Fusobacterium nucleatum subsp. polymorphum recovered from malignant and potentially malignant oral disease exhibit heterogeneity in adhesion phenotypes and adhesin gene copy number, shaped by inter-subspecies horizontal gene transfer and recombination-derived mosaicism.

Fusobacterium nucleatum is an anaerobic commensal of the oral cavity associated with periodontitis and extra-oral diseases, including colorectal cancer. Previous studies have shown an increased relative abundance of this bacterium associated with oral dysplasia or within oral tumours. Using direct culture, we found that 75 % of Fusobacterium species isolated from malignant or potentially malignant oral mucosa were F. nucleatum subsp. polymorphum. Whole genome sequencing and pangenome analysis with Panaroo was carried out on 76 F. nucleatum subsp. polymorphum genomes. F. nucleatum subsp. polymorphum was shown to possesses a relatively small core genome of 1604 genes in a pangenome of 7363 genes. Phylogenetic analysis based on the core genome shows the isolates can be separated into three main clades with no obvious genotypic associations with disease. Isolates recovered from healthy and diseased sites in the same patient are generally highly related. A large repertoire of adhesins belonging to the type V secretion system (TVSS) could be identified with major variation in repertoire and copy number between strains. Analysis of intergenic recombination using fastGEAR showed that adhesin complement is shaped by horizontal gene transfer and recombination. Recombination events at TVSS adhesin genes were not only common between lineages of subspecies polymorphum, but also between different subspecies of F. nucleatum. Strains of subspecies polymorphum with low copy numbers of TVSS adhesin encoding genes tended to have the weakest adhesion to oral keratinocytes. This study highlights the genetic heterogeneity of F. nucleatum subsp. polymorphum and provides a new framework for defining virulence in this organism.

Biological therapies and management of oral mucosal disease.

Biologic drugs are drugs made by living organisms and the term is usually limited to monoclonal antibodies or receptors targeting specific cytokines or cells that have been developed in recent decades. These drugs have had an enormous impact on the management of cancers, including head and neck cancers, and immune-mediated inflammatory conditions, for example, rheumatoid arthritis and inflammatory bowel disease. General dental practitioners will routinely be managing patients who are on these medications for a wide range of systemic conditions. These drugs also have a limited role in the management of immune-mediated oral mucosal disease. In this article, we will introduce the range of biological agents and their systemic indications and then elaborate on their use in oral mucosal disease and the disadvantages associated with their use.

Diagnostic accuracy of HPV16 early antigen serology for HPV-driven oropharyngeal cancer is independent of age and sex.

A growing proportion of head and neck cancer (HNC), especially oropharyngeal cancer (OPC), is caused by human papillomavirus (HPV). There are several markers for HPV-driven HNC, one being HPV early antigen serology. We aimed to investigate the diagnostic accuracy of HPV serology and its performance across patient characteristics. Data from the VOYAGER consortium was used, which comprises five studies on HNC from North America and Europe. Diagnostic accuracy, that is, sensitivity, specificity, Cohen's kappa and correctly classified proportions of HPV16 E6 serology, was assessed for OPC and other HNC using p16INK4a immunohistochemistry (p16), HPV in situ hybridization (ISH) and HPV PCR as reference methods. Stratified analyses were performed for variables including age, sex, smoking and alcohol use, to test the robustness of diagnostic accuracy. A risk-factor analysis based on serology was conducted, comparing HPV-driven to non-HPV-driven OPC. Overall, HPV serology had a sensitivity of 86.8% (95% CI 85.1-88.3) and specificity of 91.2% (95% CI 88.6-93.4) for HPV-driven OPC using p16 as a reference method. In stratified analyses, diagnostic accuracy remained consistent across sex and different age groups. Sensitivity was lower for heavy smokers (77.7%), OPC without lymph node involvement (74.4%) and the ARCAGE study (66.7%), while specificity decreased for cases with <10 pack-years (72.1%). The risk-factor model included study, year of diagnosis, age, sex, BMI, alcohol use, pack-years, TNM-T and TNM-N stage. HPV serology is a robust biomarker for HPV-driven OPC, and its diagnostic accuracy is independent of age and sex. Future research is suggested on the influence of smoking on HPV antibody levels.

Smoking, tooth loss and oral hygiene practices have significant and site-specific impacts on the microbiome of oral mucosal surfaces: a cross-sectional study.

