INTRODUCTION:Growing data support interactions between host-gut microbes and treatment responses in multiple myeloma (MM), where a higher abundance of Eubacterium hallii in stool samples has been found among MM patients with negative minimal residual disease after induction therapy. Here, we evaluated changes in the gut microbiome associated with daratumumab (dara) based therapy in 40 MM patients, before and after therapy. PATIENTS AND METHODS:Patients with relapsed MM and prior autologous transplantation who had received 1 to 4 prior lines of therapy were eligible. Two stool samples were collected, one within 1 week prior to dara (predara) and one immediately after 4 doses of dara (postdara). Metagenomics sequencing was conducted. Microbiome taxonomic analyses were performed using MetaPhlAn4, and microbial functional pathway analyses were conducted using HUMAnN3.6. QIIME2 was used for compositional and statistical analyses. RESULTS:Of 40 participants enrolled, there were 5 nonresponders; 35 patients achieved partial response (PR) or better (responders). Among responders, 10 patients achieved complete remission (CR), and 25 patients achieved either very good partial response (VGPR) or PR. There were no statistically significant differences between overall pre and postdara gut microbiomes. Differential abundance analysis (ANCOM-BC) showed statistically significant (q ≤ 0.05) overgrowth of Alistipes finegoldii and Acidaminococcus intestini species in responders and Ruminococcus torques, Sellimonas intestinalis and Clostridium symbiosum in nonresponders. Compared to non-CR, CR samples showed enrichment of Faecalibacterium prausnitzii; non-CR samples were enriched in Segatella copri and Faecalimonas umbilicata. DISCUSSION/CONCLUSION:Our results suggest differences in species between clinical responders and nonresponders, but larger prospective studies are needed to confirm these results.

503 Background: CBM588 has shown potential to improve clinical outcomes when combined with immune checkpoint inhibitors (ICIs) in untreated mRCC (Dizman et al Nat Med 2022; Ebrahimi et al Nat Med 2024). Since no significant treatment-related adverse events were related to CBM588 in previous studies, we sought to determine if CBM588 capsules, a novel formulation of this live biotherapeutic, designated MO-03, could be delivered at higher doses with similar tolerability and greater biologic effect. Methods: Patients (pts) with treatment-naïve mRCC and any international mRCC Database Consortium (IMDC) risk were treated with nivo (3 mg/kg q3w ×4, then 480 mg q4w) and ipi (1 mg/kg q3w ×4) plus CBM588 at one of three dose levels (4 × 10⁸, 1.2 × 10⁹, or 4 × 10⁹ CFU BID) in a 3+3 dose-escalation design. The primary endpoint was safety, with secondary endpoints including response rate and progression-free survival. Stool samples collected at baseline and Week 13 were analyzed by metagenomic sequencing to assess microbial composition across CBM588 dose levels. Plasma samples collected at baseline and Cycle 3 were analyzed for soluble mucosal addressin cell adhesion molecule-1 (sMAdCAM-1), a biomarker associated with dysbiosis (Fidelle et al Science 2023). Results: Twelve pts were treated between 6/26/2024 and 9/15/2025. Median age was 69 (range 48-80). All had clear cell histology without sarcomatoid features; two had rhabdoid features. Half the cohort had ≥3 metastatic sites, most commonly involving the lung, lymph nodes, and contralateral kidney. Most patients had favorable (50%) or intermediate (42%) IMDC risk. No dose-limiting toxicities (DLTs) were observed at any dose level, and no dose-dependent increase in immune-related toxicity was noted with CBM588 escalation. Notably, Bacteroides thetaiotaomicron showed a dose-dependent increase in relative abundance across escalating CBM588 levels (P = 0.0043 for Level 3 vs Level 1 and P = 0.0047 for Level 3 vs Level 2, Mann–Whitney U), suggesting a selective modulation of gut microbial composition. Among 11 pts with evaluable disease, 3 were responders (1 complete response, 2 partial responses), 6 had stable disease, and 2 had progressive disease. Baseline sMAdCAM-1 levels were similar between responders (Rs) and non-responders (NRs) (median: 205,196 vs. 206,462 pg/mL). However, percent change from baseline to Cycle 3 demonstrated a diverging pattern, with an early increase in Rs and relative stabilization in NRs (median: +8.2% vs –0.1%). Conclusions: No DLTs were encountered with CBM588 capsules at the highest dose level (MO-03) in combination with nivo/ipi, supporting the dosing strategy in the upcoming phase III SWOG study S2419 (BioFront). Translational studies indicate a dose effect upon microbiome composition, and sMAdCAM-1 dynamics suggest a potential association with response. Clinical trial information: NCT06399419 .

