Stool Microbiome Signature Predicts Response to Neoadjuvant Pembrolizumab in Muscle-Invasive Bladder Cancer - Filippo Pederzoli & Massimo Alfano

May 8, 2024

Alicia Morgans hosts a discussion with Massimo Alfano and Filippo Pederzoli on the promising link between the stool microbiome and the effectiveness of neoadjuvant pembrolizumab in treating muscle-invasive bladder cancer. This intriguing connection explores how the microbiome could serve as a predictive biomarker for immunotherapy outcomes. Dr. Alfano credits the genesis of their research to prior clinical and preclinical studies, combined with the unique multidisciplinary collaboration at San Raffaele Hospital. They probe the microbiome’s role in modulating immune response, potentially enhancing the effectiveness of treatments like immune checkpoint inhibitors which have shown notable results in some bladder cancer patients. Dr. Pederzoli discusses the design and findings of the PURE-01 trial, which substantiated the microbiome's impact on treatment efficacy, setting the stage for future interventions that could manipulate gut bacteria to boost immunotherapy responses.


Filippo Pederzoli, MD, Postdoctoral Associate in Pathology and Laboratory Medicine, Weill Cornell University, New York, NY

Massimo Alfano, PhD, MSc, Researcher, Biologist, Group Leader of the Extracellular Microenvironment Unit, IRCCS Ospedale San Raffaele, Milan, Italy

Alicia Morgans, MD, MPH, Genitourinary Medical Oncologist, Medical Director of Survivorship Program at Dana-Farber Cancer Institute, Boston, MA

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Alicia Morgans: Hi, I'm so excited to be here today with Professor Massimo Alfano, who's joining me from the San Raffaele Hospital in Milano, Italy. Also, Filippo Pederzoli, who is joining me from Cornell University in New York, but was previously in Milan as well with that group, together today to talk about the stool microbiome and the signature that seems to be associated with response to neoadjuvant pembrolizumab in patients with muscle-invasive bladder cancer, recently published in European Urology. Thank you, both, so much for being here with me today.

Massimo Alfano: Thank you for the invitation.

Filippo Pederzoli: Thank you.

Alicia Morgans: Wonderful. So, the stool microbiome has been a really interesting aspect of care and of sincere interest by many people in the urologic community, as well as beyond. I wonder if you could tell us, Massimo, what really prompted you to be interested in this question of the gut microbiome, and immunotherapy, and bladder cancer? And really, what was it that made you think that there might be an association that you could investigate in the PURE-01 trial, where you looked into this?

Massimo Alfano: Okay. So, thank you, Alicia, for the question. Basically, we started the project about gut microbiome, immunotherapy, and bladder cancer based on information that was available from clinical and preclinical studies. And also, by the combination of expertise that is available in my institute, where clinicians and basic scientists work together.

And basically, in the last few years, there have been several studies showing that immunotherapy, and in particular, the introduction of immune checkpoint inhibitors, demonstrated efficacy against solid tumors, including bladder cancer. So indeed, we and others demonstrated that the use of immune checkpoint inhibitors drastically improved the cure of metastatic bladder cancer patients, that compared to conventional chemotherapy, induce a complete pathological response in about 30% of the patients. Meaning that, at the end of the day, these patients do not have disease. Do not have residual disease upon the completion of therapy with immune checkpoint inhibitors. So, we asked ourselves why the immune checkpoint inhibitors work in 30% of the patients, but not in the other 70%?

And, we reasoned... I mean, we are aware that the efficacy of immunotherapy is based on the status of the immune system of the patient. And that most of the immune system is located in the intestine. And another issue is that, in the last 10 to 15 years, it has been demonstrated in vitro and in vivo, that the intestinal microbiome regulates the maturation and differentiation of the immune cells that are resident in the intestine. And that, gut microbiome was also... and also, that modification of the intestinal microbiome was associated with response and non-response to immunotherapy, in particular, in melanoma patients. So we asked ourselves if the intestinal microbiome could also be a marker predictive of response to immune checkpoint inhibitors also in bladder cancer patients? So, this is why we started this project.

