 All right, we're getting a little bit behind in our timeline. So let me go ahead and introduce David Fredricks. Dr. Fredricks is faculty member here at Fred Hutch, and also the University of Washington, a member of the Hutch School Professor of the University, and a member of the, let's call it the IBD, Vaccine Infectious Disease Division here at the Hutch, Infectious Disease Division at the University, and a long-term expert in studying the human microbiome, and as we touched on earlier, that's becoming a really hot area in terms of research, and the possible implications in overlap with immunotherapy drugs being applied to cancer. So I'm very excited to hear what Dr. Fredricks has to say for us this morning. Great. Thanks, Scott. It's a pleasure to talk about our fellow travelers and the human microbiome today with you. So first of all, what exactly is the microbiome? The microbiome is the collection of microbes and their associated genes that exist on and in the human body, forming in essence a second genome, extending the genetic and functional capabilities of human beings. Humans are really super organisms. They are a conglomeration of human and microbial genes. There are about as many microbial cells in your body as there are human cells. So you may think you're sitting alone in that chair, but you're actually sitting with 39 trillion microbes. There are about 19,000 genes in the human body. There are more than 3 million microbial genes in the human body. And those genes code for properties that we don't need to code for. For instance, in your gut, there are numerous enzymes that your bacteria make that help you to digest your food. Without those, we would lose many of those nutrients. Many of the microbes that exist in the human body remain uncultivated and are new to science. The other key point is that there's a different human-associated microbial community at every body site. So the gut microbiota is different than the microbiota that you find in the mouth, which is different than the microbiota that you find in the skin, which is different from the microbiota that you find in the genital tract. One of the areas that I study is the human vagina. And the vagina is unique in that it's one of the most acidic places in the human body outside of the stomach. It has a pH of around 3.5 to 4. And that leads to a different microbial community in the vagina, for instance, than what you have in the gut, where it's dominated by lactobacillus species, which in part produce acid that helps to drive down the pH and make it inhospitable for the other microorganisms to exist in that niche. So human beings have different microbial communities in each body site, but even within a given body site, different human beings are different. So this is data from the American Gut Project where people sent in stool samples and had the microbial community profiles characterized using broad-range 16S ribosomal RNA gene PCR with high throughput sequencing as a way of looking at the barcodes of the bacteria that are present, for instance, in stool. And what we find is that there are some subjects where we see that there's high abundance of this formicides phylum, which are a class of bacteria present in the gut. There's others that have low abundance in their formicides in red. Others with high abundance of the bacteroidities and others with low abundance of the bacteroidities. So different people have different abundances of these different bacteria in the same body site. And this is the same when you look at multiple body sites across humans. This has led to the notion that each individual may have their own personalized microbiome, which is a fascinating concept, because that also means these differences in mycobic communities may affect your health or your propensity to develop disease. This is just some data from eight subjects here at the Fredhouse Cancer Research Center where we looked at their stool samples and characterized their gut mycobic communities. Again, these are the different phyla bacteria that are present in these eight subjects. And again, what you can see across individuals is that each bar here represents a different species of bacteria that's present in their stool sample. Each bar represents a different bacterium that's present in the stool sample of that subject. And so for instance, this subject, for instance, you have these blue bars, a characteristic of lactosporaceae, whereas in this subject here it's missing that class of bacteria. So again, different subjects have different types of mycobic communities present. What are these microbes doing in the gut? Well, these microbes are taking things like fiber that's present in our diet and converting it to short chain fatty acids such as butyrate. And that butyrate is absorbed into the portal circulation, goes to the liver where it's converted into fats and sugars, which we use for energy. So in part, these microbes are releasing enzymes that help to break down fiber and other dietary elements into things that we can absorb and metabolize. This is a schematic showing what some of these bacteria are doing. And for instance, one bacterium, fecalibacterium prusnitsii, is a bacterium that digests the fiber in our diet and converts it to butyrate. Butyrate is a short chain fatty acid that's used by the intestinal epithelial cells as a primary energy