Inverse Simpson Index Research Articles

Navigating the microbial community in the trachea-oropharynx of breast cancer patients with or without neoadjuvant chemotherapy (NAC) via endotracheal tube: has NAC caused any change?

We compare the diversity and niche specificity of the microbiome in the trachea-oropharynx microbiome of malignant breast neoplasm with or without neoadjuvant chemotherapy (NAC) via NGS analysis. We prospectively collected a total of 40 endotracheal tubes intubated from subjects, of whom 20 with NAC treated breast cancer (NAC group) and 20 with breast cancer without NAC (Non-NAC group). We generated 16S rRNA-based microbial profiles in IlluminaTM platform and alpha diversity indices were compared between groups. For the comparison of taxa abundance, linear discriminant analysis effect size method with Kruskal-Wallis test was used. The distribution of variables between the two groups was compared using the Mann-Whitney test. For beta diversity analysis, PERMANOVA was used. Among the diversity indices, the NAC group showed significantly lower Chao1, Inverse Simpson, and Shannon indices than the Non-NAC group. The three most frequent taxa of all two groups were Streptococcus (20.4%), followed by Veillonella (11.9%), and Prevorella (10.4%). This order was the same in NAC and non-NAC groups. Here, we provide the first comparison data of the respiratory tract microbiome of breast cancer patients with or without NAC via NGS analysis. This study ultimately seeks to contribute to future studies on the lower respiratory tract in cancer patients with cytotoxic chemotherapy by establishing reliable control data.

A Whole Foods Plant-Based Weight Loss Intervention Improves Quality of Life, Metabolic, Microbiome and Immune Profile in MGUS/SMM As Well As Progression Trajectory in a Subset - the Nutrivention Trial

Introduction Obesity, low adiponectin, high leptin, high insulin, and diets lacking plant-based foods are risk factors for plasma cell disorders (PCDs). Patients (pts) with monoclonal gammopathy (MGUS) and smoldering myeloma (SMM) and an elevated body mass index (BMI) are twice as likely to progress to myeloma (MM). There is evidence of microbiome dysbiosis in pts with PCDs and dietary changes can induce large microbial shifts. Therefore, there is a rationale to study a whole food plant-based dietary (WFPBD) intervention to improve modifiable biomarkers and disease trajectory. Methods This was a pilot, single-arm trial of WFPBD (self-selected prepared frozen lunch/dinner provided by Plantable with breakfast/snack guidance and no calorie restriction) for 12-weeks (w) and health coaching for 24w (by Plantable coach and a research dietitian) in pts with MGUS/SMM and BMI ≥25 (NCT04920084). The primary endpoint was feasibility (mean BMI reduction ≥5% and mean dietary adherence [kcal % unprocessed plant food intake] ≥70%) at 12w; secondary endpoints included quality of life (QoL) (EORTC QLQ C30 survey using a linear-mixed effect model), metabolic markers (plasma insulin, adiponectin leptin (AL) ratio via ELISA), gut microbiome (inverse Simpson index α-diversity and relative abundance of butyrate producers (RABP) via stool 16S sequencing), peripheral blood immune profile (leukocyte subsets via 36 color flow cytometry), secreted biomarkers (plasma via Olink inflammation panel) and change in monoclonal (M)-spike concentrations. Rate of change of M-spike/year (y) measured by slope with 95% CI was calculated for up to 20 months (m) pre-intervention and 20m post intervention start (baseline (BL)). A p-value for difference in M-spike rates was calculated. Results The study enrolled 23 pts of which 20 completed the 12w intervention and 16 completed 1y. The median age was 62y with 43% male, 43% non-White, 52% MGUS, 74% obese, and 26% prediabetic/diabetic. The study met feasibility endpoints with 90% mean and 92% median adherence during 12w intervention and 77% median at 24w. There was an 8.3% mean and 6.6% median BMI reduction at 12w and 8.6% median at 24w. There was an improvement in global health status/QoL (median increase 16.7 points, p=0.03), a reduction in dyspnea (median decrease -33.3 points, p=0.001), fatigue (median decrease -11.1 points, p=0.06), and insulin (median decrease 0.791 mU/L; p=0.01), and an increase in AL ratio (median increase 0.09; p=0.0002), α-diversity (median increase 6.18 to 8.42; p= 0.03) and RABP (median increase 0.03 to 0.08; p=0.042) at 12w. Prelim results at 12w suggest a measurable, albeit not significant change in immune subsets such as an increase in monocytes, regulatory T cells, and a decrease in dendritic cells, trends previously associated with anti-inflammatory diets, as well as increase in butyrate. A decrease in proinflammatory cytokines IL8 (p=0.065), IL12B (p=0.068), and TNFB (p=0.082) and an increase in FGF21 (p=0.043) (promoter of insulin sensitivity) was also observed. Butyrate is known to reduces pro-inflammatory cytokines and increase anti-inflammatory proteins, including FGF21. Further immune and microbiome analysis is ongoing. Of 16 pts followed for 1y, 2 with significant BMI reduction had significant improvement in M-spike trajectory: Case 1: Mayo Int Risk IgGκ/IgGλ MGUS achieved a 1y BMI reduction of 19%. Pre-intervention M-spike changed +0.28g/dL/y (6 M-spike values), and during intervention M-spike changed +0.03g/dL/y (11 M-spike values); p=0.008. BL M-spike 1.2g/dL and bone marrow plasma cells (BM PC) <5%; 1.5y BM PC 5-9%. Case 2: IMWG Int Risk IgGκ SMM achieved a 1y BMI reduction of 13%. Pre-intervention M-spike changed +0.11g/dL/y (8 M-spike values), and during intervention M-spike changed -0.05g/dL/y (8 M-spike values); p=0.04. BL M-spike 1.2g/dL and BM PC 20-30%; 1y BM PC 10-15%. Two pts without significant BMI reduction (-4% and -1% at 1y) had rising M-spikes. The remaining 12 pts had median 7.5% BMI reduction at 1y with stable M-spike. Conclusions This is the first dietary intervention trial in PCDs with insights into the mechanisms by which a WFPBD may delay progression. The intervention improves QoL, metabolic (BMI, insulin resistance), microbiome (α-diversity and butyrate producers) as well as immune (reduced inflammation) profile with potentially slowing progression trajectory in a subset. A larger trial is underway (NCT05640843).

