Baseline Stool Samples Research Articles

O88 REDUCTION IN SYSTOLIC BLOOD PRESSURE FOLLOWING DIETARY FIBRE INTERVENTION IS DEPENDENT ON BASELINE GUT MICROBIOTA COMPOSITION

Background/Objective: Dietary interventions high in fibre have been shown to lower blood pressure (BP). This effect is via the gut microbiota, via fermentation of fibre and production of metabolites called short-chain fatty acids (SCFAs). Not all patients respond to dietary fibre interventions for unclear reasons. We aimed to determine if we can identify fibre-responders based on their gut microbiota and production of SCFAs. Methods: We performed a phase II cross-over placebo-controlled randomised clinical trial in untreated hypertensive patients. Twenty treatment-naive participants with hypertension received 40 g per day of fermentable fibre delivered as acetylated and butyrylated high amylose maize starch (HAMSAB) or the placebo for 3 weeks. Blood pressure was monitored at baseline and endpoint by ambulatory BP monitoring, with those experiencing a reduction of ≥2 mmHg in systolic BP classified as responders. The microbiota was determined by sequencing the V4-V5 region of the 16S rRNA gene in baseline stool samples. The relationship between baseline gut microbiota and response to dietary intervention was assessed with the MaAsLin2 package. Plasma SCFAs levels were quantified by mass spectrometry. Results: We observed a placebo-subtracted mean 24-hour systolic BP of –6.1 mmHg (p=0.03, reported in Jama et al., 2023 Nature Cardiovascular Research), with 14 (70%) individuals classified as responders and 6 (30%) as non-responders. Genera significantly enriched in responders included Dialister (β=1.29, q=1.921x10-134), Coprococcus (β=1.26, q=3.282x10-121), Bifidobacterium (β=1.67, q=1.11x10-81), Ruminococcus (β=0.161, q=1.11x10-8), and Roseburia (β=0.82, q=4.275x10-2). Responders also produced higher levels of the SCFAs butyrate (mean±SEM, non-responders 4.95±2.4 vs responders 51.15±14.7, p=0.0096), but no difference in other SCFAs. Conclusions: Participants who experienced a decrease in systolic BP following a dietary fibre intervention had increased bacterial genera known to contain species that produce SCFAs (Bifidobacterium, Roseburia, Ruminococcus) at baseline, and were able to produce more butyrate. These data suggest that baseline microbiota composition contributes to the response to dietary fibre intervention trials in people with hypertension, likely via increased capacity to produce butyrate.

Reduction in systolic blood pressure following dietary fibre intervention is dependent on baseline gut microbiota composition

Uncontrolled hypertension is a primary cause of non-communicable diseases and death globally(1). The gut microbiota plays a role in hypertension and dietary interventions high in fibre have been shown to lower blood pressure (BP)(2). Not all participants respond to dietary fibre interventions, for reasons which are unclear. Here we aimed to identify responders of a high fibre intervention based on their baseline gut microbiome. Twenty treatment-naive participants with hypertension received either placebo or 40g per day of prebiotic acetylated and butyrylated high amylose maize starch (HAMSAB) supplementation for 3 weeks in a phase II randomised cross-over double-blind placebo-controlled trial. Blood pressure was monitored at baseline and each endpoint by 24-hour ambulatory BP monitoring, with those experiencing a reduction between timepoints of ≥ 2 mmHg classified as responders. Baseline stool samples were collected and the V4-V5 region of the 16S gene sequenced. Taxonomy was assigned by reference to the SILVA database. The MaAsLin2 package was used for assessing the relationship between baseline gut microbiota and response to dietary intervention. Overall participants had significant reduction in 24-hour systolic BP (–6.1 mmHg, p = 0.03), with 14 individuals classified as responders and six individuals as non-responders. 13 genera were found to be differentially abundant between responders and non-responders. Genera significantly enriched in responders included Dialister (β = 1.29, q = 1.921x10-134), Coprococcus (β = 1.26, q = 3.282x10-121), Bifidobacterium (β = 1.67, q = 1.11x10-81), Ruminococcus (β = 0.161, q = 1.11x10-8) and Roseburia (β = 0.82, q = 4.275x10-2). Participants who experienced a decrease in systolic BP following a dietary fibre intervention had increased level of bacterial genera known to contain species that produce short-chain fatty acids (e.g. Bifidobacterium, Roseburia and Ruminococcus) at baseline. These data suggest that baseline microbiota composition contributes to the response to dietary fibre intervention trials in people with hypertension.

