Salivary microbiome profiles of oral cancer patients analyzed before and after treatment

Abstract

Background: Treating oral squamous cell carcinoma (OSCC) introduces new ecological environments in the oral cavity. This is expected to cause changes in the oral microbiome. The purpose of this study was to gain new information on the salivary microbiome of OSCC patients in order to improve the aftercare of OSCC patients. The aims of this study were to investigate possible changes in the salivary microbiome profiles of OSCC patients before and after cancer treatment and to compare these changes with the profiles of healthy controls.

Patients and methods: Paraffin-stimulated whole saliva samples were collected, and the salivary flow rate was measured from 99 OSCC patients prior to surgical resection of the tumor and other adjuvant therapy. After treatment, 28 OSCC patients were re-examined with a mean follow-up time of 48 months. In addition, 101 healthy controls were examined and sampled. After DNA extraction and purification, the V4 hypervariable region of the 16S rRNA gene was amplified and sequenced using Illumina MiSeq. The merged read pairs were denoised using UNOISE3, mapped to zero-radius operational taxonomic units (zOTUs), and the representative zOTU sequences were assigned a taxonomy using HOMD. Descriptive statistics were used to study the differences in the microbial profiles of OSCC patients before and after treatment and in comparison to healthy controls.

Results: At baseline, the OSCC patients showed a higher relative abundance of zOTUs classified as Streptococcus anginosus, Abiotrophia defectiva, and Fusobacterium nucleatum. The microbial profiles differed significantly between OSCC patients and healthy controls (F = 5.9, p < 0.001). Alpha diversity of the salivary microbiome of OSCC patients was decreased at the follow-up, and the microbial profiles differed significantly from the pre-treatment (p < 0.001) and from that of healthy controls (p < 0.001).

Conclusions: OSCC patients' salivary microbiome profile had a higher abundance of potentially pathogenic bacteria compared to healthy controls. Treatment of the OSCC caused a significant decrease in alpha diversity and increase in variability of the salivary microbiome, which was still evident after several years of follow-up. OSCC patients may benefit from preventive measures, such as the use of pre- or probiotics, salivary substitutes, or dietary counseling. Video Abstract.

Keywords: Cancer treatment; Oral cancer; Oral microbiome.

Fig. 1

Comparison of OSCC patients and healthy controls. A Principal component analysis of all OSCC patients versus healthy controls, p = 0.0001, F = 5.9—PERMANOVA. B Shannon Diversity Index of the OSCC samples versus healthy controls, p = non-significant, independent samples t test. C Shannon Diversity Index in OSCC patients versus healthy controls by alcohol consumption, independent samples t test. D Shannon Diversity Index of OSCC patients versus healthy controls by salivary flow rate (SFR): low—< 0.5 ml/min, normal—≥ 0.5 ml/min, independent samples t test

Fig. 2

Discriminatory zOTUs between OSCC patients and healthy controls on biased A and unbiased B data. *zOTU1 Streptococcusdentisani/infantis/mitis/oralis/sp. oral taxon 058/sp. oral taxon 061/sp. oral taxon 064/sp. oral taxon 070/sp. oral taxon 423/sp. oral taxon 431/tigurinus (assigned taxonomy shortened in the figure). ^¤zOTU4 Streptococcusaustralis/parasanguinis I/parasanguinis II/sp. oral taxon 057/sp. oral taxon 066 (assigned taxonomy shortened in the figure)

Fig. 3

Comparison of the salivary microbiome from 28 OSCC patients before and after treatment. A Principal component analysis, p < 0.001, F = 2.3—restricted PERMANOVA. B Bray–Curtis distance, p < 0.001—Mann–Whitney test. C Shannon diversity index, p = 0.008—paired samples t test

Fig. 4

Discriminatory zOTUs between baseline and post-therapy samples in 28 OSCC patients (output of LEfSe analysis). *zOTU1 Streptococcus dentisani/infantis/mitis/oralis/sp. oral taxon 058/sp. oral taxon 061/sp. oral taxon 064/sp. oral taxon 070/sp. oral taxon 423/sp. oral taxon 431/tigurinus (assigned taxonomy shortened in the figure)