source. And it's essential for the function of those epithelial cells and helps to repair epithelial cells when they're injured, for instance, after colitis. That butyrate not only helps to support the function of these intestinal epithelial cells, but it diffuses into the deep mucosa of the intestine where it interacts with immune cells such as T regulatory cells. And this butyrate helps to promote an anti-inflammatory environment by promoting the replication and function of T regulatory cells. So these bacteria are producing a compound which is affecting the immune system of the host and inflammation. If you're not consuming a high-fiber diet and you're missing some of these bacteria such as fecalibacterium prusnitsii, what happens is the bacteria begin to eat the mucous layer that surrounds the intestine. They then degrade that layer and then begin invading into the intestinal mucosa where they stimulate toluic receptors and other immune receptors that cause inflammation. And the absence of this short chain fatty acid butyrate leads to a loss of the down-regulating immune response from the T regulatory cells, again promoting an inflammatory response. So this is why your mother has told you to eat your fruits and vegetables, to have some of that fiber in your diet that helps to feed the microbiota that's in your gut that provides this health benefit to you. Now what happens when instead of eating your high-fiber diet you eat a diet that's high in meat and milk products? Well, it turns out that the microbes in your gut can also produce compounds that are noxious for your health. And for instance, some microbes that exist in your colon will take the carnitine in red meat or the choline in milk and cheese and convert it into a compound called trimethylamine. The trimethylamine is absorbed into your bloodstream. It goes to the liver where it's converted into another chemical called trimethylamine oxide. It turns out that trimethylamine oxide inhibits reverse cholesterol transport, causes aggregation of platelets, and leads to an increase in cardiovascular disease, including heart attacks, stroke, atherosclerosis. Interestingly, trimethylamine also has effects on the kidney where it can lead to fibrosis and injury to the kidney. And when people have chronic kidney disease with renal insufficiency, that in turn leads to higher levels of trimethylamine oxide, which then further aggravate this risk towards cardiovascular disease. So again, showing microbes not only help promote health, but also can contribute to diseases such as cardiovascular disease and atherosclerosis. And in fact, there's a lot of interest in trying to inhibit some of these enzymes in microbes that produce trimethylamine oxide as a way of reducing risk of cardiovascular disease. So why study the microbiome? There are many compelling reasons. The first is to identify microbes that are associated with health. For instance, how do microbes contribute to normal human physiology, immunity, and develop? Second, to identify microbes associated with disease. For instance, how do changes in the human microbiota contribute either directly or indirectly to disease or increase risk for disease, such as cancer? We normally think about pathogens as being a specific microbe like mycobacterium tuberculosis that's associated with TB. But we need to move beyond that simplistic concept and consider the possibility that disease may be produced by microbial communities, not just by individual microbes, working in concert, for instance, to produce a pathological state. And this leads to the notion of a dysbiosis or an alteration in microbial communities associated with disease. The other reason to study the human microbiota is that it's changeable. And we can change the microbiota by giving probiotics, which are live microbes administered to humans in order to change the colonization state, prebiotics, which are nutrients used to promote the growth of specific microbes within the human body, symbiotics, which are a combination of prebiotics and probiotics, where the probiotic uses the prebiotic in order to grow. We can use antibiotics to manipulate the microbiota and eliminate pathogens, and then we can even use fecal microbiota transplants, where we take stool from a healthy person and give it to somebody who's unhealthy to try and restore the normal microbiota diversity that's present in the colon. So how does the gut microbiota impact our health? We depend on our gut microbes to produce vitamin K. This is an essential nutrient that's essential for normal blood clotting. We don't make it. Our gut microbes do. The microbiota is essential for the normal development of both innate and adaptive immunity in the human body. Mice that are raised under germ-free conditions have completely abnormal immune systems. The microbiota plays a critical role in the function of gut epithelial cells, such as by producing butyrate. And even the microbiota plays a role in the metabolism of drugs that we take into our body. It is possible to raise mice under germ-free conditions, where they're born by cesarean section, then raised in isolator cages where all of the food and water that goes into those cages is radiated and free of microbes. And when mice are raised under those conditions, what we find is that they require 30% more nutrients in order to maintain a normal body