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Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for several hematologic (and non-hematologic) diseases, though it carries the risk of complications that are potentially fatal, such as infection and graft-versus-host disease (GvHD).1
Acute GvHD (aGvHD) is a common complication of allo-HSCT that is characterized by diarrhea, abdominal pain, anorexia, hyperbilirubinemia, and maculopapular rash.1 This clinical syndrome occurs due to inflammatory cytotoxic activity against healthy host tissue—including the gut—by donor T cells and is influenced by the donor source and conditioning regimen.1 The first step in the pathogenesis of aGvHD is thought to be increased permeability of the intestines caused by the conditioning regimen, leading to extraintestinal movement of bacteria and resulting in bacteremia.2
Loss of diversity of the intestinal microbiota (IM) has been associated with both the risk and intensity of aGvHD as well as aGvHD-related mortality, and data have suggested that the influence of certain bacterial species following allo-HSCT may promote the development of aGvHD.1 Bacteria from the oral cavity have also been shown to translocate to the gut, driving IM dysbiosis.2
Heidrich and colleagues recently published a study that aimed to directly evaluate the effect of allo-HSCT on the oral microbiota (OM) and the influence of OM dysbiosis on the risk of aGvHD as a way to further understand how the bacterial compositions of the OM and IM impact the development of aGvHD in the post-allo-HSCT setting.1 Similarly, Ingham and colleagues have just reported their work on long-term microbiota dynamics of the gut, oral, and nasal cavities in pediatric patients who had undergone allo-HSCT.2
Thirty patients who underwent allo-HSCT for hematologic disorders from a single center were consecutively enrolled, and supragingival biofilm samples were collected from all patients at three phases: at preconditioning, at aplasia, and at engraftment. Bacterial cells were recovered, and DNA extraction and sequencing were performed.
Cumulative incidence (CMI) rates were calculated for aGvHD (Grade II‒IV) and severe aGvHD (Grade III–IV) with death as a competing event, and relative risks for developing aGvHD and severe aGvHD were estimated and adjusted for graft source and intensity of the conditioning regimen.
Most patients received reduced-intensity conditioning (60%) and grafts from peripheral blood (67%), and the most common underlying disease was acute leukemia (60%). Clinical characteristics are shown in Table 1.
Table 1. Clinical characteristics*
Clinical characteristics, % (unless otherwise stated) |
Total (n = 30) |
---|---|
Sex (male) |
53 |
Median age (range), years |
50 (19–73) |
Underlying disease† |
|
Acute leukemia |
60 |
Other |
40 |
Conditioning intensity |
|
Reduced intensity |
60 |
Total body irradiation |
37 |
Pretransplant T-cell depletion |
50 |
Graft source |
|
Bone marrow |
33 |
Peripheral blood |
67 |
Donor |
|
Matched sibling |
30 |
Haploidentical |
33 |
Matched unrelated |
30 |
Mismatched unrelated |
7 |
GvHD prophylaxis |
|
MMF + CsA |
37 |
MTX + CsA |
33 |
MMF + CsA + PTCy |
30 |
Median follow up (range), months |
37 (25–46) |
CsA, cyclosporin A; GvHD, graft-versus-host disease; MMF, mycophenolate mofetil; MTX, methotrexate; PTCy, posttransplant cyclophosphamide. |
Dental biofilm microbiota (DBM) alpha diversity was assessed using the Shannon index, and a statistically significant decrease in DBM alpha diversity was observed during allo-HSCT:
Median alpha diversity value was used to stratify patients into equal-sized high- and low-diversity cohorts to evaluate the association between DBM diversity and aGvHD risk. No association was found between DBM diversity and the risk of aGvHD at preconditioning, aplasia, or engraftment.
Only genera present at relative abundance ≥0.1% in at least 25% of samples were considered in the evaluation of whether abundance of specific genera was associated with risk of aGvHD at any of the three time points. Patients were then stratified into equal-sized high- and-low abundance cohorts.