285 Effects of Dietary Lysozyme Supplementation on Nitrogen Balance, Microbiota Diversity and Growth Performance of Growing Pigs

Abstract Lysozyme is an enzyme with antimicrobial properties that might improve gut health. This study aimed to evaluate the effect of lysozyme supplementation on growth performance, nitrogen (N) balance, and microbiota diversity of pigs and to determine the optimal inclusion level of this enzyme. Barrows [n = 72; 40.6 kg ± 2.6 body weight (BW); Yorkshire x Landrace] were used for this experiment. Pigs were distributed in a completely randomized design with 12 replicates within six dietary treatments (0, 16, 32, 48, 64, and 80 mg of lysozyme/kg of diet). Pigs were housed in the same pen, and individual transponders allowed feeders to identify the pigs, record their feed intake, and provide feed according to the individual assigned treatment. Animal growth performance was evaluated through average daily feed intake (ADFI, kg/d), average daily gain (ADG, kg/d), and gain-to-feed ratio (G:F, kg/kg) over 21 days. The N balance was estimated by dual-energy X-ray on days 1 and 21. The digesta in the jejunum segment was collected to study microbiota alpha and beta diversity. The linear-plateau and quadratic-plateau models were used to determine the optimal dietary lysozyme inclusion level as a function of ADG. The data were analyzed as completely randomized, the main fixed effect was lysozyme dose, and the scanning day was considered a random effect. The ADFI decreased linearly (P ≤ 0.01), while ADG, G: F and N utilization efficiency increased linearly (P ≤ 0.01) with the inclusion of lysozyme. Based on the non-linear models estimates, the minimal dietary lysozyme concentration maximizing ADG was 40 mg/kg, and no ADG response was observed above 50 mg/kg of supplementation. Shannon and inverse Simpson indices showed significant differences between cecum and jejunum (P ≤ 0.05) and due to the lysozyme, whereas treatments did not impact Chao1, ACE indexes and observed features, suggesting a treatment effect on species evenness but not on species richness. The treatments did not affect any of the beta diversity matrices analyzed. Based on these results, lysozyme supplementation up to 50 mg/kg may affect the species evenness in jejunum digesta while improving N utilization efficiency and ADG.

Unraveling the impact of primary immunodeficiency disorders on the microbiota of dental caries in children through 16S rRNA gene-based metagenomic analysis.

Dental caries is a frequently occurring and multifactorial chronic disease in children resulting from the interaction of cariogenic bacteria and host susceptibility. The aim of this study was to elucidate the impacts of primary immunodeficiency disorders (PIDs) on microbiota of dental caries in children by 16S rRNA gene-based metagenomic analysis. Enrolled in this study were 15 children with primary PID with caries (PID group) and 15 healthy children with caries as a control (CG). The DMFT index, saliva flow rate, and buffering capacity of each participant were assessed before the metagenomic analyses were conducted. For taxonomic profiling, the reads were obtained by high-throughput sequencing of the V3-V4 hypervariable region of 16S rRNA. The DMFT score, saliva flow rate, and buffering capacity of the groups were similar. The flow rate and buffering capacity had no correlation with the number of species with 95% confidence. The metagenomic analysis resulted in the identification of 2440 bacterial species in all of the samples. Among the 50 most prevalent species present at ≥1% relative abundance, Prevotella melaninogenica and Prevotella salivae were differentially more abundant in the PID group. The PID group and CG showed similar species richness and evenness, but 4 of the 5 samples with the highest Shannon-Weiner and Inverse Simpson indices belonged to the PID group. The Spearman test results for correlation of the species in the PID subgroups showed that Prevotella oris had a positively correlated relationship with both Scardovia wiggsiae and Saccharibacteria genera incertae sedis. This study provided insight into the caries microbiota of children with immunodeficiency diseases. Differentially abundant species, novel bacterial associations, and unique bacterial species were disclosed in the PID samples, indicating the role of the immune system in altering the caries microbiota. The prominent bacterial species and associations in the PID group should be suspected in regard to their link with present or future diseases.

