Abstract 1533: 16S rRNA saliva analysis unveils differences in the head and neck squamous cell carcinoma microbiome before and after surgical resection
Authors: Guerrero-Preston Rafael E. , Godoy-Vitorino Filipa , González Herminio , Michailidi Christina , Jedlicka Anne , Dziedzic Amanda , Thangavel Rajagowthamee , Hadar Tal , Noordhuis Maartje G. , Westra William , Koch Wayne , Sidransky David 
Published: August 2015

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 compared the saliva microbiome in DNA isolated from 38 patients and 25 normal oral cavity epithelium controls to characterize the HNSCC microbiota before and after surgical resection.
PCR amplification of the 16S rRNA V3-V5 gene region was performed using the 357F/926R primer set prior to multiplexing on the Roche/454 GS Junior sequencing platform. Data were screened for chimeric sequences and contaminant chloroplast DNA after pre-processing. Passing sequences were characterized for diversity and taxonomic composition using QIIME and R before cross-tabulation analyses were performed.
After preprocessing 142,887 reads were obtained with an average length of 491 bp. The number of sequences per sample was rarefied at 3,487 to guarantee equal depth. Bacteroidetes, Firmicutes, and Proteobacteria dominated the microbiome in our sample set with less frequent presence of Actinobacteria and Fusobacteria members. At lower taxonomic levels, the most abundant genera observed were Streptococcus, Prevotella, Haemophilus and Veillonella with lower numbers of Citrobacter and Neisseraceae genus Kingella.
We found that 46 OTUs changed significantly in HNSCC patients (p&lt;0.05) when compared to the controls mainly due to the loss of Neisseria and Aggregatibacter (Proteobacteria), Leptotrichia (Fusobacteria) and Veilonella (Firmicutes) with an increase in some Lactobacillus (Firmicutes). Within bacteroidetes, Prevotella OTUs were found more abundant in control samples. HNSCC patients had a significant loss in richness and diversity (p&lt;0.05) compared to the controls. HPV positive samples were more diverse (higher Shannon values and richness) than HPV negative samples.
Longitudinal analyses (3 time periods) 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. We also observed statistically significant differences (p&lt;0.05) at the phyla (Actinobacteria and Fusobacteria), and genus (Veillonella and Prevotella) levels. Interestingly, in one patient whose HPV status shifted from HPV positive to HPV negative after surgery, the abundance of Lactobacillus OTUs decreased, and Streptococcus (OTU 1009) increased significantly, being also associated with an HPV negative status in another patient.
We are the first to observe that OTUs and several microbial communities at different taxonomic levels discriminate HNSCC from control samples; HPV positive and HPV negative samples; and pre- vs postsurgical treatment samples. Future work will determine the correlation of microbial communities in paired tissue and saliva HNSCC samples, as well as their link to treatment response and survival.
Citation Format: Rafael E. Guerrero-Preston, Filipa Godoy-Vitorino, Herminio González, Christina Michailidi, Anne Jedlicka, Amanda Dziedzic, Rajagowthamee Thangavel, Tal Hadar, Maartje G. Noordhuis, William Westra, Wayne Koch, David Sidransky. 16S rRNA saliva analysis unveils differences in the head and neck squamous cell carcinoma microbiome before and after surgical resection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1533. doi:10.1158/1538-7445.AM2015-1533