223 Background: CRC is the third leading cause of cancer related deaths worldwide with age and diet among the strongest risk factors. Emerging evidence suggests gut microbiome plays important role in CRC and is highly impacted by the exposome primarily the food intake. Since diet and age plays an important role in gut microbiome diversity, the present study aimed to investigate the alterations in the gut microbiome in young versus old mice harboring CRC allografts and fed with different diets. Methods: Two cohorts of C57BL/6 including young (n = 9, age = 6 weeks) and old (n = 9, age = 20-24 months) mice were implanted with 1x10 6 MSI (microsatellite instable) CRC MC38 tumor cells. Mice in both cohorts were randomized into three different diet groups: normal diet (ND; standard chow), high-fat (HF) and calorie-restricted (CR: 30% reduction in total calories). Mice were housed individually under standard laboratory conditions. Mice fecal samples were collected and subjected to DNA isolation (ZymoBIOMICS-96 MagBead DNA Kit), followed by metagenomic shotgun and whole genome sequencing (ZymoBIOMICSe and Illumina NovaSeq, respectively). P ≤ 0.05 were considered as statistically significant. Results: Fecal microbiome analysis showed that old microbiome has higher alpha diversity compared to the young mice. No statistically significant differences in were found in microbiome diversities between the diet groups. Pairwise permanova results showed statistically significant differences between microbiomes of old vs. young groups ( P : 0.001), CR vs. HF groups ( P : 0.012) and HF vs. ND groups ( P : 0.021). ANCOM-BC analysis determined the differentiating features and microbial functional pathways in the young mice compared to the old mice group and in different diet groups with relative abundance differences of larger than log 10 2. Bacteroides thetaiotaomicron ( P : 1.43E-39) and Parabacteroides goldsteinii ( P : 2.20E-23) were enriched in young and old groups, respectively. Akkermansia muciniphila ( P : 0.008) was significantly enriched in ND compared to CR group. Lactococcus lactis ( P : 9.94E-160) and Lachnospiraceae bacterium A4 ( P : 4.95E-10) were significantly enriched in HF compared to ND diet groups and vice-versa, respectively. Among the functional pathways, CMP-legionaminate biosynthesis I ( P : 2.38E-13) and L-arginine biosynthesis IV (archaebacteria) ( P : 4.77E-11) were the most significantly enriched in young and old groups, respectively. Conclusions: Our findings highlight the differential diversity and microbial features of gut microbiome in age- and diet-induced CRC progression. Collectively, alteration in diet and age of the host lead to changes in gut microbiota which has a direct impact on activation of specific signaling pathways, metabolism and local and systemic immune responses, which may in turn affect chemosensitivity and outcome in CRC

Colorectal Cancer (CRC) is the third most prevalent malignancy, leading to significant morbidity and mortality globally. Epidemiological studies suggest that chronological age and diet are among the major contributing factors correlated with the incidence of CRC. Our study aimed to provide insights into the association between age, diet, and gut microbiome in CRC using molecular techniques including RNA sequencing, cytokine analysis, and metagenomic analysis. We used syngeneic MC38 mice model divided into two age groups (old and young) and three diet groups (standard chow, calorie-restricted and high-fat). The major findings of this study are that age and diet impact intratumoral gene signaling (nuclear and mitochondrial), and hub genes we identified are associated with prognosis in CRC. Fecal microbiome analysis showed that old microbiomes have higher alpha diversity compared to young mice. Our results demonstrate that interactions between host (age) and external (diet) factors regulate tumor growth mediated by cytokines, mitochondrial derived proteins, and the gut microbiome. Collectively, our findings advance current understanding of the mechanisms by which aging, diet and gut microbiota impact CRC onset and progression though further investigation is warranted.

543 Background: We previously reported that combining CBM588 ( Clostridium butyricum MIYAIRI588), a live bacterial product, with cabozantinib (cabo) and nivolumab (nivo) enhanced clinical benefit in treatment-naïve patients with mRCC (Ebrahimi et al ; Nature Medicine 2024). The current study provides updated clinical data to further evaluate the potential benefits of CBM588 in combination with cabo/nivo. Methods: This open-label, randomized trial enrolled patients aged ≥18 years old with a Karnofsky performance status ≥70% and histologically verified (clear-cell, papillary or sarcomatoid component) advanced or mRCC with no prior systemic therapy for metastatic disease. Patients were randomized in a 1:2 ratio to receive either cabo/nivo (40mg PO QD and 480mg IV monthly, respectively) alone or with CBM588 (80mg PO BID). This analysis provides updated secondary clinical endpoints with extended follow-up, including overall response rates (ORR), progression-free survival (PFS), and toxicity. Clinical benefit was defined as complete response, partial response, or stable disease, per RECIST 1.1. The association between treatment arm and ORR was evaluated using Fisher’s exact test, and PFS was estimated using the Kaplan-Meier method. Results: A total of 30 patients (20:10 M:F) were recruited, with a median age of 65 years (range, 36-84). Five patients (17%) had sarcomatoid features, and two (7%) had predominant papillary histology. As of June 1, 2024, the median follow-up was 25.8 months (interquartile range, 19.2-28.1) in the overall cohort. The ORR was significantly higher in the CBM588-containing arm compared to cabo/nivo alone arm (79% versus 20%, P =0.004). In the CBM588 arm, 17 (89%) patients, and in the control arm, 8 (80%) patients had a reduction in target lesion size, with median decreases of 51% (range, 17-94%) and 22% (range, 13-100%), respectively. Clinical benefit for at least 6 months was achieved in 80% of patients treated in experimental arm and 60% patients in the control arm. The median PFS was not reached in patients receiving CBM588, compared to 13.4 months in the control arm. The median OS was not reached in either of the arms at the time of data cutoff. Grade 3 or higher treatment-related adverse events (TRAEs) were observed in 45% of the CBM588 arm compared to 40% in the control arm. The most common TRAEs in the overall cohort were transaminitis (10%), hypertension (7%), and diarrhea (7%), with no significant differences between treatment arms. No new safety signals were detected. Conclusions: The addition of CBM588 to cabo/nivo continues to show promising efficacy in mRCC, with an improved PFS and ORR. The safety profile remains consistent with previous findings, supporting further exploration in larger trials. Further translational efforts are underway to characterize the mechanism through which CBM588 augments clinical activity. Clinical trial information: NCT05122546 .