Gut Microbiome in Patients With Early-Stage and Late-Stage Melanoma

The gut microbiome modulates the immune system and responses to immunotherapy in patients with late-stage melanoma. It is unknown whether fecal microbiota profiles differ between healthy individuals and patients with melanoma or if microbiota profiles differ among patients with different stages of melanoma. Defining gut microbiota profiles in individuals without melanoma and those with early-stage and late-stage melanoma may reveal features associated with disease progression. To characterize and compare gut microbiota profiles between healthy volunteers and patients with melanoma and between patients with early-stage and late-stage melanoma. This single-site case-control study took place at an academic comprehensive cancer center. Fecal samples were collected from systemic treatment-naive patients with stage I to IV melanoma from June 1, 2015, to January 31, 2019, and from healthy volunteers from June 1, 2021, to January 31, 2022. Patients were followed up for disease recurrence until November 30, 2021. Fecal microbiota was profiled by 16S ribosomal RNA sequencing. Clinical and pathologic characteristics, treatment, and disease recurrence were extracted from electronic medical records. Fecal microbiome diversity, taxonomic profiles and inferred functional profiles were compared between groups. A total of 228 participants were enrolled (126 men [55.3%]; median age, 59 [range, 21-90] years), including 49 volunteers without melanoma, 38 patients with early-stage melanoma (29 with stage I or melanoma in situ and 9 with stage II), and 141 with late-stage melanoma (66 with stage III and 75 with stage IV). Community differences were observed between patients with melanoma and volunteers. Patients with melanoma had a higher relative abundance of Fusobacterium compared with controls on univariate analysis (0.19% vs 0.003%; P < .001), but this association was attenuated when adjusted for covariates (log2 fold change of 5.18 vs controls; P = .09). Microbiomes were distinct between patients with early-stage and late-stage melanoma. Early-stage melanoma had a higher alpha diversity (Inverse Simpson Index 14.6 [IQR, 9.8-23.0] vs 10.8 [IQR, 7.2-16.8]; P = .003), and a higher abundance of the genus Roseburia on univariate analysis (2.4% vs 1.2%; P < .001) though statistical significance was lost with covariate adjustment (log2 fold change of 0.86 vs controls; P = .13). Multiple functional pathways were differentially enriched between groups. No associations were observed between the microbial taxa and disease recurrence in patients with stage III melanoma treated with adjuvant immunotherapy. The findings of this case-control study suggest that fecal microbiota profiles were significantly different among patients with melanoma and controls and between patients with early-stage and late-stage melanoma. Prospective investigations of the gut microbiome and changes that occur with disease progression may identify future microbial targets for intervention.

Compositional shifts within the denture-associated bacteriome in pneumonia - an analytical cross-sectional study.

Introduction. Bacterial pneumonia is a common cause of morbidity and mortality in elderly individuals. While the incidence of edentulism is falling, approximately 19 % of the UK population wear a full or partial removable denture. Despite advances in denture biomaterials, the majority of dentures are fabricated using polymethyl-methacrylate. Growing evidence suggests that colonization of the oral cavity by putative respiratory pathogens predisposes individuals to respiratory infection, by translocation of these microorganisms along the respiratory tract.Hypothesis/Gap Statement. We hypothesized that denture surfaces provide a susceptible colonization site for putative respiratory pathogens, and thus could increase pneumonia risk in susceptible individuals.Aim. This study aimed to characterize the bacterial community composition of denture-wearers in respiratory health compared with individuals with a confirmed diagnosis of pneumonia.Methodology. This was an analytical cross-sectional study, comparing frail elderly individuals without respiratory infection (n=35) to hospitalized patients with pneumonia (n=26). The primary outcome was the relative abundance of putative respiratory pathogens identified by 16S rRNA metataxonomic sequencing, with quantitative PCR used to identified Streptococcus pneumoniae.Results. There was a statistically significant increase in the overall relative abundance of putative respiratory pathogens (P<0.0001), with a greater than 20-fold increase in the bioburden of these microorganisms. In keeping with these findings, there were significant shifts in bacterial community diversity (Chao index, P=0.0003) and richness (Inverse Simpson index P<0.0001) in the denture-associated microbiota of pneumonia patients compared with control subjects.Conclusion. Within the limitations of this study, our evidence supports the role of denture acrylic biomaterials as a potential colonization site for putative respiratory pathogens, which may lead to an increased risk of pneumonia in susceptible individuals. These findings support prior observational studies which have found denture-wearers to be at increased risk of respiratory infection. Further research is needed to confirm the sequence of colonization and translocation to examine potential causal relationships.

The effect of vitamin D supplementation on the gut microbiome in older Australians – Results from analyses of the D-Health Trial

ABSTRACT Observational studies suggest a link between vitamin D and the composition of the gut microbiome, but there is little evidence from randomized controlled trials of vitamin D supplementation. We analyzed data from the D-Health Trial, a randomized, double-blind, placebo-controlled trial. We recruited 21,315 Australians aged 60–84 y and randomized them to 60,000 IU of vitamin D3 or placebo monthly for 5 y. Stool samples were collected from a sample of 835 participants (417 in the placebo and 418 in the vitamin D group) approximately 5 y after randomization. We characterized the gut microbiome using 16S rRNA gene sequencing. We used linear regression to compare alpha diversity indices (i.e. Shannon index (primary outcome), richness, inverse Simpson index), and the ratio of Firmicutes to Bacteroidetes between the two groups. We analyzed between-sample (beta) diversity (i.e. Bray Curtis distance and UniFrac index) using principal coordinate analysis and used PERMANOVA to test for significant clustering according to randomization group. We also assessed the difference in the abundance of the 20 most abundant genera between the two groups using negative binomial regression model with adjustment for multiple testing. Approximately half the participants included in this analysis were women (mean age 69.4 y). Vitamin D supplementation did not alter the Shannon diversity index (mean 3.51 versus 3.52 in the placebo and vitamin D groups, respectively, p = 0.50). Similarly, there was little difference between the groups for other alpha diversity indices, the abundance of different genera, and the Firmicutes-to-Bacteroidetes ratio. We did not observe clustering of bacterial communities according to randomization group. In conlusion, monthly doses of 60,000 IU of vitamin D supplementation for 5 y did not alter the composition of the gut microbiome in older Australians.