We investigated bacterial colonisation patterns of healthy mucosa (buccal, tongue, palate and floor of mouth) in a cohort of adults in order to determine how smoking, tooth loss, plaque levels and oral hygiene practices impacted on mucosal colonisation. A total of 322 swabs were recovered from 256 participants, of whom 46% were current smokers. We analysed colonization by sequencing the V1-V3 regions of the 16S rRNA gene. Palate and tongue microbiomes generally exhibited greater biodiversity than buccal and floor of mouth. Although Neisseria, Lautropia and Haemophilus spp. showed reduced abundance in smokers, buccal mucosa specifically showed a significant increase in Prevotella spp., whereas tongue and floor of mouth tended towards increased abundance of Streptococcus spp. Unexpectedly, tooth brushing frequency had a greater impact on mucosal community structure than plaque levels. Tooth loss was associated with significant reductions in mucosal biodiversity and had site-specific impacts, with buccal communities showing increased abundance of periodontitis-associated species and Rothia mucilaginosa, whereas tongue communities exhibited increased abundance of several streptococcal OTUs and reduced abundance of Haemophilus spp. This study highlights the complex relationship between mucosal colonisation and host factors, highlighting the need for careful consideration of these factors in mucosal microbiome studies.

Influence of site and smoking on malignant transformation in the oral cavity: Is the microbiome the missing link?

The tongue and floor of the mouth are high-risk sites for oral squamous cell carcinoma (OSCC), while smoking is its most significant risk factor. Recently, questions have been raised as to the role of the oral microbiome in OSCC because of a wealth of evidence demonstrating that the microbiome of OSCC differs from that of healthy mucosa. However, oral site and smoking also have a significant impact on oral microbial communities, and to date, the role these factors play in influencing the dysbiotic microbial communities of OSCC and precursor lesions has not been considered. This review aims to examine the influence of site and smoking on the oral microbiome and, in turn, whether these microbiome changes could be involved in oral carcinogenesis.

A risk prediction model for head and neck cancers incorporating lifestyle factors, HPV serology and genetic markers.

Head and neck cancer is often diagnosed late and prognosis for most head and neck cancer patients remains poor. To aid early detection, we developed a risk prediction model based on demographic and lifestyle risk factors, human papillomavirus (HPV) serological markers and genetic markers. A total of 10 126 head and neck cancer cases and 5254 controls from five North American and European studies were included. HPV serostatus was determined by antibodies for HPV16 early oncoproteins (E6, E7) and regulatory early proteins (E1, E2, E4). The data were split into a training set (70%) for model development and a hold-out testing set (30%) for model performance evaluation, including discriminative ability and calibration. The risk models including demographic, lifestyle risk factors and polygenic risk score showed a reasonable predictive accuracy for head and neck cancer overall. A risk model that also included HPV serology showed substantially improved predictive accuracy for oropharyngeal cancer (AUC = 0.94, 95% CI = 0.92-0.95 in men and AUC = 0.92, 95% CI = 0.88-0.95 in women). The 5-year absolute risk estimates showed distinct trajectories by risk factor profiles. Based on the UK Biobank cohort, the risks of developing oropharyngeal cancer among 60 years old and HPV16 seropositive in the next 5 years ranged from 5.8% to 14.9% with an average of 8.1% for men, 1.3% to 4.4% with an average of 2.2% for women. Absolute risk was generally higher among individuals with heavy smoking, heavy drinking, HPV seropositivity and those with higher polygenic risk score. These risk models may be helpful for identifying people at high risk of developing head and neck cancer.

Classification of cytological samples from oral potentially malignant lesions through Raman spectroscopy: A pilot study.

The potential of Raman microspectroscopy of exfoliated cells has been demonstrated for oral cancer diagnosis. In this study, brush biopsies were collected from the buccal mucosa/tongue of healthy donors (n = 31) and from oral mucosal dysplastic lesions (n = 31 patients). Raman spectra were acquired and subjected to partial least squares-discriminant analysis (PLS-DA). The patient samples could be differentiated from healthy donor samples with 96% sensitivity and 95% specificity. Furthermore, PLS-DA models were developed based on cytopathological and histopathological assessment. Low and high grade dysplasia could be discriminated with 64% sensitivity and 65% specificity based on cytopathological assessment, while 81% sensitivity and 86% specificity could be achieved when histopathological assessment was within six months of the brush biopsy sampling. Therefore, this explorative study has successfully demonstrated that Raman spectroscopy may have a role in monitoring patients with dysplasia and may reduce the need for multiple biopsies.

