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The intestinal microbiota is essential for the fermentation of dietary fiber into short-chain fatty acids (SCFA) such as butyrate, acetate, and propionate. SCFAs can bind to the G-protein-coupled receptors GPR43 and GPR109A (HCAR2), with varying affinities to promote cellular effects in metabolism or changes in immune function. We explored the role of GPR109A as the main receptor for butyrate in mouse models of allogeneic hematopoietic cell transplantation (allo-HCT) and graft-versus-host disease (GVHD). Deletion of GPR109A in allo-HCT recipients did not affect GVHD, but transplantation of T cells from GPR109A knockout (KO) (Gpr109a−/−) mice into allo-HCT recipient mice significantly reduced GVHD morbidity and mortality compared with recipients of wild-type (WT) T cells. Recipients of Gpr109a−/− T cells exhibited less GVHD-associated target organ pathology and decreased proliferation and homing of alloreactive T cells to target tissues. Although Gpr109a−/− T cells did not exhibit immune deficits at a steady state, following allo-activation, Gpr109a−/− T cells underwent increased apoptosis and were impaired mitochondrial oxidative phosphorylation, which was reversible through antioxidant treatment with N-acetylcysteine (NAC). In conclusion, we found that GPR109A expression by allo-activated T cells is essential for metabolic homeostasis and expansion, which are necessary features to induce GVHD after allo-HCT.

We previously described clinically relevant reductions in fecal microbiota diversity in patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT). Recipients of high-dose chemotherapy and autologous HCT (auto-HCT) incur similar antibiotic exposures and nutritional alterations. To characterize the fecal microbiota in the auto-HCT population, we analyzed 1161 fecal samples collected from 534 adult recipients of auto-HCT for lymphoma, myeloma, and amyloidosis in an observational study conducted at 2 transplantation centers in the United States. By using 16S ribosomal gene sequencing, we assessed fecal microbiota composition and diversity, as measured by the inverse Simpson index. At both centers, the diversity of early pretransplant fecal microbiota was lower in patients than in healthy controls and decreased further during the course of transplantation. Loss of diversity and domination by specific bacterial taxa occurred during auto-HCT in patterns similar to those with allo-HCT. Above-median fecal intestinal diversity in the periengraftment period was associated with decreased risk of death or progression (progression-free survival hazard ratio, 0.46; 95% confidence interval, 0.26-0.82; P = .008), adjusting for disease and disease status. This suggests that further investigation into the health of the intestinal microbiota in auto-HCT patients and posttransplant outcomes should be undertaken.

The intestinal microbiota undergoes major perturbations during allogeneic hematopoietic stem cell transplantation (allo-HCT), and low microbiota diversity during this period is associated with an increased risk of graft-versus-host disease and mortality. Identifying the environmental variables that might impact intestinal microbiota could inform strategies to maintain and restore a healthy microbiota state. However, understanding microbial dynamics is challenging due to the high-dimensional nature of microbiota data. Here, we simplified complex microbiota communities into clusters and investigated the dynamics under different conditions in terms of transition probabilities in a large dataset of allo-HCT fecal specimens (Fig. a). The bacterial compositions of 7,930 samples from 1,076 allo-HCT patients were determined by 16S rRNA deep-sequencing. Samples were then clustered into 10 distinct states by k-means clustering of a Bray-Curtis β-diversity matrix (Fig. b). These clusters captured variations in diversity and microbiota compositions (Fig. c-d). Cluster 1 represented a high-diversity state, and Lachnospiraceae and other Clostridiales were the most commonly observed taxa in this cluster. The low-diversity clusters 9 and 10 consisted mostly of Streptococcus-dominated and Enterococcus-dominated samples, respectively. We utilized a regression-based predictive approach to model cluster transition probabilities in terms of a weight for remaining in the same cluster over time (self-weight) and a weight for attracting transitions from other clusters over time (attractor-weight). Controlling for the effect of time, the weights