Comparative analysis of vaginal microbiota sampling using menstrual cups and high vaginal swabs in pregnant women living with HIV-1 infection.

Menstrual cups (MCs) are increasingly used to collect cervicovaginal secretions to characterise vaginal mucosal immunology, in conjunction with high vaginal swabs (HVS) for metataxonomics, particularly in HIV transmission studies. We hypothesised that both methods of collecting bacterial biomass are equivalent for 16S rRNA gene sequencing. Cervicovaginal fluid (CVF) samples from 16 pregnant women with HIV-1 (PWWH) were included to represent the major vaginal bacterial community state types (CST I-V). Women underwent sampling during the second trimester by liquid amies HVS followed by a MC (Soft disc™) and samples were stored at -80°C. Bacterial cell pellets obtained from swab elution and MC (500 µL, 1 in 10 dilution) were resuspended in 120 µL PBS for DNA extraction. Bacterial 16S rRNA gene sequencing was performed using V1-V2 primers and were analysed using MOTHUR. Paired total DNA, bacterial load, amplicon read counts, diversity matrices and bacterial taxa were compared by sampling method using MicrobiomeAnalyst, SPSS and R. The total DNA eluted from one aliquot of diluted CVF from an MC was similar to that of a HVS (993ng and 609ng, p=0.18); the mean bacterial loads were also comparable for both methods (MC: 8.0 log10 16S rRNA gene copies versus HVS: 7.9 log10 16S rRNA gene copies, p=0.27). The mean number of sequence reads generated from MC samples was lower than from HVS (MC: 12730; HVS:14830, p=0.05). The α-diversity metrices were similar for both techniques; MC Species Observed: 41 (range 12-96) versus HVS: 47 (range 16-96), p=0.15; MC Inverse Simpson Index: 1.98 (range 1.0-4.0) versus HVS: 0.48 (range 1.0-4.4), p=0.22). The three most abundant species observed were: Lactobacillus iners, Lactobacillus crispatus and Gardnerella vaginalis. Hierarchical clustering of relative abundance data showed that samples obtained using different techniques in an individual clustered in the same CST group. These data demonstrate that despite sampling slightly different areas of the lower genital tract, there was no difference in bacterial load or composition between methods. Both are suitable for characterisation of vaginal microbiota in PWWH. The MC offers advantages, including a higher volume of sample available for DNA extraction and complimentary assays.

Pathogenesis of flares in liver inflammation in hepatitis B virus-infected women during and after pregnancy

Abstract Women with chronic hepatitis B (CHB) are at risk of increased liver inflammation during pregnancy. To investigate the pathogenesis of these hepatic flares we studied immune responses and gut microbiota in 19 women with CHB (12 without and 7 with antiviral (NUC) treatment) and 3 uninfected controls in each trimester and at two and six months postpartum. Increased serum alanine aminotransferase (ALT) activity as a liver inflammation marker was defined as a value greater than the upper limit of normal and twice the baseline value. While increased plasma levels of soluble CD163, inflammatory cytokines and chemokines generally occurred during episodes of liver inflammation, increases of IL-22 and IFN-γ levels during pregnancy preceded a postpartum ALT increase. In contrast, levels of IL-27, a cytokine produced by the trophoblast, increased also in women with normal ALT levels. The fecal microbiota of HBV-infected patients with and without NUC treatment had lower α-diversity (inverse Simpson index) than those of uninfected controls. 16srRNA amplicon analysis and PCA showed patient-specific clustering of microbiota. To identify factors predictive of a postpartum ALT increase, we compared 16srRNA DNA data from patients with a postpartum ALT increase to those with normal postpartum ALT levels (four patients per group). Combining all time points, the gut microbiota of those with postpartum ALT increase differed significantly from those with normal ALT activities (UMAP, PERMANOVA p&amp;lt;0.003). In conclusion, the composition of the fecal microbiota was patient-rather than time point-specific and differed between patients with and without postpartum ALT increase. Supported by NIH Bench-to-Bedside Award (B.R. and D.T.Y.L.)

DNA metabarcoding on repeat: Sequencing data of marine macrobenthos are reproducible and robust across labs and protocols

DNA metabarcoding can be used in marine environmental monitoring if results are reproducible between labs and robust against modifications to the lab protocol. In this interlaboratory study, we conducted a ring test where subsamples of blended macrobenthos samples were distributed to four laboratories located in Belgium, the Netherlands, Germany and Denmark. Samples were processed by a standardized lab protocol and by an adapted protocol, and the resulting datasets were analyzed with the same bioinformatics pipeline. Different biodiversity indicators were calculated. Our results show that bulkDNA metabarcoding of marine macrobenthos offers a highly reproducible assessment of alpha diversity patterns when using a standardized protocol, since comparable species numbers, Shannon indices and Inverse Simpson indices were found between laboratories. Especially high abundant species and species with large body sizes were shared between the laboratories. The need for using a standardized protocol to enhance comparability in alpha diversity between different studies was shown. Beta diversity patterns are less subjected to changes in the metabarcoding protocol and were almost identical between different laboratories, as the main clustering was always based on the macrobenthic community, independent of the used protocol or the laboratory that conducted the work. We conclude that DNA metabarcoding for marine environmental monitoring is an appropriate method when the aim is to study changes in community patterns and advocate its implementation in routine monitoring programs of national and European authorities, providing that a standardized protocol is implemented and/or a detailed description of the protocol is available.

