Baseline Salivary Microbiota Predicts Severe Oral Mucositis after Allogeneic Hematopoietic Cell Transplantation

Oral mucositis (OM) is a common early complication of allogeneic hematopoietic cell transplantation (alloHCT), causing pain, infections, swallowing/speech impairment, and prolonged hospitalization. As there are no approved prophylactic or therapeutic approaches for alloHCT-associated OM, management remains supportive and limited largely to narcotic analgesics, ice chips, and total parenteral nutrition. A better understanding of OM pathogenesis may yield novel targets for intervention. We hypothesized that the oral microbiota may mediate OM pathogenesis.

We conducted a prospective single-center study including longitudinal mucositis assessment using the Oral Mucositis Assessment Scale (OMAS) and multi-site oral sample collection by calibrated oral medicine specialists. Mucositis assessment was performed at baseline, day +7, day +14, day +21, day +28, and day +84. Total mucositis score (range 0-45) at each timepoint was calculated as the sum of all scores (ulceration and erythema) across all sites and used to classify OM severity (total score ≤ median vs. > median across all examinations). Saliva (baseline and days +14, +28, and +84), supragingival plaque (baseline and days +14, +28, and +84), and subgingival plaque (baseline) samples underwent shotgun metagenomic sequencing, targeting a sequencing depth of 20M read pairs per sample. MetaPhlAn4 was used for species-level taxonomic assignment. MaAsLin2 (Microbiome Multivariable Association with Linear Models) was used to identify microbiota predictors of OM severity at days +7, +14, and +21. Species abundances in different oral sites at the same or the closest preceding OM assessment timepoint for the same patient were included in the model. A significance threshold of 0.05 was considered for P values, or Benjamini-Hochberg q values in case of multiple testing.

Fifty-two patients were enrolled (median age 43, M:F ratio 1). Despite the original plan at the time of consent, 9 patients received reduced-intensity conditioning due to newly discovered comorbidities; these patients were included. Mucositis severity increased rapidly until day +7, followed by a slower rise to a peak at day +14. It then rapidly improved until day +21. Further improvement occurred until day +28, followed by complete resolution by day +84 in most patients. Significant clinical predictors of worse OM were male sex for day +7 (P = 0.01), myeloablative conditioning for day +14 (P = 0.01), older age for day +21 (P = 0.01), and diseases other than acute leukemia for day +84 (P = 0.03). Salivary flow rate was not associated with OM (Wilcoxon’s P for baseline, day +14, day +28, and day +84 timepoints: 0.47, 0.68, 0.30, and 0.80, respectively).

A total of 455 samples were analyzed. The strongest associations were between day +14 OM and baseline salivary microbiota: Prevotella nanceiensis (more severe OM; q = 0.07), an unclassified actinomyces species (sp. ph3) (less severe OM; q = 0.007), and an unclassified schaalia species (SGB17158, less severe OM; q = 0.07). A composite baseline salivary score calculated as Prevotella nanceiensis - (Actinomyces sp. ph3 + Schaalia SGB17158) was significantly higher in patients with more vs. less severe OM at day +14 (P = 2x10^-5), with a high prediction accuracy (AUC 85%; 95%CI 75%-96%). This score was not associated with other baseline characteristic such as age (P = 0.69), sex (P = 0.78), disease (P = 0.34), salivary flow rate (P = 0.24), or exposure to antibiotics with ≥10% exposure rate within 2 weeks before the baseline sample (P for TMP-SMX and fluoroquinolones 0.25 and 0.73, respectively). When the same score was calculated at different timepoints, no change was apparent over time, despite post-HCT antibiotic exposures and other microbiota insults.

In summary, we have found a predictive biomarker of OM after alloHCT using baseline salivary microbiota. The score identified here is a remarkably stable, intrinsic, subject-level characteristic of the oral microbiota, predisposing the patient to more severe OM. This finding introduces novel opportunities targeting the microbiota to prevent OM.