measured the contribution of different environmental exposures to intestinal microbial behaviors. A negative parameter coefficient indicates cluster destabilization or decreased cluster transition likelihood in the case of self-weights and attractor-weights, respectively. We evaluated the impact of the 3 most commonly used non-prophylactic antibacterial drugs using 2359 daily samples from 385 allo-HCT patients collected between day -14 to 7 relative to transplant. High-diversity cluster 1 was significantly destabilized by piperacillin-tazobactam (pip-tazo) exposure (β = -0.87, P < 0.05). Meanwhile, exposure to cefepime and meropenem did not have a significant effect on cluster 1 stability (Fig. e). Exposure to pip-tazo also increased the transition probability to the Streptococcus-dominated cluster 9 (β = 1.83, P < 0.001), while cefepime (β = 2.69, P < 0.05) and meropenem (β = 1.96, P < 0.01) exposure favored transitions to the Enterococcus-dominant cluster 10. These results suggest that antibiotic exposures are associated with different composition outcomes depending on patient microbiota states during transplant period. In a small subset of 242 daily samples from 46 allo-HCT patients with detailed daily dietary information, we observed that an increase in total protein intake (range = 0-137.4g; median = 36g) was associated with low self-maintenance of cluster 1 (β = -1.29, P < 0.05), while an increase in total fat intake (range = 0-183.3g; median = 34.5g) improved cluster 1 stability (β = 1.44, P < 0.05). Overall, dietary intakes could also modulate transition probabilities between microbial communities in allo-HCT patients. While prior studies have assessed specific bacterial taxa or diversity indices as biomarkers of clinical outcomes, here we considered the entire intestinal communities and demonstrated that various environmental exposures were associated with changes in microbiota composition during allo-HCT. Using a regression-based approach that predicts cluster transitions in response to environmental conditions, we found that pip-tazo exposure was associated with destabilization of a high-diversity state and increased transitions to a Streptococcus-dominated state, while cefepime and meropenem exposure did not disrupt high-diversity microbial community. Furthermore, increased protein intake was also associated with disruption to the high-diversity cluster, while increased fat intake strengthened the maintenance of a diverse and healthy microbial community. Ultimately, this computation framework aims to inform strategies to optimize treatment plans for allo-HCT patients to maximize a healthy gut microbiota state and clinical outcomes. Disclosures Gomes: Seres Therapeutics: Other: Part of Salary. Peled:Seres Therapeutics: Research Funding. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Clurman:Seres Therapeutics: Research Funding. Giralt:Celgene: Consultancy, Research Funding; Takeda: Consultancy; Sanofi: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. Perales:Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Medigene: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Kyte/Gilead: Research Funding; Miltenyi: Research Funding; MolMed: Membership on an entity's Board of Directors or advisory committees. Pamer:MedImmune: Honoraria; Seres Therapeutics: Honoraria, Patents & Royalties; Bristol Myers Squibb: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Ferring Pharmaceuticals: Honoraria. van den Brink:Acute Leukemia Forum (ALF): Consultancy, Honoraria; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Flagship Ventures: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Evelo: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria; Therakos: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Merck & Co, Inc.: Consultancy, Honoraria; Juno Therapeutics: Other: Licensing; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees.