Abstract 5910: Prospective platform to define microbiome correlates of metastasis and therapy resistance in early onset and average onset gastrointestinal cancers

Abstract Background: The incidence of early onset (EO) gastrointestinal (GI) cancers prior to age 50 is rising, and the etiology is unknown. The gut microbiome may contribute to GI cancer pathogenesis, though how bacteria drive metastasis and treatment resistance is not known. Purpose: To define the microbiome contribution to EO-GI cancers and metastasis by analyzing longitudinal samples from previously untreated patients with GI cancers. Methods: We designed a prospective biospecimen collection platform. We selected patients with newly diagnosed, previously untreated colorectal cancer (CRC) and esophagogastric cancer (EGC). We collected stool, biopsy or surgical tissue, and peripheral blood mononuclear cells (PBMCs) at baseline prior to treatment and at each restaging scan for patients with metastatic disease. For those with locally advanced disease, we collected samples at baseline and after each treatment phase (surgery, radiation, chemotherapy). Stool samples were analyzed using shotgun sequencing. Tissue samples were banked for further analysis. Clinical data was manually curated. Relative abundances of bacteria at the species level were compared between groups. Alpha diversity was calculated using the inverse Simpson index and compared between groups using the Wilcoxon signed-rank test. Beta diversity was analyzed using the Bray-Curtis dissimilarity matrix and compared using PERMANOVA. Multivariate association between species abundance and clinical covariates was performed using MaAsLin2 R package. Results: We analyzed a total of 150 stool samples from 76 patients (colorectal n=53, esophagogastric n=23), including up to 6 samples from a single patient over time. Mean alpha diversity did not differ significantly by primary site or age at diagnosis in patients with CRC but was higher in stage IV compared with stage I disease (P=0.054). Beta diversity was significantly different between samples from patients with right- compared with left-sided CRC (P=0.005) but did not differ significantly by diagnosis age (&amp;lt;50 vs. &amp;gt; 50). Among those with EGC, mean alpha diversity was not significantly different in samples from patients with esophagus or gastroesophageal junction (proximal) tumors compared with gastric (distal) tumors and did not differ significantly by age group. Beta diversity was significantly different between patients with EGC over 50 compared with those under 50 (P=0.002). When CRC baseline samples were examined, several bacterial species were associated with age &amp;lt;50 at diagnosis, including Streptococcus anginosus group (P=0.001), Solobacterium moorei (P=0.013), and Firmicutes bacterium CAG83 (P=0.016). Conclusions/Future Directions: Microbiome composition may cluster by primary tumor site and age at diagnosis in patients with previously untreated GI cancers. Functional analysis and is ongoing and will be presented. Citation Format: Melissa A. Lumish, Asha R. Saxena, Nicholas Waters, Anqi Dai, Saskia Hartner, Teng Fei, Matthew Drescher, Jonathan Bermeo, Dorina Ismailgeci, Maggie Fox, Yelena Y. Janjigian, Luis A. Diaz, Martin R. Weiser, Jonathan Peled, Marcel van den Brink, Karuna Ganesh. Prospective platform to define microbiome correlates of metastasis and therapy resistance in early onset and average onset gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5910.

Abstract P1-04-02: Gut microbiome diversity correlates with tumor PD-L1 status in metastatic triple negative breast cancer (mTNBC): correlative analysis of gut microbiome and tumoral biomarkers

Abstract Background: Greater gut microbiome diversity is observed in patients with a clinical response to immune checkpoint inhibitor (ICI) therapy in solid tumors and in preclinical models targeting the gut microbiome is a promising strategy to improve ICI efficacy. The investigation of the immunomodulatory effects of the gut microbiome and tumor biology is in its infancy and whether interindividual microbiome variations correlate with tumor biology in breast cancer is unknown. Here we report a correlative analysis of gut microbiome diversity with tumor biomarkers: PD-L1, tumor immune cell density, tumor infiltrating lymphocytes (TILs), MutSig3, and interferon (IFN) gene signatures in a cohort of mTNBC patients treated with olaparib and durvalumab of the Adaptive Multi-Drug Treatment of Evolving Cancers (AMTEC) trial (NCT03801369). Methods: AMTEC participants undergo a baseline biopsy (bx) and fecal sample collection, then start olaparib monotherapy on a 28-day cycle. Following the first cycle, an on-treatment (on-tx) bx and fecal sample are collected, then as part of enrollment to Arm 1, durvalumab is added to the treatment regimen. Ten patients with stool collections and tissue biopsies were used in this analysis, eight had paired pre- and on-tx collections. At interim-analysis, participants were categorized as responders (PR/SD) or non-responders (PD). Gut microbiome composition in fecal samples collected pre- and on-olaparib monotherapy was assessed by 16S rRNA sequencing and alpha diversity was quantified by observed diversity, Fisher, Inverse Simpson, Pielou, and Shannon indices. Response, PDL1 expression (Positive CPS &amp;gt;1), multiplex immunohistochemistry (mIHC) immune cell density and immune cell signature, TILs, RNA signatures including interferon (INF) family signatures, and mutational significance (MutSig3) were correlated to alpha diversity by T-test. Results: Average baseline alpha diversity of the fecal microbiome did not vary based on response (6 non-responses and 4 responses). However, in the on-tx samples, Fisher (CB: mean = 21.81, std. dev. = 3.6; PD: mean = 17.04, std. dev. = 2.3; p=0.048) and Observed alpha diversity (CB: mean = 260, std. dev. = 38.8; PD: mean = 209, std. dev. = 25.8; p = 0.049) were higher in responders. Average baseline alpha diversity of the gut microbiome as measured across all examined indices was significantly lower in participants with PD-L1 expressing tumors (p &amp;lt; 0.05 for each, T-test). Among on-tx samples, there was a trend toward lower alpha diversity in PD-L1 expressing tumors, but PD-L1 expression did not correlate with clinical response. There was a notable trend toward greater alpha diversity in on-tx bx and high TILs. Alpha diversity did not differ by immune cell densities by mIHC (CD8+ T cell, B cell, and regulatory T cells), hypo-inflamed/pro-inflammatory signature, IFN gene signature, nor MutSig3 score. Conclusion: Our preliminary data reveal that higher alpha diversity following olaparib monotherapy correlated with response to therapy with combination olaparib and durvalumab and correlated with tumor PD-L1 status. Whether gut microbiome features represent a meaningful biomarker for ICI treatment of mTNBC warrants further study. Enrollment to stage 2 of AMTEC Arm 1 is ongoing. Citation Format: Brie Chun, Shaun Goodyear, Travis Rice-Stitt, Lisa Karstens, Erin Dahl, Allen Li, Evthokia Hobbs, Mitri Zahi. Gut microbiome diversity correlates with tumor PD-L1 status in metastatic triple negative breast cancer (mTNBC): correlative analysis of gut microbiome and tumoral biomarkers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-04-02.