4550 Background: In two randomized phase I trials, Clostridium butyricum MIYAIRI588 (CBM588), a live biotherapeutic, demonstrated preliminary activity in modulating the gut microbiome, enhancing systemic immune responses, and improving clinical outcomes in patients receiving first-line nivolumab/ipilimumab and nivolumab/cabozantinib for mRCC (Dizman et al. and Ebrahimi et al. Nature Medicine). Herein, we present the long-term follow-up data for nivolumab/ipilimumab with or without CBM588. Methods: Newly diagnosed patients with mRCC, clear cell and/or sarcomatoid histology, and International mRCC Database Consortium intermediate/high risk were randomized to receive nivolumab/ipilimumab with or without CBM588 in a 2:1 ratio. Response outcomes were assessed using RECIST 1.1. Clinical outcomes were secondary endpoints. Objective response rate (ORR; complete response [CR] or partial response [PR]), disease control rate (DCR; CR, PR, or stable disease [SD] > 6 months), progression-free survival (PFS), and overall survival (OS) outcomes were compared across arms. Results: Twenty-nine patients were included in the final analysis: 19 in the nivolumab/ipilimumab with CBM588 arm and 10 in the nivolumab/ipilimumab arm. The median age was 66.2 years, 72% were male, 83% had IMDC intermediate risk and 93% had clear cell histology. Baseline characteristics were similar across arms. ORR and DCR were 58% and 79% in nivolumab/ipilimumab with CBM588 arm versus 20% and 20% in nivolumab/ipilimumab arm, respectively (p = 0.06 and p = 0.004). At a median follow-up of 60.0 (95% CI 51.9-68.1) months, the median PFS was 38.2 (95% CI 23.6-52.8) months in the nivolumab/ipilimumab and CBM588 arm versus 19.3 (95% CI 0-41.9) months in the nivolumab/ipilimumab arm (Hazard ratio [HR] 0.24, 95% CI 0.09-0.61 p = 0.003). At the time of data cutoff, 9 (47.4%) and two (20%) patients were alive in the nivolumab/ipilimumab with CBM588 and nivolumab/ipilimumab arms, respectively. The median OS with nivolumab/ipilimumab with CBM588 was 55.0 (95% CI 10.5-75.5) months versus 39.0 (95% CI 23.7-54.3) months with nivolumab/ipilimumab (HR 0.438 [95% CI 0.17-1.1] p = 0.09). Conclusions: Although limited by the sample size, the combination of nivolumab/ipilimumab with CBM588 demonstrated superior clinical activity over nivolumab/ipilimumab in our cohort. Additionally, ORR, PFS and OS with nivo/ipi/CBM588 exceeded those observed with nivolumab and ipilimumab in historical datasets (Motzer et al. NEJM). Larger efforts investigating the impact of CBM588 on clinical outcomes are underway. Clinical trial information: NCT03829111 .

The microbiome is key to understanding endometrial cancer (EC) etiology and prevention strategies, implicated in the regulation of estrogen in estrogen-driven cancers. Utilizing robust methodologies in the QIIME 2 platform, we examined 16S rRNA vaginal and rectal microbiome data from an EC cohort: 192 women with benign gynecologic conditions, endometrial hyperplasia, or endometrial cancer. Distinct microbial compositions and community networks specific to EC were identified and related to histological grade with adjustments for EC risk factors. Vaginal health-associated Lactobacillus and Limosilactobacillus, and rectal Prevotella and Peptoniphilus, were depleted in EC, while detrimental vaginal Anaerococcus, Porphyromonas, Prevotella, Peptoniphilus, and rectal Buttiaxella were enriched. Significant bacterial features were shared between rectal and vaginal sites in EC, such as Prevotella timonensis and Peptoniphilus A. Vaginal Lactobacillus abundance contributed to less feature sharing from the rectum. Putative microbial metabolic analysis identified dysregulation of amino acid, complex carbohydrate, and hormone metabolism amongst patients with EC.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited treatment options and poor survival. This study explores how a medically supervised ketogenic diet (MSKD) impacts the gut microbiome and treatment response in patients with newly diagnosed metastatic PDAC receiving gemcitabine, nab-paclitaxel, and cisplatin chemotherapy. As part of a randomized Phase II trial, 32 evaluable patients (median age 65.9 years; 53% male), 16 were randomized to either MSKD or standard diet (SD) combined with chemotherapy. Stool samples were collected from 4 patients in the MSKD group (13 samples) and 8 patients in the SD group (23 samples). Deep shotgun metagenomic sequencing was conducted using NovaSeq (Illumina). Taxonomical and functional microbiome characterization was performed using MetaPhlAn 4.0 and HUMAnN 3.6 (The Huttenhower Lab, Harvard). Comprehensive bioinformatics analyses, using QIIME2, were performed to analyze microbiome composition and function over time. Patients in the MSKD group showed better treatment responses overall. Microbiome profiles between the two groups remained distinct throughout therapy. Though not statistically significant due to sample size, MSKD patients showed lower alpha diversity (Shannon diversity; p-value=0.1339), likely reflecting reduced intake of fermentable fiber. Differential abundance analysis using the ANCOM-BC method revealed a significant enrichment of beneficial taxa in the MSKD group compared to the SD group (p<0.05, log-fold change [lfc]≥2), including Akkermansia muciniphila (linked to improved response to immune checkpoint inhibitors; p=0.017931, q=1), Intestinimonas butyriciproducens (a short-chain fatty acid [SCFA] and butyrate producer; p=3.09E-05, q=0.017), and Streptococcus thermophilus (a supporter of gut mucosal health; p=1.15E-05, q=0.006). During the course of the study, only MSKD patients showed sustained increases in Roseburia hominis (SFCA/butyrate producer; p=3.94E-05, q=0.021) and Lacticaseibacillus rhamnosus (with anti-inflammatory and immune-modulating properties; p=7.28E-19, q=3.98E-16), along with a marked reduction in potentially pro-inflammatory Actinomyces spp.(p≤4.47E-05, q≤0.024). Functional profiling showed increased abundance of cancer-relevant microbial pathways in MSKD patients, including phosphatidate metabolism (PWY-7039; involved in lipid signaling and immune regulation; p=4.36E-04, q=0.20) and allantoin degradation (PWY-5705; linked to redox balance; p=9.16E-06, q=0.004). Conversely, L-ascorbate biosynthesis V (PWY-6415) was reduced (p=3.80E-04, q=0.17), likely due to decreased D-galacturonate substrate availability from fiber restriction. These findings suggest that an MSKD may beneficially modulate the gut microbiome in ways that support and enhance therapeutic response in patients with PDAC. Further validation in a larger cohort is warranted to confirm these results and improve analytical resolution. Keehoon Lee, Derek Cridebring, Gayle S. Jameson, Denis J. Roe, Erkut Borazanci, Diana L. Hanna, Caroline G.P. Roberts, Meredith S. Pelster, Richard C. Frank, Angela T. Alistar, Alan M. Miller, J. Erin Wiedmeier-Nutor, Sandra D. Algaze, Alison R. Zoller, Sarah J. Hallberg, Betsy C. Wertheim, Joshua D. Rabinowitz, Stephen Gately, Jennifer Keppler, Sunil Sharma, Drew W. Rasco, Daniel D. Von Hoff. Ketogenic Diet Modulates Gut Microbiome Composition and Enhances Treatment Response in Patients with Advanced Pancreatic Cancer: A Phase II Study [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B094.