Abstract 6204: Differential gut microbiome alpha diversity in endometrial and ovarian carcinoma patients

Abstract Background: Emerging research underscores the gut microbiome's critical role in cancer development, progression, and response to treatment. As a part of the “ASAP” study, which focuses on the implementation of comprehensive molecular profiling and deep clinical annotation of electronic health records in participants diagnosed with or at risk of developing cancer, we have established a microbiome-associated biobank and corresponding clinical dataset to systematically explore the dynamics of the gut microbiome as patients progress through cancer treatment. We describe a pilot project where we explore the baseline microbiome samples collected from ASAP participants, particularly from those with endometrial and ovarian carcinomas. Methods: Stool samples from patients were collected at the time of enrollment in the ASAP study and sampling is repeated every three months to align with the additional biosampling procedures. Microbial DNA was extracted from the baseline stool samples and subjected to sequencing of the V4 region of the 16S rRNA gene. After generating species level operational taxonomic units (OTUs), alpha diversity measures were generated in the form of the inverse Simpson index. In order to determine whether there was a difference in alpha diversity between individuals with ovarian (N = 37) and endometrial (N = 35) carcinomas, we employed a t-test with 100,000 permutations to derive an empirical p-value. Results: We present sequencing results from the initial n=297 samples. Our comparison of the alpha diversity between ASAP study participants with ovarian and endometrial carcinomas showed a significant difference between the two groups, with the ovarian cancer patients having a lower alpha diversity (empirical p-value = 0.03915). Due to the uniform sampling, biobanking, and analytical procedures, our study allows the ability to explore microbiome features amongst various cancer diagnoses while minimizing laboratory and analytical differences that plague microbiome reproducibility. Conclusions: We observed a significant difference in alpha diversity between patients with endometrial and ovarian carcinomas. Although the current results are preliminary, the wealth of information available in the ASAP protocol will allow us to continue exploring the association of the gut microbiome with cancer and the associated treatment. Citation Format: Casey T. Finnicum, Tobias Meissner, Christel Davis, Sarah Viet, Jason L. Petersen, McKenna Deaton, Benjamin Solomon, William Spanos, David Starks, Rachel Elsey, Erik A. Ehli, Casey Williams. Differential gut microbiome alpha diversity in endometrial and ovarian carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6204.

Abstract 3805: Intestinal microbiome with chemotherapy response and RAS mutation in patients with advanced or metastatic colorectal cancer: a pilot, exploratory study

Abstract Intestinal microbiomes interact with the tumor microenvironment, participating in inflammation and immune regulation, and are associated with the response to anticancer therapy. However, there has been limited research exploring the relationship between changes in the microbiome and treatment response in patients with metastatic colorectal cancer (mCRC) during chemotherapy and radiation therapy.This study included all 15 patients with mCRC treated with first-line systemic therapy between October 2021 and February 2023. We conducted 16S rRNA sequencing on baseline stool samples and investigated the association of intestinal microbiome with chemotherapy response and RAS mutation. The QIIME2 and R packages were used for analysis. As a results, Median age was 61 years (range, 35-73), and there were 8 patients with RAS-mutated mCRC. All patients were treated with first-line systemic therapy; FOLFOX (5-fluorouracil and oxaliplatin)-based (n=13) and FOLFIRI (5-fluorouracil and irinotecan)-based chemotherapy (n=2). Of them, 2 patients received short-course radiotherapy (ScRTx) to primary tumor before systemic chemotherapy, where pre- and post-ScRTx stool samples were collected. Among 15 patients, 9 patients (60%) achieved responses. Non-responders tend to exhibit higher alpha diversity (Chao1, Observed ASV) compared to responders. Firmicutes, Lactobacillus, Faecalibacterium, and the [Luminococcus]_gauvreauii_group were more prevalent in responders, whereas Bacteroidota, Prevotella, DTU089, Alistipes, Christensenellaceae_R-7_group, and Porphyromonas in non-responders. Despite no statistical significance, alpha diversity decreased in post-ScRTx compared to pre-ScRTx samples. Akkermansia, Prevotella, and Peptostreptococcus were more prevalent in the post-ScRTx compared to pre-ScRTx samples. Regarding the association of intestinal microbiome with RAS mutation, Bray curtis and weighted unifrac analyses revealed significant differences in beta diversity; higher incidence of Faecalibacterium, Lactobacillus, Prevotella, Anaerofilum, and UCG-005 in the RAS-wild type mCRC, while Bacteroides, Collinsella, Holdemanella, and Anaerostipes in RAS-mutated mCRC.This pilot study suggests the prognostic value of gut microbiomes and its association with RAS mutation in patients with mCRC treated with first line systemic therapy. A microbial functional study with immune cytokine change is planned to confirm these findings in future. Citation Format: Jwa Hoon Kim, Boyeon Kim, Soohyeon Lee. Intestinal microbiome with chemotherapy response and RAS mutation in patients with advanced or metastatic colorectal cancer: a pilot, exploratory study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3805.

