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.

Microbiome studies show altered microbiota in head and neck squamous cell carcinoma (HNSCC), both in terms of taxonomic composition and metabolic capacity. These studies utilized a traditional bioinformatics methodology, which allows for accurate taxonomic assignment down to the genus level, but cannot accurately resolve species level membership. We applied Resphera Insight, a high-resolution methodology for 16S rRNA taxonomic assignment that is able to provide species-level context in its assignments of 16S rRNA next generation sequencing (NGS) data.Resphera Insight applied to saliva samples from HNSCC patients and healthy controls led to the discovery that a subset of HNSCC saliva samples is significantly enriched with commensal species from the vaginal flora, including Lactobacillus gasseri/johnsonii (710x higher in saliva) and Lactobacillus vaginalis (52x higher in saliva). These species were not observed in normal saliva from Johns Hopkins patients, nor in 16S rRNA NGS saliva samples from the Human Microbiome Project (HMP). Interestingly, both species were only observed in saliva from Human Papilloma Virus (HPV) positive and HPV negative oropharyngeal cancer patients. We confirmed the representation of both species in HMP data obtained from mid-vagina (n=128) and vaginal introitus (n=121) samples.Resphera Insight also led to the discovery that Fusobacterium nucleatum, an oral cavity flora commensal bacterium linked to colon cancer, is enriched (600x higher) in saliva from a subset of HNSCC patients with advanced tumors stages.Together, these high-resolution analyses on 583 samples suggest a possible role for bacterial species in the therapeutic outcome of HPV positive and HPV negative HNSCC patients.

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Previous microbiome studies at the genus level have described altered microbiota in head and neck squamous cell carcinoma (HNSCC), both in terms of taxonomic composition and metabolic capacity. We applied high-resolution microbiome profiling (Resphera Insight) to analyze 16S rRNA sequencing data in saliva and tissue samples from HNSCC patients and healthy controls. DNA extraction and amplicon library preparation was performed for saliva samples from HNSCC (n=38) and controls (n=25), as well as tissue samples from HNSCC (n=25) and controls (n=8). Raw sequences were processed for quality and length, screened for chimeras and filtered for contaminant human and chloroplast DNA. High-quality passing sequences were submitted to Resphera Insight for species-level taxonomic assignments, followed-by differential abundance analysis with the DESeq package. Samples from a subset of HNSCC patients were significantly enriched with commensal species from the vaginal flora, including Lactobacillus gasseri/johnsonii (710x higher in saliva and 1990x higher in tissue) and Lactobacillus vaginalis (52x higher in saliva). These species were not observed in normal saliva or tissue samples from Hopkins patients (n=33) nor in normal saliva samples (n=292) from the Human Microbiome Project (HMP). Interestingly, both species were only observed in saliva from Human Papilloma Virus positive (HPV+) and HPV negative (HPV-) oropharyngeal cancer patients, and we confirmed their representation in vaginal samples from the HMP (n=249). We also found that Fusobacterium nucleatum (F.nucleatum), an oral cavity flora commensal bacterium linked to colon cancer, is enriched (600x higher in saliva and 51x higher in tissue) in a subset of HNSCC patients with advanced tumors (T3 or above). F. nucleatum was detected in samples obtained before and after treatment with chemo-radiation, but not with surgery alone. Interestingly, we identified upregulation of the oncogenic Wnt/Beta catenin pathway (Wnt7B, FZD6, SFRP4) and down regulation of immune system pathways (TLR10, IRF8) with genome-wide mRNA arrays (Affymetrix) in HNSCC samples enriched for F.nucleatum. Using fluorogenic quantitative PCR we confirmed that F.nucleatum and Fusobacterium spp. are enriched in saliva samples collected prior to treatment in another cohort of HNSCC patients, and in post-treatment saliva samples obtained from HNSCC patients treated with PD-1 checkpoint blockade. We also found enrichment of HPV+ oropharyngeal tumors with F.nucleatum and Fusobacterium spp., prior to therapy, while some recurrent HPV- oropharyngeal tumors are enriched only with Fusobacterium spp. Together, these results suggest that bacteria may impact therapy in HPV+ and HPV- oropharyngeal and oral cavity cancer by altering oncogenic and immune system pathways. Citation Format: Rafael E. Guerrero-Preston, James Robert White, Filipa Godoy-Vitorino, Herminio Gonzalez, Arnold Rodríguez-Hilario, Kelvin Navarro, Gustavo A. Miranda-Carboni, Christina Michailidi, Anne Jedlicka, Stephanie Hao, Sierra Canapp, Jessica Bondy, Amanda Dziedzic, Barbara Mora Lagos, Gustavo Rivera-Alvarez, Winston Timp, William Westra, Wayne Koch, Hyunseok Kang, Luigi Marchionni, Young Kim, David Sidransky. High-resolution microbiome profiling and genome wide arrays uncover bacteria driven alterations of oncogenic and immune pathways in head and neck cancer patients treated with surgery, chemo-radiation and PD-1 checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1018. doi:10.1158/1538-7445.AM2017-1018

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