title: The microbiome profile of oral squamous cell carcinoma tissues in a group of Sri Lankan male patients
Creators: name: Perera, Manosha
Publisher: name: Griffith University
Year: 2017
Subjects: subject: Microbiome
subject: Oral squamous
subject: Cell carcinoma tissues
subject: Sri Lankan male patients

description: Background: Findings of previous metagenomic studies on the association between the nature of the oral microbiome and oral squamous cell carcinoma (OSCC) remain indecisive due to inherent methodological limitations and variations. Furthermore, except one recently published study, investigations on the complete oral microbiome (mycobiome, bacteriome, and some putative oncogenic viruses, in the present study HPV, EBV and HHV8) in oral cancer, assessed in a single study have not been reported. Objective: To ascertain the microbiome profile of oral squamous cell carcinoma (OSCC) tissues in a group of Sri Lankan male patients. Methods: An unmatched case-control study was performed, comprised of 134 cases of clinically diagnosed OSCC, subsequently subjected to histopathological confirmation and 134 clinically diagnosed controls with benign oral mucosal lesions namely; Fibro-epithelial polyps FEPs, lipomas, keratosis without dysplasia and mucoceles. From this main sample, a sub-sample consisting of 29 Oral Squamous Cell Carcinoma (OSCC) cases, similar in clinical presentation and in the associated risk factors to the overwhelming majority of OSCC cases in Sri Lanka, and 25 FEP controls were selected. All subjects were &gt; 40 years old, and were not on antibiotics in the 2 months prior to of data collection. The affected sites were buccal mucosa or tongue. Incisional biopsies of cases and excisional biopsies of controls were collected, transported, stored and dispatched as frozen tissues at -800C. DNA was extracted from frozen specimens using Gentra Puregene Tissue kit (Qiagen, Germany), solid tissue protocol according to the manufacturer’s instructions. Few modifications were made to this protocol to ensure complete lysis of Gram positive cell walls of bacteria to obtain maximum yield of microbial DNA. The DNA extracts were stored at -800C. Subsequently, extracted DNA samples were subjected to sequencing by Illumina’s 2x300 bp chemistry at ACE (Australian Centre for Ecogenomics). High quality non-chimeric merged reads were classified to the species level using a prioritized BLASTN-algorithm for bacteriome and BLASTN-algorithm with UNITE’s named species sequences as reference for mycobiome. Downstream compositional analysis was performed using QIIME. Differentially abundant taxa were identified by linear discriminant analysis Effect Size (LEfSe) and G-test for bacteriome and mycobiome. PICRUSt (pylogenetic investigation of communities by reconstruction of unobserved states) was utilized for prediction of functional genes of bacteriome. Differences in compositional bacterial profile, functional bacteriome profile and compositional fungal profile in OSCC tissues and FEP tissues were compared by Mann-Whitney test of statistical significance. Statistical significance was set at p &lt;0.05. The analysis was conducted based on KEGG orthologs (KO) and pathways. Variations in genes and pathways between the cases and controls were investigated using LEfSe. Nested PCR was performed to detect HPV and real time PCR was performed to detect EBV and HHV 8 in these OSCC cases and FEP controls. A pre-tested interviewer administered questionnaire was used for data collection which predominantly comprised of socio-demographic information, tooth cleaning habits, information on established risk factors: areca nut/betel chewing, tobacco smoking and chewing, and alcohol consumption, daily fruit and vegetable consumption. Clinical data were extracted from clinical records and biopsy reports. The clinical oral examinations of these cases and controls were conducted by a Specialist in Dental Public Health comprising recording decayed teeth, missing teeth, filled teeth and mobile teeth. Oral hygiene status was assessed with the simplified oral hygiene index (OHI-S) of Green &amp; Vermillion 1964 and periodontal disease status was assessed by probing pocket depth (PD) and clinical attachment loss (CAL) at 4 sites per anterior teeth and 6 sites per posterior teeth.Periodontal disease status was classified according to Case Definitions for Periodontitis developed by Centre for Disease Control (CDC) Periodontal Disease Surveillance Work Group (Page &amp; Eke, 2007). These data were entered and analysed using SPSS-21 Statistical Package. Percentage distributions were presented as descriptive statistics and Chi-Square Test of Statistical Significance was used as inferential statistics to compare groups with regard to differences in socio-demographic attributes, risk habit profiles, tooth cleaning practices, oral hygiene status and periodontal disease status. Furthermore, t-test and Fisher’s exact test to compare groups (cell counts &lt;5) were used to compare means with regard to duration of risk habits, missing teeth, mobile teeth, decayed and filled teeth among cases and controls. Results: 1074 bacterial species representing 274 genera and 21 phyla were recognized with Streptococcus and Rothia comprising of the approximately 28% and 18% of average bacteriome respectively.The species richness and diversity were significantly decreased in OSCC. Capnocytophaga, Pseudomonas and Atopobium were the marker genera associated with OSCC cases with significantly different relative abundance compared to FEP controls. Conversely, Streptococcus and Rothia were significantly abundant keystone genera in controls.Citrobacter koseri, Fusobacterium nucleatum subsp.polymorphum, Streptococcus dysgalactiae and Pseudomonas aeruginosa were dominated in OSCC bacteriome profile. In contrast, Rothia mucilaginosa, Streptococcus mitis, Gemella