Abstract 6684: Impact of fecal microbiome on the efficacy of neoadjuvant chemo-immunotherapy and colitis adverse events: Findings from the NADIM II Trial

Abstract Introduction: The balance and composition of the gut microbiome play a pivotal role in modulating the host immune system, and they have been associated with survival outcomes in patients undergoing immunotherapy-based treatments and immune-related adverse events. In this study, our objective was to analyze the fecal microbiota of non-small-cell lung cancer (NSCLC) patients with locally advanced disease undergoing neoadjuvant chemo-immunotherapy (IO-CT). Methods and patients: Baseline stool samples were collected from 86 resectable stage IIIA/B NSCLC patients enrolled in the NADIM II clinical trial (NCT03838159). The patients were randomly assigned to receive either neoadjuvant nivolumab plus chemotherapy (CT) or CT alone. DNA extraction was performed using the QIAamp PowerFecal DNA Kit (Qiagen), and the samples were analyzed using the Ion AmpliSeq Microbiome Health Research assay (ThermoFisher). The NGS analysis utilized the AmpliSeq Microbiome Health w1.3 workflow. Alpha diversity was assessed using the Simpson and Chao1 Index. Results: Among the 86 collected stool samples, 77 were deemed valid for analysis. Bacteroides were the most common genus with a relative percentage (RP) of 18.9% ± 14.8 followed by Faecalibacterium (13.4% ± 7.8). The presence of bacteria from the genera Bifidobacterium, and Faecalibacterium was associated with improved progression-free survival (PFS) [HR = 0.4 (0.16-0.98), and HR = 0.08 (0.01-0.72), respectively] and overall survival (OS) [HR = 0.19 (0.06-0.62)], and HR = 0.04 [0.01-0.43], respectively), in the experimental arm. The detection of Faecalibacterium prausnitzii (validated by qPCR) was associated with improved PFS [HR = 0.17 (0.04-0.76)] and OS [HR = 0.05 (0.01-0.29)] in the experimental. On the other hand, detection of bacteria from the genera Synergistes, Lactobacillus, and Anaerotruncus was associated to higher risk of disease progression [HR = 9.9 (1.2-85), HR = 3.7 (1.5-9.1), and HR = 12 (1.4-110)] and death [HR = 51 (3.2-820), HR = 6.7 (2-23), and HR = 25 (2.3-280)] in this set of patients.In the entire cohort, patients whose samples were in the highest tertile of alpha diversity, had significantly prolonged PFS compared to those with intermediate or low diversity [HR = 0.39 (0.16-0.94)]. Finally, 25 colitis adverse events (CAE) occurred in 18 patients, including two Grade 3 (2.3%) and four Grade 2 (4.6%) events. A higher RP of Bacteroides were found in patients who did not suffer colitis events (P = 0.016), whereas higher RP of Coriobacterium were detected in patients who suffered CAE (P = 0.023). Conclusions: Our findings suggest that composition of the gut microbiome may play a crucial role in the effectiveness of neoadjuvant IO-CT in NSCLC patients. Specifically, the presence of F. prausnitzii may enhance the antitumor activity of these treatments. Citation Format: Roberto Serna-Blasco, Alejandro Rodriguez Festa, Sandra Sanz-Moreno, Lucía Robado de Lope, Pilar Mediavilla Medel, Ernest Nadal, Jose Luis González Larriba, Alex Martínez-Martí, Reyes Bernabé, Joaquim Bosch-Barrera, Joaquín Casal-Rubio, Virginia Calvo, Amelia Insa, Santiago Ponce, Noemí Reguart, Javier de Castro, Joaquín Mosquera, Manuel Cobo, Andrés Aguilar, Carlos Camps, Rafael López de Castro, Teresa Morán, Isidoro Barneto, Delvys Rodríguez-Abreu, Carlos Aguado de la Rosa, Ramón Palmero, Florentino Hernando-Trancho, Javier Martín-López, Alberto Cruz-Bermúdez, Bartomeu Massuti, Miguel Ángel Molina Vila, Atocha Romero, Mariano Provencio. Impact of fecal microbiome on the efficacy of neoadjuvant chemo-immunotherapy and colitis adverse events: Findings from the NADIM II Trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6684.

The Predictive Value of Gut Microbiota Composition for Sustained Immunogenicity following Two Doses of CoronaVac.

CoronaVac immunogenicity decreases with time, and we aimed to investigate whether gut microbiota associate with longer-term immunogenicity of CoronaVac. This was a prospective cohort study recruiting two-dose CoronaVac recipients from three centres in Hong Kong. We collected blood samples at baseline and day 180 after the first dose and used chemiluminescence immunoassay to test for neutralizing antibodies (NAbs) against the receptor-binding domain (RBD) of wild-type SARS-CoV-2 virus. We performed shotgun metagenomic sequencing performed on baseline stool samples. The primary outcome was the NAb seroconversion rate (seropositivity defined as NAb ≥ 15AU/mL) at day 180. Linear discriminant analysis [LDA] effect size analysis was used to identify putative bacterial species and metabolic pathways. A univariate logistic regression model was used to derive the odds ratio (OR) of seropositivity with bacterial species. Of 119 CoronaVac recipients (median age: 53.4 years [IQR: 47.8-61.3]; male: 39 [32.8%]), only 8 (6.7%) remained seropositive at 6 months after vaccination. Bacteroides uniformis (log10LDA score = 4.39) and Bacteroides eggerthii (log10LDA score = 3.89) were significantly enriched in seropositive than seronegative participants. Seropositivity was associated with B. eggerthii (OR: 5.73; 95% CI: 1.32-29.55; p = 0.022) and B. uniformis with borderline significance (OR: 3.27; 95% CI: 0.73-14.72; p = 0.110). Additionally, B. uniformis was positively correlated with most enriched metabolic pathways in seropositive vaccinees, including the superpathway of adenosine nucleotide de novo biosynthesis I (log10LDA score = 2.88) and II (log10LDA score = 2.91), as well as pathways related to vitamin B biosynthesis, all of which are known to promote immune functions. In conclusion, certain gut bacterial species (B. eggerthii and B. uniformis) and metabolic pathways were associated with longer-term CoronaVac immunogenicity.