Background: Based on preclinical data demonstrating a synergistic effect of combining immune checkpoint inhibitors (ICIs) with CDK4/6 inhibitors, we conducted a phase I/II study of palbociclib, endocrine therapy, and pembrolizumab in patients (pts) with HR+/HER2- MBC. We previously reported that the gut microbiome can predict clinical response to this ICI combination. Emerging evidence also suggests that the gut microbiome is closely linked to the development of immune-related adverse events (irAE), and that the onset of irAE may be associated with improved ICI response. Here, we explore the role of the gut microbiome on the onset of severe irAE in this cohort of pts with HR+/HER2- MBC treated with palbociclib, endocrine therapy, and pembrolizumab. Methods: Pts with stage IV HR+/HER2- MBC were enrolled and treated with palbociclib, endocrine therapy, and pembrolizumab. Baseline stool samples were collected at Day -28 to C1D1 (N=28 pts). Next on-treatment sample was used if baseline unavailable. Gut microbiota composition was assessed using deep metagenomic sequencing. Taxonomic profiling was conducted using MetaPhlAn4. Functional profiling was performed using HUMAnN3. Correlative studies assessed the association between the gut microbiome and severe (grade >3) irAE. Results: Between March 2017 and May 2022, 40 pts (39 female/1 male) were accrued, with a median age of 51 (range 39-75). With a median follow-up of 35.8 months (mos) (95% CI: 26.1, 63.5), the median progression-free survival (PFS) was 27.8 mos (95% CI: 11.2, NR). The incidence of severe irAE was 11/40 (27.5%). There was no difference in PFS between pts with and without severe irAE (P=0.6). The median PFS was 27.8 mos (95% CI: 6.7, NR) and 27.1 mos (95% CI: 8.7, NR) in pts with and without severe irAE, respectively. ANCOM-BC (Analysis of compositions of microbiomes with bias correction) identified Dialister invisus (log fold change [LFC] 2.45 [P=0.02]), Blautia caecimuris (LFC 2.43 [P=0.01]), Flavonifractor plautii (LFC 2.40 [P=.01]), and Eisenbergiella massiliensis (LFC 2.12 [P=0.001]) in greater abundance in pts without severe irAE, while Coprococcus eutactus (LFC -3.94 [P=0.0009]) was in greater abundance in pts with severe irAE. Beta diversity using Bray-Curtis analysis showed significant differences in gut microbiome composition between irAE groups (P=0.04). Although alpha diversity using the Shannon diversity analysis showed no differences across irAE groups (P=0.3), Shannon diversity analysis showed significant differences in functional genetic pathways between irAE groups (P=0.03). ANCOM-BC identified several distinguishing metabolic pathways between irAE groups, including the CMP-pseudoaminate biosynthesis pathway which was enriched in pts without severe irAE (LFC 2.37 [P=0.0002]). Conclusions: Certain gut bacteria can predict the development of severe irAE in a cohort of pts with HR+/HER- MBC treated with palbociclib, endocrine therapy, and pembrolizumab. Several metabolic pathways were enriched between irAE groups, which suggests differences in immune activation that can lead to the onset of irAE. This study suggests that the gut microbiome can serve as a risk factor and biomarker of severe irAE in breast cancer pts, which warrants further evaluation. Citation Format: Alexis LeVee, Keehoon Lee, Colt Egelston, Susan Yost, Nora Ruel, Paul Frankel, Christopher Ruel, Daniel Schmolze, Peter Lee, Christina Yeon, Yuan Yuan, James Waisman, Sumanta Pal, Joanne Mortimer. Impact of the gut microbiome on immune-related adverse events (irAE) in HR+/HER2- locally advanced or metastatic breast cancer (MBC) patients (pts) receiving palbociclib, endocrine therapy, and pembrolizumab [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-05-24.