Introduction Translocation of intestinal bacteria across impaired mucosal barriers has long been believed to occur following exposure to chemotherapy. Consistent with this, we and others have previously reported that expansions of potentially pathogenic bacteria within the gastrointestinal microbiome precedes bloodstream infection. While the blood microbiome represents a rich area for investigation, detailed unbiased characterization of the blood microbiome has not previously been possible. Here, we sought to investigate the relationship between the blood and stool microbiome in patients undergoing allogeneic hematopoietic stem cell transplantation (HCT) who are at high risk for gut barrier dysfunction, severe infection, and the immunological complications of transplantation including graft-versus-host disease (GVHD). We show preliminary data suggesting that the blood microbiome may hold biomarkers for gut integrity. Subsequently, it may offer microbiological data supporting causative organisms in "culture-negative" fevers, or in itself, perform an immunomodulatory role acting as a damage/pathogen associated molecular pattern (DAMP/PAMP). Methods We sequenced serially-collected plasma and stool samples (n = 61 unique samples of each type) from a cohort of 19 patients who underwent allogeneic HCT at our center. Microbial cell-free DNA (mcfDNA) was extracted from plasma and sequenced using a next generation sequencing assay (Karius, Inc, Redwood City, CA). After sequences are processed, mcfDNA abundances are reported in molecules per microliter. Stool samples underwent were profiled using 16S-targeted sequencing (V4-V5 region) on the Illumina MISEQ platform and analyzed using the DADA2 pipeline. Association of blood microbial burden with clinical factors was assessed using a Wilcoxon rank sum test. Results We confirmed a high sequence homology between the DNA found in plasma and stool samples, thus demonstrating that circulating mcfDNA is gut-derived in these patients. By comparing microbial sequences from paired plasma and stool samples collected equivalent time points during the neutropenic nadir after HCT, we observed a correlation between the abundance of bacterial mcfDNA in plasma and DNA from the same microbes in stool. Of note, this occurred independently of conditioning regimen intensity, and was observed in patients who had received myeloablative, non-myeloablative and reduced intensity therapy. We assessed the association of bacterial mcfDNA burden (only considering sequences common to stool sequences) with various clinical factors. Translocation was significantly higher in patients who experienced pre-engraftment mucositis (any observed grade) compared with those who did not (p = 0.02, n = 5 in the mucositis group, any grade, and 12 in the mucositis-free group), but there was no relationship between the degree of translocation and HCT-CI, conditioning intensity, donor type, age or pre-engraftment fevers. We next asked whether translocation events were associated with acute GVHD (aGVHD) by assessing a subgroup of patients who received unmodified grafts (peripheral blood stem cell or bone marrow; n = 10) and tracked the degree of translocation from the gut to the blood stream pre-transplant, during the neutropenic nadir, and following engraftment. As shown in Figure 1, we observed low pre-transplant translocation (as quantified by the total bacterial DNA abundance in plasma where sequences were also shared in stool samples), and a marked increase during neutropenic nadir. Remarkably, even in this small cohort, we observe a higher burden of translocation in the post-engraftment period in patients who subsequently develop aGVHD, while it decreased to baseline levels in those who do not (n = 7 in the aGVHD group; n = 3 aGVHD-free; p = 0.05). Conclusions Here we demonstrate the first use of a culture-free molecular assay to track the blood microbiome and identify features of the circulating mcfDNA that correlate with the microbial DNA in stool samples. Despite the small sample size, these data suggest that the maintenance of a high mcfDNA-burden beyond the neutropenic nadir is associated with subsequent GVHD development, and provides some evidence for early gut barrier dysfunction that permits translocation in these patients. Disclosures Blair: Karius, Inc: Employment. Peled:Seres Therapeutics: Other: IP licensing fees, Research Funding. Giardina:Seres Therapeutics: Other: Salary funding. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Hollemon:Karius, Inc: Employment. Ho:Karius, Inc: Employment. Bercovici:Karius, Inc: Employment. Ahmed:Karius, Inc: Employment. Hong:Karius, Inc: Employment. Giralt:Celgene: Consultancy, Research Funding; Takeda: Consultancy; Sanofi: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. van den Brink:Therakos: Consultancy, Honoraria; Merck & Co, Inc.: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Acute Leukemia Forum (ALF): Consultancy, Honoraria; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Flagship Ventures: Consultancy, Honoraria; Juno Therapeutics: Other: Licensing; Evelo: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria.

