16S rRNA amplicon sequencing identifies microbiota associated with oral cancer, human papilloma virus infection and surgical treatment

Abstract

Systemic inflammatory events and localized disease, mediated by the microbiome, may be measured in saliva as head and neck squamous cell carcinoma (HNSCC) diagnostic and prognostic biomonitors. We used a 16S rRNA V3-V5 marker gene approach to compare the saliva microbiome in DNA isolated from Oropharyngeal (OPSCC), Oral Cavity Squamous Cell Carcinoma (OCSCC) patients and normal epithelium controls, to characterize the HNSCC saliva microbiota and examine their abundance before and after surgical resection.The analyses identified a predominance of Firmicutes, Proteobacteria and Bacteroidetes, with less frequent presence of Actinobacteria and Fusobacteria before surgery. At lower taxonomic levels, the most abundant genera were Streptococcus, Prevotella, Haemophilus, Lactobacillus and Veillonella, with lower numbers of Citrobacter and Neisseraceae genus Kingella. HNSCC patients had a significant loss in richness and diversity of microbiota species (p<0.05) compared to the controls. Overall, the Operational Taxonomic Units network shows that the relative abundance of OTU's within genus Streptococcus, Dialister, and Veillonella can be used to discriminate tumor from control samples (p<0.05). Tumor samples lost Neisseria, Aggregatibacter (Proteobacteria), Haemophillus (Firmicutes) and Leptotrichia (Fusobacteria). Paired taxa within family Enterobacteriaceae, together with genus Oribacterium, distinguish OCSCC samples from OPSCC and normal samples (p<0.05). Similarly, only HPV positive samples have an abundance of genus Gemellaceae and Leuconostoc (p<0.05). Longitudinal analyses of samples taken before and after surgery, revealed a reduction in the alpha diversity measure after surgery, together with an increase of this measure in patients that recurred (p<0.05). These results suggest that microbiota may be used as HNSCC diagnostic and prognostic biomonitors.

Keywords: 16s rRNA; human papilloma virus (HPV); microbiome; oral cancer; oropharyngeal cancer.

Figure 1. Beta diversity comparisons by Principal Component Analysis (PCA) using Non-metric multidimensional scaling (NMDS), with Euclidean distances, discriminated HNSCC (n=17) from Control samples (n=25)

A. PCA reveals that the microbial communities in HNSCC patients are significantly different to those seen in Control samples. B. NMDS shows that the microbial communities in HPV negative (HPV-) oropharyngeal samples are significantly different from the ones seen in HPV-oral cavity patients.

Figure 2. Taxonomic profiles at the phyla and genus levels, of 59 saliva samples according to tumor histology, HPV status and sampling site

There were a total of 13 microbial communities identified at the phyla-level in our patient population A. The top 5 (>1% relative abundance) communities were, Firmicutes, Bacteroidetes, Proteobacteria, Actinoabcteria and Fusobacteria B. At the genus-level, Streptococcus spp. prevail across all samples, followed by Veillonella, Prevotella, Lactobacillus and Haemophilus spp C-D.

Figure 3. Nodes in Operational Taxonomic Units (OTUs) network significantly discriminate between head and neck squamous cell carcinoma (HNSCC) and normal samples in saliva

The figure was created using QIIME and imported to Cytoscape. Significant OTUs p< 0.05 were plotted in the OTU Network. Pie charts were created showcasing taxa distinguishing samples by Tumor Histology and HPV status. A. represents taxa that differed significantly in relative abundance (p<0.05) when comparing saliva from normal patients with patients with HNSCC, as well as HPV negative and HPV positive patients. The OTU network shows that the total abundance of genus Streptococcus, Dialister, and Veillonella can be used to discriminate tumor samples from control samples B. Paired taxa within family Enterobacteriaceae together with genus Oribacterium, clearly distinguish OCSCC samples C. Paired taxa within family Enterobacteriaceae together with genus Oribacterium, clearly distinguish OCSCC samples (from OPSCC and normal samples D. and E.. Similarly, only HPV positive samples have an abundance of genus Gemellaceae and Leuconostoc.

Figure 4. Rarefaction curves of species richness and diversity between Controls and HNSCC samples based on chao1 richness estimator and Faith's diversity measure (PD)

Next-generation sequencing (NGS) has revealed a large microbial diversity that was previously concealed with culture-dependent methods. Although the true microbial diversity is limited to the number of samples, species richness and sample diversity can be estimated using diversity indices and species richness estimators. The chao1 index estimates total species richness based on all species actually discovered, including species not present in any sample. This approach uses the numbers of singletons (single appearance) and doubletons (that appeared twice) to estimate the number of missing species due to undetected species information is mostly concentrated on low frequency counts. Faith's phylogenetic diversity (PD) measure estimates the relative feature diversity of any nominated set of species by the sum of the lengths of all phylogenetic branches required to span a given set of taxa on the phylogenetic tree. A. shows the chao1 index estimates when comparing tumor versus control. B. shows the PD estimates when comparing tumor versus control. C. shows the chao1 index estimates when comparing tumor site and HPV status. D. shows the PD estimates when comparing tumor site and HPV status. Microbial communities of samples obtained from Control patients display significant higher richness (p=0.001, ANOVA) and significant higher diversity (p=0.001, ANOVA) than HNSCC samples. When considering HPV status and both sampling sites we found that the diversity from the oral cavity (HPV negative) HNSCC samples was higher than in both HPV positive (p=0.003, ANOVA) and negative (p=0.006, ANOVA) HNSCC samples from the oropharynx.

Figure 5. Differentially enriched microbiota OTUs in HNSCC when compared to control samples

A. Histograms of the 42 statistically significant differences between OTUs abundance show a significant abundance of Streptococcus spp. in HNSCC samples according to G-tests results (p<0.05) By default, OTUs unclassified at the genus level are plotted as N/A by Phyloseq. B. Taxonomic Heatmap using Spearman's distance, combined with Ward clustering for 30 most statistically significant OTUs between Control and HNSCC samples (p<0.05, ANOVA). Heatmap color pallete used was “RdBu” with red and blue representing respectively low and high abundance. Ward's clustering method involves an agglomerative clustering algorithm that treats a cluster analysis as an analysis of variance, used to analyze the differences among group means and their associated classes. Streptococcus and Lactobacillus spp. are significantly associated with HNSCC samples according to G-tests and ANOVA results.

Figure 6. LDA Effect Size (LEfSe) algorithm was used on genus level OTU tables to determine taxa that best characterize each biological class: A. Comparison of HNSCC with normal samples found that Streptococcus and Lactobacillus spp

are significantly associated with HNSCC samples. Haemophilus and Neisseria spp. are related to Control samples. B. Comparison of HPV positive and HPV negative HNSCC found that Lactobacillus spp. is significantly associated with HPV positive samples in the oropharynx. Similarly, Eikenella and Neisseria spp. are associated with HPV negative samples. Patients treated with surgery had significant enrichment for Haemopilus, Neisseria, Aggregatibacter and Leptotrichia, while patients treated with CRT/Surgery had significant enrichment for Lactobacillus and Lactobacillaceae C.

Figure 7. Time-series analyses of HNSCC patients (n=11) for whom we had repeated saliva samples, according to both sampling sites, HPV status and TNM staging

Bacterial communities were noticeably different between T1-2N2AM0 and T3N2BM0 TNM stage. Lactobacillus spp. were significantly more abundant in patients with T3-2N2BM0 staging (p<0.01, G-test). Legend indicates genus that show a relative abundance higher than 1%.