TPS611 Background: The combination of nivolumab and ipilimumab is currently one of the standard-of-care treatments for patients with metastatic renal cell carcinoma (mRCC). However, in the vast majority of patients, disease progression eventually occurs. Thus, novel methods to increase the efficacy of nivolumab/ipilimumab are being explored. Recent evidence suggests that the gut microbiome can remarkably influence the efficacy of immune checkpoint inhibitors (ICI) in patients with mRCC, particularly through the modulation of microbial diversity and the presence of key bacterial species (Routy et al . Science 2018; Salgia et al . Eur Urol 2020). The current study explores the use of CBM588, Clostridium butyricum MIYARI588, which has demonstrated the potential to enhance clinical outcomes when combined with ICI therapy (Dizman et al . Nature Medicine 2022; Ebrahimi et al . Nature Medicine 2024). This ongoing phase I study aims to determine the maximum tolerated dose (MTD) of CBM588 in combination with nivolumab/ipilimumab in previously untreated mRCC patients, and to evaluate the biological effects of CBM588. Methods: This investigator-initiated, phase I, open-label, single-arm, dose-escalation study is being conducted in patients with untreated, histologically confirmed mRCC. Eligible patients must be ≥18 years old, have an ECOG performance status ≤2, and be diagnosed with clear cell and/or sarcomatoid histology. The study is open to all IMDC risk groups. Patients must have measurable disease by RECIST 1.1 and no prior systemic therapy for metastatic disease. The study follows a 3+3 dose-escalation design with three planned dose levels for CBM588 (4 × 10 8 CFU, 1.2 × 10 9 CFU, and 4 × 10 9 CFU), administered twice daily orally. Nivolumab (3 mg/kg every 3 weeks for four cycles, followed by 480 mg every 4 weeks) and ipilimumab (1 mg/kg every 3 weeks for four cycles) are administered intravenously. The primary objective is to assess the safety and tolerability with escalating doses of CBM588 in combination with nivolumab and ipilimumab. Secondary objectives include evaluating changes in the gut microbiome, modulation of immune pathways, alterations to the systemic metabolome, and clinical outcomes such as overall response rate (ORR) and progression-free survival (PFS). Responses are assessed by CT every 12 weeks. Dose level one has been completed without dose-limiting toxicities. Enrollment at dose level two began in October 2024. Clinical trial information: NCT06399419 .

4550 Background: Two recent randomized phase I clinical trials have provided compelling evidence that CBM588, a Clostridium butyricum-based live biotherapeutic, holds potential to enhance clinical outcomes in patients with mRCC receiving frontline ICI combinations (Dizman et alNature Medicine 2022; Ebrahimi et alASCO 2023). We examined the impact of CBM588 on gut microbiome composition in a combined cohort of these two studies to further investigate its impact on gut microbiome. Methods: We analyzed stool samples from two phase I randomized clinical trials that enrolled patients with mRCC treated with (1) nivolumab/ipilimumab (nivo/ipi) +/- CBM588 and (2) cabozantinib/nivolumab (cabo/nivo) +/- CBM588. We compared gut microbiome diversity and composition at baseline and week 12 between patients in the standard of care (SOC) arms (nivo/ipi or cabo/nivo) and those who received CBM588 in combination with a SOC regimen (SOC/CBM). Taxonomic profiling was performed using MetaPhlan v4, and changes in the abundances of clinically relevant microbial species from baseline to week 12 were assessed using the Wilcoxon matched pairs test. The ratio of Firmicutes/Bacteroidetes, a measure of gut dysbiosis, was computed across time points in the two cohorts. Results: Among 58 patients included in the analysis, 38 received SOC/CBM588 as first-line treatment. The median age of the overall cohort was 60 years (range: 36-90). The majority were male (71%), had clear cell mRCC (88%), and intermediate/poor risk disease (79%). In both the SOC and SOC/CBM cohorts, there were no statistically significant differences in alpha and beta diversity between baseline and week 12. Among clinically relevant species compared between baseline and week 12, Alistipes senegalensis decreased in both the SOC and SOC/CBM cohorts (log fold change [LFC] -0.82 [P=0.004] and LFC -0.36 [P=0.007], respectively), while Eubacterium siraeum decreased only in the SOC cohort (LFC -1.75 [P=0.005]). The Firmicutes/Bacteroidetes ratio increased from 89.0% to 96.4% in the SOC cohort, whereas a notable decrease was observed in this ratio from 100.0% to 75.7%. in the SOC/CBM cohort. Conclusions: Supplementation with CBM588 leads to a marked correction of gut dysbiosis and prevents the depletion of species previously associated with ICI response (i.e., Eubacterium siraeum). These findings provide a plausible mechanism for the enhanced clinical outcome with CBM588 now seen across two small, randomized trials. A phase III study is planned within the cooperative groups to evaluate the clinical activity and gut microbiome modulation capacity of CBM588 in combination with ICIs in mRCC.