Introduction Cellular therapy with chimeric antigen receptor (CAR) T cells has fundamentally changed the treatment of many cancers. Unfortunately, not all patients who receive this therapy have a favorable response. Additionally, some patients may develop toxicity due to cytokine release syndrome (CRS) or neurotoxicity. Recent studies have found a relationship between the intestinal microbiome and the response to immunotherapy with checkpoint blockade. We propose the intestinal microbiota as a factor that influences the efficacy and toxicity of CAR T cells. We hypothesize that the differences in outcomes of patients who receive CAR T cells are related to the composition of their intestinal microbiota at baseline. We report a single-center analysis of pre-CAR T cell infusion microbiota composition. Methods We collected stool samples from recipients of CAR T cells at Memorial Sloan Kettering Cancer Center (MSKCC). A baseline sample was collected prior to CAR T cell infusion. Samples were submitted for 16S RNA sequencing of the V4-V5 region on the Illumina MiSeq platform and the operational taxonomic units (OTUs) were classified using the NCBI Reference Sequence Database. Clinical response to assess efficacy was classified as either complete response (CR) or no complete response. Given the sample size, toxicity was pooled to encompass Grade 1 to 4 CRS and Grade 1 to 4 neurotoxicity. Linear discriminant analysis effect size (LEfSe) was used to identify microbial biomarkers for efficacy and toxicity between groups using relative abundances with a linear discriminant analysis score threshold >2.5. Results We analyzed 24 baseline samples from 24 patients treated at MSKCC. The patients were all adult recipients of cellular therapy with CAR T cells. The patients varied in conditioning regimen, CAR construct and underlying diagnosis, which included solid tumors and hematologic malignancies. First, we assessed the 16S relative abundance of the intestinal microbiota of the patients at baseline. We found that the composition of the microbiota prior to CAR T cell infusion was diverse, as defined by an Inverse Simpson >4 in all of the patients, although the level of diversity amongst the patient samples varied (Fig A). An assessment of the efficacy of CAR T cells with LEfSe analysis found increased abundance in several families of the Clostridiales order (Firmicutes phylum), including Oscillospiraceae, Ruminococcacaeae, and Lachnospiraceae, in those patients who achieved a CR. For the patients who did not achieve a CR, we found an increased abundance of a family in the Clostridiales order (Firmicutes phylum), Peptostreptococcaceae. Patients who experienced toxicity, either CRS or neurotoxicity, had an increased abundance of families within the Clostridiales or Lactobacillales order (Firmicutes phylum), which included Lachnospiraceae and Lactobacillaceae. Finally, patients who did not experience toxicity also had an increased abundance of a family in the Clostridiales order (Firmicutes phylum), Peptostreptococcaceae. Conclusion We demonstrate that our subset of patients had diverse microbial composition prior to receiving CAR T cell therapy despite the fact that many of them were heavily pre-treated. Additionally, we observe the abundance of the family Lachnospiraceae in the patients who achieved a CR and those who experienced toxicity. Many Lachnospiraceae are butyrate producers, whose presence has been found to be protective against Clostridium difficile infection in recipients of allogeneic hematopoietic cell transplant but whose abundance is lower in colon cancer. Conversely, we observe an abundance of the family Peptostreptococcaceae in patients who did not achieve a CR or who did experience toxicity. Peptostreptococcaceae has been found to be more abundant in the intestines of patients with colon cancer. Of note, the intestinal micriobiota that we identify are not congruent with the specific bacteria that have been found to promote anti-tumor immunity to checkpoint blockade. Our data suggests a role for the intestinal microbiota in mediating the response to CAR T cells and proposes that the baseline microbial composition may correlate with efficacy and toxicity. Further studies will investigate biochemical mechanisms to understand the interplay of the intestinal microbiota and the immune system to improve patient outcomes following CAR T cell therapy. Disclosures Park: Adaptive Biotechnologies: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; AstraZeneca: Consultancy; Kite Pharma: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Shire: Consultancy. O'Cearbhaill:Juno: Research Funding. Mailankody:Juno: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Physician Education Resource: Honoraria. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Palomba:Pharmacyclics: Consultancy; Celgene: Consultancy. Riviere:Fate Therapeutics Inc.: Research Funding; Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Introduction: Increasing evidence suggests that the intestinal microbiota is involved in the development of acute graft-vs.-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). We previously reported in single center studies that Enterococcus a) is associated with GVHD (Holler et al., BBMT 2014) and b) can dominate the post-transplant gut microbiota in up to 50% of allo-HCT patients resulting in a 9-fold increased risk of bacteremia (Taur et al, CID 2012). To further investigate the hypothesis that Enterococcus can trigger the development of GVHD, we studied both allo-HCT patients and pre-clinical mouse transplant models. Methods and Results: Stool samples from 1240 allo-HCT patients at 4 different transplant centers in the U.S., Germany and Japan were collected approximately weekly during inpatient hospitalization. The V4-V5 region of the bacterial 16S rRNA genes from 6718 samples was sequenced at one central site on the Illumina platform. We observed Enterococcus mono-domination (relative abundance > 30%) in post-transplant samples ranging from 20 to 60% of patients at different centers (Fig. A, left). This