Oral Microbiome Dynamics in Patients with Acute Lymphoblastic Leukemia and Oral Mucositis

Abstract

The oral microbiome of patients with acute lymphoblastic leukemia (ALL) undergoes changes caused by the neoplasia as well as the antimicrobial activity of chemotherapy (CTX), which promotes the development of oral mucositis (OM). This study aimed to analyze the oral microbiome dynamics and salivary cytokine production in pediatric ALL patients before and during CTX, comparing children who did and did not develop OM. We conducted a longitudinal, observational, and analytical study including 32 newly diagnosed pediatric ALL patients (ages 2-16 years) undergoing CTX. Oral rinse and non-stimulated saliva samples were collected at baseline (day 0), day 14, and day 21 of induction of CTX, with an additional sample taken during OM episodes when possible. Microbiome analysis was performed using 16S rRNA sequencing on an Illumina MiSeq platform, and salivary cytokines were measured using a Luminex multiplex assay. The most pronounced microbiome changes occurred on day 14, particularly in patients who developed OM, characterized by higher α diversity, increased abundance of opportunistic taxa, and elevated IL-6 concentrations. In contrast, patients who did not develop OM exhibited a more stable microbial composition. Overall, these findings indicate that temporal oral dysbiosis and increased IL-6 may serve as early markers and potential predictors of OM development during chemotherapy in pediatric ALL patients.

Keywords: acute lymphoblastic leukemia; chemotherapy; oral microbiome; pediatric oral mucositis.

Figure 1

Samples were collected on day 0 (first sample), day 14 (second sample), and day 21 of chemotherapy (third sample). A sample was also collected during the mucositis phase (fourth sample), although only four children presented with symptoms during the sample collection period. Patient follow-up was subsequently reviewed in clinical records to determine how many patients developed mucositis during the remaining phases of chemotherapy. OM: during oral mucositis; No: patients who did not develop oral mucositis; Yes: patients who developed oral mucositis.

Figure 2

Alpha diversity based on the Shannon Index (A) by time point: on days 0 (n = 30), 14 (n = 29) and 21 (n = 27) of chemotherapy, and during oral mucositis, where no statistically significant difference was identified (ANOVA p = 0.66). (B) When comparing the alpha diversity of the groups according to whether they developed oral mucositis, it was observed that the Shannon index on day 14 was significantly higher in patients who did develop oral mucositis (ANOVA No p = 0.60; Yes p = 0.19. t test: day 0 p = 0.24; day 14 ** p = 0.01; day 21 p = 0.81) (No: day 0 n = 18, day 14 n = 16, day 21 n = 15; Yes: day 0 n = 12, day 14 n = 13, day 21 = 14). OM: during oral mucositis; No: patients who did not develop oral mucositis; Yes: patients who developed oral mucositis. The colors represent the different time points: Day 0 (blue), Day 14 (purple), Day 21 (pink), and samples taken during active mucositis (green).

Figure 3

Relative abundance (A) at the phylum level, where it is observed that the relative abundances are similar between days 0, 14 and 21 of chemotherapy, with a predominance of Firmicutes. On the other hand, at the time of oral mucositis, the phylum Bacteroidota predominated. (B) At the genus level by time point (days 0, 14 and 21 of chemotherapy), important variations are also observed, especially during oral mucositis. (C) The relative abundance at the genus level is shown in patients who developed oral mucositis and those who did not develop it (based on the medical records) and by time point (days 0, 14 and 21 of chemotherapy). Note that the RA of microorganisms in patients who did not develop mucositis is more significantly altered on day 14 of chemotherapy. In the case of patients who developed mucositis, RA varies with the days of chemotherapy. By day 21, the RA of microorganisms in patients who did and did not develop oral mucositis was somewhat similar, compared with days 0 and 14. OM: during oral mucositis; No: patients who did not develop oral mucositis; Yes: patients who developed oral mucositis.

Figure 4

Differential abundance analysis. (A) Representative microorganisms of each group are shown by time point (day 0, 14, 21, and during oral mucositis). On day 0, the genera Pectobacterium and Bacillus can be mentioned (Kruskal–Wallis: * p < 0.05; ** p < 0.01). On day 14, Haemophilus was found to be the most representative genus, and on day 21, it was Treponema. (B) The same analysis was performed on day 14 in patients who did or did not develop oral mucositis based on their records, and it was found that Selenomonas and Streptococcus, among others, were the representative microorganisms of the group of patients who did develop oral mucositis (Kruskal–Wallis: * p < 0.05. ** p < 0.01). OM: during oral mucositis; No: patients who did not develop oral mucositis; Yes: patients who developed oral mucositis.

Figure 5

IL-6 concentration in saliva. (A) by time point (days 0, 14, and 21 of chemotherapy and during oral mucositis), where a statistically significant increase in the concentration of this interleukin was identified as the days of chemotherapy increased (Kruskal–Wallis; * p < 0.05; ** p < 0.01). (B) when comparing according to the development or not of oral mucositis, it was identified that patients who did develop oral mucositis presented a statistically significant increase in IL-6 from day 0 to day 14 of chemotherapy (Kruskal–Wallis * p = 0.03). No: patients who did not develop oral mucositis; Yes: patients who developed oral mucositis. Data normalized to a logarithmic scale of 10. The colors represent the different time points: Day 0 (blue), Day 14 (purple), Day 21 (pink).

Figure 6

Correlation networks (day 14 of CTX) between clinical variables (green), laboratory variables (blue), salivary cytokines (pink), and bacteria (purple). The Pearson correlation coefficient shows whether there is a positive correlation (red line) or a negative correlation (blue line) between variables. A p-value less than or equal to 0.05 was considered significant, and r = 0.4 was the cutoff. (A) in patients who developed mucositis and (B) in patients who did not develop oral mucositis.