Gut Biome and Graft-versus-Host Disease: Do Organisms in the Gut Contribute to GVHD?

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Gut Biome and  Graft-versus-Host Disease: Do Organisms in the Gut Contribute to GVHD? 

Thursday, May 2, 2024

Presenter: Robert Jenq MD,  Director of MD Anderson Microbiome Core Facility and Associate Professor in the Department of Genomic Medicine, MD Anderson Cancer Center

The presentation is 30 minutes long followed by 17 minutes of Q & A.

Many thanks to Incyte and Ironwood Pharmaceuticals whose support helped make this workshop possible.

Summary: The overall health of the gut microbiome and its organisms can influence the prevalence and severity of graft-versus-host disease. This presentation reviews the causes and mechanisms of how organisms in the gut can affect GVHD and reviews potential strategies to minimize this problem.

Highlights:

• Having a stem cell transplant can temporarily reduce microbiome diversity.
• Patients with more diverse microbiome have a decreased risk of death due to GVHD
• Avoiding antibiotics that reduce diversity in the gut microbiome and supporting the gut biome with a well-balanced diet can help prevent a sequence of events that can worsen GVHD.

Key Points:

(08:10): Patients with more diverse microbiomes have improved overall survival.

(08:47): This improved survival probably reflects differences in GVHD related mortality.

(09:15): Certain antibiotics that are used in transplant patients can decrease the diversity of the microbiome.

(11:05): In one study, patients receiving a strong antibiotic called meropenem developed GVHD at much higher rates than those that did not receive that medication.

(14:57): A bacteria called bacteroides thetaiotaomicron can thin mucus in the intestinal layers and lead to more severe GVHD.

(17:58): One study found that patients with more bacteroides in their stool responded better to steroid treatment for GHVD than those who had less bacteriodies.

(20:41): A sub-type of bacteria called B. ovatus was found to prevent thinning of mucus in the gut.

(25:08): In human patients receiving a stem cell transplant, chemotherapy can suppress appetite and impair nutrition which also impairs the gut bacteria.

(28:13): Probiotics are not very beneficial in restoring the bacteria in microbiome needed to reduce the risk of severe GVHD.

(29:45): Fecal transplantation via capsules has more promise for restoring or maintaining a healthy gut biome.

Transcript of Presentation:

(00:01): [Michala O’Brien]:  Introduction. Welcome to the workshop Gut Biome and Graft-versus-Host Disease: Do Organisms in the Gut Contribute to GVHD? My name is Michala O'Brien, and I will be your moderator for this workshop.

(00:14): It's my pleasure to introduce today's speaker, Dr. Robert Jenq. Dr. Jenq is an Associate Professor in the Department of Genomic Medicine at the MD Anderson Cancer Center in Houston, Texas. He is the director of the MD Anderson's Microbiome Core Facility, which examines the microbiome's impact on cancer treatment, including stem cell transplant, bone marrow transplantation, and cancer immunotherapies. Please join me in welcoming Dr. Robert Jenq.

(00:48): [Dr. Robert Jenq]:  Overview of Talk. Thanks to Michala and Marla and Susan and all the other folks that make this amazing symposium possible. So the title of my talk is Gut Biome and Graft-versus-Host Disease or GVHD: Do Organisms in the Gut Contribute? and here are some of my disclosures. I do advise some companies that are in the microbiome space, and I have a patent that we receive royalties from Seres Therapeutics.

(01:39): I do want to acknowledge work from a talented postdoc in my laboratory, Eiko Hayase. She's a physician scientist. Here she is at a recent tandem meeting by some giant GVHD block letters, and she's done a lot of the work that I'll be presenting in a little bit.

(01:57): So I think for this audience, I don't need too much background about GVHD, but here's one slide showing some of the colonoscopy findings that can be seen in acute GI GVHD. So GVHD can definitely afflict the lower intestinal tract. And this is the type of GVHD that's most modulated by the microbiome or the bacteria that live within us.

(02:23): The study of the microbiome dates back to the 1920s and studies of germ-free rodents. A little bit of history about why we think the microbiome might be involved. So here I'm showing you this metal sterilizable isolator. This is a research instrument that allowed scientists in the 1920s and beyond to study germ-free rodents. I think they were originally rabbits or Guinea pigs. Later rats and mice were born and bred and maintained completely germ-free and they lived in these containers. The researchers would use these gloves to manipulate the mice or other rodents, and they could look through these glass windows at the top. So, this kind of looks like a steam engine, right? This is pretty bulky and difficult to work with.

