Ashish Kamat: Welcome to UroToday's Bladder Cancer Center of Excellence. I'm Ashish Kamat from Houston, Texas, at MD Anderson Cancer Center. It's my pleasure to welcome today, Dr. Sita Vermeulen, who's joining us from Nijmegen, the Netherlands. Sita is Associate Professor of Genetic Epidemiology and is well-entrenched within the Department for Health Evidence Cancer Epidemiology Group, and has done a lot of work over the years in the field of bladder cancer, especially as it relates to BCG and its effects on patients that have a diagnosis of bladder cancer.

Sita recently was lead in the work that went on with trained immunity as a molecular mechanism for BCG immunotherapy in bladder cancer. Dr. Vermeulen, it's really our pleasure to welcome you today to present your work. So the stage is yours.

Sita Vermeulen: Okay, thank you, Ashish, for your introduction, and thank you to UroToday for giving me the opportunity to speak about the perspective that we recently published on immunity as a molecular mechanism for BCG immunotherapy in bladder cancer. This perspective was written by Jelmer van Puffelen, a PG student supervised by myself, and all of our Team C colleagues, Professors Joosten and Netea. They are both experts in the field of immunology and infection, and they lead the founding lab of trained immunity, or TRIM, in short, and non-specific memory of the innate immune system. And I will explain that in more detail later.

So their labs showed that BCG vaccination against tuberculosis induces TRIM, and reached out a number of years ago to our [inaudible], to collaboratively study the trained immunity effects of BCG instillations in bladder cancer. And as a part of that collaboration, we wrote this perspective.

So my presentation will basically follow the outline of the perspective. But here, I've added some slides containing preliminary results of the TRIBUTE study that we are currently performing.

BCG is a vaccine-derived from the live attenuated strain of mycobacterium bovis, and used for vaccination against tuberculosis. It's also the gold standard adjuvant therapy for high-risk nonmuscle-invasive bladder cancer. The treatment schedule consists of six weekly inductions, it's the six weekly induction cycle, followed by multiple three weekly maintenance cycles, during one, two, three years.

There are multiple studies that have shown its favorable effects on recurrence progression and complete response rates, in comparison to a transurethral resection of the bladder tumor only, or TURBT plus chemo installations. There've also been many studies focused on describing and understanding the induced immune response, and the antitumor activity of BCG instillations. And I guess that we can say that the immune response entails a very broad activation. It's complex, and it's reasonably described, but the actual antitumor activity is, however, less well understood.

Reverse BCG response is proposed to tell a number of steps. First, the attachment of BCG to the urothelium. And second, the uptake by residents, macrophages and cancer cells, and urothelial cells, though there is unclarity about the latter. So subsequently, a strong innate immune response follows, including the secretion of pro-inflammatory innate cytokines, including IL-1β, TNF, IL6, and GM-CSF. This was also new recruitment of a variety of immune cells, including neutrophils, monocytes, macrophages, TMV cells, and natural killer cells, to the bladder. And also the formation of granulomas, which mainly consist of an aggregation of macrophages.

The involvement of the adaptive immunity is crucial for BCG response, and it's triggered by BCG antigen presentation by MHC-II antigen-presenting cells, and that results in a pro-inflammatory T helper 1 immune response. So based on the release of interferon-gamma, also enables cytotoxic CD8 T-cell function, and they enhanced the cytotoxicity of NK cells. So when all the immune response is proposed to results in the local presence of a variety of potentially cytotoxic immune cells and soluble factors, including, for instance, [inaudible]. And they all could be involved in the tumor cell killing.

One of the aspects of the innate immunity that has not been a topic of attention and research in BCG and bladder cancer is TRIM, or train immunity, the nonspecific, or long-term memory of the innate immune response. And the concept of TRIM is briefly explained in this figure.

So here, we see a naïve monocyte that has been exposed to BCG. It has uptake of BCG and subsequent production of pro-inflammatory innate cytokines. The exposure to BCG also causes epigenetic rewiring via histone modifications, including the enrichment of activating trimethylation of histone 3, [inaudible] 4, and promoters of pro-inflammatory cytokine genes, including TNF and IL6, as depicted here.

The exposure to the pathogen also causes changes in the intracellular metabolism, including, for instance, increased glucose consumption. So as a result of the metabolic and epigenetic rewiring, BCG trained innate immune cells produced more pro-inflammatory cytokines than non-BCG trained cells when they are challenged by the second stimulus, that is either related or unrelated to the initial training stimulus, in this case, BCG. And PAMP stands for Pathogen Associated Molecular Pattern, and for damaged, or dangerous, associated molecular pattern. So these are the three hallmarks of trained immunity.

The first indication for the existence of TRIM came from the observed broad protection of BCG vaccination, beyond its protection from tuberculosis. TRIM is a very active field of study, and more is learned about the exact mechanisms, continuously. I think that for now, it's important to mention that it has become evident that BCG imprints a persistent transcriptomic myelin bias on human hematopoietic progenitor STEM cells, and that increased immune response persists for at least a number of months.