Patients with high Veillonella relative abundance at preconditioning had a lower CMI of aGvHD, which was significant even after adjusting for graft source and intensity of conditioning regimen. Patients with high Streptococcus or Corynebacterium relative abundance at preconditioning had a higher CMI of aGvHD, though only Streptococcus remained significantly associated with aGvHD risk after adjusting for graft source and intensity of the conditioning regimen (Table 2).
Table 2. Risk analyses for the association of acute graft-versus-host disease with relevant microbiota variables*
|
Adjusted |
|||||||
---|---|---|---|---|---|---|---|---|
Non-adjusted |
Veillonella at P |
Streptococcus at P |
Corynebacterium at P |
|||||
HR |
p value |
HR |
p value |
HR |
p value |
HR |
p value |
|
Graft source (bone marrow) |
0.95 |
0.92 |
1.42 |
0.38 |
0.75 |
0.64 |
1.42 |
0.59 |
Conditioning intensity (myeloablative) |
0.74 |
0.59 |
0.50 |
0.37 |
0.79 |
0.7 |
0.79 |
0.73 |
Veillonella at P (high vs. low) |
0.24 |
0.009 |
0.21 |
0.006 |
- |
- |
- |
- |
Streptococcus at P (high vs. low) |
2.89 |
0.036 |
- |
- |
3.17 |
0.03 |
- |
- |
Corynebacterium at P (high vs. low) |
2.74 |
0.04 |
- |
- |
- |
- |
2.79 |
0.053 |
HR, hazard ratio; P, preconditioning. |
Veillonella and Streptococcus had the highest relative abundance at preconditioning, and patients with a Veillonella/Streptococcus ratio ≥1 at preconditioning had a lower CMI of aGvHD (p = 0.004). Notably, the association between this ratio and aGvHD risk was stronger than the association seen for each genus separately, and it remained significant after adjusting for graft source and conditioning regimen intensity (p = 0.005). There was no risk between the Veillonella/Streptococcus ratio at aplasia or engraftment and aGvHD risk.
The final analysis conducted by the investigators was to determine whether bloom—defined as the sudden expansion of a genus from near absence to dominance—of potentially pathogenic bacteria seen during allo-HSCT was associated with aGvHD risk. During allo-HSCT, 23 blooms involving 12 genera were observed, affecting 20 patients, 3 of whom experienced more than one blooming event.
The study included 29 pediatric patients at a single hospital center. Each patient underwent a myeloablative conditioning regimen prior to allo-HSCT, and the patients were grouped into categories based on conditioning regimen:
Sampling timepoints were pre-examination, around the start of conditioning, at the time of allo-HSCT, and weekly during the first 3 weeks after transplantation. Fecal samples (212), buccal swabs (248), and anterior naris swabs (249) were collected from patients at each sampling timepoint for a total of 709 patient samples. Severity of aGvHD was grouped into Grades 0‒I and II‒IV and was graded via daily clinical assessment of skin, liver, and gastrointestinal manifestations.
The gut microbiota of the patients at pre-examination was compared with age-matched healthy children and demonstrated an alpha diversity 2.4-fold lower in the patient group, potentially due to treatment given to these children prior to enrollment in the study. Bacterial composition also differed between the two groups, with several taxa that were significantly more abundant in the patient group compared with healthy controls, including Lactobacillus, Enterococcus, Erysipelotrichaceae, and Klebsiella. Contrastingly, Prevotella, Ruminococcus, and Akkermansia were more abundant in the healthy controls.
A tree-based sparse linear discriminant analysis (LDA) was performed to characterize samples from the gut, oral, and nasal sites at different time points, identifying three partly entwined phases:
Samples from the oral and nasal cavities in phases I and III overlapped, which suggested that microbial communities from later timepoints had possibly returned to a state similar to that prior to HSCT. Interestingly, the nasal community composition at Month +12 was different from the composition from the nasal cavity at Week +1, though both had low alpha diversity.
