Discerning the effect of pharmacological exposures on intestinal bacterial communities in cancer patients is challenging. Here, we deconvoluted the relationship between drug exposures and changes in microbial composition by developing and applying a new computational method, PARADIGM (parameters associated with dynamics of gut microbiota), to a large set of longitudinal fecal microbiome profiles with detailed medi-cation-administration records from patients undergoing allogeneic hematopoietic cell transplantation. We observed that several non-antibiotic drugs, including laxatives, antiemetics, and opioids, are associated with increased Enterococcus relative abundance and decreased alpha diversity. Shotgun metagenomic sequencing further demonstrated subspecies competition, leading to increased dominant-strain genetic convergence during allo-HCT that is significantly associated with antibiotic exposures. We integrated drug-microbiome associations to predict clinical outcomes in two validation cohorts on the basis of drug ex-posures alone, suggesting that this approach can generate biologically and clinically relevant insights into how pharmacological exposures can perturb or preserve microbiota composition.The application of a computational method called PARADIGM to a large dataset of cancer patients' longitu-dinal fecal specimens and detailed daily medication records reveals associations between drug exposures and the intestinal microbiota that recapitulate in vitro findings and are also predictive of clinical outcomes.

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.

BackgroundDysgeusia is a common but understudied complication in patients undergoing autologous hematopoietic cell transplantation (auto‐HCT). We assessed the feasibility of using chemical gustometry (CG) to measure dysgeusia and explored its associations with symptom burden, nutrition, chemotherapy pharmacokinetics (PK), and the oral microbiome.MethodsWe conducted a single‐center, prospective feasibility study (NCT03276481) of patients with multiple myeloma undergoing auto‐HCT. CG was performed longitudinally testing five flavors (sweet, sour, salty, bitter, umami) to calculate a total taste score (maximum score, 30). We measured caloric intake and patient‐reported symptoms, assessing their correlation with oral microbiota composition and salivary and blood melphalan PK exposure.ResultsAmong all 45 patients, 39 (87%) completed at least four (>60%) and 22 (49%) completed all six CG assessments. Median total CG scores remained stable over time but were lowest at day +7 (27, range 24–30) with recovery by day +100. Symptom burden was highest by day +10 (area under the curve, 2.9; range, 1.0–4.6) corresponding with the lowest median overall caloric intake (1624 kcal; range, 1345–2267). Higher serum/salivary melphalan levels correlated with higher patient‐reported dysgeusia and lower caloric intake. Oral microbiota α‐diversity was stable early and increased slightly by day +100.ConclusionsAssessment of dysgeusia by CG is feasible after auto‐HCT. Most dysgeusia, symptom burden, and lowest caloric intake occurred during the blood count nadir. Higher melphalan concentrations correlated with more dysgeusia and poorer caloric intake. Future studies will aim to modulate melphalan exposure by PK‐targeted dosing and characterize patient taste preferences to personalize diets for improved nutritional intake.Lay summary Taste changes after cancer treatments are very common. We used chemical gustometry (taste testing) to study taste changes and to better understand why patients with multiple myeloma experience this symptom after autologous hematopoietic cell transplantation. We found that taste testing was feasible, taste changes peaked when blood counts were lowest, and most patients recovered their taste by 100 days after transplantation. Taste changes correlated with lower food intake and with higher levels of chemotherapy in the body. Future work will focus on using personalized chemotherapy doses to reduce taste changes and to match patients' individual taste preferences with their diets.

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.

Studies of the relationship between the gastrointestinal microbiota and outcomes in allogeneic hematopoietic stem cell transplantation (allo-HCT) have thus far largely focused on early complications, predominantly infection and acute graft-versus-host disease (GVHD). We examined the potential relationship of the microbiome with chronic GVHD (cGVHD) by analyzing stool and plasma samples collected late after allo-HCT using a case-control study design. We found lower circulating concentrations of the microbe-derived short-chain fatty acids (SCFAs) propionate and butyrate in day 100 plasma samples from patients who developed cGVHD, compared with those who remained free of this complication, in the initial case-control cohort of transplant pa-tients and in a further cross-sectional cohort from an independent transplant center. An additional cross-sectional patient cohort from a third transplant center was analyzed; however, serum (rather than plasma) was available, and the differences in SCFAs observed in the plasma samples were not recapitulated. In sum, our findings from the primary case-control cohort and 1 of 2 cross-sectional cohorts explored suggest that the gastrointestinal microbiome may exert immuno-modulatory effects in allo-HCT patients at least in part due to control of systemic concentrations of microbe-derived SCFAs.

