Ashish Kamat: Hello, and welcome to UroToday's Bladder Cancer Center of Excellence. I'm Ashish Kamat, Professor of Urologic Oncology and Cancer Research at MD Anderson Cancer Center in Houston, Texas, and it's a pleasure to welcome today, Dr. Laura Bukavina, who's currently a fellow at Fox Chase Cancer Center. Laura's going to share with us her presentation, which she gave a GU ASCO, which is so well received and won the award, on Microbiome of Bladder Cancer. Laura, you've been doing a lot of great things, both in urology and bladder cancer, and also across the globe with your work at the Ukraine border, currently, so it's a real pleasure to have you here today. And with that, I'm going to the stage over to you. Take it away.

Laura Bukavina: Thank you so much. Thank you, Dr. Kamat, and thank you, UroToday, for having me speak a little bit about the research that we have been doing at Fox Chase, as well as Case Western looking at microbiome of bladder cancer and how this can really help our patients with bladder cancer. First of all, I do have a disclosure. This is funded by P30 Microbiome Grants.

The way to think about microbiome is really the new frontier in medicine. 99% of our genome is actually made up not of human DNA, but actually made up of microbiome, including bacteria and fungi in our gut, as well as our tissues. While majority of people who think about microbiome, think about it as in terms of gut, really, any part of your body has specific microbiome that it harbors, including tissue, including bladder cancer tissue, including urine, and that's really something new that we have been sort of discovering over the last decade, and that's we do because of finer techniques to the lab and our ability to discriminate, to really use 16S technology to look at this microbiome within tissue and urine.

What's critically important is, not only are we looking at what's normal within microbiome, but we're also looking see within disease process, even within cancer or other disease process, such as diabetes or obesity, what changes does that lead to your microbiome? And vice versa. Does microbiome lead to potentially predisposition to some of these chronic diseases or potentially cancer?

Speaking of cancer, there's several landmark studies across 100 different types of disease processes, including cancer. But if you look within urology, really, there's only about two studies focusing on bladder cancer, looking at only about 100 patients, 100 samples. So we certainly have been behind on the microbiome and utilizing this to our advantage.

There's different ways to think about microbiome, and I think there's really a couple schools of thought about this. Does a microbiome actually cause the cancer themselves? Or, is there a change in your microbiome based on whatever disease process that is going on, that's actually causing a random association, is what people who call it, of the microbiome to your disease? We pretty much are all familiar what one microbiome and one disease model is, and that's very typical of the H. pylori example causing gastric cancer. Well, we know there's one microbiome and it has certain effects on the tissue and it causes that malignancy or that disease.

But, what we really have gotten to know more all the last decade is that majority of the microbiome is not actually one bug, one disease process. In fact, it's a collaboration between the host, between the microbiome, and the immune reaction that goes on. Particularly, over the last 10 years, we have been incredibly interested in how microbiome affects these, what we call, small molecules that potentially could be modifying disease process either through suppressing cancer or through activating cancer.

One of these things is small-chain fatty acids, which have been looked at in kidney, prostate, and bladder cancer. And what they look at is looking to see how these byproducts of fermentation of fiber really affect your gut microbiome and affect the spread of disease. They also had looked at whether these short-chain fatty acid affect your immune modulation, and there's multiple mouse models, as well as some early human work within melanoma, showing that yes, in fact, your microbiome, and particularly the production of short-chain fatty acids, does affect your cytotoxic T lymphocytes. This is just another example, showing that the higher the level of short-chain fatty acids, the more likely you are to have activation of cytotoxic T cells.

Now, back to our work. I already mentioned that we really don't know much about microbiome and bladder cancer. There's small studies, 30 to 40 patients, but before we can even start on thinking of ways to modulate microbiome to help our patients and to improve any sort of efficacy to treatment, we need to be able to characterize this correctly across multiple patients and disease processes. And that's exactly what we did.

Our lab has looked at microbiome characterization within gut, which means fecal studies, as well as urine, to evaluate, what do bladder cancer have or have not within their stool and urine that's different from that of healthy controls? And what we find is that the microbial diversity, meaning how diverse your population of microbiome on both the order species and genus level, is compared to healthy controls. This has been replicated in multiple other cancers, but what we know is that your microbiome is incredibly different if you are diagnosed with bladder cancer.

What I really wanted to really point out is what's called the beta diversity of bladder cancer patients compared to healthy controls. And what you can see is bladder cancer patients on the left are denoted here on by red dots. Not only do they have a different composition of their microbiome, but if you particularly split the bladder cancer patients up into smokers and non-smokers, which is the graph on the right side, the smokers, even if they've had a diagnosis of bladder cancer, are incredibly different from the non-smokers with bladder cancer. These non-smokers are actually what we call intermediate microbiome group, meaning they sort of fit in between their microbial composition between the healthy cohorts and the bladder cancer cohorts.

So this led us to thinking, are there potential different populations of what we call enterotypes, which is a way to classify people based on a combination of microbiome. Not just one bug or the other, but really looking at a combination of different bacteria and calling it an enterotype. And this is not a term that we came up with, this is something that's been funded for about 10 new years. And any human, a healthy human, particularly falls within three different enterotypes in this world, and that depends on your geography, depends on your food, diet, and smoking status.

But we wanted to see, could we replicate in enterotype within bladder cancer patients? And what we found is that, yes, there are particular enterotypes within bladder cancer patients that are not only related to smoking, but also they have different bacterial compositions. And unlike the healthy people who have about three different enterotypes, within bladder cancer there's only two different enterotypes. Why is important? Especially, why is this important to my patients?