weight compared to mice raised under normal conditions. And what this shows is that the gut microbiota is actually essential for extracting nutrients from our body. The microbiota plays a critical role in organ size development in intestine. These mice raised under germ-free conditions have massive hypertrophy of the intestines and is quite abnormal. Again, showing that the microbes are playing a key role in maintaining a healthy environment. And even mice that are raised under germ-free conditions have abnormal behavior, where they engage in more searching and locomotor activity when raised germ-free. So this is a slide that shows the microbiota of one subject over time. And on the x-axis is days after hematopoietic cell transplantation. And on the y-axis we see the relative abundance of different bacterial species shown here in this legend on the right. And what this graph is meant to display is how dynamic the gut microbiota can be in response to antibiotics. So this is a patient who came to transplant with a very abnormal microbiota because he had clostridium difficile colitis and had a microbiota dominated by streptococcal species and then was put on levofloxin, which is an antibiotic that we give to some of our cancer patients who are neutropenic in order to prevent them from developing infections. And under the pressure of this antibiotic we see a bloom in enterococcal species in this subject under that antibiotic pressure. And then look what happens at this time point right here when the levofloxin was stopped. We see this bloom, this explosion of these bacteria in yellow here. And again an explosion, a radiation of these bacteria in blue of the lactose paraceae showing that when you remove that antibiotic selection pressure you get an expansion of all these different classes of bacteria present in the gut. And then also interestingly an increase in some gram negative rods that were present in the subject. And then he was put back on levofloxin where we see a collapse and a disappearance of these gram negative rods. He was later put on urnipenem where another antibiotic where again we see a collapse and loss of numerous bacterial species. So it just goes to show you the impact that antibiotics can have on our gut microbiota which is important when we talk about that in just a moment. So what's the connection between microbes and cancer? Well some microbes may protect against cancer such as by metabolizing genotoxic agents which may cause DNA damage. Some microbes play a direct role in inducing cancer. Microbes such as helicobacter pylori, an organism that lives in the stomach of certain individuals and is associated with gastric cancer in those individuals. Or human papillomavirus, a virus that's associated with cervical cancer. Some microbes may facilitate the treatment of cancer by enhancing the host immune response and we'll talk a little bit about those in a moment. Now this is a study just from this week which I pulled from July 5th looking at combination immune checkpoint inhibitor therapy for the treatment of metastatic renal cell carcinoma. And what's interesting about this study and some of the studies that Scott has mentioned previously is that these immune checkpoint inhibitors show a distinct role in the treatment of renal cell carcinoma. But in the study about 40% of people responded to the immune checkpoint inhibitors and a larger percentage of people didn't respond to these immune checkpoint inhibitors which begs the question why? What is different about those individuals for instance that aren't responding to the immune checkpoint inhibitors? Well, this is another kind of cartoon slide to augment what Scott showed you and essentially these T cells have this PD1 receptor and when bound to the PD1 receptor it acts as a break to reduce its inflammatory potential. And so even though the T cell may bind to an antigen on a tumor cell that would allow it to kill that tumor cell, the breaks are on and it can't actually kill that tumor cell unless you block this PD1 which then releases this break and some of these immune checkpoint inhibitors really work in this way by releasing this break and allowing the T cell to then kill off the tumor cell. Now, why should you care about the microbiota in somebody who has kidney cancer? Well, there were some fascinating studies that recently were published in the journal Science that linked the microbiota to response to immune checkpoint inhibitors and what they showed in several animal models is that if you take mice and you give them melanoma and then give them an immune checkpoint inhibitor, it works fine as long as you've got an intact microbiota but if they have a disturbed microbiota, the immune checkpoint inhibitors no longer work and in fact, if you take these mice and you raise them under germ-free conditions the immune checkpoint inhibitors don't work but if you give back specific bacteria to these mice you now reactivate the ability of these immune checkpoint inhibitors to have an antitumor response against the melanoma and for instance in this study published here what they showed was that bifodobacterium species a bacterium that's present in the gut of humans and very common in the gut of children is associated with having an active immune response when given the immune checkpoint inhibitors and in this study, another study, again