BACKGROUND:Relationships between microbiota composition and clinical outcomes after allogeneic hematopoietic-cell transplantation have been described in single-center studies. Geographic variations in the composition of human microbial communities and differences in clinical practices across institutions raise the question of whether these associations are generalizable.METHODS:The microbiota composition of fecal samples obtained from patients who were undergoing allogeneic hematopoietic-cell transplantation at four centers was profiled by means of 16S ribosomal RNA gene sequencing. In an observational study, we examined associations between microbiota diversity and mortality using Cox proportional-hazards analysis. For stratification of the cohorts into higher- and lower-diversity groups, the median diversity value that was observed at the study center in New York was used. In the analysis of independent cohorts, the New York center was cohort 1, and three centers in Germany, Japan, and North Carolina composed cohort 2. Cohort 1 and subgroups within it were analyzed for additional outcomes, including transplantation-related death.RESULTS:We profiled 8767 fecal samples obtained from 1362 patients undergoing allogeneic hematopoietic-cell transplantation at the four centers. We observed patterns of microbiota disruption characterized by loss of diversity and domination by single taxa. Higher diversity of intestinal microbiota was associated with a lower risk of death in independent cohorts (cohort 1: 104 deaths among 354 patients in the higher-diversity group vs. 136 deaths among 350 patients in the lower-diversity group; adjusted hazard ratio, 0.71; 95% confidence interval [CI], 0.55 to 0.92; cohort 2: 18 deaths among 87 patients in the higher-diversity group vs. 35 deaths among 92 patients in the lower-diversity group; adjusted hazard ratio, 0.49; 95% CI, 0.27 to 0.90). Subgroup analyses identified an association between lower intestinal diversity and higher risks of transplantation-related death and death attributable to graft-versus-host disease. Baseline samples obtained before transplantation already showed evidence of microbiome disruption, and lower diversity before transplantation was associated with poor survival.CONCLUSIONS:Patterns of microbiota disruption during allogeneic hematopoietic-cell transplantation were similar across transplantation centers and geographic locations; patterns were characterized by loss of diversity and domination by single taxa. Higher diversity of intestinal microbiota at the time of neutrophil engraftment was associated with lower mortality. (Funded by the National Cancer Institute and others.).

Abstract Introduction Chronic graft-versus-host disease (cGVHD) affects up to 50% of the long-term survivors of allogeneic hematopoietic stem cell transplantation (HCT), and is the leading cause of mortality in patients who survive to two years post-transplant. Unlike acute GVHD (aGVHD), the primary pathology in cGVHD is fibrotic, affecting the skin, and lacrimal and salivary glands, and shares many features with the autoimmune conditions Sjogrens syndrome and systemic sclerosis. Certain gastrointestinal microbiota compositions have been associated with these autoimmune conditions, and we thus hypothesized that the configuration of microbial communities would also be associated with cGVHD, and may then serve as predictive biomarkers or offer mechanistic insights into cGVHD pathogenesis. Methods We identified a cohort of 55 patients with cGVHD as confirmed by a formal clinical consensus process applying NIH criteria, transplanted at MSKCC between January 2013 and August 2017. 47% of patients underwent transplantation for acute leukemia, with the remainder for non-hodgkin lymphoma (25%), myelodysplastic syndrome (13%), chronic leukemia (6%), myeloma and myeloproliferative disorder (each 2%). The majority received unmodified peripheral blood stem cell grafts (60%) with the remainder receiving CD34- selected grafts (20%), and marrow or cord blood grafts (20%). 55% of the cohort developed acute GVHD prior to day 100. 532 stool samples were available from 55 cGVHD cases, and were compared with 1462 samples from 165 control patients (matched for graft-source from the MSK allo-HCT Fecal Biobank). The median day of cGVHD onset in the cases was d194. The patient characteristics in the cases and controls were equivalent, including the rate of aGVHD prior to day 100 (55% vs 46%; p = 0.27). In addition, we compared the cGVHD cases with a separate cohort of patients with grade 3-4 aGVHD who were selected from the database on the basis of aGVHD diagnosis and a failure to develop cGVHD (n = 71 patients, contributing 835 samples), and an additional control group (matched for graft-source, n = 213 patients, 1786 samples) who did not develop any GVHD. All stool samples underwent 16S-targeted sequencing (V4-V5 region) on the Illumina platform. Results No differences in a-diversity as a function of time peri-HCT were observed, nor did we observe clustering of community characteristics in tSNE-space, or differences using the linear discriminant effect size (LEfSE) pipeline when we specifically examined samples collected pre-transplant, peri-engraftment, or peri-d100. Targeted analysis of genera reported to be enriched in intestinal communities of patients with autoimmune diseases however, revealed higher relative abundance in Prevotella prior to BMT in patients who went on to develop chronic GVHD (cGVHD case vs source-matched control p < 0.0001; cGVHD case vs cGVHD case vs no-GVHD control p <0.0001; Fig 1A). When we analyzed the relative abundance of genera known to be relevant in GVHD, we found increased relative abundance of Akkermansia and Streptococcus at day 100 in the cGVHD cases compared to controls (p =0.017; Fig 1B). Conclusion Here, we report for the first time, genus-level intestinal microbial signatures that are associated with cGVHD, identifiable at pre-transplant (Prevotella) and peri-d100 (Akkermansia, Streptococcus) time points, prior to cGVHD onset. Interestingly, at species level, Prevotella copri abundance has been mechanistically associated with new-onset rheumatoid arthritis, and we have previously associated Akkermansia muciniphilia, a commensal bacteria with known mucus-degrading capacity, with aGVHD in pre-clinical studies. Thus, the cGVHD-associated dysbiosis we have identified may have value as both a biomarker for cGVHD risk and mechanistic relevance. Figure 1. Figure 1. Disclosures Perales: Merck: Other: Personal fees; Takeda: Other: Personal fees; Novartis: Other: Personal fees; Abbvie: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support. Peled:Seres Therapeutics: Research Funding.