(03:17): By the 1960s, technology had advanced and they were using a flexible film isolator that you can see on the left. Finally, on the right, is a modern isolator that scientists are using to handle these germ-free rodents.

(03:34): When germ-free mice received transplants, they developed almost no graft-versus-host disease (GVHD). So the reason why I'm bringing this up is there were some early studies done in the 1970s, and here's one of them by Dr. Jones and colleagues, where they did a bone marrow transplant in mice. And this was well known to produce graft-versus-host disease, just like in our patients. Interestingly, in the setting of being germ-free, these mice had almost no GVHD to speak of. So this was a very, very interesting, very provocative finding.

(04:10): Antibiotics also reduced the prevalence of GVHD in mice that weren’t germ-free. This was followed a few years later by another group by Dr. van Bekkum and colleagues where they found that they could basically get the same results in mice that weren't germ-free by giving them antibiotics in their drinking water. So they gave the mice a mixture of three or four different antibiotics with the goal of trying to kill off all the bacteria in the GI tract, and they found that these mice also did better from a GVHD perspective.

(04:40): So this was actually translated into clinical practice. Here I'm showing you a picture of a pediatric patient being transplanted at the Fred Hutch Cancer Center in Seattle from the 1980s. And you can see that the patient is in a flexible film isolator similar to the one that I showed you earlier that we used for the rodents. It's bigger, of course, but the patients would stay in the hospital and stay in these isolators starting with their chemotherapy and continuing until a month or maybe even two months after the transplant. This was standard practice until around the early '90s. There was an initial study published in the New England Journal, one of the most prestigious journals, demonstrating that this actually led to lower rates of GVHD and improved survival.

(05:39): However, later studies showed that there wasn't necessarily as clear benefit as was seen in this first study. Some of the reasons might be that newer preventive medications for graft-versus-host disease became available. So drugs like cyclosporine or tacrolimus, for example. Nevertheless, there was this potential for the gut microbiome and how it could be impacting on GVHD that was tantalizing and didn't reach fulfillment.

(06:15): Recently new technology has allowed the study of how the microbiome changes in people during transplant hospitalization. More recently, groups have been taking the latest technologies that allow us to characterize the bacteria in samples more easily and cheaply using deep sequencing methods. And this led to a recent new paper in the New England Journal. This came out a couple years ago. And what they were looking at was in four different cohorts, so one in Sloan Kettering in New York City, one in Duke in North Carolina, a cohort in Regensburg, Germany, and a cohort in Hokkaido, Japan. There were almost 1,400 patients distributed in these four different sites. What the researchers were interested in is how does the microbiota or microbiome change during the transplant hospitalization, and then what is the potential association or impact on how the patients did.

(07:14): It’s now well-known that a more diverse fecal sample is associated with better microbiome health. And so what I'm showing you here is there's something called diversity. And so this is like an ecological concept. It lets you characterize how many different species are in a sample and how evenly balanced they are. And so folks in the microbiome field have found that a more diverse fecal sample is associated with better microbiome health.

(07:37): Having a transplant temporarily reduces microbiome diversity. And what we see in our transplant patients across the globe is that patients come in with high diversity, but that diversity goes down pretty dramatically over the course of the hospitalization, and then towards the tail end starts to come back up again. That's one takeaway message.

(07:56): The other takeaway message is that the gray dots show you all the different samples that were collected. And so there's a lot of heterogeneity or spread among all the patients. Not all patients are the same.

(08:10): Patients with more diverse microbiomes have improved overall survival. It turns out that diversity quantification, that measurement, is predictive. And so if you separate the patients into higher diversity or lower diversity, on the left is the New York City cohort, the higher diversity patients had improved overall survival compared to the lower diversity half. And that same analysis held up in the other three cohorts that were combined into a cohort two. So in these retrospective clinical studies, we always try to see if findings are reproducible. If they're reproducible, they're more believable.

(08:47): This improved survival probably reflects differences in GVHD-related mortality. And so this is a very nicely reproduced finding. It turns out the reason why folks are doing better in terms of surviving better is probably related to GVHD-related mortality or dying or succumbing to GVHD. The patients in the black line are the patients with more diversity in their microbiome, and you can see that they have a lower rate of mortality over time.