Abstract Background We have previously demonstrated that the orally administered, live bacterial product CBM588 can augment clinical outcomes with cabo/nivo in pts with mRCC (Ebrahimi et al ASCO 2023). As it is postulated this effect may be mediated through changes in systemic immunity, we sought to characterize changes in serum cytokine profile induced by CBM588. Methods Pts with previously untreated mRCC with clear cell, papillary or sarcomatoid histology were enrolled. Key eligibility criteria included Karnofsky performance status ≥ 70% and measurable disease. Pts were randomized to receive cabo/nivo on a standard schedule alone or the same regimen with CBM588 at 80 mg bid. Pts had blood collection performed at baseline and at weeks 9, 13, 17 and 25. Samples underwent processing within 4-6 hrs of collection, and a total of 30 cytokines and growth factors were assessed using the Luminex Flexmap 3D system (Biotechne). Changes in circulating cytokine levels between baseline and week 13 (± 7 days) were examined across the treatment arms to investigate the impact of CBM588 on the immune system. The Wilcoxon matched-pairs test was used to compare the levels of cytokines at the two timepoints. Results A total of 30 pts were enrolled; the majority were male (67%) and had intermediate- or poor-risk disease (60%). Most pts had clear cell histology (87%); 5 pts (17%) had sarcomatoid features. As previously reported, pts receiving cabo/nivo/CBM588 had a significantly higher response rate as compared to pts receiving cabo/nivo alone (74% vs 20%; P=0.01). Across baseline and week 13, a total of 53 samples were available for assessment. In pts receiving cabo/nivo/CBM588, there was a significant change in the level of IL-12, IL-13, eotaxin, interferon-γ, and granulocyte-macrophage colony stimulating factor (GM-CSF); this was not observed in the cabo/nivo alone arm. Additional flow cytometric analyses characterizing relevant CD4- and CD8-positive subsets will be presented. Conclusions Taken together with our previous study evaluating nivolumab/ipilimumab with CBM588 (Dizman et al Nat Med 2022), these results suggest that the augmented clinical outcome seen with the addition of CBM588 to cabo/nivo may be related to systemic immunomodulation. Combined analyses of the cohorts across studies are planned.

1038 Background: Based on preclinical data highlighting synergy between CDK4/6 inhibitors and immune checkpoint inhibitors (ICIs), we conducted a phase I/II study of palbociclib, pembrolizumab, and endocrine therapy (ET) in pts with HR+/HER2- MBC. Given evidence suggesting a correlation between ICI response and stool microbiome composition in a wide array of cancers, we sought to determine if this biomarker could segregate responders in our cohort. Methods: Pts with stage IV HR+/HER2- MBC were enrolled and treated with palbociclib, pembrolizumab, and ET. Cohort 1 enrolled pts with stable disease on palbociclib + ET for at least 6 months. Cohort 2 and 3 enrolled pts in the first-line (1L) setting, with cohort 3 designed with a palbociclib + ET lead-in to evaluate the immune potentiating effect of palbociclib (not included in this report). The primary endpoint was objective response rate (ORR); secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity. Correlative studies assessed the association between the gut microbiome and response using previously published methods (1). Results: Between March 2017 and May 2022, 40 pts were accrued, with 4, 19 and 17 pts in cohorts 1, 2 and 3, respectively. There were 39 females and 1 male, with a median age of 51 (range 39-75). The ORR in cohorts 1, 2 and 3 were 2/4 (50%), 11/19 (58%) and 13/17 (76%), respectively. Median PFS across cohorts 2 and 3 (reflecting 1L therapy) was 25 mo (95% CI 21- not reached), and at a median follow-up of 35 mo, OS was 76% (95% CI 0.60-0.96). The most common grade 3/4 adverse events across all cohorts included neutropenia (83%), leukopenia (56%), elevated alanine aminotransferase (13%), elevated aspartate aminotransferase (13%), and thrombocytopenia (10%). Gut microbial profiling (total N=24 pts; Arm 2 N=7, Arm 3 N=17) demonstrated multiple species higher in abundance in pts with a response (p < 0.05), with notable species belonging to the families Lachnospiraceae, Ruminococcaceae, and Rikenellaceae. Several metabolic pathways were enriched in responders (p-value < 0.05), including pathways related to fatty acid biosynthesis and the tricarboxylic acid (TCA) cycle. Conclusions: Pembrolizumab added to palbociclib + ET in the 1L setting demonstrated a higher response rate compared to palbociclib + ET alone in HR+/HER2- MBC. Specific gut profiles were associated with response, with similar taxa of bacteria associated with ICI response in other tumor types (2). Enriched metabolic pathways may generate microbiota-derived metabolites that have an immunomodulatory effect. These results suggest that the gut microbiome may be associated with response to ICI combination therapy in MBC, which warrants further evaluation. 1. Dizman et al.,Nature Med 2022. 2. Spencer et al Science 2021. Clinical trial information: NCT02778685 .