Intestinal microbiota composition is strongly associated with HCT patient outcomes. We have reported associations between antibiotic exposures and microbiota diversity and GVHD-related mortality. However, understanding microbiota-composition dynamics in response to specific perturbations is challenging due to the high-dimensional nature of microbiota data. This study identified clusters of intestinal microbiota compositions and investigated their dynamics using transition probabilities in a large dataset of allo-HCT fecal specimens.The bacterial compositions of 7,930 samples from 1,076 allo-HCT patients were determined by 16S rRNA deep-sequencing and visualized by tSNE (Fig. a). Samples were clustered into 10 distinct microbiota configurations by k-means clustering of a b-diversity matrix (Fig. b). Visually, clusters reflected monodominant taxonomic groups: cluster 5 overlaps with the Enterococcus group, while cluster 10 overlaps with the Streptococcus group. These clusters also captured variations in diversity, as clusters 1-2 and 5-10 represented high- and low-diversity states, respectively (Fig. c).Clusters also exhibited dynamic behaviors: high-diversity clusters 1-2 were common in pre-HCT samples, while most post-HCT samples belonged to low-diversity clusters 5-10 (Fig. d). The temporal behaviors of microbiota composition per patient could be modeled by cluster transition probabilities from 1 week pre- to 1 week post-HCT. Without piperacillin-tazobactam (pip-tazo) administration, patients who had a diverse composition pre-HCT were most likely to maintain their diverse state (probability P = 20%), and had a low chance of transitioning to the Enterococcus cluster (cluster 5, P = 5%) and to the Streptococcus cluster (cluster 10, P = 0%) post-HCT. However, pip-tazo exposure was associated with increased transition to the Enterococcus cluster (P = 15%) and to the Streptococcus cluster (P = 9%), and decreased diversity maintenance (P = 14%) (Fig. e). The occurrence of each cluster at peri-engraftment time (days 7-21) was used to estimate its association with patient survival. The Streptococcus cluster was associated with increased mortality, highlighting a disadvantageous cluster transition under pip-tazo exposure (Fig. f).While prior studies have associated bacterial taxa or diversity indices with biomarkers of clinical outcomes, here we considered the entire intestinal communities and demonstrated that post-HCT mortality risk can be predicted by the global microbiota composition at days 7-21. This computational framework can be used to predict cluster transitions in response to various clinical variables such as specific drug exposures and clinical events by means of a high-resolution transition matrix, ultimately informing strategies to optimize treatment plans for HCT patients to maximize a healthy gut microbiota state and clinical outcomes. Intestinal microbiota composition is strongly associated with HCT patient outcomes. We have reported associations between antibiotic exposures and microbiota diversity and GVHD-related mortality. However, understanding microbiota-composition dynamics in response to specific perturbations is challenging due to the high-dimensional nature of microbiota data. This study identified clusters of intestinal microbiota compositions and investigated their dynamics using transition probabilities in a large dataset of allo-HCT fecal specimens. The bacterial compositions of 7,930 samples from 1,076 allo-HCT patients were determined by 16S rRNA deep-sequencing and visualized by tSNE (Fig. a). Samples were clustered into 10 distinct microbiota configurations by k-means clustering of a b-diversity matrix (Fig. b). Visually, clusters reflected monodominant taxonomic groups: cluster 5 overlaps with the Enterococcus group, while cluster 10 overlaps with the Streptococcus group. These clusters also captured variations in diversity, as clusters 1-2 and 5-10 represented high- and low-diversity states, respectively (Fig. c). Clusters also exhibited dynamic behaviors: high-diversity clusters 1-2 were common in pre-HCT samples, while most post-HCT samples belonged to low-diversity clusters 5-10 (Fig. d). The temporal behaviors of microbiota composition per patient could be modeled by cluster transition probabilities from 1 week pre- to 1 week post-HCT. Without piperacillin-tazobactam (pip-tazo) administration, patients who had a diverse composition pre-HCT were most likely to maintain their diverse state (probability P = 20%), and had a low chance of transitioning to the Enterococcus cluster (cluster 5, P = 5%) and to the Streptococcus cluster (cluster 10, P = 0%) post-HCT. However, pip-tazo exposure was associated with increased transition to the Enterococcus cluster (P = 15%) and to the Streptococcus cluster (P = 9%), and decreased diversity maintenance (P = 14%) (Fig. e). The occurrence of each cluster at peri-engraftment time (days 7-21) was used to estimate its association with patient survival. The Streptococcus cluster was associated with increased mortality, highlighting a disadvantageous cluster transition under pip-tazo exposure (Fig. f). While prior studies have associated bacterial taxa or diversity indices with biomarkers of clinical outcomes, here we considered the entire intestinal communities and demonstrated that post-HCT mortality risk can be predicted by the global microbiota composition at days 7-21. This computational framework can be used to predict cluster transitions in response to various clinical variables such as specific drug exposures and clinical events by means of a high-resolution transition matrix, ultimately informing strategies to optimize treatment plans for HCT patients to maximize a healthy gut microbiota state and clinical outcomes. Figure 1.

IntroductionDysgeusia commonly influences nutritional intake and quality of life after HCT, yet its pathobiology is poorly understood and methods to study it are not established. We report interim results of a prospective feasibility study of chemical gustometry (CG) to measure dysgeusia in pts with multiple myeloma (MM) undergoing high-dose melphalan and autologous HCT (mel-AHCT). We evaluated associations between dysgeusia and symptoms, nutrition, mel pharmacokinetics (PK), and oral microbiota composition. We hypothesized that mel penetration into saliva contributes to dysgeusia.MethodsCG was performed by the Henkin method pre-AHCT and on days (d) -1, +7, +14, +30 testing 5 flavors (sweet, sour, salty, bitter, umami) to calculate a total taste score (max value 30). CG was considered feasible if > 60% of pts could complete > 60% of all evaluations. Caloric intake (kcal) was calculated with 24-hr recall using the USDA 5-Step Multiple-Pass Method, and pt-reported symptoms were assessed using the MD Anderson Symptom Inventory (MDASI) at the above times and on d +3 and +10. Salivary microbiome diversity was assessed longitudinally by 16S sequencing and the inverse Simpson index. 6-point serum mel concentrations were used to calculate an area under the curve (AUC) and peak salivary mel was obtained 75 min post-infusion. Spearman rank-sum correlation was used to assess associations.Results22 mel-AHCT MM pts have follow-up through d +30. 18 pts (82%) completed > 60% of all assessments. Median age was 62 (range 41-75). 11 pts (50%) were male. Mel 200 mg/m2 and 140 mg/m2 was given in 19 (86%) and 3 (14%) pts, respectively. Median serum mel AUC was 12.6 mg*h/L (range 5.8-17.5); median salivary mel was 119 ng/ml (range 25-662). Higher serum mel AUC correlated with higher salivary mel levels, p=0.004, and associated with poorer caloric intake from pre-ASCT to d +30, p=0.04. Higher serum mel AUC associated with higher MDASI scores (worse symptom burden) from pre-AHCT to d +30, p=0.005, but not with taste scores. Higher salivary mel associated with poorer caloric intake from pre-AHCT to d +30, p=0.02. MDASI scores peaked d +10, coincident with nadirs in taste scores and caloric intake on d +7 and +10, respectively. Taste scores partially recovered by d +30, but often did not return to baseline (Fig 1A-C). Oral microbiota diversity was stable early after AHCT but increased by d+100. D+7 diversity did not correlate with serum mel AUC, p=0.4, or salivary mel, p=0.84 (Fig 2A-C).ConclusionCG is a feasible method to evaluate dysgeusia after AHCT. Dysgeusia peaked by d +7 and often persisted through d +30. Oral microbiota diversity was stable early after AHCT. Higher serum and salivary mel levels did not correlate with microbiota diversity but were associated with poorer caloric intake and higher MDASI scores suggesting that using targeted mel PK dosing may improve caloric intake and limit symptom burden after AHCT.

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.

Patients with multiple myeloma (MM) who achieve minimal residual disease (MRD) negativity after upfront treatment have superior outcomes compared with those who remain MRD+ Recently, associations have been shown between specific commensal microbes and development of plasma cell disorders. Here, we report the association between intestinal microbiota composition and treatment outcome in MM. Microbiota composition of fecal samples collected from 34 MM patients after induction therapy and at the time of flow cytometry-based bone marrow MRD testing was determined by 16S ribosomal RNA sequencing. We observed a higher relative abundance of Eubacterium hallii in the 16 MRD- patients relative to the 18 MRD+ patients. No association was observed between microbial relative abundance and autologous stem cell transplantation history or MM paraprotein isotype. No differences in microbiota α diversity were observed between MRD- and MRD+ patients. The potential association of microbiota composition with treatment response in MM patients is an important parameter for additional correlative and clinical investigation.

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).

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.

Intestinal microbiota injuries in allo-HCT patients are characterized by loss of a-diversity and domination of microbial communities by single organisms. These injury patterns are associated with poor survival after allo-HCT and are likely attributable to antibiotic exposure, nutritional alterations, and regimen-related mucosal injury. As recipients of auto-HCT patients have similar exposures, we hypothesized that similar patterns of dysbiosis occur in auto-HCT patients. We present the first analysis of intestinal microbiota composition in auto-HCT patients at two independent institutions.From a prospectively collected cohort, we retrospectively identified 365 patients (median age 60) who received auto-HCT (May 2009 to Feb. 2018) at two transplant centers (MSK n = 316; Duke n = 49) with heterogeneous conditioning regimens, pre-HCT remission status, and diagnoses: 179 (49%) myeloma, 153 (42%) lymphoma, and 33 (9%) other diseases. 857 samples collected approximately weekly peri-transplant were 16S sequenced (V4-V5 region, Illumina platform) at a central laboratory. Stool samples from 17 volunteers at MSK and a publicly available dataset of 313 subjects from the Human Microbiome Project (HMP) served as healthy-control cohorts.The median pre-auto-HCT a-diversity during day -10 to 0 (as measured by Simpson reciprocal index, S) at both centers was significantly lower than healthy controls (Fig A) (HMP vs MSK auto-HCT, S=12.05 vs. 9.19, p<0.005; HMP vs Duke auto-HCT, S=12.05 vs 6.91, p<0.005, Wilcoxon test). Pre-HCT diversity was comparable in between auto-HCT and allo-HCT at each center (MSK auto-HCT vs MSK allo-HCT, S = 12.05 vs 8.74, p=0.53). Overall (days -10 to +30) diversity decreased comparably after auto-HCT and allo-HST across both centers, while auto-HCT patients demonstrated a slightly more rapid recovery at day +30 compared to allo-HCT patients (Fig 1B). Monodomination was observed in the samples (Fig 1C), with Streptococcus and Enterococcus as the most common genus, as defined by any single taxon comprising >30% of bacterial abundance. The cumulative incidence of intestinal domination by any organism was >50% by day 0 and was >75% by day +14.Microbial diversity is reduced prior to transplant in both auto-HCT and allo-HCT patients compared with healthy volunteers. Loss of diversity after auto-HCT occurs across transplant centers and the degree of injury is comparable to the dysbiosis observed in allo-HCT patients. We previously reported that intestinal monodomination increases the risk of bacteremia with corresponding organisms in allo-HCT patients. We now report these events are a common occurrence after auto-HCT, which suggests prevention or repair of microbiota injury as a strategy to reduce the toxicity of auto-HCT. Intestinal microbiota injuries in allo-HCT patients are characterized by loss of a-diversity and domination of microbial communities by single organisms. These injury patterns are associated with poor survival after allo-HCT and are likely attributable to antibiotic exposure, nutritional alterations, and regimen-related mucosal injury. As recipients of auto-HCT patients have similar exposures, we hypothesized that similar patterns of dysbiosis occur in auto-HCT patients. We present the first analysis of intestinal microbiota composition in auto-HCT patients at two independent institutions. From a prospectively collected cohort, we retrospectively identified 365 patients (median age 60) who received auto-HCT (May 2009 to Feb. 2018) at two transplant centers (MSK n = 316; Duke n = 49) with heterogeneous conditioning regimens, pre-HCT remission status, and diagnoses: 179 (49%) myeloma, 153 (42%) lymphoma, and 33 (9%) other diseases. 857 samples collected approximately weekly peri-transplant were 16S sequenced (V4-V5 region, Illumina platform) at a central laboratory. Stool samples from 17 volunteers at MSK and a publicly available dataset of 313 subjects from the Human Microbiome Project (HMP) served as healthy-control cohorts. The median pre-auto-HCT a-diversity during day -10 to 0 (as measured by Simpson reciprocal index, S) at both centers was significantly lower than healthy controls (Fig A) (HMP vs MSK auto-HCT, S=12.05 vs. 9.19, p<0.005; HMP vs Duke auto-HCT, S=12.05 vs 6.91, p<0.005, Wilcoxon test). Pre-HCT diversity was comparable in between auto-HCT and allo-HCT at each center (MSK auto-HCT vs MSK allo-HCT, S = 12.05 vs 8.74, p=0.53). Overall (days -10 to +30) diversity decreased comparably after auto-HCT and allo-HST across both centers, while auto-HCT patients demonstrated a slightly more rapid recovery at day +30 compared to allo-HCT patients (Fig 1B). Monodomination was observed in the samples (Fig 1C), with Streptococcus and Enterococcus as the most common genus, as defined by any single taxon comprising >30% of bacterial abundance. The cumulative incidence of intestinal domination by any organism was >50% by day 0 and was >75% by day +14. Microbial diversity is reduced prior to transplant in both auto-HCT and allo-HCT patients compared with healthy volunteers. Loss of diversity after auto-HCT occurs across transplant centers and the degree of injury is comparable to the dysbiosis observed in allo-HCT patients. We previously reported that intestinal monodomination increases the risk of bacteremia with corresponding organisms in allo-HCT patients. We now report these events are a common occurrence after auto-HCT, which suggests prevention or repair of microbiota injury as a strategy to reduce the toxicity of auto-HCT. Figure 1.

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, patients may develop toxicity due to cytokine release syndrome (CRS) or neurotoxicity. We hypothesized that the composition of the intestinal microbiota prior to CAR T cell infusion is correlated with efficacy and toxicity.We collected fecal samples from recipients of CAR T cells at Memorial Sloan Kettering Cancer Center (MSKCC) pre-CAR T cell infusion. Microbiota composition was profiled by 16S sequencing. Shotgun metagenomic sequencing was performed on a subset of the samples. Clinical response to assess efficacy was classified as complete response (CR) or no CR. Toxicity was defined as CRS or neurotoxicity of Grade 1 to 4. Linear discriminant analysis effect size (LEfSe) identified differentially abundant bacteria between groups with a linear discriminant analysis (LDA) score threshold >2.5.We analyzed baseline samples from 25 patients treated at MSKCC. The patients were adult recipients of CAR T cells who varied in regard to conditioning regimen, CAR construct and underlying diagnosis, which included hematologic and solid malignancies. LEfSe revealed increased representation of bacterial taxa in the microbiome of CR versus no CR, with Oscilliospiraceae, Ruminococcacaeae and Lachnospiraceae enriched in CR and Peptostreptococcaceae more abundant in no CR (A). A higher abundance of Lachnospiraceae was found in those who experienced toxicity, while Peptostreptococcaceae was more abundant in patients who did not have toxicity (B). We explored shotgun metagenomic sequences from 19 of 25 samples that were functionally annotated using the shortBRED pipeline. We inspected abundances of genes assigned to three pathways that we hypothesized may be immunologically relevant: B vitamin synthesis, bile acid biosynthesis, and short-chain fatty acid production. We observed increased abundance of genes associated with B vitamin biosynthesis in patients who had no CR (C) or toxicity (D).We observe differential abundance of microbiota in patients who achieved a CR or experienced toxicity as compared to those who did not achieve a CR or experience toxicity. We observe that increased B vitamin gene abundance is associated with no CR or toxicity. Overall, this data indicates that features of the microbiota may correlate with outcomes to CAR T cell therapy. 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, patients may develop toxicity due to cytokine release syndrome (CRS) or neurotoxicity. We hypothesized that the composition of the intestinal microbiota prior to CAR T cell infusion is correlated with efficacy and toxicity. We collected fecal samples from recipients of CAR T cells at Memorial Sloan Kettering Cancer Center (MSKCC) pre-CAR T cell infusion. Microbiota composition was profiled by 16S sequencing. Shotgun metagenomic sequencing was performed on a subset of the samples. Clinical response to assess efficacy was classified as complete response (CR) or no CR. Toxicity was defined as CRS or neurotoxicity of Grade 1 to 4. Linear discriminant analysis effect size (LEfSe) identified differentially abundant bacteria between groups with a linear discriminant analysis (LDA) score threshold >2.5. We analyzed baseline samples from 25 patients treated at MSKCC. The patients were adult recipients of CAR T cells who varied in regard to conditioning regimen, CAR construct and underlying diagnosis, which included hematologic and solid malignancies. LEfSe revealed increased representation of bacterial taxa in the microbiome of CR versus no CR, with Oscilliospiraceae, Ruminococcacaeae and Lachnospiraceae enriched in CR and Peptostreptococcaceae more abundant in no CR (A). A higher abundance of Lachnospiraceae was found in those who experienced toxicity, while Peptostreptococcaceae was more abundant in patients who did not have toxicity (B). We explored shotgun metagenomic sequences from 19 of 25 samples that were functionally annotated using the shortBRED pipeline. We inspected abundances of genes assigned to three pathways that we hypothesized may be immunologically relevant: B vitamin synthesis, bile acid biosynthesis, and short-chain fatty acid production. We observed increased abundance of genes associated with B vitamin biosynthesis in patients who had no CR (C) or toxicity (D). We observe differential abundance of microbiota in patients who achieved a CR or experienced toxicity as compared to those who did not achieve a CR or experience toxicity. We observe that increased B vitamin gene abundance is associated with no CR or toxicity. Overall, this data indicates that features of the microbiota may correlate with outcomes to CAR T cell therapy. Figure 1.

Abstract 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, p<0.005; HMP vs DUMC AHCT, S=12.05 vs 6.91, p<0.005) and reduced in both AHCT patients and allo-HSCT patients (MSKCC AHCT vs MSKCC allo-HSCT, S = 12.05 vs 8.74, p=0.53). In samples taken from days -10 to +30 after transplant, diversity decreased comparably after AHCT and allo-HSCT across both centers, while AHCT patients demonstrated a more rapid recovery at day +30 compared to allo-HSCT patients (Fig 1B). Finally, monodominance was observed in the samples (Fig 1C), with Streptococcus as the most common genus. The cumulative incidence of intestinal domination by any organism was >50% 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.

Abstract 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.

Bacground: Increasing evidence suggests that the patientsÕ gut microbiota plays an important role in the development of acute GVHD. Enterococcus faecium dominates the gut microflora of a substantial portion of allo-HCT patients after transplant, and this domination can precede blood stream infections. Its relevance for GVHD, however, is poorly defined. Methods & Results: Extending our previously published observation (Taur et al., Clin Infect Dis 2012), we observed a posttransplant monodomination of the gut microbiome with Enterococcus spp. in 21% allo-HCT patients within a cohort of 637 patients at our center (Figure 1A). This Enterococcus domination of the gut microbiota from allo-HCT patients was confirmed in samples from transplant centers at Duke University and the University Hospital Regensburg (17% and 8% of allo-HCT patients). The Enterococcus monodomination was significantly associated with moderate to severe acute GVHD (Figure 1B). In MHC-matched (Figure 1C) and -disparate (Figure 1D) mouse models of acute GVHD we also found a gut flora domination with E. faecalis early after transplant in mice with acute GVHD. This bloom is accompanied by a significant translocation of Enterococci into mesenteric lymph nodes (Figure 1E). Gavaging mice a laboratory E. faecalis-strain after transplant significantly aggravated acute GVHD (Figure 1F). Early results from transplant experiments with gnotobiotic mice harboring an altered Schaedler flora spiked with Enterococcus faecalis also showed aggravated acute GVHD compared to gnotobiotic mice without Enterococcus spike. IgA-coated gut microbes have been shown to be relevant for colitis. We FACS-sorted IgA-coated bacteria from mice with acute GVHD, and found Enterococci highly enriched in the IgA-coated fraction (Figure 1G). Conclusion: Studying both human and mouse gut microbial ecologies in acute GVHD, we report a causative role for Enterococcus spp. is an important risk factor for acute GVHD in allo-HCT patients.