A15 LEVERAGING CULTURE-DEPENDENT AND -INDEPENDENT METAGENOMICS TO IDENTIFY ENTEROBACTERIACEAE GENES ASSOCIATED WITH ACTIVE ULCERATIVE COLITIS

Abstract Background Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) that is restricted to the large intestine and is characterized by mucosal inflammation. There is evidence that pathogenic bacteria contribute to UC pathogenesis in at least some patients and the bacterial family Enterobacteriaceae are prime suspects. Some strains of Enterobacteriaceae carry virulence genes important for colonizing the gut (e.g. fimH) and disrupting the intestinal epithelium (e.g. hemolysins). We hypothesize that key virulence genes carried by strains of many Enterobacteriaceae species contribute to disease activity in some UC patients. As we predict virulence genes are strain-specific, the detection of the species alone (e.g. Escherichia coli) is insufficient to predict pathogenic potential and this has confounded previous studies to identify infectious agents in UC. Aims We aim to use culture-dependent and -independent sequencing approaches to identify Enterobacteriaceae genes in the UC gut microbiome associated with active disease. Methods UC patient stool samples collected throughout enrolment in randomized control trials of fecal microbiota transplantation (FMT) for adult UC (n=10) and microbiome studies in early-onset pediatric UC (n=25) were cultured on MacConkey (MAC) agar to enrich for Enterobacteriaceae. Strains were isolated from baseline stool samples cultured on MAC agar for whole genome sequencing and virulence gene characterization. Baseline and post-treatment stool samples cultured on MAC agar were sent for metagenomic sequencing. Using a subset of 11 pediatric patients, I developed a metagenomic pipeline to identify Enterobacteriaceae genes enriched during active UC compared to periods of remission or milder disease. Results Known virulence genes, including fimH and hemolysins, were present in some strains across multiple Enterobacteriaceae species. Using an unbiased metagenomic approach with the pediatric cohort, we identified 42 genes enriched at baseline with the criteria they must be elevated in at least three of the 11 patients. The majority of the 42 genes were distributed into six gene clusters, including a small plasmid. Conclusions Our approach allows us to identify genes enriched in active UC that have not been previously described in the literature and our analysis will be repeated with our adult cohort. Genes that are enriched in active UC in the pediatric and/or adult cohort will be validated in publicly available metagenomic datasets that consist of both IBD patients and healthy controls. Furthermore, culture-dependent approaches allow us to test mechanisms in vivo that are informed by our bioinformatics. Leveraging microbiome and clinical data provides a unique window to guide future diagnostics and treatments to improve outcomes for UC patients. Funding Agencies CIHROGS

Gut microbiome predicts gastrointestinal toxicity outcomes from chemoradiation therapy in patients with head and neck squamous cell carcinoma

ObjectivesChemoradiation (CRT) in patients with locally advanced head and neck squamous cell cancer (HNSCC) is associated with significant toxicities, including mucositis. The gut microbiome represents an emerging hallmark of cancer and a potentially important biomarker for CRT-related adverse events. This prospective study investigated the association between the gut microbiome composition and CRT-related toxicities in patients with HNSCC, including mucositis. Materials and methodsStool samples from patients diagnosed with locally advanced HNSCC were prospectively collected prior to CRT initiation and analyzed using shotgun metagenomic sequencing to evaluate gut microbiome composition at baseline. Concurrently, clinicopathologic data, survival outcomes and the incidence and grading of CRT-emergent adverse events were documented in all patients. ResultsA total of 52 patients were included, of whom 47 had baseline stool samples available for metagenomic analysis. Median age was 62, 83 % patients were men and 54 % had stage III-IV disease. All patients developed CRT-induced mucositis, including 42 % with severe events (i.e. CTCAE v5.0 grade ≥ 3) and 25 % who required enteral feeding. With a median follow-up of 26.5 months, patients with severe mucositis had shorter overall survival (HR = 3.3, 95 %CI 1.0–10.6; p = 0.02) and numerically shorter progression-free survival (HR = 2.8, 95 %CI, 0.8–9.6; p = 0.09). The gut microbiome beta-diversity of patients with severe mucositis differed from patients with grades 1–2 mucositis (p = 0.04), with enrichment in Mediterraneibacter (Ruminococcus gnavus) and Clostridiaceae family members, including Hungatella hathewayi. Grade 1–2 mucositis was associated with enrichment in Eubacterium rectale, Alistipes putredinis and Ruminococcaceae family members. Similar bacterial profiles were observed in patients who required enteral feeding. ConclusionPatients who developed severe mucositis had decreased survival and enrichment in specific bacteria associated with mucosal inflammation. Interestingly, these same bacteria have been linked to immune checkpoint inhibitor resistance.