Abstract PD11-05: PD11-05 Gut microbiome signatures correlate with overall survival among patients receiving eribulin with or without pembrolizumab for hormone receptor-positive metastatic breast cancer

Abstract Background: The gut microbiome modulates response and resistance to immune checkpoint inhibitors (ICI) across different cancer types. The objective of this study was to explore the association between fecal microbiome profiles and clinical outcomes in patients with hormone receptor-positive (HR+) metastatic breast cancer (MBC) treated with eribulin (E) + pembrolizumab (P) or E alone as part of a randomized phase II study (NCT03051659). Patients and Methods: Metagenomic shotgun sequencing was performed on fecal samples collected prior to randomization from a subset of 26 participating patients (E+P, n = 15; E, n = 11) collected prior to randomization. Sequencing data were processed with MetaPhlAn3 to obtain diversity scores and taxonomic composition profiles. Associations between microbiome diversity, taxonomic composition, and clinical outcomes including survival metrics were assessed using Kaplan-Meier estimation methods and Cox proportional hazard models. Results: In this subset, median follow-up was 21.6 months and median overall survival (OS) was 17.9 months. OS was not statistically significant between two treatment arms as observed in the overall cohort. Metagenomic microbiome analysis revealed higher alpha diversity by inverse Simpson Index values associated with longer survival among all patients (p = 0.004), and in patients in the E + P arm (p = 0.006) but not in the E arm (p = 0.2). At the taxonomic level, longer OS among all patients was associated with a baseline lower relative abundance of Blautia wexlerae (≤0.2%vs &amp;gt;0.2%) (22.1 vs 16.6 months; p = 0.01) and a higher relative abundance of Odoribacter splanchnicus, a common short-chain fatty acid producing gut bacterium (&amp;gt;0.35% vs ≤0.35%) (22.3 vs 11.3 months; p &amp;lt; 0.0001). These associations remained significant after controlling for age, ECOG-PS status, and use or not of prior lines of chemotherapy in the metastatic setting. Conclusions: These results suggest high diversity and composition of the gut microbiome are associated with prolonged OS in HR+ MBC pts receiving eribulin with pembrolizumab. Additional and larger clinical studies are needed to validate these findings and preclinical models are needed to interrogate the mechanisms by which gut microbiome diversity and composition may impact therapeutic response and clinical outcomes in this patient population. Citation Format: Romualdo Barroso-Sousa, Tianyu Li, Ashish V. Damania, Molly K. DiLullo, Tanya Keenan, Gerburg M. Wulf, Laura M. Spring, Elizabeth A. Mittendorf, Jennifer A. Ligibel, Nadim J. Ajami, Jennifer Wargo, Nabihah Tayob, Sara Tolaney. PD11-05 Gut microbiome signatures correlate with overall survival among patients receiving eribulin with or without pembrolizumab for hormone receptor-positive metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD11-05.

Dynamics and recovery of forest understory biodiversity over 17 years following varying levels of retention harvesting

Abstract Retention harvesting is advocated as an alternative to intensive timber harvesting, such as clear‐cutting, to better maintain or facilitate recovery of biodiversity and other ecological values in managed forests. However, it is not clear how long the benefits of retention harvests persist. We investigated responses of understory vascular plant cover, richness, diversity (inverse Simpson index) and composition to a gradient in dispersed retention (2% [clear‐ cut], 10%, 20%, 50% and 75% retention; unharvested reference [100% retention]) at 3, 6, 11, and 17 years after harvest, in four boreal mixedwood forest types (deciduous (broadleaf)‐dominated, deciduous‐dominated with conifer understory, mixed, and conifer‐dominated) in western Canada. Understory cover and richness tended to increase in the short‐term (3 years), peaked at 6–11 years with differences following the gradient of harvesting intensity, then plateaued or declined in the second decade (17 years), by which time there were minimal or no differences among harvesting levels, including the reference. Responses for diversity were minimal. In contrast, composition varied along the gradient of harvesting intensity and showed little recovery towards the unharvested condition over the 17‐year period. Generally, for plant community composition, clear‐cut and lower retention treatments (10%, 20%) were similar to one another but differed from the higher retention and unharvested reference treatments. Synthesis and applications: Retention harvests can moderate the negative impacts of harvesting and facilitate the recovery of biodiversity. Our results suggest that for the cover, richness and diversity of understory vascular plants, this moderating influence is weak and short‐lived. However, higher levels of retention can temper changes in understory composition relative to the unharvested forest, but full recovery is likely to be slow and will be complicated by post‐harvest regeneration dynamics.