mono-domination was primarily attributable to E. faecium, and was associated with a significantly increased risk for grade 2-4 acute GVHD (Fig. A, right). In three different mouse models we found a transient bloom of E. faecalis around 7 days after transplant in allo-HCT recipients with GVHD (Fig. B; C57BL/6 -> 129SV model). This bloom did not occur in allo-HCT recipients of a T cell depleted allograft without GVHD. To further investigate this Enterococcus bloom, we treated mice with an experimental E. faecalis-strain on days 4 to 6 after transplant and found significantly increased lethal GVHD. Colonizing germ-free mice with a minimal gut flora also lead to increased lethal GVHD when enterococci were added to the gnotobiotic flora (Fig. C). The allo-HCT recipients with Enterococcus-containing flora had also increased serum IFNg levels. Short chain fatty acids (SCFA) can be protective against GVHD and gut inflammation through maintenance of epithelial homeostasis and increases in anti-inflammatory regulatory T cells in the gut. In BMT mouse models, we found that Enterococcus-dominated allo-HCT recipients with GVHD have significantly less cecal butyrate, a major SCFA. Similarly, Enterococcus domination after allo-HCT also leads to a decrease in fecal SCFAs in patients (Fig. D). Next, we hypothesized that intestinal IgA might have a protective role against this pathogen in mice. 16S sequencing of flow sorted IgA-coated vs. non-coated bacteria from fecal samples of allo-HCT patients and transplanted mice revealed no specific IgA-coating pattern of enterococci both before or after transplant rather excluding the hypothesis that IgA might have a protective role against Enterococci. E. faecalis and E. faecium use the disaccharide lactose as a major carbohydrate source for growth and expansion as observed by analyses of the Enterococcus genome and in vitro growth experiments. In mice, we observed that a lactose-free diet significantly decreases the Enterococcus bloom after transplant in allo-T cell recipients and in first survival experiments attenuates lethal GVHD (Fig. E). Conclusion: Our studies in mouse and man demonstrate that the abundance of Enterococcus in the intestinal flora plays a role in the development of GVHD and the prevention of Enterococcus growth with a lactose-free diet can ameliorate GVHD. Disclosures Peled: Seres Therapeutics: Research Funding.

Intestinal microbiota composition is associated with important outcomes after allo-HCT including survival, relapse, GVHD, and infections. These observations have been made almost exclusively by characterizing the microbiota in the first weeks after transplantation, and in single-center studies. We previously reported that intestinal diversity measured peri-neutrophil engraftment is predictive of overall survival in a multicenter cohort. Here, we hypothesized that pre-HCT microbiota configuration may also be an important determinant of post-transplantation outcomes. We report a multicenter analysis conducted at 4 independent international institutions to test this hypothesis. We collected 1922 stool samples ~weekly from 991 unique allo-HCT patients at four international transplant centers: Cohorts 1 and 2 in the US, cohort 3 in Europe, and cohort 4 in Japan. The patients-all adult recipients of allo-HCT-varied in underlying diagnosis, donor-graft sources, conditioning intensity, and GVHD prophylaxis. Samples from all 4 centers were sequenced and analyzed at a central laboratory using the V4-V5 region of 16S rRNA. For patients with multiple samples within the pre-HCT sampling period, which we defined as day -20 to 0, median values were used. On average, patients from all 4 transplantation centers had reduced microbiota diversity pre-HCT, as measured by median α-diversity (inverse Simpson) values that were 1.7-to-2.5-fold lower than those of healthy volunteers. This comparison was made both in volunteers whose samples we sequenced ourselves and in a publicly available dataset (Fig A, p We next asked how similar these pre-HCT communities are across geography. We found that Bray-Curtis distances between institutions were reproducibly much smaller in magnitude than the changes observed over time during transplantation (Fig C, p In order to characterize these clinically relevant low-diversity phenotypes, we defined domination as a microbiota injury in which any operational taxonomic unit comprises >30% of bacterial abundance. The dominating taxa belonged to multiple genera, the most common being Enterococcus, Streptococcus, Lactobacillus, Escherichia, and Klebsiella (Fig E) as annotated by Greengenes and NCBI databases. At all four institutions, the cumulative incidence of intestinal domination by any organism was >50% by day 0 and was >87% by day +28 (Fig F). We performed additional analyses in the largest cohort and found that the low-diversity state is associated with exposure to broad-spectrum antibiotics, conditioning intensity, and low calorie intake. We demonstrate that HCT patients at 4 institutions on 3 continents presented with microbiota configurations that were similar to one another and distinct from those of healthy individuals. Severe microbiota injury as revealed by domination is a common event whose development begins before allograft infusion, and pre-HCT microbiota injury predicts poor overall survival. These observations suggest the pre-HCT period as a window of opportunity to (a) assess microbiota injury as part of comorbidity evaluation, (b) inform selection of antibiotic prophylaxis, gut-decontamination, GVHD-prophylaxis, or conditioning regimens, and (c) intervene with microbiota injury-remediation or prevention strategies. Figure. Figure. Disclosures Peled: Seres Therapeutics: Research Funding. Perales:Merck: Other: Personal fees; Takeda: Other: Personal fees; Abbvie: Other: Personal fees; Novartis: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support. Jenq:MicrobiomeDx: Consultancy; Ziopharm Oncology: Consultancy; Seres Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees; Seres Therapeutics, Inc.: Patents & Royalties.