(09:15): Some stronger antibiotics that are used in transplant patients can decrease the diversity of the microbiome. There's one more takeaway from the study that I wanted to highlight, and it's getting at the potential explanation for why some patients lose their microbiome diversity or why they get an injured microbiome. And the answer seems to be antibiotics. And interestingly, it's not all antibiotics. So cefepime is here towards the top. This is an antibiotic that we commonly use for treating neutropenic fever. And then same with meropenem and piperacillin-tazobactam. Those are also used to treat neutropenic fever.

(09:45): They're thought to be pretty much equivalent by the infectious disease doctors, but there are some, meropenem and piperacillin-tazobactam that are stronger in terms of killing bacteria that can't survive in the setting of oxygen, so these anaerobic bacteria. These antibiotics are a little broader or stronger in terms of their spectrum of activity, and those are the two antibiotics that were associated with losing more diversity. So it seems like a stronger antibiotic, at least in terms of your microbiome, might not be such a good thing.

(10:24): So for the rest of the talk, I'm going to turn to research from my group here at MD Anderson. When I got here about seven years ago, we went back in time and looked in the patient charts and we found almost 300 patients that had either AML or MDS and underwent an allotransplant at our center.

(10:44): And we looked at the antibiotics that these patients received. It turns out that about a quarter didn't need any antibiotics, about a little over a third received cefepime, that's that antibiotic that I showed you earlier that was not associated with losing diversity, and then some of the patients received either meropenem or meropenem and cefepime.

(11:05): In one study, patients receiving the strong antibiotic, meropenem, developed GVHD at much higher rates than those that did not receive that medication. Here's how these patients did at MD Anderson. The light dotted blue line shows you how the patients did if they didn't receive any antibiotics. I'm showing you that their incidence of developing lower GI GVHD was quite low, maybe around 10% of the patients developed GVHD at one year. If they received cefepime, they had a very similar incidence of developing GVHD. Whereas the patients that received meropenem, I think you can tell from the two red lines, these patients were developing GVHD at a much higher rate, up to a quarter of the patients were developing GVHD. So 25% versus 10% is a pretty substantial difference in terms of risk.

(11:54): So we wanted to get at whether these associations were really causal or not. And the only way to really get at that without doing a clinical trial in patients is to do animal experimentation. And so in my lab and many other labs, we can do these bone marrow transplants in mice just like they were doing in the 1970s. And what we did here was we added on top meropenem, that same antibiotic that our patients are getting. We gave it to the mice after their allotransplant. And so in the blue line here are mice that are unfortunately starting to die from GVHD starting around day 30.

(12:35): These mice don't get any kind of prevention medication for GVHD. This is sort of like natural GVHD that is uncontrolled. It turns out if the mice received meropenem, then they start to die much more rapidly for more severe cases of GVHD.

(12:58): Antibiotics can do different things. They can kill off some bacteria, but they can also select or make space for other bacteria. You could ask, is the meropenem killing off beneficial bacteria or could the meropenem be selecting for harmful bacteria?

(13:15): It appeared that meropenem was promoting harmful bacteria in the mice. And so we tried to get at that question by adding additional antibiotics. This is called ‘decontamination’. Wwe added more antibiotics to the drinking water. And it turns out that those mice did better, very similar to those really early studies in the 1970s. So it turns out if you kill off most of the microbiome, the mice actually do pretty well. And so meropenem we think is doing something to select for the harmful bacteria. That was our interpretation of this.

(13:47): So using these new sequencing methods, we could characterize how many different bacteria and how much of each bacteria is in each poop sample from the mice. On the left are mice that don't have GVHD, in the middle are mice with sort of normal or mild GVHD, and on the right are mice that have GVHD that's been aggravated by the antibiotic meropenem. And I think you can see that these mice had a lot more of these purple bacteria in these bars. Those bacteria are called bacteroides.

(14:23): So we're going to get a little bit in the weeds in terms of bacteriology here for a few minutes. So bear with me. So these mice are colonized with this bacteria called bacteroides thetaiotaomicron. This is not a bacteria that typically causes serious infections in humans, not normally, unless maybe you're getting abdominal surgery or something like that. But otherwise, it doesn't really get into the bloodstream. It can't tolerate oxygen. It'll die if it's exposed to oxygen. So it doesn't really cause blood stream or pneumonia-type infections.

(14:57): A bacteria called bacteroides thetaiotaomicron can thin mucus in the intestinal layers and lead to more severe GVHD. But one thing it can do is eat mucus as a source of food. And so mucus is really important. It lines the intestinal layers and it protects us as a barrier separating our gut bacteria from our intestinal tissues.