While a logical hypothesis insinuates that two gene instillations in the bladder induce a TRIM phenotype in bladder cancer as well. But there are, of course, differences, compared to intradermal BCG vaccination; including the systemic versus the local application, and the HM profile of the vaccinees, versus the bladder cancer patients.

One of the main goals of our perspective was to assess the presence of a TRIM phenotype after bladder instillations with BCG in bladder cancer by evaluating the existing literature. So data on epigenetic rewiring, or metabolic configuration, was lacking, to our knowledge. But what we did find were three papers that reported on cytokine production of ex-vivo stimulated monocytes, from obesity treated bladder cancer patients. We also identified one paper that reported on the relative mRNA levels in whole blood during BCG treatment. And we could refer to five papers that report on changes in urinary cytokine levels throughout BCG therapy. The result of this paper actually aligned with the expectation for TRIM, namely an increased cytokine response after an increased number of BCG instillations.

So in the perspective, we also propose a mechanism of BCG-induced TRIM in bladder cancer. Locally, it starts with the uptick of BCG, and then the monocytes, and of the subsequent differentiation of the monocyte into the macrophage after the production of cytokines by the monocyte. And then there are these epigenetic and metabolic reconfigurations that I've explained in one of the earlier slides. So there's ample opportunity for the required second stimulus in the microenvironments of the bladder. And these include the vesicant pathogen-associated molecular patterns originating from, well, either that, or residing material from BCG itself, but also dams could function as good stimulants. And you can think of molecular patterns that are released by necrotic cells. So there's a systemic level, BCG induces trained immunity in hematopoietic STEM cells, and multiphoton progenitors in the bone marrow, and that this results in the circulating epigenetically and transcriptionally modified monocytes and macrophages that have a strong pro-inflammatory phenotype.

In order to study the TRIM phenotype in BCG, we took bladder cancer patients, we set up the TRIBUTE studies a few years ago. That study is coordinated by Jelmer van Puffelen and performed in the Radboud UMC, and Erasmus MC, in the Netherlands. So in this study, we collected blood samples at nine different time points during the first year of BCG treatment. And we used these blood samples to measure epigenome and transcriptome profiles in isolated PBMCs. And we also measured cytokine production and ex-vivo stimulated PBMCs, and measured an information protein panel in the plasma.

So between June 2018, and March 2020, we were able to include 17 high-risk BCG-naïve nonmuscle-invasive bladder cancer patients that fulfilled the eligibility criteria. And we've just finalized the first analyses, and I will share some preliminary results in the next few slides.

In this slide, we see the TNF levels in supernatants of PBMCs that have been stimulated for 24 hours with either LPS or PAM 3 cysts. Each row in this figure represents a single patient and each column a different time point during the BCG treatment. The color of the squares indicates the full change of the TNF level over the pre-BCG levels.

What we can see here is that almost all patients show a TRIM phenotype already after a single BCG instillation. You can also see that there's a high variation in TRIM responses between patients. What's also noticeable is that there is an increased response also at the beginning of the maintenance cycles, indicating a long-term TRIM phenotype. What we also see is a decrease at the end of the induction and the maintenance cycles, potentially indicating tolerance. What is also visible here is that the observed effects are independent of the used stimulants, as they are pretty comparable for LPS and [inaudible].

We also performed ChIP-seq for the activating H3K4 trimethylation mark in seven patients at five different time points. Some basic results shown here show that there are 375 statistically different peaks compared to pre-BCG. And that the differential peaks are especially visible during the maintenance cycle.

A pathway analysis using all genes that show differential peak changes between any of the time points indicates developments of the logical, plausible candidates' biological processes, and that these include the immune response and leukocyte activation.

So the question, of course, rises whether TRIM is also important for the anti-cure response induced by BCG in that scan core efficacy. We will look into that in the TRIBUTE study, but will not be able to fully answer that question, given the limited number of patients that we have. But a recently published study by the group of Bivalacqua, however, indicates that the efficacy of BCG indeed evolves the induction of TRIM. It showed that STING activation by a recombinant BCG, this thing stands for a stimulator of interferon genes, leads to a more potent trained epigenetic phenotype, with increased chromatin activation mark, and decreased impressive marks, and TNF and IL6, compared to wild-type BCG. That's indicated in these figures here. And they also show that this more potent TRIM phenotype, induced by the recombinant BCG, has immunotherapeutic benefits in rats and mice. So I think this is all very promising.

So in conclusion, we can say that there are strong indications for induction of a TRIM phenotype through BCG instillation, and that there is also a potential role for TRIM in BCG response. And I think this offers very welcome opportunities to optimize BCG treatment for nonmuscle-invasive bladder cancer patients. First, by exploiting TRIM in therapy, by optimizing TRIM, and second also, by the options of improving the selection of therapy type, and dosage, and frequency, for the individual patients.

I would like to end this presentation by acknowledging the authors of the prospective paper and those involved in the patient inclusion, the sample collection, the data generation, the data analysis, within the TRIBUTE study.