Well, here's the issue. If you are particularly enterotype 1 and you are diagnosed with bladder cancer and you underwent neoadjuvant chemotherapy, the chances of you responding to neoadjuvant chemotherapy noted as PT 0 at cystectomy is slim to none. None of the patients who had in enterotype 1 composition within our cohort, who underwent neoadjuvant chemo followed by cystectomy had any sort of complete response. And this is vastly different when you look at the second enterotype, where you see partial incomplete response on a regular basis. You can break this down further by individual bugs, and certainly the bugs that we see mostly associated with neoadjuvant chemotherapy response, and that goes back to the small active molecules. Are the bugs that are able to produce butyric acid, which is a great finding.

Now, people might ask, "Well, most of the action bladder cancer is in urine, so why don't you look at urine?" And we did. We wanted to see, can we find similar findings in urine? As you can see here, again, especially look at B, there's significant diversity differences between the cancer and the controls, meaning the cancer group had lower diversity, they lost a lot of the diversity of their bacteria, they lost diversity in Lactobacillus, they lost diversity in Enterobacteriales, and they had a displacement of what we call dysbiosis, meaning bugs that typically are not butyric acid producers.

Then we looked at neoadjuvant chemotherapy response based on your urine microbiome. And across species, across responders, and across non-responders, what we found is that Lactobacillus, particularly in males and females, not only by order, species, or phylum, Lactobacillus, persistently was associated with a better response to neoadjuvant chemotherapy. And if you look across about 100 different bugs, Lactobacillus was the one that consistently came up and the one that was associated with better chemotherapy response.

So, these findings are all-new, these findings are all exciting, but we certainly need to do more in terms of validating these and really figure out the mechanism of how this potentially can be manipulated to help our patients. Can we use this data, find the mechanism, and use it for our advantage in terms of micromodulation, and potentially microbial modulation, to improve response to chemotherapy and improve response to immunotherapy in our bladder cancer patients?

Ashish Kamat: That was great, Laura, very nice present, and congratulations on some really good work. People sometimes joke about the microbiome to say that your gut is not Las Vegas. What happens in your gut does not stay in your gut. It affects things all over the body. And there's a widespread recognition that the microbiome, in fact, affects things all the way from of Alzheimer's disease to cardiovascular disease and clearly cancer, so it's not surprising that with the tools that we have nowadays, people are able to come up with the distinctions that you did in your study. And I really want to, again, congratulate for actually doing this.

Could you, based on your work that you've done so far, and I'm sure you've done a lot more than what you presented today, share some of your thoughts on whether you think that the microbiome in the cancer and non-cancer subjects is a dynamic process? And if so, what would you postulate is the appropriate time point at which that assay or the assessment should be made to make that differentiation?

Laura Bukavina: That's a great question. And then not only were we curious whether microbiome is a dynamic process, but also we're curious, is microbiome also a process of your environment? So, is this the process that correlates to your significant other? And we have looked at this. We have looked at tangential time points during their neoadjuvant chemotherapy to look at changes in microbiome. And then we did the same thing with their partners. People that they live with, eat the same diet, have same exposures on a daily basis, and we looked at their microbiome to potentially see other factors outside of what we see in the hospital, in these bladder cancer patients that could be affecting our outcomes.

What we found is that even if they're living in the same household, they're eating the same food, and they're being exposed to the same environment, their microbiome is still different. And patients that are going through neoadjuvant chemotherapy, right before their second treatment at the start of neoadjuvant chemotherapy is sort of their defining point. So it's not before neoadjuvant chemotherapy, it's not right after, it's right after that first cycle where we're able to determine their baseline microbiome.

Ashish Kamat: That's interesting. So what you found was that it wasn't the inducible, but it was almost, in other words, after they start the chemotherapy, what's starting to happen with the microbiome that is correlated, let's say, and so we validate it's not necessarily.

Laura Bukavina: Right.

Ashish Kamat: No, it's correlated with their response. Any mechanistic ideas as to why that might be the case?

Laura Bukavina: It could be. We started thinking about potentially different microbial compositions and how resistant they are to chemotherapy. Are there byproducts of chemotherapy that certain bugs utilized for metabolism? And then we started thinking about other ways that, potentially, some of these bugs are actually working with the chemotherapy, such as Lactobacillus. Back 15, 20 years ago, Lactobacillus was one of the first immunomodulating bugs that we used in bladder cancer, and that's because it's known to initiate a CD8 response. So is it that the chemotherapy itself is starting a cytotoxic immune reaction to cancer and having a certain microbial composition works synergistically with chemotherapy, is sort of our ongoing hypothesis at this point.

Ashish Kamat: Right? And then, again, this is work that has been done, especially in colorectal cancer or gastric cancers, and there's a good correlation, especially between, as you mentioned, the Bacteroides and the Fusobacteriota and response to checkpoint inhibitors and chemotherapy, as well. So it makes perfect sense that we would see this in other cancers. And like I said, it's always great to have results that fit in with what we and others and you would have hypothesized.

In the interest of time, let me sort of flip it back over to you with maybe some high-level thoughts that you want to share with our audience on things that you are planning to do with the data you've collected, future directions you're taking it in?

Laura Bukavina: Sure. Well, what we're planning to do next is switch from just characterizing microbiome, and we're working on more animal models, murine models, to see if we can harvest potential fecal microbiome and micromodulation in mice and change the course of their treatment response by modulating the composition of their gut and urine tract.

Ashish Kamat: Great. Thank you once again, Laura, for taking the time and spending it with us today, and come congratulations again on your award.

Laura Bukavina: Thank you so much, Dr. Kamat.