Pre-transplant and longitudinal changes in faecal microbiome characteristics are associated with subsequent development of chronic graft-versus-host disease.

The role of the gastrointestinal microbiome in predisposing to chronic graft-versus-host disease (cGVHD), an immune-mediated haematopoietic cell transplant (HCT) complication, is not well defined. We examined the relationship of the host faecal microbiome with subsequent cGVHD development by analysing baseline stool samples as well as post-HCT changes in microbiome composition and metabolite pathway analyses. We analysed pre-transplant baseline samples from 11 patients who subsequently developed cGVHD compared to 13 controls who did not develop acute GVHD or cGVHD at any time. We found a significant differential abundance of multiple taxa at baseline between cGVHD versus controls, including the Actinobacteria phylum and Clostridium genus. A subgroup analysis of longitudinal samples within each patient revealed a greater loss of alpha diversity from baseline to post-engraftment in patients who subsequently developed cGVHD. Metabolic pathways analysis revealed that two pathways associated with short-chain fatty acid metabolism were enriched in cGVHD patient microbiomes: β-oxidation and acyl-CoA synthesis, and γ-aminobutyrate shunt. In contrast, a tryptophan catabolism pathway was enriched in controls. Our findings show a distinct pattern of baseline microbiome and metabolic capacity that may play a role in modulating alloreactivity in patients developing cGVHD. These findings support the therapeutic potential of microbiome manipulation for cGVHD prevention.

A grape seed and bilberry extract reduces blood pressure in individuals at risk of developing type 2 diabetes: the PRECISE study, a double-blind placebo-controlled cross-over intervention study.

Type 2 Diabetes Mellitus (T2DM) is a major risk factor for the development of cardiometabolic diseases. T2DM prevention is largely based on weight-loss and whole diet changes, but intervention with dietary plant bioactives may also improve metabolic health. To assess whether supplementation with bilberry and grape seed extract for 12 weeks improves cardiometabolic outcomes in individuals at risk of developing T2DM, and to determine whether individual treatment response is associated with differences in gut microbiota composition and levels of phenolic metabolites in blood and feces. In the randomized, double-blind, placebo-controlled, cross-over PRECISE intervention study, 14 participants, aged ≥45 years, with a BMI >28 kg/m2, and having an increased risk of T2DM, received a supplement containing 250 mg of bilberry plus 300 mg of grape seed extract, or 550 mg of a control extract, per day, for 12 weeks each. Blood samples were obtained for the assessment of HbA1c, fasting glucose, oral glucose tolerance tests, insulin, glucagon levels, total, LDL and HDL cholesterol, and phenolic acids. We also assessed advanced glycation end products in the skin, ambulatory 24 hours blood pressure, 7-day dietary intake by weighed food diaries, fecal levels of phenolic metabolites using LC-MS/MS and gut microbiota composition using 16S rRNA gene sequencing analysis. The combined bilberry and grape seed extract did not affect glucose and cholesterol outcomes, but it decreased systolic and diastolic ambulatory blood pressure by 4.7 (p < 0.001) and 2.3 (p = 0.0009) mmHg, respectively. Eight out of fourteen participants were identified as blood pressure 'responders'. These responders had higher levels of phenylpropionic and phenyllactic acids in their fecal samples, and a higher proportional abundance of Fusicatenibacter-related bacteria (p < 0.01) in their baseline stool samples. Long-term supplementation with bilberry and grape seed extract can improve systolic and diastolic blood pressure in individuals at risk of T2DM. Individual responsiveness was correlated with the presence of certain fecal bacterial strains, and an ability to metabolize (epi)catechin into smaller phenolic metabolites.Clinical trial registry number: Research Registry (number 4084).

Neoadjuvant immunotherapy with ipilimumab plus nivolumab and radiologically and pathologically quantifiable responses through modulation of the tumour microenvironment in resectable hepatocellular carcinoma.