Alicia Morgans: And so interesting... as you said, there has been this association between treatment effect in other solid tumors that are very interesting to investigate in bladder cancer. And potentially, an area where we might be able to alter the gut microbiome over time as studies continue, and actually have an intervention around the basic information that you're learning from your studies. So, thank you for that. Very, very exciting and thought-provoking. Now I wonder, Filippo, can you remind us what the design of the PURE-01 trial was? And what did you really investigate? And I know you had both an investigation within PURE-01, but you also had a preclinical model that you investigated as well.

Filippo Pederzoli: Sure. Thank you very much for the question. So the PURE-01 trial, as you may know, is one of the landmark trials that marked the advent of immunotherapy in bladder cancer. Led by Professor Andrea Necchi in Milan, the single-arm phase two clinical trial tested three courses of neoadjuvant pembrolizumab, an anti-PD-1 immunotherapy drug in patients with organ-confined muscle-invasive bladder cancer, before radical cystectomy as definitive surgical treatment.

The primary endpoint of the trial was the pathologic complete response rate in the radical cystectomy surgical specimen, which was reached in about 40% of patients. So very interesting results for our patients. With the increasing number of available drugs targeting different molecules and pathways in the immune response, and in the immune response against cancer, huge efforts are actively devoted to the search for the best biomarker or the best combination of biomarkers that might assign the right treatments to the right patients, to maximize the likelihood of response and survival.

In the context of the PURE-01 trial, many biomarker translational studies have been conducted by Dr. Necchi and colleagues, including us. We have learned that those with the highest pretreatment immune gene signature scores, those with high expression of the PD-L1 biomarker, and those harboring the highest tumor mutational burden levels, yielded the highest probability of benefiting from neoadjuvant pembrolizumab.

Of course, among the rising biomarkers in cancer immunotherapy, the microbiome, the intricate ecosystem of microbes residing in our bodies has emerged as a critical factor for the success of immunotherapy. Research indicates that the diversity and composition of specific bacterial species present in the microbiome can profoundly influence the immune system's response to therapy. Serving both as a predictive biomarker, or as we were saying before, a modifiable element to try to boost and increase the efficacy of immunotherapy.

For those reasons, we decided to investigate the microbiome, also in the context of the PURE-01 trial.

Alicia Morgans: So interesting. And I wonder, what did you find in your investigation that really warranted publication in such a phenomenal journal, European Urology? We'd love to hear.

Filippo Pederzoli: So, we performed microbiome analysis of pembrolizumab. Samples before the starting of immunotherapy, from 42 patients from the PURE-01 studies. Among them, 23 of these patients showed a pathologic response, either complete or partial. Of course, we focus mostly on the intestinal microbiome as defined through the stool samples, because that's the area of our body where there is more interaction between the immune system and the resident microbiome.

Among all the identified bacteria, 16 bacterial taxa... taxa, are the names we give to bacteria in a hierarchical way... in the taxonomic way, 16 were differentially expressed between responders and non-responders. Notably, the genus Suterella was more abundant in responders. Whereas the species Ruminococcus bromii was more abundant in non-responders. We also decided to test these 16 differentially expressed taxa for their association with event-free survival. Which is a survival estimate that has been recently reported for the whole PURE-01 clinical trial, at a three-year median follow-up. Among the identified taxa, the phylum Proteobacteria and the species Ruminococcus bromii showed the best association with event-free survival, after dichotomizing the patients into low and high abundance for each taxa.

Then we decided to do something more to try to validate, or at least, to get supporting evidence about our findings from human samples. And to do so, we first investigated whether a widely used preclinical model of bladder cancer, namely the orthotopic MB49 mouse model, could be a valid prototype to study the interaction between the microbiome, immunotherapy, and bladder cancer. To this end, we decided to test if enriching the microbiome of the mice with Ruminococcus bromii, the bacterial species that we identified from the human samples linked to worse response in humans, would decrease the efficacy of anti-PD-1 immunotherapy. And the data we got from the mouse studies suggested the evidence that the enrichment of the murine gut microbiome with Ruminococcus bromii actively decreased anti-PD-1 efficacy in the mice.