haemolysans andStreptococcus sp. oral taxon 070 were the predominant members of the FEP bacteriome. It was possible to predict important metabolic pathways of bacterial communities by PICRUSt bioinformatics resource extant computer algorithms. These metabolic pathways included biosynthesis of lipopolysaccharides (LPS), energy metabolism, replication recombination, carbon fixation and nitrotoluene degradation which were significantly higher in OSCC tissues compared to FEP tissues (p &lt;0.05). Moreover, membrane and intracellular structural molecules, chromosomes, peptides and repair proteins were predicted to be significantly higher in OSCC tissues compared to FEP tissues (p &lt; 0.05) Thus, inflammatory and bacterial carcinogenic metabolite production such as nitrotoluene by nitroreductase enzyme activity was predicted to be significantly higher (p &lt; 0.05) in the bacteriome of OSCC tissues compared with FEP tissues. In contrast, bacteria performing, glycolysis / glucogenesis, base excision repair, protein kinases, C5 branched dibasic acid metabolism and metabolism of xenobiotic molecules by cytochrome P450 were significantly higher in the bacteriome of FEP tissues compared with OSCC tissues (p &lt;0.05) according to the same prediction algorithms. 364 fungal species representing 160 genera and 2 phyla (Ascomycota and Basidiomycota) were identified, with Candida and Malassezia making up 48% and 11% of the average mycobiome, respectively. However, only 5 species and 4 genera were detected in ≥50% of the samples. The species richness and diversity were significantly lower in OSCC. At the genus level, Candida, Hannaella and Gibberella were overrepresented in OSCC while Alternaria and Trametes were more abundant in FEP. Species-wise, C. albicans, C. etchellsii and Hannaella luteola-like species were enriched in OSCC while Malassezia restricta, Aspergillus tamarii, Alternaria alternate, Cladosporium halotolerans, and Hanseniaspora uvarum-like species were the most significantly abundant in FEP. HPV was detected in 4/54 (7.4 %) of overall samples and only 1/29 (3.5%) of OSCC cases. HPV was present in 3/25 (12.0%) of FEP controls. HPV 12, HPV 42, HPV 32, HPV 31 type and HPV α9 were detected in 4 subjects. One FEP control subject was infected with multiple HPV types. Of the types identified from this FEP tissue, HPV α9 and HPV 31, considered as high risk family and type respectively. EBV was detected in 21/27( 77.8%) of OSCC cases and in 13/26 (50%) of FEP controls. Thus, prevalence of EBV was significantly higher (p &lt; 0.05) in OSCC cases when compared with FEP controls. HHV 8 was not detected in any sample. According to socio-demographic profile 51.7% OSCC cases were employed as farmers and another 41.4% as skilled/unskilled labourers in significantly higher proportions than FEP controls (p &lt;0.05). Moreover, 20.7% and 69.0% OSCC cases had poor and fair oral hygiene status respectively compared to 12.0% and 36.0% FEP controls. Furthermore, the overwhelming majority (96.0%) of controls used tooth paste and tooth brush for tooth cleaning while the majority of OSCC cases reported less optimal methods of tooth cleaning such as use of finger and charcoal/tooth powder and use of charcoal with the tooth brush. Furthermore, 34.5% and 51.7% of OSCC cases had severe and moderate periodontal status while 8.0% and 24.0% FEP controls belonged to those two categories respectively. All aforementioned differences among OSCC cases and FEP controls were statistically significant (p &lt;0.05). In addition, significantly higher frequency of practicing betel chewing, alcohol consumption and significantly lower daily consumption of fruits and vegetables (&lt; 5 portions) were reported by OSCC cases compared to FEP controls (p &lt;0.05). Conclusions: A dysbiotic, inflammatory and carcinogenic metabolite-producing bacteriome dominated by Citrobacter koseri, Fusobacterium nucleatum subsp.polymorphumPseudomonas aeruginosa as well as a dysbiotic mycobiome dominated by Candida albicans, was found at the advancing front of OSCC compared with FEP tissues. A plethora of factors related to tumour microenvironment such as nutrient availability, pH of the environment, competition among species for binding sites, inter-species antagonism or cooperation and the differences in receptors present on OSCC tissues could have resulted in this specific OSCC associated bacteriome.Moreover, life-style related risk habits such as betel chewing and periodontal disease status of the cases could have influenced oral bacterial colonization in OSCC tissues. Against this backdrop, it is reasonable to speculate that the tumour micro- environment influences the community structure and function of the microbiome associated with OSCC as evident from the present study. However, present findings do not exclude the possibility of bacteriome contributing to carcinogenesis. Overall, fungi of the genera Candida, Hannaella and Gibberella were significantly more abundant amongst OSCC samples compared with FEP controls (p&lt;0.05). Among viruses, a higher prevalence of EBV was found in OSCC cases compared with controls and this difference was statistically significant (p&lt;0.05). As poor oral hygiene status, sub optimal tooth cleaning habits, burden of periodontal disease, frequency of betel chewing and alcohol consumption were significantly higher (p&lt;0.05) among OSCC cases compared with FEP controls is reasonable to argue such differences could have had influenced aforementioned findings in two distinct microbiome profiles of OSCC cases and FEP controls. Thus, functional metagenomic studies with cohort study design (including adequate sample size, controlling for confounding effects of established risk factors) is warranted to determine possible signature bacteria and fungi which correlate with OSCC as well as to explore their exact role in oral carcinogenesis.