here looking at the PD1 inhibitor this is a different immune checkpoint inhibitor, the CTLA4 molecule which works in a similar way to the PD1 connection of proteins that again, having this bacterium bacteroides fragilis in the gut of these mice protects them and allows them to have an antitumor response Is that at all relevant to renal cell carcinoma? Well, there was an abstract that was recently presented at a genetic urinary cancer meeting highlighting that those patients with renal cell carcinoma who had had previous antibiotic therapy had a reduction in progression free survival compared to those who hadn't received antibiotics suggesting that the gut microbiota may be impacting the response to these immune checkpoint inhibitors and here in this particular study, the survival and those that had received antibiotics was 2.3 months where it was 8 months and those that hadn't received antibiotics What this study didn't show was whether there were specific bacteria that were associated with this response to immune checkpoint inhibitors Question in the back That hasn't been determined and furthermore, there's also a potential for confounding here which is as possible that those patients that got antibiotics were sicker than those patients that hadn't received antibiotics but we need to do more studies looking at whether there are specific antibiotics whether there are specific changes in the microbiota that predict your response to immune checkpoint inhibitors There are other examples where microbes can play a role in cancer therapy and one example is with bladder cancer and for decades, we've used essentially a bacterial immunotherapy for the treatment of bladder cancer and here, what physicians do is they instill a mycobacterium, basill comet garong which is a relative of tuberculosis into the bladder of people with resected bladder cancer and what happens is that those tumor cells take up the mycobacteria in green into those cells and in fact, the same mutations that allow the cell to turn into a cancer cell also promote the uptake of these mycobacteria within the cell these mycobacteria cause direct damage to the cell, killing them but also help to recruit in immune cells that participate in controlling the bladder cancer that's present in the wall of that bladder so this is an early example of immune therapy using microbes to enhance the immune response so how can we manipulate the microbiota of humans? Well, we can give probiotics, things like lactobacillus and even bifidobacteria the problem with a lot of these probiotic formulations is that they were formulated because the bacteria grow well in milk and they're not necessarily the bacteria that are healthy for your intestine on the other hand, bifidobacteria are common members of probiotic formulations and given its connection with enhanced immune checkpoint response in mice it's possible that, for instance, bifidobacteria probiotics could play a role in enhancing immune checkpoint inhibitor response in humans those studies haven't been done yet there's bacteria therapy or also known as fecal mycobacter transplantation we are doing this to patients with clostridium difficile colitis here at the center and at the University of Washington in many places across the United States people with clostridium difficile colitis have an absence of healthy bacteria in their gut and this is one way of reconstituting a healthy microbiota where we have a healthy donor, we collect stool from that donor make sure they don't have any infections like HIV, hepatitis, etc and after screening that donor and their stool we then take that stool and inject it via colonoscope into the colon of that patient and it's actually more effective than antibiotic therapy for the treatment of clostridium difficile and helps to restore bacterial diversity it's possible in the future that if we know that there's a certain microbial community profile associated with response to your anti-tumor therapy we could manipulate the microbiota in this way using fecal mycobacter transplantation another more appealing way of altering the microbiota is to use engineered microbial communities and there are numerous companies that are creating freeze dried capsules containing microbes from the human gut which they've grown up to high concentration created spores for these bacteria you ingest the capsule, the capsule dissolves it goes into your colon and releases those spores where those bacteria can replicate that's another appealing way of altering the microbiota of humans in order to enhance health of subjects so my take home message is that humans are colonized with trillions of microbial cells that help to aid digestion, stimulate immune responses and shape our chemical environment some cancers are caused by microbes such as helicobacter pylori and stomach cancer there have been very few studies that have examined this in patients with renal cell carcinoma although there's at least one epidemiological study that's demonstrated that patients with a history of urinary tract infection have about a two fold increased risk of renal cell carcinoma there are differences in microbial communities across humans risk of disease and response to treatment such as with renal cell carcinoma and micro induced inflammation has been harnessed to treat bladder cancer and there may be opportunities to