The 12 most abundant families at each site were examined to provide a more detailed view of the abundance dynamics:
Individual discriminating amplicon sequencing variants (ASVs) were examined to determine which specific gut taxa drove the differences between samples seen in the LDA. This analysis revealed 19 clades in the gut that best separated the samples based on timepoint. The two most discriminating clades with positive LDA coefficients were Enterococcaceae and Lactobacillaceae, which increased in abundance from the day of HSCT (Enterococcaceae) and Week +1 (Lactobacillaceae); each clade decreased in abundance from Month +3 to levels similar to pre-examination (Table 3). The two most discriminative clades with negative LDA coefficients were two individual ASVs, one Lachnospiraceae clade, and two Ruminococcaceae clades; the abundance of these clades decreased in Week +1 and recovered after Month +3. Enterococcaceae were more abundant in phase II samples, and Lachnospiraceae and Ruminococcaceae were more abundant in phase I and III samples.
Table 3. Distinct discriminatory lineages*
|
Positive LDA coefficients |
Negative LDA coefficients |
||
---|---|---|---|---|
Clades |
Most abundant ASVs |
Clades |
Most abundant ASVs |
|
Gut |
Enterococcaceae |
E. faecium |
Lachnospiraceae |
Blautia wexlerae |
Oral cavity |
Actinomycetaceae |
A. viscosis |
Prevotellaceae |
P. melaninogenica |
Nasal cavity |
Corynebacteriaceae |
C. propinquum |
- |
- |
ASV, amplicon sequencing variant; LDA, linear discriminant analyses. |
Ten clades of 71 total ASVs were identified in the oral cavity, and 30 discriminating nasal clades of 36 total ASVs were revealed in the nasal cavity (Table 3).
Patients with Grades 0–I aGvHD had lower relative abundances of Tannerellaceae in the gut pre-HSCT compared with patients with Grades II-IV aGvHD, particularly at pre-examination and start of conditioning. In addition, three predictive ASVs were identified in the gut demonstrating that high abundances of ASV 128 (Parabacteroides distasonis, Tanerellaceae, p < 0.01), ASV 268 (Lachnospiraceae NK4A136 group sp., Lachnospiraceae, p = 0.01) and ASV 3 (Lactobacillus sp., Lactobacillaceae, p < 0.01) pre-HSCT were associated with development of aGvHD Grades II–IV.
The bacterial community of the oral cavity pre-HSCT in patients with Grades II–IV aGvHD was characterized by a lower relative abundance of Neisseriaceae and higher relative abundances of Aerococcaceae and Prevotellaceae when compared with Grades 0‒I aGvHD, particularly at the pre-examination and start of conditioning timepoints. High abundances of the ASVs 568 (Actinomyces sp., Actinomycetaceae), 226 (Prevotella melaninogenica, Prevotellaceae), and 500 (Pseudoproprionibacterium propioninum, Proprionobacteriaecae) (all p < 0.001) pretransplantation predicted the development of aGvHD Grades II–IV after HSCT.
In the nasal cavity, the proportion of Neisseriaceae pretransplant was higher in patients with aGvHD Grades 0–I compared with Grades II–IV, while Actinomycetaceae and Corynebacteriaceae were more abundant in patients with aGvHD Grades II–IV compared with Grades 0–I. Regarding specific ASVs, the partial 16S rRNA gene sequence of ASV 66 (with a high sequence similarity to Actinomyces viscosus) predicted development of Grades II–IV aGvHD (p = 0.03) when occurring in abundance pre-HSCT. In addition, pre-HSCT levels of this organism were 2.3-times higher in patients with aGvHD Grades II–IV compared with Grades 0–I. High pre-HSCT abundance of the partial 16S rRNA gene sequence of ASV 47 (exhibiting a high sequence similarity to Rothia aeria), on the other hand, predicted that a patient would not develop aGvHD (p = 0.03).
aGvHD is a significant cause of mortality after allo-HSCT, and the current first-line therapy—corticosteroids—has a response rate ranging from 40% to 70%, highlighting the importance of being able to predict aGvHD risk and develop preventive therapy.1 Heidrich et al. and Ingham et al. have identified that changes in the oral, nasal, and gut microbiomes, and their immune environments prior to and during allo-HSCT, may be predictive of aGvHD after transplantation, though it is important to note that these were single-centered studies with limited sample sizes. These studies indicate that future research may allow for the development of more precise treatment strategies in this area.
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