Germline determinants of humoral immune response to HPV-16 protect against oropharyngeal cancer.

Although several oropharyngeal cancer (OPC) susceptibility loci have been identified, most previous studies lacked detailed information on human papillomavirus (HPV) status. We conduct a genome-wide analysis by HPV16 serology status in 4,002 oral cancer cases (OPC and oral cavity cancer (OCC)) and 5,256 controls. We detect four susceptibility loci pointing to a distinct genetic predisposition by HPV status. Our most notable finding in the HLA region, that is now confirmed to be specific of HPV(+)OPC risk, reveal two independent loci with strong protective effects, one refining the previously reported HLA class II haplotype association. Antibody levels against HPV16 viral proteins strongly implicate the protective HLA variants as major determinants of humoral response against L1 capsid protein or E6 oncoprotein suggesting a natural immune response against HPV(+)OPC promoted by HLA variants. This indicates that therapeutic vaccines that target E6 and attenuate viral response after established HPV infections might protect against HPV(+)OPC.

Raman spectral cytopathology for cancer diagnostic applications.

Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol takes 2 h 20 min for sample preparation, measurement and data preprocessing and up to 8 h for a complete analysis. A key feature of the protocol is that it uses the same sample preparation procedure as commonly used in diagnostic cytology laboratories (i.e., liquid-based cytology on glass slides), ensuring compatibility with clinical workflows. Our protocol also covers methods to correct for the spectral contribution of glass and sample pretreatment methods to remove contaminants (such as blood and mucus) that can obscure spectral features in the exfoliated cells and lead to variability. The protocol establishes a standardized clinical routine allowing the collection of highly reproducible data for Raman spectral cytopathology for cancer diagnostic applications for cervical and lung cancer and for monitoring suspicious lesions for oral cancer.

Biomedical applications of vibrational spectroscopy: Oral cancer diagnostics.

Vibrational spectroscopy, based on either infrared absorption or Raman scattering, has attracted increasing attention for biomedical applications. Proof of concept explorations for diagnosis of oral potentially malignant disorders and cancer are reviewed, and recent advances critically appraised. Specific examples of applications of Raman microspectroscopy for analysis of histological, cytological and saliva samples are presented for illustrative purposes, and the future prospects, ultimately for routine, chairside in vivo screening are discussed.

Alcohol drinking and head and neck cancer risk: the joint effect of intensity and duration.

BACKGROUND: Alcohol is a well-established risk factor for head and neck cancer (HNC). This study aims to explore the effect of alcohol intensity and duration, as joint continuous exposures, on HNC risk. METHODS: Data from 26 case-control studies in the INHANCE Consortium were used, including never and current drinkers who drunk ≤10 drinks/day for ≤54 years (24234 controls, 4085 oral cavity, 3359 oropharyngeal, 983 hypopharyngeal and 3340 laryngeal cancers). The dose-response relationship between the risk and the joint exposure to drinking intensity and duration was investigated through bivariate regression spline models, adjusting for potential confounders, including tobacco smoking. RESULTS: For all subsites, cancer risk steeply increased with increasing drinks/day, with no appreciable threshold effect at lower intensities. For each intensity level, the risk of oral cavity, hypopharyngeal and laryngeal cancers did not vary according to years of drinking, suggesting no effect of duration. For oropharyngeal cancer, the risk increased with durations up to 28 years, flattening thereafter. The risk peaked at the higher levels of intensity and duration for all subsites (odds ratio = 7.95 for oral cavity, 12.86 for oropharynx, 24.96 for hypopharynx and 6.60 for larynx). CONCLUSIONS: Present results further encourage the reduction of alcohol intensity to mitigate HNC risk.

Raman microspectroscopic study for the detection of oral field cancerisation using brush biopsy samples.

Field cancerisation (FC) is potentially an underlying cause of poor treatment outcomes of oral squamous cell carcinoma (OSCC). To explore the phenomenon using Raman microspectroscopy, brush biopsies from the buccal mucosa, tongue, gingiva and alveolus of healthy donors (n = 40) and from potentially malignant lesions (PML) of Dysplasia Clinic patients (n = 40) were examined. Contralateral normal samples (n = 38) were also collected from the patients. Raman spectra were acquired from the nucleus and cytoplasm of each cell, and subjected to partial least squares-discriminant analysis (PLS-DA). High discriminatory accuracy for donor and PML samples was achieved for both cytopalmic and nuclear data sets. Notably, contralateral normal (patient) samples were also accurately discriminated from donor samples and contralateral normal samples from patients with multiple lesions showed a similar spectral profile to PML samples, strongly indicating a FC effect. These findings support the potential of Raman microspectroscopy as a screening tool for PML using oral exfoliated cells.