Background: Inadequate dietary intake after allo-HSCT is pervasive and associated with poor outcomes. We and others have reported associations between microbiota composition and survival, GVHD, infections, and relapse after allo-HSCT. We hypothesized that dietary perturbations contribute to microbiome changes. However, oral intake is often poorly monitored. We implemented a program to monitor intake, antibiotic, and oral/fecal microbiota data during HSCT hospitalization. Methods: Dietary intake for patients (pts) undergoing allo-HSCT was collected between January and September 2017. Pts self-documented intake at each meal on a form automatically generated by the hospital kitchen listing their selected menu items. Data were confirmed, quality-controlled and entered into a computerized nutritional-analysis program by a Registered Dietitian. Daily stool samples were collected as able for 16S sequencing. In a subset of pts, oral swish samples were collected prior to HSCT and during neutropenia. Results: Thirty-eight pts (median age 53, range 24-74) were monitored with a median length of stay of 28 days. The most frequent indications were acute leukemia (42%) or MDS (32%). Preparatory regimens and graft sources were heterogeneous, though all pts received myeloablative or reduced-intensity conditioning prior to PBSC (74%), DUCB (11%) and haploidentical (11%) or conventional BMT (5%). On admission, 71% of pts met >75% of estimated caloric needs versus 11% of the 36 surviving pts at discharge. Thirty-two pts (84%) remained on exclusively oral diets while 4 (11%) received TPN and 2 (5%) received enteral nutrition in the ICU. Intake was lowest during neutropenia, when pts met a median of 32% estimated energy needs (range 8-82%) from day 0 until engraftment (B). Median weight loss for surviving pts was 6% (range +4-13%). A single-pt example of the intensive nutritional monitoring (A) illustrates how microbiota changes can be viewed in the context of antibiotic exposure and diet. In a multivariate linear regression model of intestinal microbiota α-diversity dynamics, decreased calorie intake and exposure to piperacillin-tazobactam (pip-tazo) were significant predictors of loss of α-diversity (P < .05 and P < .01, respectively). In the absence of pip-tazo, calorie intake correlated positively with diversity (r = .22, p = 2.95x10−5, C). The oral microbiota in 12 of these pts also showed a post-HSCT decrease in α-diversity (P = .0257, D); this drop was associated with broad-spectrum antibiotic exposure (P = .016). Conclusion: Our analysis suggests that profound nutritional perturbations and antibiotic exposure are both common and are associated with intestinal microbiota injury. Intensive nutritional monitoring is feasible and can help interpret microbiota data. A microbiota injury similar to that observed in the gut, appears to occur in the oral cavity.

We hypothesized that third-party fecal microbiota transplantation (FMT) may restore intestinal microbiome diversity after allogeneic hematopoietic cell transplantation (allo-HCT). In this open-label single-group pilot study, 18 subjects were enrolled before allo-HCT and planned to receive third-party FMT capsules. FMT capsules were administered no later than 4 weeks after neutrophil engraftment, and antibiotics were not allowed within 48 hours before FMT. Five patients did not receive FMT because of the development of early acute gastrointestinal (GI) graft-versus-host disease (GVHD) before FMT (n = 3), persistent HCT-associated GI toxicity (n = 1), or patient decision (n = 1). Thirteen patients received FMT at a median of 27 days (range, 19-45 days) after HCT. Participants were able to swallow and tolerate all FMT capsules, meeting the primary study endpoint of feasibility. FMT was tolerated well, with 1 treatment-related significant adverse event (abdominal pain). Two patients subsequently developed acute GI GVHD, with 1 patient also having concurrent bacteremia. No additional cases of bacteremia occurred. Median follow-up for survivors is 15 months (range, 13-20 months). The Kaplan-Meier estimates for 12-month overall survival and progression-free survival after FMT were 85% (95% confidence interval, 51%-96%) and 85% (95% confidence interval, 51%-96%), respectively. There was 1 nonrelapse death resulting from acute GI GVHD (12-month nonrelapse mortality, 8%; 95% confidence interval, 0%-30%). Analysis of stool composition and urine 3-indoxyl sulfate concentration indicated improvement in intestinal microbiome diversity after FMT that was associated with expansion of stool-donor taxa. These results indicate that empiric third-party FMT after allo-HCT appears to be feasible, safe, and associated with expansion of recipient microbiome diversity. This trial was registered at www.clinicaltrials.gov as #NCT02733744.