Ashish Kamat: Thank you so much, Sita. That was very enlightening, and very succinct in a concise manner. Obviously, a lot of work has gone into the data that you presented, and in the interest of time, I appreciate you keeping it short and sweet. If I could ask you a couple of questions, and feel free to answer based on work that you've done before, as well. How would you use this data today to advise patients and providers as to the need to continue on maintenance therapy with BCG beyond just the initial induction?

Sita Vermeulen: Well, I think that it has already been shown, that the relevance of the maintenance cycle has been already shown in multiple studies. And I think that what we find here in the studies into TRIM is that it, also from that perspective, confirms the relevance of the maintenance cycles, especially in the results that we just showed you. So from that respect, I think we can only confirm the relevance also, from this perspective.

Ashish Kamat: Do you think that you'd be able to use data that, or assays that you are developing, or have developed, to guide the number of installations and the duration of therapy for an individual patient? Where do you see the field as far as personalizing BCG therapy is concerned?

Sita Vermeulen: If we could do that, that would be great. I think, however, that we do need some more understanding of the actual induction of TRIM immunity. It's relevant, of course, it's not the only mechanism by which BCG exerts its effects, but I think we need more data on optimizing the TRIM immunity response, and what it needs to be optimized.

I think there are now also, as I'm speaking about the trial, the NIMBUS trial that just came out, and that showed that a reduction, for instance, of the induction cycle to the first, second, and sixth instillation, only leads to an increase of recurrences. But seeing the results that we have, and I wonder whether reducing it to instillation one, two, three, four, would be sufficient. The same may hold for the maintenance cycles. I mean, we know it's necessary, but maybe two would be sufficient. I think there were a lot of questions about the ultimate frequency of the instillations.

Ashish Kamat: You make good points. Because, of course, it's been known and studied for many years now, by many groups, including ours and yours, that the cytokines and the response does tend to vary between BCG-naïve and prior exposed patients. And it seems to arise up to the fourth indefinitely, in some patients, the fifth instillation of the BCG from the induction. But when it's maintenance therapy, after the third instillation, you actually have a drop in the response because it's an immune therapy and it's following the bell-shaped curve. But, everybody's sort of trying, and the NIMBUS trial is one example of a one size fits all. And I suspect, based on some of the data that you've shown today and shared, that it's not one size fits all. Each individual patient will probably have a varied response to the immune stimulation that BCG brings, some of it based on innate immunity, some of it based on trained immunity, which clearly has implications for the treatment of patients with nonmuscle-invasive bladder cancer.

Could I ask you your thoughts on the relevance of the trained immunity when it comes to using BCG for other indications? And now, again, I'm sure you're aware of the multiple trials that are going on throughout the world that Mihai sort of initiated and helped us develop when it comes to BCG with COVID-19, for example. But if we look at COVID-19, if we look at its effects on yellow fever and other infections, how strong overall do you think BCG's trained immunity aspects play when it comes to the implications for BCG as a vaccine in the infectious disease world, number one? And number two, what do you think is its relevance when we're thinking of combination therapies, for example, with pembrolizumab, or atezolizumab?

Sita Vermeulen: I think this would be an excellent question to ask Mihai when I see him next time, your first question. Like I said, there are trials ongoing in relation to COVID. It is already known that BCG vaccination reduces also the subsequent risk of, for instance, respiratory tract infection and sepsis. So a lot is ongoing with regard to death in the field of BCG vaccination. I think now he will be very able to answer that question. What is interesting to mention in this case, is that there are, of course, patients now that receive BCG instillation, but also a question, "Am I now also protected for COVID, or for other infections as well?". I'm thinking our data are too limited at this point, to make any conclusive remarks in that respect. And your second question was?

Ashish Kamat: How do you think the TRIMs would be useful when it comes to combination therapies of BCG with, for example, checkpoint inhibitors?

Sita Vermeulen: Yeah, so that's also something I think, well, I see the induction of TRIM as sort of a long-term amplification of the innate immune response, where the body remains in full attack mode, and its tumor antigen unspecific, let's say. So it all started with a strong innate immune response that leads to a type 1 TH to help a response. So of course, it's a different mechanism from the adaptive immunity based on checkpoint inhibitor therapy. So how they exactly intertwine, it's difficult for me to say, but I do think that it's relevant in that respect, that in all these novel trials with novel therapeutics, that collecting biomaterial that would allow some measurements of TRIM phenotypes, for instance, will be very relevant to gain more information and more empirical data on that.

Ashish Kamat: Great. Once again, I want to thank you for taking the time to join us today. This has been very informative. In closing, if you want to leave the audience with one or two take-home points regarding your study, what would those be?

Sita Vermeulen: I think one of the things is to keep in mind that TRIM has been overlooked, but it is relevant, but still, of course, a lot of research needs to be done to really pinpoint the exact mechanism, and how to exploit it in the treatment of bladder cancer. And yeah, I would like to repeat the last remark. I think it's very important that, in these novel trials, people are aware of this, and collect relevant biomaterials for us to be able to answer questions in this respect.

Ashish Kamat: Great. Once again, Sita, I want to thank you for taking the time and joining us today at the Bladder Cancer Center of Excellence. Stay safe and stay well.

Sita Vermeulen: Thank you.