(15:17): We thought it was interesting, the fact that this bacteria that's known to eat mucus was increasing in the mice that had worsened GVHD. It turns out you can actually try to look at how thick the mucus is under the microscope. And so here on the top left, you can see that at the bottom of the picture are the cells of the colon, the epithelium, and on the top are the bacteria.

(15:47): Adding a mixture of antibiotics to ‘decontaminate’ the gut can restore a more normal mucus layer in mice. And that purple frothy layer in the middle is the mucus layer that's in between. And so on the right is the mouse with GVHD and that mucus layer is pretty similar, not that changed. On the bottom left here, you can see that the mouse treated with meropenem, that mucus layer is very, very thinned out. And then finally on the bottom right, if we add the mixture of antibiotics that decontaminate the mice and produce pretty much a germ-free mouse, at least temporarily, that mucus layer is close to back to normal.

(16:22): Further mice experiments confirmed the causal link between bacteroides thetaiotaomicron and more severe GVHD. The other piece of evidence that we wanted to see if we could obtain was, what if we introduce this one bacteria that we think is harmful, this B. theta bacteria. Is that enough to make the mice sick? And it turns out it is. If you decontaminate the mice, they're doing very well in the green line. But then if you reintroduce this one bacteria, then that's enough to have the mice unfortunately succumb to GVHD again. So pretty strong evidence we thought, at least in mouse models, that this bacteria is a harmful bacteria.

(17:00): One other thing that we wanted to look at is whether the bacteria was changing its behavior, because this bacteria, it's found in mice normally, and it doesn't chew up the mucus under normal circumstances. It really only starts chewing up the mucus after antibiotic treatment. We were able to do RNA sequencing, which tells us what genes the bacteria are producing or trying to make proteins of. And it turns out these B. theta bacteria in the mice that are treated with antibiotics, they're turning on some of these enzymes that are involved in breaking down mucus.

(17:40): So everything seems to fit that these bacteria, not only are they sufficient to produce the aggravated GVHD, but they're also really revving up their machinery to chew up mucus as a source of food.

(17:58): One study found that patients with more bacteroides in their stool responded better to steroid treatment for GHVD than those who had less bacteriodies. The next question we wanted to ask is, can the microbiota impact on how well a patient does when they're being diagnosed with GVHD and then treated with corticosteroids, which are the standard treatment to treat GVHD? So we did this in a smallish cohort of patients here at MD Anderson, almost 40 patients.

(18:21): These were patients that had just presented with new onset diarrhea after their allotransplant before they got a colonoscopy, or before they had been treated with steroids. And here we're looking at what their microbiome looks like. It turns out they kind of naturally cluster into two different types. There's one subset of patients here on the left that had more of the brown bacteria, which was bacteroides, and then on the right are a lot of these other colors, but they had a lot less bacteroides.

(18:54): Bacteroides is, of course, that B. theta genus that I told you about earlier, right? The bacteria that eat mucus. So on the left are patients that had more bacteroides. You would think that these patients might be doing worse, but we actually found the opposite.

(19:14): So here I'm showing you did the patients respond to treatment or not? And in cluster one, the high bacteroides cluster, these patients had almost a 90% response rate to being treated with steroids. Whereas in the bigger cluster that had very little bacteroides, these patients had less than a 50% response rate to treatment.

(19:38): Another bacteroides, called B. ovatus, may be a beneficial bacteria for people with GVHD. And so these patients were not doing as well. So why is this happening? Well, it turns out these patients in cluster one, they had a different bacteroides. It's a close relative, but it's not the same bacteria. It's not that B. theta bacteria that eats mucus. It's actually a bacteria called B. ovatus. I'll tell you a little bit more about B. ovatus, but we isolated B. ovatus and we put it into our mouse model of GVHD. So these are mice that have GVHD that's aggravated by this antibiotic called meropenem.

(20:17): And it turns out if you introduce B. ovatus to the mice by mouth, then the mice actually do better and they don't die nearly as much from GVHD. So it seems like our mouse model is reproducing or recapitulating what we saw in the patients in that B. ovatus might be a beneficial bacteria for GVHD.

(20:41): B. ovatus was found to prevent thinning of mucus in the gut. We were interested in what B. ovatus could be doing to that mucus layer that I showed you earlier, and it turns out B. ovatus can prevent the mucus from being eaten up or chewed up, and it's a lot thicker and close to normal in thickness.