Effect of red wheat, aleurone, and testa layers on colon cancer biomarkers, nitrosative stress, and gut microbiome composition in rats.

We previously found greater reduction of colon cancer (CC) biomarkers for red wheat compared to white wheat regardless of refinement state. In the present study we examined whether the phenolic-rich aleurone and testa layers are drivers of chemoprevention by red wheat and their influence on gut microbiota composition using a 1,2-dimethylhydrazine-induced CC rat model. Rats were fed a low-fat diet (16% of energy as fat), high-fat diet (50% of energy as fat), or high-fat diet containing whole red wheat, refined red wheat, refined white wheat, or aleurone- or testa-enriched fractions for 12 weeks. Morphological markers (aberrant crypt foci, ACF) were assessed after methylene blue staining and biochemical markers (3-nitrotyrosine [3-NT], Dclk1) by immunohistochemical determination of staining positivity within aberrant crypts. Gut microbiota composition was evaluated from 16S rRNA gene sequencing of DNA extracted from cecal contents. Relative to the high-fat diet, the whole and refined red wheat, refined white wheat, and testa-enriched fraction decreased ACF, while only the refined red wheat and aleurone-enriched fraction decreased 3-NT. No significant differences were observed for Dclk1. An increase in microbial diversity was observed for the aleurone-enriched fraction (ACE index) and whole red wheat (Inverse Simpson Index). The diet groups significantly modified overall microbiome composition, including altered abundances of Lactobacillus, Mucispirillum, Phascolarctobacterium, and Blautia coccoides. These results suggest that red wheat may reduce CC risk through modifications to the gut microbiota and nitrosative stress, which may be due, in part, to the influence of dietary fiber and the phenolic-rich aleurone layer.

A quality improvement study on the relationship between intranasal povidone-iodine and anesthesia and the nasal microbiota of surgery patients.

The composition of the nasal microbiota in surgical patients in the context of general anesthesia and nasal povidone-iodine decolonization is unknown. The purpose of this quality improvement study was to determine: (i) if general anesthesia is associated with changes in the nasal microbiota of surgery patients and (ii) if preoperative intranasal povidone-iodine decolonization is associated with changes in the nasal microbiota of surgery patients. One hundred and fifty-one ambulatory patients presenting for surgery were enrolled in a quality improvement study by convenience sampling. Pre- and post-surgery nasal samples were collected from patients in the no intranasal decolonization group (control group, n = 54). Pre-decolonization nasal samples were collected from the preoperative intranasal povidone-iodine decolonization group (povidone-iodine group, n = 97). Intranasal povidone-iodine was administered immediately prior to surgery and continued for 20 minutes before patients proceeded for surgery. Post-nasal samples were then collected. General anesthesia was administered to both groups. DNA from the samples was extracted for 16S rRNA sequencing on an Illumina MiSeq. In the control group, there was no evidence of change in bacterial diversity between pre- and post-surgery samples. In the povidone-iodine group, nasal bacterial diversity was greater in post-surgery, relative to pre-surgery (Shannon's Diversity Index (P = 0.038), Chao's richness estimate (P = 0.02) and Inverse Simpson index (P = 0.027). Among all the genera, only the relative abundance of the genus Staphylococcus trended towards a decrease in patients after application (FDR adjusted P = 0.06). Abundant genera common to both povidone-iodine and control groups included Staphylococcus, Bradyrhizobium, Corynebacterium, Dolosigranulum, Lactobacillus, and Moraxella. We found general anesthesia was not associated with changes in the nasal microbiota. Povidone-iodine treatment was associated with nasal microbial diversity and decreased abundance of Staphylococcus. Future studies should examine the nasal microbiota structure and function longitudinally in surgical patients receiving intranasal povidone-iodine.

ALL-079 Association of T-Cell Receptor Repertoire Diversity With L-Asparaginase Hypersensitivity in Childhood Acute Lymphoblastic Leukemia

Asparaginase is an indispensable component of therapy for childhood acute lymphoblastic leukemia (ALL) but is very commonly associated with allergy which causes morbidity and poorer outcomes. The exact aspects of T-cell immunity underpinning association with asparaginase allergy has yet to be characterized. To evaluate the associations of T-cell receptor (TCR) repertoire and its diversity with asparaginase hypersensitivity in children with ALL. Longitudinal TCR-sequencing of 180 bone marrow samples across 3 time-points (diagnosis, post-induction, post-consolidation) from 67 children with ALL treated on the frontline Ma-Spore ALL 2010 trial. 12 out of 67 children (17.9%) had allergy in our cohort, representing 16 episodes of varying severity. First, we evaluated the association of TCR-repertoire diversity early on in treatment with the occurrence of subsequent allergy. We found that a higher TCR diversity at all time-points was significantly associated with late allergy (P=0.03 for Shannon's entropy, P=0.01 for inverse Simpson's index). This TCR diversity was characterized by a higher proportion of infrequent clones occurring <0.5% (P=0.008). Patients with allergy had significantly lower proportion of shared clonotypes (P=0.003). Examining the dynamic changes of the TCR repertoire between diagnosis and week 5 for each patient, allergic patients had a much less similar clonotypic set between timepoints compared to non-allergic patients (P=0.003). In fact, patients with a similarity coefficient of <0.05 (i.e., demonstrated greater longitudinal variability in their clonotypes) had an 8.1-fold risk of allergic event (95% CI 1.7 - 39.1, P=0.001). Evaluating the TCR-repertoire before and after an allergy, we found that there was convergence of TCR towards a common antigen, where clonotypes became more closely related after an allergy. Finally, we found that allergic patients had a lower proportion of public clonotypes (i.e., public clonotypic sequences found on VDJDB database) compared to non-allergic patients (P=0.02), supporting the "hygiene hypothesis" as a predisposing factor in the development of allergy. A TCR-repertoire that is more diverse and more dynamically changing increases the chance of having a matching reactive clonotype, which increases risk of allergy. Understanding the immunological basis of T-cell response in allergy may help in developing strategies to mediate this common toxicity.

