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

// Rafael Guerrero-Preston 1, 2 , Filipa Godoy-Vitorino 3 , Anne Jedlicka 4 , Arnold Rodriguez-Hilario 3 , Herminio Gonzalez 3 , Jessica Bondy 1 , Fahcina Lawson 1 , Oluwasina Folawiyo 1 , Christina Michailidi 1 , Amanda Dziedzic 4 , Rajagowthamee Thangavel 5 , Tal Hadar 1 , Maartje G. Noordhuis 1, 6 , William Westra 7 , Wayne Koch 1 , David Sidransky 1 1 Department of Otolaryngology and Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 2 Department of Obstetrics and Gynecology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico 3 Natural Sciences Department, Microbial Ecology and Genomics Laboratory, Inter American University of Puerto Rico, Metropolitan Campus, San Juan, Puerto Rico 4 Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Public Health, Baltimore, Maryland, USA 5 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA 6 Department of Otorhinolaryngology-Head and Neck Surgery, University of Groningen, University Medical Center, Groningen, The Netherlands 7 Department of Pathology-Surgical Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Correspondence to: Rafael Guerrero-Preston, email: rguerre3@jhmi.edu David Sidransky, email: dsidrans@jhmi.edu Keywords: microbiome, 16s rRNA, oral cancer, oropharyngeal cancer, human papilloma virus (HPV) Received: February 23, 2016      Accepted: May 16, 2016      Published: May 30, 2016 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.