Conditioning Regimens are Associated with Distinct Patterns of Microbiota Injury in Allogeneic Hematopoietic Cell Transplantation

Abstract

Purpose: The gut microbiota is subject to multiple insults in allogeneic hematopoietic cell transplantation (allo-HCT) recipients. We hypothesized that preparative conditioning regimens contribute to microbiota perturbation in allo-HCT.

Experimental design: This was a retrospective study that evaluated the relationship between conditioning regimens exposure in 1,188 allo-HCT recipients and the gut microbiome. Stool samples collected from 20 days before transplantation up to 30 days after were profiled using 16S rRNA sequencing. Microbiota injury was quantified by changes in α-diversity.

Results: We identified distinct patterns of microbiota injury that varied by conditioning regimen. Diversity loss was graded into three levels of conditioning-associated microbiota injury (CMBI) in a multivariable model that included antibiotic exposures. High-intensity regimens, such as total body irradiation (TBI)-thiotepa-cyclophosphamide, were associated with the greatest injury (CMBI III). In contrast, the nonmyeloablative regimen fludarabine-cyclophosphamide with low-dose TBI (Flu/Cy/TBI200) had a low-grade injury (CMBI I). The risk of acute GVHD correlated with CMBI degree. Pretransplant microbial compositions were best preserved with Flu/Cy/TBI200, whereas other regimens were associated with loss of commensal bacteria and expansion of Enterococcus.

Conclusions: Our findings support an interaction between conditioning at the regimen level and the extent of microbiota injury.

Figure 1.. Fecal samples from 1,188 recipients of allo-HCT were analyzed.

(A) CONSORT diagram for patient inclusion. The patients were conditioned with one of the eight regimens received by >40 patients with at least one stool sample collected between days −20 to 30. (B) Flu/Cy/TBI200 was the only nonmyeloablative (NMA) regimen. Flu/Mel and Mel/Tt/Flu were the reduced-intensity conditioning (RIC) regimens; the rest of the regimens were myeloablative (MAC). (C) Not all diversity loss can be attributed to antibiotic exposures. Each point is a stool sample whose α-diversity (as measured by 16S amplicon sequencing and the inverse Simpson index) is plotted over time relative to transplantation. Lines are smoothed average by the LOESS (locally estimated scatterplot smoothing) method, in which each data point (α-diversity measurement) is considered as an independent event. The black curve averages all samples, including those collected before or after any antibiotic exposure. The purple curve ignores any sample collected after exposure to any antibacterial antibiotic; as virtually all patients commenced prophylactic antibiotics by day −2 or with the onset of neutropenia, the purple curve does not extend beyond day 0. The blue curve ignores any samples collected after exposure to non-prophylactic antibiotics, i.e., samples exposed to the prophylactic antibiotics ciprofloxacin and intravenous vancomycin are included in the blue smoothed average. Time bin t1 (day −20 to −6) and t2 (day −5 to 0) are indicated with dashed vertical lines. (D) Among samples from n = 507 patients whose samples were not exposed to any antibacterial antibiotics, α-diversity declines significantly between t1 and t2, indicating that not all diversity loss can be attributed to antibiotic exposures. t1 α-diversity is also significantly lower than those of healthy volunteers (n = 30). Only one stool sample per patient was considered in each time window. (E) Among samples from n = 507 patients whose samples were not exposed to any antibacterial antibiotics, microbiota composition is significantly different between t1 and t2, as measured by Bray-Curtis distance to a centroid of healthy volunteers (n = 28) compositions, indicating that not all composition changes can be attributed to antibiotic exposures. (F) Liquid stool samples (n=957), collected between days 0 to 10, were more commonly collected from recipients of more intense conditioning regimens, as assessed by laboratory technicians at the time of sample aliquoting. X-squared for all samples= 118.44, df = 4, p-value < 2.2 × 10⁻¹⁶. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 Abbreviations: allo-HCT – allogeneic hematopoietic cell transplantation; MAC – myeloablative conditioning; RIC – reduced-intensity conditioning; NMA – nonmyeloablative conditioning; Abx – antibiotics.

Figure 2.. Microbiota injury patterns in allo-HCT are conditioning regimen-specific.

(A) Fecal microbiota α-diversity declines most in recipients of MAC and the least in recipients of NMA conditioning (Table S2). Each point is a stool sample whose α-diversity is plotted over time relative to transplantation. The smoothed curves plots are moving averages of recipients of different regimens. (B) Distinct patterns of α-diversity dynamics can be observed among recipients of specific conditioning regimens. Five MAC regimens are plotted in the left panel, two RIC and one NMA regimen are plotted in the right panel. (C) In a patient-level pair-matched analysis of the earliest sample collected before (days −20 to −1) and earliest sample after conditioning (days 0 to 10), the NMA regimen Flu/Cy/TBI200 was associated with the least reduction in diversity (42%). In contrast, the MAC regimens were associated with the greatest reduction (69%−78%). Regimens were compared to Flu/Cy/TBI200 with the Wilcoxon signed-rank test. (D) Microbiome dissimilarity, as determined by applying multidimensional scaling to the Bray-Curtis distance matrix, between samples collected at days 0 and 12 was significantly increased in all conditioning regimens except Flu/Cy/TBI200. The greatest distance was in the myeloablative regimen Clo/Tt/Mel. (E) A multivariable MaAsLin2 model(22) adjusting for time of sampling and conditioning regimens reveals a differential association between microbial taxa and conditioning regimens. Bacteria are ordered by taxonomical ranking. ASVs that could not be classified to the genus level were analyzed at the family level, as indicated by f_Ruminococcaceae. Flu/Cy/TBI200, highlighted with a gray rounded rectangle, was associated with the preservation of members of the Clostridia class. (F) A grading scheme classifying regimens into three categories of microbiota injury (low [I], intermediate [II], and high [III]) based diversity reduction between days −20 to 30 was introduced. The estimated effect on diversity of each regimen was derived from a generalized estimating equation (Table S4) regression model, adjusting for time, age, sex, exposure to GVHD prophylaxis, antibiotics, and conditioning. (G) Patients who received unmodified grafts had a higher incidence of acute GVHD Grade II-IV with regimens categorized as having greater microbiota toxicity. CMBI hazard ratios are derived from a multivariable Cox-regression adjusting for age, sex, comorbidity burden, donor and HLA matching, and GVHD prophylaxis. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 HCT – allogeneic hematopoietic cell transplantation; MAC – myeloablative conditioning; RIC – reduced-intensity conditioning; NMA – nonmyeloablative conditioning; CMBI – conditioning-associated microbiota injury; graft-versus-host disease (GVHD);