Introduction: We have previously reported that clinically relevant, dramatic reductions occur in intestinal bacterial diversity during allogeneic hematopoietic stem cell transplant (allo-HSCT). These are likely attributable to antibiotic exposure, nutritional alterations, and intestinal mucosa injury from high-dose chemotherapy. Patients undergoing autologous hematopoietic stem cell transplantation (AHCT) also receive antibiotics and experience nutritional alterations due to mucositis and other gastrointestinal toxicities. We hypothesized that the pattern of dysbiosis seen in AHCT patients would reflect the changes in allo-HSCT patients. Here, we present a novel analysis of microbiota diversity in AHCT patients from two independent institutions. Methods: We retrospectively identified a cohort of 365 patients (median age 60 years) who underwent AHCT for treatment of hematologic malignancy between May 2009 to February 2018 at two large-volume transplant centers in the US. The population was diverse in terms of histology, conditioning regimens and remission status prior to transplant, with 179 (49%) patients diagnosed with multiple myeloma, 153 (42%) patients diagnosed with lymphoma, and 33 (9%) patients with other diseases. Stool samples from the selected patients were collected approximately weekly during inpatient hospitalization. Sequencing of the V4-V5 region of the bacterial 16S rRNA genes from all samples was performed on the Illumina platform at a central site. Microbial diversity was measured by the Simpson reciprocal a-diversity index (S). We defined the pre-AHCT period as days -10 to 0, and computed median values for patients with multiple samples within that period. We additionally defined monodomination of the microbiota as a single operational taxonomic unit comprising >30% of bacterial abundance. For comparison, we sequenced samples from 17 healthy volunteers and used a public dataset of sequences from 313 healthy volunteers from the NIH Human Microbiome Project (HMP). Median pre-transplant microbial diversity in the healthy patient and AHCT groups was compared by a pairwise Wilcox test to a retrospective cohort of allo-HSCT patients. Results: We evaluated 857 samples from 365 adult patients undergoing AHCT, with 316 patients from Memorial Sloan Kettering Cancer Center (MSKCC) and 49 patients from Duke University Medical Center (DUMC). Median pre-transplant diversity in AHCT patients from both centers was significantly lower than in normal controls (Fig 1A) (HMP vs MSKCC AHCT, S=12.05 vs. 9.19, p50% by day 0 and was >75% by day +14. Conclusion: Microbial diversity is reduced prior to transplant in both AHCT and allo-HSCT patients. Loss of diversity after AHCT occurs across centers and the degree of injury is comparable to the dysbiosis in allo-HSCT patients. Preliminary analysis suggests that lower diversity may correlate with worse progression-free survival (PFS) in myeloma patients in our diverse AHCT cohort. Given the known associations of alterations in microbiota composition with toxicities and overall survival in allo-HSCT patients, further evaluation of microbiota injury and its associations with toxicities, PFS, and overall survival (OS) in AHCT patients is warranted. Figure 1: A: The median Simpson reciprocal a-diversity index (S) of pre-transplant (days -10 to 0) samples of AHCT and allo-HSCT patients from two centers, as well as two cohorts of healthy volunteers, was plotted and a pairwise Wilcox test was performed, with p-values as indicated. B: (S) was plotted against time relative to allo-HSCT (on L) and AHCT (on R), for samples collected from day -10 to day +30. Larger values indicate greater diversity. C: Microbiota composition and changes in bacterial monodominance after transplant (days -14 to +28); the most common genus post-transplant is Streptococcus. Figure 1. Figure 1. Disclosures Peled: Seres Therapeutics: Research Funding. Sauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite: Consultancy. Shah:Amgen: Research Funding; Janssen: Research Funding. Perales:Novartis: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Merck: Other: Personal fees; Takeda: Other: Personal fees; Abbvie: Other: Personal fees. Jenq:Ziopharm Oncology: Consultancy; Seres Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees; MicrobiomeDx: Consultancy; Seres Therapeutics, Inc.: Patents & Royalties.

