ALBERT

Description: 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, p75% 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.

Description: 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.

Description: 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

Description: 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.

Description: 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, p87% 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.

Description: We have previously described clinically relevant reductions in fecal microbiota diversity of patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT). Recipients of high-dose chemotherapy and autologous hematopoietic cell transplantation (auto-HCT) incur similar antibiotic exposures and nutritional alterations. To characterize the fecal microbiota in the auto-HCT population, we analyzed 1,161 fecal samples collected from 534 adult recipients of auto-HCT for lymphoma, myeloma and amyloidosis in an observational study conducted at two transplant centers in the United States. Using 16S ribosomal gene sequencing, we assessed fecal microbiota diversity as measured by the inverse Simpson index, and composition. At both centers, early pre-transplant fecal microbiota diversity was lower than in healthy control subjects 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 allo-HCT. Above-median fecal intestinal diversity in the peri-engraftment period was associated with decreased risk of death or progression (PFS HR 0.46 [95% CI, 0.26-0.82], P=0.008), adjusting for disease and disease status. This suggests that further investigation into the health of the intestinal microbiota in auto-HCT patients and post-transplant outcomes should be undertaken.