4129 Background: Liver resection (LR) is a potentially curative option for early-stage hepatocellular carcinoma (HCC), and immune checkpoint inhibitors (ICI) are being investigated in the perioperatory setting to improve long-term relapse rates and survival outcomes. Methods: PRIME‐HCC is a phase Ib study testing nivolumab (3 mg/kg, day 1 and day 22) plus ipilimumab (1mg/kg, day 1 only) (N+I) prior to LR in resectable HCC. Primary endpoint was safety, while secondary endpoints included radiological response (per RECIST v1.1) and pathological response on resection specimens (ReS). Translational endpoints encompassed analysis of tissue samples with multiplex immunohistochemistry (MIHC) and gut microbiota profiling with high-throughput sequencing and their correlation with response. Results: As of 7th of February 2023, 25 patients (pts) were enrolled, all Child-Pugh A, mostly male (n=18, 72%), with cirrhosis (n=13, 52%), and viral hepatitis (44%, 4 HBV and 7 HCV). Any-grade adverse events (AEs) occurred in 88% pts (n=22), of which 24% (n=6) grade (G) 3. No G4-5 AEs were recorded. Any-grade treatment-related (tr)AEs were reported by 72% pts (n=18), including 8% G3 trAEs (n=1 ALT/AST elevation and n=1 dyspnea). After a median follow-up of 14.5 months (95% CI 7.0-22.0), 21 pts were assessed for radiological response and underwent surgery. Median time from treatment start to LR was 2.4 months (interquartile range 2.2-2.7). LR was delayed in 1 pt (4.8%) because of G2 tr-hypothyroidism but occurred uneventfully. Objective response rate was 29% (n=5 partial responses, n=1 complete response [CR]); disease control rate was 95% (n=20), with 1 progressor having a mixed HCC/cholangiocarcinoma. Major pathological response (MPR), defined as ≥70% tumour regression, was observed in 56% (n=9) of the pathologically evaluable ReS (n=16), with 38% pathological CR (n=6). In the MIHC analyses, pretreatment biopsies of MPR pts were significantly enriched in peritumoral CD4+ and CD8+ cells, with no intratumoral difference. When comparing baseline biopsies and ReS, MPR pts had a significant decrease of intratumoral FOXP3+/CD4+ regulatory T cells (Tregs), while peritumoral Tregs increased significantly in non-MPR ReS. In baseline stool samples, α and β diversity did not differ across responders (R) and non-responders (NR), nor it changed after ICI. However, pre-treatment stool samples of NR pts were significantly enriched in Alistipes and Colinsella genera. Conclusions: Neoadjuvant N+I is feasible and tolerable for early-stage HCC, with promising radiological and pathological response rates. Study of tissue and gut microbiota highlights tumor and host-related changes. Immune infiltrate and gut microbiota composition lend themselves as predictors of anti-tumour efficacy of N+I combination. Clinical trial information: NCT03682276 .

Gut metabolomic profiles in paediatric ulcerative colitis patients prior to and after receiving faecal microbiota transplants.

Ulcerative colitis (UC) is an immune-mediated inflammation of the colonic mucosa. Gut microbiota dysbiosis may play a significant role in disease pathogenesis by causing shifts in metabolomic profiles within the gut. To identify differences and trends in the metabolomic profile of paediatric UC patients pre- and post-faecal microbiota transplants (FMT). Forty-six paediatric patients with mild-to-moderate UC and 30 healthy paediatric patients were enrolled in this study. Baseline stool samples were collected prior to FMT initiation and at months 1, 3, 6, and 12 post-FMT. Pediatric Ulcerative Colitis Activity Index (PUCAI) scores were calculated at baseline and months 1, 3, 6, and 12 after FMT. The average Bray-Curtis dissimilarities to healthy subjects decreased after FMT. In principal coordinate analysis plots, UC patients' centroids drew nearer to healthy individuals. The variance explained by phenotype (Healthy versus UC) reduced and remained significant. From 1 to 3 months after FMT, PUCAI trends were statistically significant and decreasing. PUCAI scores remain flat starting 6 months after FMT. This study concludes that paediatric UC patients have a significantly different baseline metabolite profile than healthy controls. Although being time limited, FMT significantly altered these metabolite profiles and shifted them towards that of healthy controls.

Profiling the Fungal Microbiome after Fecal Microbiota Transplantation for Graft-versus-Host Disease: Insights from a Phase 1 Interventional Study

Disruption of the intestinal bacterial microbiota is frequently observed in the context of allogeneic hematopoietic cell transplantation (HCT) and is particularly pronounced in patients who develop graft-versus-host disease (GVHD). Donor fecal microbiota transplantation (FMT) restores gut microbial diversity and reduces GVHD in HCT recipients. The composition of the intestinal fungal community in patients with GVHD, and whether fungal taxa are transferred during FMT are currently unknown. We performed a secondary analysis of our clinical trial of FMT in patients with steroid-refractory GVHD with a focus on the mycobiota. We characterized the fecal mycobiota of 17 patients and healthy FMT donors using internal transcribed spacer amplicon sequencing. The donor who provided the majority of FMT material in our study represents an n-of-one study of the intestinal flora over time. In this donor, mycobiota composition fluctuated over time while the bacterial microbiota remained stable over 16 months. Fungal DNA was detected more frequently in baseline stool samples from patients with steroid-refractory GVHD than in patients with steroid-dependent GVHD. We could detect fungal taxa in the majority of samples but did not see evidence of mycobiota transfer from donor to recipient. Our study demonstrates the feasibility of profiling the mycobiota alongside the more traditional bacterial microbiota, establishes the methodology, and provides a first insight into the mycobiota composition of patients with GVHD.

Immunotherapy response-associated Akkermansia: canary in a coal mine?

Gut microbes can shape cancer immunotherapy responses; however, specific taxa have not been tested prospectively as biomarkers. Derosa and colleagues now demonstrate that the presence of Akkermansia muciniphila in baseline stool samples can be associated with improved outcomes and enhanced antitumor immune cell infiltrates in patients with lung cancer treated with immune checkpoint blockade (ICB).