Background: Multiple myeloma (MM) patients who achieve minimal residual disease (MRD) negative status after upfront treatment have prolonged progression-free and overall survival compared with those who remain MRD(+) (Landgren CO, Devlin SM et al. Bone Marrow Transplant. 2016;51(12):1565-8). Commensal intestinal microbial composition has been associated with treatment outcomes in cancer patients. We sought to evaluate whether the composition of the intestinal microbiota is associated with MRD status in patients with MM. Methods: Stool samples were collected prospectively from 34 patients after completion of upfront therapy for MM at the time of MRD testing. MRD was assessed with next-generation flow cytometry of bone marrow aspirates (sensitivity 10-5). Microbial analysis was performed via sequencing of 16S rRNA V4-V5 regions using the Illumina MiSeq platform and sequence data was analyzed using UPARSE (Edgar RC, Nature Methods 2013;10(10):996-8). The linear discriminant effect size method (LEfSe) (Segata N et al. Genome Biol. 2011;12(6):R60.) was used to compare detected clades among all groups and evaluate for associations with outcomes, using MRD as class and autologous stem cell transplant (ASCT) as subclass. Alpha diversity was calculated by the Inverse Simpson index and differential relative abundance were calculated using the phyloseq package and compared using the Wilcoxon rank sum test on the R statistical computing platform. Results: Among 34 patients evaluable for microbiota composition and MRD status, the median age was 62.5 years and 16 (47.1%) were MRD(-) at time of stool collection. 24 (70.6%) were treated with carfilzomib, lenalidomide, and dexamethasone as induction therapy (MRD(-): 14 (87.5%), MRD(+):10 (55.5%). 4 (28.5%) MRD(-) patients had autologous stem cell transplant(ASCT), compared with 10 (55.5%) who were MRD(+). In the cohort's samples, we observed 19 phyla, 315 genera, 654 species, and 1549 operational taxonomic units (OTUs). There was no significant difference in alpha diversity between MRD(-) (median 12.24, IQR = 8.76-13.98) and MRD(+) patients (median 12.44, IQR = 8.36 -16.23), p=0.6 by Wilcoxon rank sum test. A positive association with MRD negativity was noted with two butyrate-producing organisms, Eubacterium hallii (p=0.001) and Faecalibacterium prausnitzii (p= 0.006). To further evaluate these relationships, we performed a differential abundance analysis of these selected taxa in MRD(+) and MRD(-) patients at the genus and species level. The relative abundance of the genera Eubacterium and Faecalibacterium were higher in fecal samples from MRD(-) patients than MRD(+) patients (Eubacterium MRD(-): median 4.51% (IQR = 2.83 - 7.32%) vs. MRD(+): median 3.07% (IQR = 1.35 - 3.87%), p=0.0326; Faecalibacterium MRD(-): median 1.68% (IQR = 0.69 - 7.48%) vs. MRD(+): median 0.003% (IQR = 0 - 3.19%), p=0.022. The relative abundance of both species of interest were higher in MRD(-) patients than in MRD(+) patients: E. hallii MRD(-): median 2.67% (IQR = 2.11 - 3.98%) vs. MRD(+): median 1.01% (IQR = 0 - 2.16%), p=0.001; F. prausnitzii MRD(-): median 1.43% (IQR = 0.53 - 7.28%) vs. MRD(+): median 0.3%, (IQR = 0 - 2.54%), p=0.022. Other species of Eubacterium and Faecalibacterium were not significantly differentially abundant between the two groups. Conclusions: Intestinal microbiota containing several butyrate-producing anaerobes appear to be associated with MRD-negativity in patients with myeloma, with higher relative abundance of Eubacterium hallii and Faecalibacterium prausnitzii in MRD(-) patients compared with MRD(+) patients. Butyrate and other short-chain fatty acids are biologically active metabolites formed during microbial fermentation of dietary or host-derived carbohydrates, which supply the host with energy and also modulate immunity, including exerting anti-inflammatory functions. Microbes of the genus Eubacterium have been associated with reduced risk of relapse in several hematologic cancers after