So to sum up, our study on stool samples from the patients enrolled in the PURE-01 clinical trial highlighted different bacterial taxa associated with response or lack of response to neoadjuvant pembrolizumab. This data laid the foundation for further investigation to validate our study, to investigate the role of the gut microbiome and their potential actionability and druggability in the context of immunotherapy for bladder cancer patients.

Alicia Morgans: It's so exciting. And, as I think about where you go from here with this work, I wonder, how... and you may or may not know the answer to this, so there's no pressure here, but how manipulable or changeable are these particular bacteria in the gut? Can we drug them to get rid of the ones that are associated with poor outcomes? Can we give someone a probiotic, or a stool transplant if absolutely necessary, but otherwise a probiotic to give them the colonies that are more associated with response?

Filippo Pederzoli: You are really asking the key question around the microbiome field right now, because we have to understand that we need personalized and targeted ways to interact with the microbiome. There is not one solution that fits all problems. Indeed, we observe... in line with other studies done in melanoma, or kidney cancer, or other tumors that really respond to immunotherapy, that the administration of antibiotics in patients undergoing immunotherapy has detrimental effects. We observe that antibiotic therapy given during or immediately before the treatment with neoadjuvant pembrolizumab in the PURE-01 trial had a negative effect on the efficacy of immunotherapy itself.

In our previous studies from our group, we saw that the use of antibiotics concurrently with neoadjuvant pembrolizumab in the PURE-01 study was negatively associated with pathologic complete response rates with an odds ratio of 0.18. It was also associated with a higher recurrence rate with a hazard ratio of 2.64.

From this observation, we deduced that the more likely explanation was that antibiotics were affecting, in a detrimental way, the microbiome-immune system-cancer immunotherapy axis. And that prompted us to study the microbiome, as we are discussing right now. As we mentioned before, the microbiome can be an actionable target and we are actively studying how to do this. We can do this as suggested through prebiotics, probiotics, or fecal microbiota transplantation to optimize the gut flora. These approaches aim to cultivate a microbial environment that supports immune function, and complements the immunotherapeutic agents.

Novel ways to interact with the immune system and the immunotherapy outcome also include synthetic biology, in which there are emerging engineering bacterial strategies to modulate the immune response directly at immune sites. Potentially, increasing the precision and effectiveness of the treatment. By integrating microbiome science into oncology, in a precision medicine way, we hope to unlock new therapeutic potentials that make cancer treatment not only more effective but also tailored to individual patients' microbial and immunological profiles.

Alicia Morgans: Fantastic. This is very exciting work, and I feel it may have implications beyond urothelial carcinoma, in this particular setting alone. And, I look forward to seeing how things continue to develop. And Massimo, I will give you the final word. What is the summary of your findings and the final message to listeners as they are considering your paper and its implications?

Massimo Alfano: Okay. Thank you, Alicia, for the question. I believe that our findings are relevant to a wide range of audiences consisting of both clinical and preclinical researchers, in different fields. First, because the intestinal microbiome community could represent a marker predictive of response to immune therapies, as we and others have demonstrated.

Second, because the intestinal microbiome affects the function of the immune system. And this interaction occurs through a mechanism that is mainly through the release of bacteria metabolites. So, as Filippo mentioned already, the second message that I want to provide to the listeners is that the efficacy of immune therapy for several neoplastic diseases, but also non-neoplastic diseases, can be modulated. For example, by intervening on the composition and the richness of the intestinal microbiome.

The third message that I want to provide, according to the discussion by Filippo, is that dysbiosis, induced also by antibiotics, has a negative effect on immune therapy. So, my suggestion to the listener is that the use of antibiotics in the context of immune therapy must be clearly evaluated.

And finally, in conclusion, our findings together with the information gained by other studies, show that interaction among clinicians and preclinical researchers is strongly recommended to improve the effectiveness of the medicine and the quality of life of the patients.

Alicia Morgans: Fantastic. I continue to be amazed at the way that our understanding of therapeutics evolves beyond simply the drug itself, to understanding biomarkers of the disease. And increasingly, biomarkers of the microbiome that may influence the outcomes, the effectiveness, and even as you said, the quality of life of the patient. So, thank you both so much for this incredible work, and for taking the time to share it with us today.

Massimo Alfano: Thank you, Alicia.

Filippo Pederzoli: Thank you very much.