(20:58): B. ovatus and B. theta are two related, but different bacteroides that affect the gut biome and GVHD differently. A little bit about what's going on here. The title of my slide here is B. ovatus and B. theta - A Tale of Two Bacteroides. So bacteroides is a genus, so it's like an umbrella of different types of bacterial species. For example, humans, right? Humans are homo sapiens, but there are other species, they're all extinct, in the same genus as us. These ancient humans that we find their bones, the archaeologists find their bones and know that they're not humans for example, like Neanderthals for example.

(21:39): I guess you could say that B. ovatus and B. theta are sort of sister species and they have some things in common, but they have some things that are different. So at the top, what these researchers found was that B. theta can eat mucus, but B. ovatus cannot, which is interesting. And then towards the bottom you can see that B. ovatus can eat a lot of different types of fibers, plant fibers, but B. theta cannot break those down. And so some of these I'm showing you on the right are these fibers that are called arabinoxylans, and these are very common in whole grains. So things like wheat, barley, corn, rice, oats, and rye are very rich in arabinoxylans. And B. ovatus can eat these as a source of food and B. theta can't.

(22:35): So that led us to look at what the arabinoxylans get broken down into. So these are fibers, right? They're long chains of individual sugar molecules and they're very rich in a sugar molecule called xylose. And so what we found is that if you take mice and you introduce B. ovatus to the mice, that raises levels of the sugar xylose, in the intestine, in the fecal contents of the colon.

(23:08): So we were able to collaborate with a B. ovatus expert, a guy named Dr. Eric Martens from the University of Michigan. And so Eric, he has a strain of B. ovatus and he has the capability to go into its genes and actually knock out certain genes at will. It's kind of amazing. And so he had a version of B. ovatus that wasn't able to eat xylanases. So this is a version of B. ovatus that can't do what most B. ovatus can do, which is break down these fibers that are rich in whole grain food.

(23:46): Further research in mice found B. ovatus could reduce GVHD through its ability to break down whole grain fibers and produce xylose. And it turns out when we introduce this mutant version of B. ovatus into the mice, this version of B. ovatus can't mediate a benefit, so the mice don't do well in response, whereas the mice do well with the normal version of B. ovatus that can break down these fibers. So to us, this is pretty convincing evidence or proof that B. ovatus is benefiting the mice and reducing GVHD through its capability to break down these whole grain fibers. So finally we asked, well, is the xylose enough by itself?

(24:29): We think that B. ovatus is breaking down these whole grain fibers and producing xylose, and then the xylose is mediating a benefit in terms of GVHD. And so here we simply put xylose into the drinking water of the mice and we found that that was enough to keep the mucus layer nice and thick. And this actually showed up in terms of survival of the mice too. So in the red line are mice that are unfortunately succumbing to graft-versus-host disease in the setting of meropenem treatment, but in the green line are mice that received xylose in the drinking water and seem to be doing much better.

(25:08): In human patients receiving transplants, chemotherapy can suppress appetite and impair nutrition which also impairs the gut bacteria. So a couple of summary slides about what we think is going on. We think that the gut microbiome is pretty complicated, right, with lots of different bacteria types. But adding to that complexity is the fact that antibiotics can change the microbiome and diet can also change the microbiome. And so what we think happens to our patients is that in order to get an allotransplant, they have to receive chemotherapy, sometimes a combination of different types of chemotherapy, sometimes radiation, and all these things combine to suppress our appetite.

(25:51): Antibiotics can then cause collateral damage in the GI tract. The patients sometimes develop mucositis or mouth sores. It's very common to lose their appetite, and so they're not eating as well. This is impairing nutrition and also impairing the gut bacteria that live in us. And on top of that, patients oftentimes need antibiotics. They might develop a fever and they need antibiotics to treat infections, but the side effect, the collateral damage of those antibiotics is that it can attack the bacteria in our GI tract and hurt the microbiome.

(26:26): This can promote mucus eating bacteria and inflammation that is problematic for patients. And we think that this all leads to expansion or outgrowth of mucus degrading bacteria, and this can hurt the lining of the intestinal tract. And so here's my depiction of these mucus degrading bacteria that are turning on us and contributing to the inflammation, this lion here.

(26:47): Avoiding antibiotics when possible and eating a well-balanced diet can maintain gut health and prevent a sequence of events that can worsen GVHD. However, avoiding antibiotics if possible, if they're not necessary, as well as eating a nice balanced diet with lots of vegetables and fruit and whole grains and giving your gut bacteria all the different possible fibers and different food sources that they can utilize and break down, leads to a happy, diverse normal state of the bacteria. And so the mucus-eating bacteria are kept in check and they don't turn on us and they don't start eating the mucus. And as a result, they don't cause as much damage. They stay cute and cuddly.

(27:32): A couple other lessons learned that I just wanted to summarize here because I think these will be common questions. So I mentioned the first point already. Antibiotics are important tools for treating infections but can have side effects, including injuring the microbiome.

(27:49): The next point is diet and nutrition are very important for supporting the microbiome. And this is something that recent literature have supported from other groups as well, is that really eating a well-balanced diet is very important for supporting all the different beneficial bacteria that live in us.

(28:13): Probiotics are not very beneficial in restoring the microbiome. Probiotics, so I didn't talk about probiotics in my talk so far, but probiotics that are available from the health food store or from yogurts, they have typically bacteria that are called lactobacillus or bifidobacteria, and these are bacteria that are probably not very beneficial in restoring the microbiome.

(28:39): And it makes sense if you think about it, because these are bacteria that are designed to consume milk products. I mean, that's why they produce yogurt. But most of us and our patients, we're not consuming only milk products. We eat lots of other foods. And so these bacteria actually don't live very long in our intestinal tract. We consume them in yogurt or probiotic pills and they just tend to pass through and they don't really make that much of a difference. So they're not very good at restoring the microbiome.

(29:13): They can have a beneficial effect in terms of reducing diarrhea that can happen after antibiotics. That's been pretty well studied and shown be beneficial it’s been shown in several clinical trials that they do help for antibiotic-induced diarrhea. But I don't think that's going to be necessarily the solution for a patient who's undergoing an allotransplant who get antibiotics and want to restore their microbiome after. I don't think probiotics will necessarily be the answer.

(29:45): Fecal transplantation via capsules has more promise for restoring or maintaining a healthy biome. What's coming along, but it's still early days are companies that are working on strategies including fecal transplantation or growing specific mixtures of bacteria and then putting those into capsules, and then giving them to patients. These are studies that are still pretty early on, early phase clinical trials, but they do seem to be holding some promise. Time will tell.

(30:16): I want to thank all the folks in my research group that made this research possible, as well as, of course, Eiko, who I acknowledged in the beginning, and then sources of funding.

(30:27): And grant support from The Sabin Foundation, The Mark Foundation, The Texas Cancer Prevention Research Institute, NIH, and of course, my home institution, MD Anderson. So with that, I'll take questions.

Question and Answer Session

(30:53): [Michala O’Brien]:  Thank you, Dr. Jenq, for this excellent presentation. We'll now begin the Q&A session. The first question is, how can you have a healthy biome if you are on antibiotics for several years?

(31:22): [Dr. Robert Jenq]:   Yeah, so this is a tricky question. If you have to have antibiotics, because I would imagine some patients they have recurrent pneumonias, maybe you have chronic lung GVHD or something like that and you get recurrent infections and you need antibiotics, it can be pretty tricky to maintain your microbiome. I think having a discussion with your doctor who's prescribing you the antibiotics and letting them know that you're concerned about your microbiome can potentially lead them to pick certain antibiotics that might be more sparing of the gut microbiome.

(32:03): So not all antibiotics are equally harmful. That's one thing to remember. Just like I showed you earlier in the beginning, cefepime was not associated with loss of microbiome diversity. So not all antibiotics are equally bad. And many of the antibiotics actually that are used orally to treat pneumonia or sinusitis or something like that, like levofloxacin or azithromycin, these are actually not that harmful to the microbiome. So yeah, not all is lost if you need to be treated with antibiotics is what I'm trying to say.

(32:39): The other thing that you can potentially do is to eat a varied diet, and so a diet where you have a bunch of different leafy vegetables and other types of vegetables and fruits and whole grains. These are things that can potentially help your microbiome recover faster. Remember, children when they're born, they don't have a microbiome at all. They're born pretty much sterile, newborn babies, and they're able to pick up a full microbiome during their childhood just from their environment from what they eat.

(33:20): [Michala O’Brien]: How often should a GVHD patient with a normal colonoscopy have a repeat colonoscopy?

(33:32): [Dr. Robert Jenq]:   So this is getting at colonoscopy to evaluate GVHD, I assume. So I would say if a patient has GVHD and this was diagnosed with a colonoscopy and they've responded to treatment, then we don't typically do a repeat colonoscopy to check on the GVHD. As long as you're improving, you're clinically better, we don't take another look to confirm that things look better on the inside as well.

(34:07): If the colonoscopy is being done for other reasons like colon cancer screening, for example, then that would be the usual time frame in terms of colon cancer screening, which I think is... I mean, I'm not a gastroenterologist, but it's between five and 10 years.

(34:25): [Michala O’Brien]:   Another viewer said, "Thank you for this information. It's very valuable. Extrapolating information from some of the studies, what are your thoughts of helping the microbiome while on chronic antibiotics, FAM therapy, azithromycin?"

(34:51): [Dr. Robert Jenq]: This is one of those treatments to treat long chronic GVHD, right? And I guess I would say if you need the azithromycin, then you need the azithromycin for your lungs. And it's actually not the worst in terms of the worst perpetrators of microbiome injury. Azithromycin is not that bad.

(35:15): And what you can do is to try to eat a balanced diet in terms of different types of fruits and vegetables and whole grains. One thing that I do when I go to a salad bar is I'll sample a little bit of everything to try to get all the different types of fibers and sugars that might be in all the different types of fruits and vegetables.

(35:39): [Michala O’Brien]: Is there any specific diet you should follow before a transplant to increase your gut microbiome?

(35:52): [Dr. Robert Jenq]:  I keep repeating myself, I guess, but yeah, I think having a diet that's very varied in all these different fibers and sugars is probably really all we can recommend at this point in terms of trying to improve your microbiome before your transplant. I'll also say that most patients when they come in, they actually have a pretty healthy microbiome. I think relative to what happens later after some of these antibiotic treatments, the microbiome at the outset is usually in good shape. The typical patient that comes to transplant has a pretty intact microbiome.

(36:32): [Michala O’Brien]: There might be some repeat here, but I'll try to get through this question. It's a long one. If the microbes in the gut microbiome play a critical role in the proper function of transplanted T cells and therefore in the control of graft-versus-host disease, could you explain if a fecal transplant used to repopulate the required species that have been killed off by the treatment and the long-term use of antibiotics, considering that the oral use of probiotics will not permanently repopulate these species and a fecal transplant will?

(37:16): [Dr. Robert Jenq]: Yeah, it's a long question, and it's a pretty sophisticated question too. So this is being studied, fecal transplantation, to see if this could be a way of preventing GVHD from happening. There are a few different centers that have looked at this. Sloan Kettering has done a trial of actually an autologous fecal transplant. So it's the patient's own microbiome that was collected and set aside and stored prior to the transplant hospitalization and then reintroduced once the immune system recovers.

(37:55): There are other centers that are looking at fecal transplant from unrelated donors, like from healthy donors. These have so far been very small studies, but my understanding is they show that there could be some benefit. Fecal transplant is a little bit tricky though. It really should only be done by professionals is one thing I should say.

(38:20): There was a case, it was in the New England Journal maybe two, three years ago of a transplant patient who unfortunately received a fecal transplant that included a resistant bacteria, so like an antibiotic-resistant bacteria, and that bacteria actually got in the bloodstream and I think the patient passed and died from that infection. So fecal transplant laboratories that produce fecal transplants nowadays, they have a procedure to screen and look for any kind of antibiotic resistant bacteria.

(38:55): The other word of caution is I think there was a patient who had some type of inflammatory bowel disease. It wasn't a bone marrow transplant patient, but they did a fecal transplant from I think a roommate or a relative at home and actually gave themselves CMV, cytomegalovirus. It was a case of somebody who had done their own fecal transplant and got a viral infection as a result. Not something to be done at home.

(39:32): [Michala O’Brien]:  Is there any current research data that shows a gluten-free diet is good for lessening gut GVHD?

(39:44): [Dr. Robert Jenq]:   I'm not aware of any data that gluten-free is good for gut GVHD. In general, gluten-free is really important if you have a gluten allergy. But folks who don't have a gluten allergy, I think the gluten-free diet is pretty controversial in terms of any benefit, including GVHD.

(40:16): [Michala O’Brien]:  Here's another research question. Has there been any research to show how the gut microbiome affects other types of GVHD?

(40:26): [Dr. Robert Jenq]:  So there has been research done there and the connections there are not quite as clear. The loss of diversity that I showed you earlier in the gut microbiome, it's not very strongly associated with skin GVHD. It's not associated with liver GVHD. It's not even all that strongly associated with upper gut GVHD.