allogeneic hematopoietic cell transplantation, including MM (Peled JU, Devlin SM et al. J Clin Oncol 2017;35(15):1650-9). This is first study to our knowledge to suggest an association between gut microbiota and MRD status in patients with myeloma and supports further investigation of a potential role for intestinal microbiota in the natural history and treatment of myeloma. Disclosures Peled: Seres Therapeutics: Research Funding. Landgren:Karyopharm: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lesokhin:Squibb: Consultancy, Honoraria; Genentech: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Serametrix, inc.: Patents & Royalties: Royalties; Takeda: Consultancy, Honoraria; Janssen: Research Funding.

Introduction The composition of the intestinal microbiota is associated with important outcomes after allo-HSCT including survival, relapse, GVHD, and infections. The microbial composition changes drastically after allo-HSCT, with precipitous drops in α-diversity and domination by single organisms. Low diversity is particularly associated with poor overall survival from transplant-related mortality, including death after GVHD. These observations were all made in single-center cohorts. Here we compare-for the first time-the kinetics of microbiota diversity in allo-HSCT patients from three independent international institutions. We also explore the role of nutrition as well as antibiotics in a single-center pilot study and the recovery from microbiota injury after discharge. Methods We collected stool samples approximately weekly from adult allo-HSCT inpatients and approximately monthly post-discharge. Samples from all three centers were sequenced at MSKCC using the V4-V5 region of 16S rRNA. In the nutrition cohort, patients self-documented oral intake at each meal; data were confirmed in focused patient interviews by a registered dietician three times weekly and nutritional compositions were obtained from the hospital kitchen database. Oral swish samples were collected prior to allo-HSCT and during post-transplant neutropenia. Results 5,823 samples from 1,118 patients were compared: 5,508 samples from 947 MSKCC patients, 108 samples from 40 Duke patients, and 152 samples from 109 Regensburg, Germany patients. The patients-all adult recipients of allo-HSCT -varied in underlying diagnosis, donor-graft sources, conditioning intensity, and GVHD prophylaxis. We mapped the microbiota composition of each sample in 2-D space using t-Distributed Stochastic Neighbor Embedding (t-SNE). Samples from all three centers spread throughout t-SNE space, revealing no transplant-center-specific effect (Fig 1A). Enterococcus domination, a low-diversity state we previously associated with subsequent enterococcal bacteremia was observed in all three centers (Fig 1A). Diversity decreased comparably after allo-HSCT transplant at all centers (Fig 1B), validating our prior findings in this larger, international multicenter cohort. In a separate cohort of MSKCC patients, we explored the relative contributions of antibiotics and nutritional intake using a multivariate linear regression model of α-diversity dynamics. Exposure to piperacillin-tazobactam (pip-tazo), a drug with broad-spectrum antibacterial activity, and calorie intake were significant predictors (p Conclusion We determine that striking microbiota injuries after allo-HSCT occur generally across geographic regions. Nutritional perturbations and antibiotic exposure are both associated with microbiota injury, and recovery from the injured state occurs over several months post-discharge. Importantly, diversity at the time of neutrophil engraftment is associated reproducibly with overall survival. Disclosures Peled: Seres: Research Funding. Sung: Merck: Research Funding; Novartis: Research Funding; Cellective: Research Funding. Jenq: Seres: Research Funding. van den Brink: Jazz Pharmaceuticals: Consultancy; Seres: Research Funding; PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement.