Longitudinal and integrated analysis of oral and stool microbiome in allogeneic hematopoietic cell transplantation recipients.

Abstract

Abstract Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapy for many benign and malignant hematologic diseases. Graft-versus-host-disease (GVHD) is a significant complication and the primary cause of non-relapse mortality following allo-HCT. Studies have linked greater intestinal microbial diversity pre-transplantation and in the first 30 days post-transplantation with better survival and GVHD-related mortality. However, the impact of microbiome diversity over time on acute (aGVHD) and chronic GVHD (cGVHD) is unclear. In this study, we longitudinally assessed the composition of the fecal and oral microbiomes in allo-HCT patients prior to conditioning therapy and at several timepoints over 6 months post-transplant to better understand the microbiome's role in GVHD. Methods: We enrolled 27 patients (median age 55 years; range 27-69) receiving allo-HCT from an HLA-matched unrelated donor from 1/1/2016-1/4/2018. Fecal and oral (buccal swab and mouthwash) samples were collected at five time points: baseline (within 2 weeks before allo-HCT), absolute neutrophil count nadir (within 3 days of ANC of 0, ~day 5-7 post-allo-HCT), myeloid engraftment (within 3 days after ANC >500, ~day 14 post-allo-HCT), 3 months post-allo-HCT, and 6 months post-allo-HCT. Microbiome composition was assessed with 16S rRNA gene amplicon sequencing. Results: All patients underwent myeloablative conditioning regimens without T-cell depletion, and they received GVHD prophylaxis with Tacrolimus/Methotrexate. Levaquin was used for antibiotic prophylaxis during severe neutropenia. With each patient serving as their own internal control, we tracked the change in bacterial communities via Shannon diversity index (DI) across our time points. We observed a decreased bacterial DI in both fecal and oral (via mouthwash, not buccal swab) samples at the time of nadir: -0.59 decrease (p<0.01) in fecal DI, and -0.34 decrease (p<0.01) in oral DI. The decrease in oral bacterial diversity was maintained at engraftment (-0.38; p<0.01), with a similar apparent trend in fecal microbiome diversity (-0.62; p=0.058). At 3 months and 6 months, there was no difference in either fecal or oral microbiome diversity compared to each patient's baseline. We next compared bacterial diversity between patients with and without aGVHD. In total, 13 patients (48%) experienced aGVHD during the study. There was no difference in oral or fecal bacterial diversity prior to allo-HCT between patients with and without aGVHD: fecal DI 2.92 vs 3.17 (p=0.20), buccal swab DI 2.50 vs 2.39 (p=0.75), and mouthwash DI 2.47 vs 2.63 (p=0.82). Interestingly, at count nadir, fecal microbiome diversity was significantly reduced in aGVHD patients (DI 2.00 vs 3.05; p<0.01). This difference was not significant at time of engraftment; however, at 3- and 6-months post allo-HCT, fecal diversity remained lower in the aGVHD group (3-mon: DI 2.78 vs 3.29; p=0.03; 6-mon: DI 2.75 vs 3.45; p=0.028). Oral samples demonstrated no statistically significant difference in bacterial diversity related to aGVHD. Amongst patients with gastrointestinal aGVHD, there was an enrichment in the proteobacteria phylum including the Enterobacteriaceae family which has been associated with decreased overall survival in allo-HCT. We also observed enrichment of Clostridia in patients without aGVHD, specifically Lachnospiraceae which have been associated with increased production of butyrate and improved outcomes in allo-HCT. Finally, we observed a decrease in bacterial diversity in patients who developed oral cGVHD at 3 months (buccal swab; p=0.047), with an enrichment of Firmicutes, specifically Lactobacillaceae. There was no difference in diversity when grouping by all patients with cGVHD (p=0.085). Conclusions: Together, these results show that allo-HCT decreases both fecal and oral microbiome diversity and highlight the role of the fecal microbiome composition in aGVHD. While studies have linked lower fecal microbiome diversity within 30 days post-transplant to poor outcomes, to our knowledge this is the first longitudinal study to link ongoing fecal microbiome diversity loss in the first 6 months post allo-HCT with higher aGVHD. Importantly, we observe a decrease in oral microbiome diversity in patients with oral cGVHD at 3 months but not broadly across all cGVHD patients. Our findings underscore the need for further research on the microbiome's impact on allo-HCT patients.