Abstract Background Two recent randomized phase I clinical trials have provided compelling evidence that CBM588, a Clostridium butyricum-based live biotherapeutic, holds potential to enhance clinical outcomes in patients with mRCC receiving frontline ICI combinations (Dizman et al Nature Medicine 2022; Ebrahimi et al ASCO 2023). We examined the impact of CBM588 on gut microbiome composition in a combined cohort of these two studies to further investigate its impact on gut microbiome Methods We analyzed stool samples from two phase I randomized clinical trials that enrolled patients with mRCC treated with (1) nivolumab/ipilimumab (nivo/ipi) +/- CBM588 and (2) cabozantinib/nivolumab (cabo/nivo) +/- CBM588. We compared gut microbiome diversity and composition at baseline and week 12 between patients in the standard of care (SOC) arms (nivo/ipi or cabo/nivo) and those who received CBM588 in combination with a SOC regimen (SOC/CBM). Taxonomic profiling was performed using MetaPhlan v4, and changes in the abundances of clinically relevant microbial species from baseline to week 12 were assessed using the Wilcoxon matched pairs test. The ratio of Firmicutes/Bacteroidetes, a measure of gut dysbiosis, was computed across time points in the two cohorts. Results Among 58 patients included in the analysis, 38 received SOC/CBM588 as first-line treatment. The median age was 60 years (range: 36-90) and the majority of patients were male (71%), had clear cell mRCC (88%), and intermediate/poor risk disease (79%). In both the SOC and SOC/CBM cohorts, there were no statistically significant differences in alpha and beta diversity between baseline and week 12. Among clinically relevant species compared between baseline and week 12, Alistipes senegalensis was found to decrease in both the SOC and SOC/CBM cohorts (log fold change [LFC] -0.82 [P=0.004] and LFC -0.36 [P=0.007], respectively), while Eubacterium siraeum decreased only in the SOC cohort (LFC -1.75 [P=0.005]). The Firmicutes/Bacteroidetes ratio increased from 89.0% to 96.4% in the SOC cohort, whereas a notable decrease was observed in this ratio from 100.0% to 75.7%. in the SOC/CBM cohort. Conclusions CBM588 leads to a marked correction of gut dysbiosis and prevents the depletion of species previously associated with ICI response (i.e., Eubacterium siraeum). These findings provide a plausible mechanism for the enhanced clinical outcome with CBM588 now seen across two small, randomized trials. A phase III study is planned within the cooperative groups to evaluate the clinical activity and gut microbiome modulation capacity of CBM588 in combination with ICIs in mRCC.

Emerging research into the human microbiome, an intricate ecosystem of microorganisms residing in and on our bodies, reveals that it plays a pivotal role in maintaining our health, highlighting the potential for microbiome-based interventions to prevent, diagnose, treat, and manage a myriad of diseases. The objective of this review is to highlight the importance of microbiome studies in enhancing our understanding of rare genetic epilepsy and related neurological disorders. Studies suggest that the gut microbiome, acting through the gut–brain axis, impacts the development and severity of epileptic conditions in children. Disruptions in microbial composition can affect neurotransmitter systems, inflammatory responses, and immune regulation, which are all critical factors in the pathogenesis of epilepsy. This growing body of evidence points to the potential of microbiome-targeted therapies, such as probiotics or dietary modifications, as innovative approaches to managing epilepsy. By harnessing the power of the microbiome, we stand to develop more effective and personalized treatment strategies for children affected by this disease and other rare neurological diseases.

Background: The gut microbiota produce a variety of metabolites, which signal metabolic pathways influencing metabolism and immunity. There are growing data to support interactions between host-gut microbes and treatment responses in various malignancies. A prospective study of serial changes in gut microbiomes in patients with MM after autologous stem cell transplantation (ASCT) showed that reduced alpha diversity at engraftment was associated with a lesser response to ASCT. Another study in patients with MM after an induction therapy showed that minimal residual disease (MRD) negativity was associated with a higher abundance of Eubacterium hallii in stool samples. Our study was designed to assess changes in the gut microbiome associated with daratumumab (Dara) based therapy. Methods: Patients with relapsed MM and prior autologous transplantation who had received 1-4 prior lines of therapy were eligible. Two stool samples were collected, one within 1 week prior to Dara (pre-Dara) and one immediately after 4 doses of Dara (post-Dara). Stool samples were uniformly collected using the Zymo DNA/RNA Shield Fecal Collection Tubes (Zymo Research, Irvine) and stored at -80C until processing by TGen North Clinical Microbiome Services Center. Metagenomics sequencing was conducted by Illumina NovaSeq X. Microbial functional pathways were analyzed using HUMAnN3 and QIIME2. Results: Forty patients with MM were enrolled, with a median prior lines of therapy of 2 (range: 1-4). In this study, 60% of the patients were female, the median age was 63 (range: 43-78), and 35% of patients had high risk disease. There were no statistical differences between overall Pre- and Post-Dara gut microbiomes in all patients. Five patients did not respond (non-responders), and 35 patients achieved partial response (PR) or better (responders). ANCOM-BC analysis showed statistically significant (p ≤ 0.05) overgrowth of Alistipes finegoldii and Acidaminococcus intestini species in responders and Clostridium symbiosum, Enterocloster clostridioformis and Blautia producta in non-responders. No differentiating functional pathways were detected between Pre- and Post-Dara samples and between responders and non-responders. Adjustment for additional therapy to Dara (pomalidomide [12], lenalidomide [11] and others [5]) was analyzed, after which we found no statistically significant difference between single agent Dara and combination groups. For prophylaxis during Dara therapy, all patients received acyclovir, and 7 patients received sulfamethoxazole and trimethoprim for prophylaxis. Three patients took oral antibacterial antibiotics (levofloxacin and azithromycin) within 4 weeks before the second collection. Levofloxacin, azithromycin and sulfamethoxazole/trimethoprim did not significantly affect microbiome diversity. Conclusion: To our knowledge, this is among the first studies to evaluate the role of microbiota in clinical outcomes among relapsed MM treated with Dara. Despite our somewhat limited sample size, our preliminary results suggest statistically significant differences in specific species between clinical responders and non-responders. Future trials with an expanded number of patients are needed to confirm our findings and to further explore potential differences by pathways. If our results are confirmed, it could inform potential future trials to evaluate the use of probiotics to encourage growth of predominant species in responders to promote better responses to CD-38 directed antibody treatment. Alternatively, probiotic trials among MM patients to inhibit growth of dominant microbes in non-responders can potentially be considered.