P711 Stool microbiome communities predict remission in pediatric Crohn’s disease patients even after start of treatment

Abstract Background Early relapse in children with Crohn’s Disease (CD) after reaching remission is associated with a more severe disease course that significantly impairs quality of life. We have previously shown that a Bayesian approach predicted clinical course in children with CD following the first year after diagnosis with high accuracy when ensuring samples were truly treatment-naïve. Here, we aimed to assess the impact on the accuracy when taking a broader timeframe of stool collection to baseline, in order to facilitate use in clinical trials and eventually daily practice. Methods We selected de novo paediatric CD patients with PCDAI&amp;gt;10 from the RISK cohort with baseline stool samples within 14 days after start of induction treatment. We assessed if they sustained remission at 12 months (PCDAI≤10). Using QIIME2 sequences were demultiplexed, joined, and denoised (deblur) to obtain amplicon sequence variants (ASVs). The ASVs were classified (classify-sklearn) using a pre-trained SILVA database. We used hierarchical Bayesian model for microbial community structure (BioMiCo), previously trained on treatment-naïve stool samples to predict treatment outcomes at 6 months according to baseline gut microbiome differences. Results Patient metadata and 16S rRNA amplicon data were available from 197 stool samples of newly diagnosed paediatric CD patients as part of the RISK cohort. Previous analysis of 42 truly treatment-naïve samples lead to prediction of samples in patients maintaining remission without early treatment escalation and those that did not in 81% and 75% (AUC=0.79). In this analysis, we selected 13 samples of children with PCDAI that were taken within 14 days after start of induction therapy. PCDAI varied from 15–47.5 at baseline. Therapy regimens started within the first 14 days were EEN, 5ASA, corticosteroids, immunomodulators and antibiotics. The Bayesian model predicted 12-month outcome of patients that maintained remission with a positive predictive value of 75% and negative predictive value of 60% (AUC 0.76). Conclusion Using treatment-naïve faecal samples only, a Bayesian approach predicted clinical course in treatment-naïve children with CD over the first year after diagnosis with high accuracy. When taking a broader timeframe of stool collection after start of treatment, the accuracy of the model decreased only slightly. Further exploration of microbiome signatures and potential use in practice should therefore emphasize the importance of treatment naivety of samples, but not necessarily rule them out for prediction of treatment outcome.

701. An Open-label Phase 2a Study of Ibezapolstat, a Unique Gram-positive Selective Spectrum (GPSS) Antibiotic, for Patients with Clostridioides difficile Infection

BackgroundIbezapolstat, a DNA polymerase IIIC inhibitor, currently in Phase 2 clinical development for treatment of C. difficile infection (CDI). Its unique mechanism of action targets low G+C content Gram-positive bacteria primarily Firmicutes including C. difficile. Phase I healthy volunteer results demonstrated a favorable microbiome profile suggestive of an anti-recurrence effect. The purpose of this study was to report clinical outcomes, pharmacokinetics, and microbiome changes from this Phase 2a clinical study and to continue to test for anti-recurrence microbiome properties.MethodsIbezapolstat 450 mg was given twice daily for 10 days to patients with mild-moderate CDI defined as diarrhea plus a positive C. difficile toxin test. Test of cure was evaluated at day 12 and sustained clinical cure at day 38. Stool samples were evaluated for C. difficile cultures and microbiome changes.ResultsTen subjects (female: 50%) aged 50 ±15 years were enrolled. All ten subjects experienced a clinical cure by the test of cure visit at day 12 and all 10 subjects experienced a sustained clinical cure at the day 38 visit. Ibezapolstat was well tolerated with 1 adverse event (nausea) probably related to drug. Ibezapolstat systemic exposure was minimal with no plasma level reaching 1 ug/mL any time during therapy. Ibezapolstat colonic concentrations averaged 400 ug/g stool at day 3 and greater than 1,000 ug/g by day 10 of dosing. Six of the seven available baseline stool samples grew toxigenic C. difficile of various ribotypes including RT078-226 and RT014-020 (Ibezapolstat MIC range: 0.25-1 ug/mL). Follow-up cultures were no growth starting from day 3 stool cultures. Microbiome changes included overgrowth of Actinobacteria and/or Firmicute phylum species while on therapy.ConclusionFavorable clinical efficacy and safety results were observed in ibezapolstat patients with CDI including 100% clinical cure and sustained clinical cure. These results begin to validate our approach to ibezapolstat development in that the favorable microbiome effects seen in healthy Phase 1 volunteers may be predictive of beneficial patient outcomes, including low rates of recurrence. These results support the continued clinical development of ibezapolstat.Disclosures Kevin W. Garey, Pharm.D., M.S., FASHP, Summit Therapeutics (Research Grant or Support) Michael Silverman, MD, Acurx Pharmaceuticals (Consultant)

Combined Use of Fecal Biomarkers in Inflammatory Bowel Diseases: Oncostatin M and Calprotectin.