(40:49): It was really the most strongly associated just with lower gut GVHD. There's also, of course, chronic GVHD. And there haven't been that many studies yet, but there was one study that suggested that microbiome injury might be associated with more chronic GVHD, especially if some of the bacterial metabolites are decreased. But I think more work to be done there in terms of the chronic GVHD setting.

(41:21): [Michala O’Brien]:  Is there anything patients can do to offset the effects of years of antibiotics and other prokaryotic medications impacting gut microbiome while on immunosuppressants?

(41:36): [Dr. Robert Jenq]: So most immunosuppressants are not thought to hurt the microbiome. That's my understanding, and I think it makes sense. The medications that really hurt the microbiome are antibiotics because antibiotics are designed to kill bacteria, and so they can through this bystander collateral damage effect hurt our microbiome. Immune suppressants by and large are not that harmful to the microbiome.

(42:11): [Michala O’Brien]:  Can you talk a little bit more about maybe some clinical data on the long-term implications for GVHD and gut microbiome and maybe some treatments down the line or in a pipeline?

(42:31): [Dr. Robert Jenq]:  I'm very happy to give the talk and it's what I study, so it's like near and dear to me, but it might be a little bit frustrating because there's nothing that you can act on quite yet other than what I'm telling you is eat lots of different fruits and vegetables and whole grains. I mean, that's almost obvious, right? And so in the horizon, the field is asking questions like what's the best antibiotic to give a patient who needs an antibiotic to treat their neutropenic fever or their infection during their hospitalizations and which antibiotic is best to still protect the gut microbiome, for example.

(43:14): So that's one area of investigation. There are also folks that are asking, is it possible to try to restore the gut microbiome after injury? And so can you do that with a fecal transplant? Can you do that with bacteria that you grow in the laboratory and put into a capsule? There are other folks that are asking the question, are there ways to supplement the diet that can help the microbiome recover faster or can help the bacteria that you introduce grow better?

(43:53): That's the area of prebiotics, so different types of fibers and other foods that can help feed your gut microbiome. And so that's an active area of research as well.

(44:09): [Michala O’Brien]:  This is a question about other medications like metformin and omeprazole on the microbiome. What is its implications?

(44:25): [Dr. Robert Jenq]:  Yeah, so metformin can change the microbiome. I think there are folks that have studied that. It can change the composition of the microbiome a bit. It's not as dramatic as an antibiotic, and it's also a little bit hard to tease apart the fact that metformin can actually cause loose stools to some degree. And so by changing the consistency of the stools, that can actually just change the microbiome composition.

(44:55): It's kind of like an indirect effect, right? It's not a direct effect. But I would say metformin is relatively mild relative to antibiotics. Omeprazole and other drugs in that category, PPIs or proton pump inhibitors, that's actually an interesting question, and I think it probably needs to be studied more. When you have stomach acid, stomach acid has a natural role in terms of sterilizing food and saliva and other things that we're swallowing.

(45:32): Our mouths are actually full of bacteria and usually stomach acid sterilizes all those bacteria that we're swallowing. But omeprazole prevents production of stomach acid, and so it's known that these medications can increase bacterial densities in the upper GI tract. Whether that has any effect on graft-versus-host disease or something like that I think is an unknown question. My guess is probably not.

(46:05): We've done studies looking at all the different types of antibiotics and other medications that our patients receive to see if any of these medications could be associated with GVHD or not or developing GVHD or not. And other than antibiotics, certain antibiotics being harmful and a medication called ursodiol being beneficial, we haven't really seen that other strong associations. So I think the chances that PPIs contribute to GVHD is pretty low, but not really well studied.

(46:46): [Michala O’Brien]:  I think this is going to have to be our last question. Do long-term use of antivirals such as acyclovir reduce gut diversity? Acyclovir, sorry.

(47:01): [Dr. Robert Jenq]:  Acyclovir. Acyclovir is used to prevent zoster and other herpes viruses. I think it's very unlikely that acyclovir does anything to the microbiome. I would have to look it up to check to be sure. But because viruses are so different than bacteria, I think it's very unlikely that acyclovir is bad for your microbiome.

(47:26): [Michala O’Brien]:  Closing. Well, on behalf of BMT InfoNet and our partners, I'd like to thank Dr. Jenq for a very informative and helpful presentation, and thank you to the audience for your excellent questions. Please contact BMT InfoNet if we can help you in any way and enjoy the rest of the symposium.