460 Background: Our team has previously demonstrated in two prospective studies that the live bacterial product CBM588 may enhance clinical outcomes in patients with mRCC (Ebrahimi et al ASCO 2023, Dizman et al Nature Med 2022). In the current study, we sought to determine if gut microbial functionality is associated with clinical outcomes in patients with mRCC treated with cabo/nivo with or without CBM588. Methods: Pts ≥18 yrs old with histologically verified (clear-cell, papillary, or sarcomatoid component) mRCC and no prior systemic therapy for metastatic disease were enrolled and randomized 1:2 to receive either cabo/nivo at the standard dose/schedule alone or with CBM588 dosed at 80mg PO BID. Whole metagenome sequencing was performed on stool specimens collected at baseline and week 12 of treatment. Taxonomic profiling was conducted using MetaPhlAn 4, and functional profiling was performed using HUMAnN 3. HUMAnN 3 annotates open reading frames and provides highly accurate information on metabolic pathways and other molecular functions from metagenomic or metatranscriptomic sequencing data. The ANCOM-BC was used to detect the taxonomic/genetic features with differential abundance between two time-points within the same treatment arm. Results: A total of 30 (20:10 M:F) pts were enrolled with a median age of 65 (36-84). 5 pts (17%) had sarcomatoid features, and 2 pts (7%) had predominant papillary histology. Objective response was achieved in 20% and 65% of the pts in the cabo/nivo and cabo/nivo/CBM588 arm, respectively. Significant changes in 9 metabolic pathways (1 upregulation, 8 downregulation) in the control arm and 7 metabolic pathways (2 upregulation, 5 with downregulation) in the experimental arm were identified. Superpathways of biosynthesis of different forms of menaquinole, a reversible redox component of the electron transfer chain, were depleted with cabo/nivo treatment. In contrast, the biosynthesis of menaquinol-8 and 1,4 dihydroxy-6-naphthoate (an intermediate of the menaquinone pathway) were upregulated in cabo/nivo/CBM588 arm. Conclusions: Our interrogation of metabolic dynamics and pathways in patients receiving CBM588 suggests key differences in biosynthesis pathways of menaquinone between control and experimental arms. Menaquinones (vitamin K2 derivatives) have been previously reported to induce apoptosis in many cancer cell types and also increase the objective response rate to sorafenib in patients with hepatocellular carcinoma. Our findings provide mechanistic evidence for the effect of the addition of CBM588 to cabo/nivo on gut microbiome function and the resultant improvement in clinical outcomes in mRCC, potentially through enhancing the enteric production of vitamin K2. Clinical trial information: NCT05122546 .

Supplementation with CBM588, a bifidogenic live bacterial product, has been associated with improved clinical outcomes in persons with metastatic renal cell carcinoma (mRCC) receiving nivolumab and ipilimumab. However, its effect on those receiving tyrosine kinase inhibitor-based combinations is unknown. In this open-label, randomized, investigator-initiated, phase 1 study, 30 participants with locally advanced or mRCC with histological confirmation of clear cell, papillary or sarcomatoid component were randomized in a 2:1 fashion to receive cabozantinib (an inhibitor of vascular endothelial growth factor receptor, MET and AXL) and nivolumab (anti-programmed cell death protein 1) with or without CBM588 as first-line treatment. Metagenomic sequencing was performed on stool samples to characterize their gut microbiome at baseline and 13 weeks into treatment. The primary endpoint was a change in the relative abundance of Bifidobacterium spp.; secondary endpoints included objective response rate (ORR), progression-free survival (PFS) and toxicity profile. The primary endpoint of the study was not met and the addition of CBM588 to cabozantinib and nivolumab did not result in a difference in the relative abundance of Bifidobacterium spp. or alpha diversity (as measured by the Shannon index). However, ORR was significantly higher in participants treated with CBM588 compared to those in the control arm (14 of 19, 74% versus 2 of 10, 20%; P = 0.01). PFS at 6 months was 84% (16 of 19) and 60% (6 of 10) in the experimental and control arms, respectively. No significant difference in toxicity profile was seen between the study arms. Our results provide a preliminary signal of improved clinical activity with CBM588 in treatment-naive participants with mRCC receiving cabozantinib and nivolumab. Further investigation is needed to confirm these findings and better characterize the underlying mechanism driving this effect. ClinicalTrials.gov identifier: NCT05122546 In a randomized phase 1 trial, the addition of a live Clostridium species-containing product to a tyrosine kinase inhibitor and anti-programmed cell death protein 1 treatment combination did not increase bacterial abundance of Bifidobacterium spp. but enhanced clinical responses in participants with metastatic renal cell carcinoma.