorine. So this could be very exciting. We actually don’t know what the use of 18 label Fluorine ketone body will do. But it is the third major radio label metabolic tracer after FDG and F-18 label glutamine and it could be for potential widespread use. Here is an example of an image that she obtained and where it’s going is just where you ‘d expect. So you see some in the brain, okay, the eyes always light up, the bone, and the marrow lights up very typically.But here’s a tumor. This is actually an MCF-7 tumor, which is an ER positive tumor. One that we’re studying as well animal models, I’ll tell you in a minute. This is a bone uptake, not as intense, but we’re gon na be playing with that to determine how to manipulate the images and not the images, but the actual physiology, just the black and white version of the same. From the side this is not optimally shown for imaging. This is the sternum in front but the heart is behind. So the heart also takes up. So this is just where you ‘d expect beta hydroxybutyrate to go. It goes to heart, goes to brain and it goes to tumor. So we believe this is the tracer, we believe this is actually an accurate analog beta hydroxybutyrate. So to be explored with this tracer, our first, first thing you have to do is find out normative image findings under different dietary conditions. ‘Cause you have to know actually how it behaves. And we’re in the process of doing that.And because it goes to these areas, it goes to the brain, it goes to the heart and goes to cancers, its distribution is similar to fluorodeoxyglucose but perhaps it will be complimentary in terms of the information for specific applications. We don’t know yet until we try it out and it may permit exploration of individual mechanisms of ketone bodies and cancer therapies and perhaps identification in advance of susceptible patients.And the final thing I ‘d like to discuss briefly is the use of diet and drugs and mouse models. We’re studying particularly two different mouse models and her collaborator Yuzu CD1 nude mice MCF again, ER positive breast cancer xenograft in five mice per group and we also have a spontaneous breast cancer tumor model just to see what the difference is, you know both all animal models have limitations. So we’re gon na try both. And the idea is to couple diet and again rapamycin. Rapamycin as (mumbles) points out, well, he didn’t mention rapamycin, but you mentioned the pathways. Rapamycin is a drug as discovered on Easter Island actually, and it inhibits mTOR and I was described as some more than the most of us who know nothing about alphabet soup, but when I was growing up, there was something called alphabet soup. And that’s what all these signaling molecule sound like xmTOR and AKT and that sort of thing but anyway, mTOR is a growth molecule. It stimulates growth and rapamycin inhibits mTOR. So it’s been conceived of as an anti cancer drug or to be used as one.But sadly, like the PI3 kinase inhibitors that Dr. Cantley developed, it also causes counter regulatory effects and namely causes hypoglycemia. So we’ve been planning to do this for a number of years, or crushingly disappointed that Dr. Mukherjee and Dr. Cantley beat us to it, but we’re in the process of doing it now at least. And so we’re looking at a spontaneous tumor mouse model and CD1 nudes and comparing a standard diet standard diet plus rapamycin versus a ketogenic diet, and ketogenic diet plus rapamycin. And we’re looking at changes in tumor growth, cancer death markers, and overall survival. And we’re obviously interested in testing whether a ketogenic diets synergizes with drug therapy to increase overall efficacy at lower drug doses and lower toxicities. So that’s the basic idea. Just wanted to tell you very briefly about CD1. I stumbled on CD1 about 10 years ago, when I was doing a study for someone entirely other purpose.I picked it because I figured one day I’ll eventually be doing animal research in cancer. And I looked at regular CD1s and put them on a ketogenic diet, and I was stunned to find out that it is a mouse. It develops about 2 millimo ketosis on a ketogenic diet. That’s very unusual for a mouse. So this was terrific. I tried two different diet variants, 10% protein and 16% protein for reasons too long to discuss on the time that I have left, but basically, the amount of ketosis was the same at both. So we’re still working on which one we’re about to use. Animals have now been ordered. We hope to start the animal study to evaluate the dietary, you know, plus drug regimen I just described. And so that’ll be the fourth and final thing I discuss.Thank you very much for your attention. (audience clapping)

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