BackgroundFecal biomarkers have emerged as one of the most useful tools for clinical management of inflammatory bowel disease (IBD). Oncostatin M (OSM), like fecal calprotectin (FC), is highly expressed in the inflamed intestinal mucosa which may have potential usefulness. We aimed to evaluate the additional utility of these two fecal biomarkers for IBD diagnosis, activity, and prediction of infliximab response over FC alone.MethodsIn group 1, 236 IBD patients (145 Crohn’s disease, 91 ulcerative colitis), 50 disease controls, and 32 healthy controls were recruited for IBD diagnosis and activity. In group 2, baseline stool samples were collected from 62 patients to predict infliximab response at week 28 and 52. The performance of fecal biomarkers for IBD management was assessed by the area under the receiver operating characteristic curve (AUC).ResultsFecal OSM and FC levels were increased in IBD patients and were positively correlated with clinical and endoscopic activity. Their combination showed a better ability for disease diagnosis (AUC = 0.93) and slightly improved the capability to identify mucosal healing (AUC = 0.923). Baseline OSM and FC levels were elevated in non-responders at week 28 and 52. The AUCs of OSM, FC, and their combination to predict therapeutic response were 0.763, 0.834, and 0.859 at week 28, 0.638, 0.661, and 0.704 at week 52, respectively. Combined use of fecal and blood biomarkers improved predictive accuracy with an AUC of 0.919 at week 28 and 0.887 at week 52.ConclusionIn addition to FC, OSM is a novel fecal biomarker, and their combination is more beneficial for disease diagnosis and prediction of infliximab response but not for disease activity in IBD patients. Further larger-scale studies are required to confirm our findings.

Histo-Blood Group Antigens, Enteropathogen Carriage and Environmental Enteropathy in Stunted Zambian Children.

Stunting, the most common form of childhood undernutrition, is associated with environmental enteropathy (EE). Enteric infections are believed to play a role in the pathophysiology of EE and stunting though the exact mechanism remains undetermined. The FUT2 (secretor) and FUT3 (Lewis) genes have been shown to be associated with some symptomatic enteric infections in both children and adults. These genes are responsible for the presence of histo-blood group antigens (HBGAs) in various secretions and epithelial surfaces.We evaluated whether the secretor and Lewis status influences asymptomatic enteric infections and thus EE severity on duodenal biopsies of stunted children. In this case-control study, we used saliva samples to determine the secretor and Lewis status of stunted children (cases, n = 113) enrolled in a nutritional rehabilitation program and from their well-nourished counterparts (controls, n = 42). Where available, saliva was also collected from the mothers. Baseline stool samples were used to detect asymptomatic enteropathogen carriage. Duodenal biopsies were collected from a subgroup of stunted children (n = 77) who had an upper gastrointestinal endoscopy done as part of the evaluation process for their non-response to nutritional therapy. The proportion of secretors was similar between the cases and the controls (82% vs 81%, P = 0.81). The stunted children had significantly higher rates of carrying multiple enteropathogens, but this was not associated with their secretor status nor that of their mothers. The secretor status was also not associated with mucosal morphometry of duodenal biopsies. This case-control analysis in Zambian children does not support the hypothesis that fucosylation status determines asymptomatic enteropathogen carriage in stunting.

Impact of cranberry juice consumption on gut and vaginal microbiota in postmenopausal women

AbstractCranberries have long been purported to provide protection against urinary tract infections. There is a line of evidence suggesting that causal pathogens might be seeded from the bacteria reservoirs in the intestinal and vaginal tracts. We tested the hypothesis whether cranberry intake would reshape bacteria taxa in the gut, as well as the vaginal ecosystem. A total of 25 postmenopausal women were enrolled into a randomized, double‐blind, placebo‐controlled study. Stool samples and vaginal swabs were collected at baseline and after 15 days of consumption of placebo or cranberry beverages, microbiota analyses were performed by Illumina Miseq sequencing following a double‐index 16S rRNA gene amplicon. All baseline stool samples generally fell in theBacteroidesenterotype. Significant increases ofPrevotella(P = 0.04),ClostridiumXIVa members (P = 0.04),Eggerthella(P = 0.03), andBifidobacterium(P = 0.02) were shown following the cranberry juice intervention; this indicates modulation of the gut microbiota by cranberry components. Baseline vaginal microbiotas fell in three distinct patterns—Lactobacillusdominant, diversified microbiome, andStreptococcusdysbiosis. Compared with the placebo, the cranberry intervention significantly reduced the abundance of pathogenicStreptococcus(P = 0.04) in the dysbiosis group and increased commensal bacteriaAnaerococcus,Finegoldia,Actinomyces, andCorynebacteriumin the diversified microbiome and dysbiosis groups. Overall, these data suggest that cranberry consumption may improve vaginal microbiota composition in individuals with dysbiosis. Gut‐borne taxa stimulation by the combination of cranberry oligosaccharides and polyphenols present in the cranberry product potentially mediates these beneficial properties.

Gut Bacterial Diversity Associates with Efficacy of Anti-CD19 CAR T-Cell Therapy in Patients with Large B-Cell Lymphoma

Gut Bacterial Diversity Associates with Efficacy of Anti-CD19 CAR T-Cell Therapy in Patients with Large B-Cell Lymphoma