harness this in the future for treating other cancers I'll end there and open it up to any questions you have yes you mentioned that process yes is there a norm like there is for blood tests or is it that we treat them as long as the body is in good shape the body finds its own normal yeah and that's what's so interesting is that there are different states of normal you know there isn't one size fits all what we do know is that normal is associated with diversity which is that we find lots of different bacterial species and typically you have about 100 or 200 different bacterial species present in the gut it's abnormal just to have one okay but what happens you may ask well how do you acquire your microbiota and the fact of the matter is that you acquire that in infancy and the first two years of life are incredibly dynamic with bacterial species moving in and then leaving and then other species moving in and leaving but after about two to three years of life it begins to stabilize towards a more adult like microbiota with lots of diversity and stability of the microbiota so we know that normal means that there's different people with different types of microbiota communities we still need to understand though whether some of what we call normal is associated with health and whether there are certain things that are more healthy than other types of what we now call normal question so it's pretty new and a lot of this has been driven in the last 20 years by advances in sequencing technology many of these microbes that exist for instance in the human colon haven't been propagated in the laboratory they're difficult to grow and so if you can't grow them how do you study them there's a way of studying them without growing them which is to look at their DNA and so what we do is instead of trying to grow them in a petri dish in the laboratory what we do is we take that stool sample we break open all of the cells and we sequence through the DNA that's there as a way of identifying all the microbes that are present in that community and that high throughput sequencing technology has only been available for the last about 10 years which has allowed us to really characterize the full diversity of microbes that are present and that's led to an explosion in the field and this interest in microbiome science and so we're really still beginning to understand who's there but more importantly how they're interacting with each other the other microbes and with the human host to either promote health or lead to disease question white yeah and so first of all there's no evidence that that stuff harms you okay unlike some other things that we could give you and so it's fine to take activity or kombucha or kefir or sauerkraut or other things that contain microbes and in fact we did a study here at the cancer research center where we looked at patients who develop bacteremia with some of the bacteria that we typically think of as probiotic type of bacteria like lactobacillus and what we find is even in our cancer patients who are immunocompromised they develop bacteremia with these organisms they never die of it which is just goes to the point that these are non-pathogenic low pathogenicity organisms and so I would say to take some of these probiotics now the bigger question though is does it provide a benefit and that's harder to prove that it's providing some distinct health benefit and the other point that's interesting is that in studies where we've given probiotics to people where they get billions and billions of cells of some microbe every day it doesn't actually displace your normal microbiota yeah it's disappointing you would think that by giving two trillion cells of a specific microbe every day like lactobacillus ramnosus which is a probiotic you would completely change the microbiota it doesn't it does change what those microbes are doing it changes their gene expression profile when you take those and it may change how the human host is interacting with those microbes but it's not like you're completely re-engineering your microbiota by taking a probiotic which is also why I think that it's relatively safe to take and for some there is some evidence that probiotics can help so for instance in patients who are receiving antibiotics it tends to prevent them from developing C. diff it doesn't really help that much well if they've already developed C. diff but for patients who don't have C. diff and are at risk for C. diff there's some benefit to taking probiotics for instance it produces colitis so it's an antibiotic associated diarrhea where you get an overgrowth of one bacterium that's in your gastrointestinal tract and it produces a toxin that causes damage to the gut and diarrhea and we treat that with antibiotics but all of those antibiotics then lead to a collapse in diversity which then paradoxically increases your risk of recurrence of the C. diff which is why some of the new treatments are instead of giving more antibiotics you give bacteria therapy to restore the normal bacterial diversity and it turns out by restoring diversity those other microbes produce compounds that inhibit the growth of the C. diff so again suggesting this holistic approach to treatment so bottom line is I don't think the probiotics are going to hurt you we need to collect more evidence of how they're helping should we switch over