Fecal Microbiota Comparison Between Healthy Teaching Horses and Client-Owned Horses

The objective of this study was to compare the fecal microbiota of two healthy teaching horse herds with that of client-owned horses from the same geographic areas. The fecal microbiota of client-owned horses from Ontario Canada (n = 15) and Florida, USA (n = 11) was compared with that teaching horses from the University of Guelph, Ontario, Canada (n = 10) and the University of Florida, Florida, USA (n = 15). The fecal microbiota was characterized by sequencing of bacterial DNA using the V4 hypervariable region of the 16S rRNA gene. The diversity (inverse Simpson index) of the fecal microbiota was significantly higher in teaching than client owned horses from the same geographical area (P < .05). The community membership (Jaccard Index) and structure (Yue and Clayton index) of teaching horses was also significantly different from that of client owned horses from the same geographical area (AMOVA P < .001). The bacterial membership and structure of the fecal microbiota of Ontario and Florida teaching horses were significantly different, while the bacterial membership, but not the structure of Ontario and Florida client owned horses was significantly different (AMOVA P < .001). In all four groups of healthy horses, Lachnospiraceae, Ruminococcaceae, Bacteroidales, Clostridiales, and Treponema were detected in high relative abundance. The fecal microbiota of healthy horses from teaching herds kept in the same environment with identical management practices differs significantly from that of horses housed in different facilities with dissimilar management practices. Our results suggest an effect of the environment and management practices on the gastrointestinal microbiota. Researchers should attempt to include healthy horses from the same farm with similar management as control groups when comparing with diseased horses.

Dynamics of gut microbiota in patients suffering from hematologic malignancies after allogeneic hematopoi-etic stem cell transplantation

In this study, we described the dynamics of intestinal microbiota of 32 patients after allogeneic hematopoietic stem cell transplantation (HSCT). After HSCT, on 14th day and 30th day, the inverse Simpson index value was more than 3 times lower compared to the pre-HSCT period. Biodiversity became more similar to the pre-HSCT level by 100th day. The correlation between diversity of microbiota and development of post-HSCT complications wasn’t observed. Our findings indicated individual-specific changes in the taxonomic structure of patients’ microbiota after HSCT. Changes in the relative abundance of the bacterial families Streptococcaceae, Actinomycetaceae, Acidaminococcaceae, Akkermansiaceae, Desulfovibrionaceae, Prevotellaceae, genera Streptococcus, Clostridioides, Phascolarctobacterium, Paraprevotella, Bilophila, Akkermansia, Coprococcus, Sutterella, Alistipes appear to be associated with the development of post-HSCT complications. High abundance of phylum Proteobacteria, families Actinomycetaceae and Streptococcaceae, genus Streptococcus before HSCT can be considered as a potential predictor of graft-versus-host disease development in the post-HSCT period.

Importance of fish species in the bottom and near-bottoml biotopes of the Far-Eastern Seas and Pacific waters of Russia

Importance of fish species in the benthal ichthyocoenoses (up to depths of 2000 m) is discussed for the Russian waters in the Far-Eastern Seas and adjacent North-West Pacific on the data of bottom trawl surveys conducted by Pacific Fish. Res. Inst. (TINRO) in 1977–2010. The inverse Simpson index (or polydominance index) was chosen as a measure of dominance; Sorensen-Chekanovsky similarity index was used for comparative analysis of the dominance structure in ichthyocoenoses. The importance is determined for the top 20 species ranked by biomass (94.9 % of the total biomass of all demersal fish on the shelf and 95.8 % — at the continental slope, on average). Poor evenness under strong domination of 2–4 species is noted in the fish communities within the range of 0–2000 m. Walleye pollock Theragra chalcogramma dominate in all regions whereas the subdominants are pacific herring Clupea pallasii, pacific cod Gadus macrocephalus, atka mackerel Pleurogrammus monopterygius, or okhotsk atka mackerel P. azonus, depending on the region. The pollock domination is interrupted at the continental slope of the Bering Sea, where giant grenadier Albatrossia pectoralis dominate. The subdominants at the continental slope of all regions are greenland halibut Reinhardtius hippoglossoides matsuurae, popeye grenadier Coryphaenoides cinereus, pacific herring, pacific sleeper shark Somniosus pacificus, scale-eye plaice Acanthopsetta nadeshnyi, and blackfin flounder Glyptocephalus stelleri. Generalized graphs of the ichthyocoenoses similarity between the studied regions (Okhotsk Sea, Bering Sea, Japan Sea, and Pacific waters at Kamchatka and Kuril Islands) by species structure and composition of the dominant species are presented, separately for the shelf and continental slope.