Microbiota as Predictor of Mortality in Allogeneic Hematopoietic-Cell Transplantation

Abstract

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.).

Copyright © 2020 Massachusetts Medical Society.

Figures

Figure 1 (facing page).. Intestinal Diversity during Transplantation Period and Association with Overall Survival.

Intestinal microbiota diversity, as measured by 16S ribosomal RNA gene sequencing and the inverse Simpson index, declined similarly during the course of allogeneic hematopoietic-cell transplantation (HCT) at all four institutions (Panel A). Each point represents a stool sample, color coded according to institution. Curves are loess-smoothed averages. Between each patient’s baseline sample (earliest sample obtained on day −30 to −6) and the periengraftment period (median value of samples obtained from day 7 to 21), the median diversity decreased by a factor of 4.3 at Memorial Sloan Kettering Cancer Center (MSK); by a factor of 3.3 at Duke University Medical Center, Durham, North Carolina; by a factor of 1.7 at University Medical Center, University Hospital Regensburg, Regensburg, Germany; and by a factor of 2.5 at Hokkaido University Hospital, Sapporo, Japan (Fig. S2). Overall survival was longer among patients with higher intestinal diversity in periengraftment samples, both in cohort 1, which comprised patients from MSK (Panel B), and in cohort 2, which comprised patients from the other three sites (Panel C). Tick marks indicate censored data. The median diversity value in cohort 1 (2.64) was used as the stratification cutoff in these landmark analyses (day 21). In cohort 1, there were 104 deaths among 354 patients in the higher-diversity group and 136 deaths among 350 patients in the lower-diversity group; in cohort 2, there were 18 deaths among 87 patients in the higher-diversity group and 35 deaths among 92 patients in the lower-diversity group. CI denotes confidence interval. The cumulative incidences of transplantation-related death and of relapse or progression of disease are shown for cohort 1 (Panel D). There were 52 transplantation-related deaths in 354 patients in the higher-diversity group and 82 such deaths in 349 patients in the lower-diversity group. There were 84 relapse events in 354 patients in the higher-diversity group and 81 relapse events in 349 patients in the lower-diversity group. Panel E shows the subgroup analysis involving recipients of T-cell–replete (unmodified) grafts or T-cell–depleted grafts in cohort 1 (Fig. S4). The numbers of patients at risk in the analyses shown in Panels D and E are provided in Table S4. Subsets of the data plotted in Panels A and D have been reported previously.,

Figure 2 (facing page).. Global Spectrum of Microbiota Disruption in Patients Undergoing Allogeneic HCT.

The microbiota composition of 8767 samples from 1362 patients from all four institutions are shown according to the t-distributed stochastic neighbor embedding (t-SNE) algorithm. Each point represents a single stool sample, and the axes (t-SNE1 and t-SNE2) have arbitrary units. The more similar the samples are, the closer together they appear on the t-SNE plot. Earlier samples (Panel A, left side) are enriched for higher-diversity configurations (Panel B, left side), whereas later samples are enriched for lower-diversity configurations (Panel B, right side). Samples from all four institutions were well distributed across t-SNE space (Panel C). A quantitative comparison of beta-diversity distances between samples from different institutions is shown in Figure S6A. Color coding according to the most abundant taxon in each sample shows that the early cluster was characterized by Clostridia (brown, orange, and pink), Bacteroidetes (teal), and Actinobacteria (purple) (Panel D). Some low-diversity states were characterized by domination with the genera enterococcus (dark green), streptococcus (light green), klebsiella (bright red), and escherichia (dark red). Among patients who had at least one sample obtained both before and after hematopoietic-cell transplantation, the fraction of patients who had had at least one instance of domination each week, defined as a relative abundance of at least 30% for any taxonomic unit, is shown in Panel E. The fraction of samples with domination in each 7-day sliding window is shown in Panel F. The odds over time of a sample being dominated was similar in three of the cohorts and was slightly higher at Hokkaido (Fig. S6B). The taxa that contributed to domination events in cohort 1 are shown in Panel G. Domination was defined at the level of operational taxonomic units; color coding is at higher taxonomic ranks (see color legend in Panel D).

Figure 3.. Microbiota Injury before HCT and Association with Transplantation Outcomes.

The diversity of the initial pretransplantation samples from patients (obtained on day −30 to −6) is lower than the diversity of 313 samples from 212 participants of the Human Microbiome Project (HMP) and 1 sample obtained from each of 34 healthy adult volunteers at MSK (healthy controls) (Panel A). A total of 993 patients were included in the analysis at MSK, 95 at Duke, 40 at Regensburg, and 45 at Hokkaido. The horizontal line in each box represents the median, the lower and upper boundaries of the boxes the interquartile range, the ends of the whisker lines the minimum and maximum values within 1.5 times the interquartile range, and the dots the individual data points. The proportions of samples from healthy volunteers and initial samples from patients that could be classified according to the enterotype scheme are plotted (Panel B). Samples obtained before HCT were less likely to be classified as belonging to an enterotype than were samples obtained from healthy volunteers. In cohort 1, overall survival was longer among patients with higher intestinal diversity in the initial samples than among those with lower diversity (Panel C). There were 72 deaths among 250 patients in the higher-diversity group and 101 deaths among 251 patients in the lower-diversity group (Table S7). Tick marks indicate censored data. Combined analyses of diversity before transplantation and during the periengraftment period are shown in Table S8 and Figure S7.