A pilot study for early detection of oral premalignant diseases using oral cytology and Raman micro-spectroscopy: Assessment of confounding factors.

This study demonstrates the efficacy of Raman micro-spectroscopy of oral cytological samples for differentiating dysplastic, potentially malignant lesions from those of normal, healthy donors. Cells were collected using brush biopsy from healthy donors (n = 20) and patients attending a Dysplasia Clinic (n = 20). Donors were sampled at four different sites (buccal mucosa, tongue, alveolus, gingiva), to ensure matched normal sites for all lesions, while patient samples were taken from clinically evident, histologically verified dysplastic lesions. Spectra were acquired from the nucleus and cytoplasm of individual cells of all samples and subjected to partial least squares-discriminant analysis. Discriminative sensitivities of 94% and 86% and specificity of 85% were achieved for the cytoplasm and nucleus, respectively, largely based on lipidic contributions of dysplastic cells. Alveolar/gingival samples were differentiated from tongue/buccal samples, indicating that anatomical site is potentially a confounding factor, while age, gender, smoking and alcohol consumption were confirmed not to be.

Acetaldehyde production by Rothia mucilaginosa isolates from patients with oral leukoplakia.

Rothia mucilaginosa has been found at high abundance on oral leukoplakia (OLK). The ability of clinical isolates to produce acetaldehyde (ACH) from ethanol has not been investigated. The objective of the current study was to determine the capacity of R. mucilaginosa isolates recovered from OLK to generate ACH. Analysis of R. mucilaginosa genomes (n = 70) shows that this species does not normally encode acetaldehyde dehydrogenase (ALDH) required for detoxification of ACH. The predicted OLK metagenome also exhibited reduced ALDH coding capacity. We analysed ACH production in 8 isolates of R. mucilaginosa and showed that this species is capable of generating ACH in the presence of ethanol. The levels of ACH produced (mean = 53 µM) were comparable to those produced by Neisseria mucosa and Candida albicans in parallel assays. These levels were demonstrated to induce oxidative stress in cultured oral keratinocytes. This study shows that R. mucilaginosa can generate ACH from ethanol in vitro at levels which can induce oxidative stress. This organism likely contributes to oral ACH levels following alcohol consumption and the significance of the increased abundance of R. mucilaginosa in patients with potentially malignant disorders requires further investigation.

Laryngeal Cancer Risks in Workers Exposed to Lung Carcinogens: Exposure-Effect Analyses Using a Quantitative Job Exposure Matrix.

INTRODUCTION: Various established occupational lung carcinogens are also suspected risk factors for laryngeal cancer. However, individual studies are often inadequate in size to investigate this relatively rare outcome. Other limitations include imprecise exposure assessment and inadequate adjustment for confounders. METHODS: This study applied a quantitative job exposure matrix (SYN-JEM) for four established occupational lung carcinogens to five case-control studies within the International Head and Neck Cancer Epidemiology Consortium. We used occupational histories for 2256 laryngeal cancer cases and 7857 controls recruited from 1989 to 2007. We assigned quantitative exposure levels for asbestos, respirable crystalline silica, chromium-VI, and chromium-VI and nickel combined (to address highly correlated exposures) via SYN-JEM. We assessed effects of occupational exposure on cancer risk for males (asbestos, respirable crystalline silica, chromium-VI, and chromium-VI and nickel combined) and females (asbestos and respirable crystalline silica), adjusting for age, study, tobacco smoking, alcohol consumption, and asbestos exposure where relevant. RESULTS: Among females, odds ratios (ORs) were increased for ever versus never exposed. Among males, P values for linear trend were <0.05 for estimated cumulative exposure (all agents) and <0.05 for exposure duration (respirable crystalline silica, chromium-VI, and chromium-VI and nickel combined); strongest associations were for asbestos at >90th percentile cumulative exposure (OR = 1.3, 95% confidence interval [CI] = 1.0, 1.6), respirable crystalline silica at 30+ years duration (OR = 1.4, 95% CI = 1.2, 1.7) and 75th-90th percentile cumulative exposure (OR = 1.4, 95% CI = 1.1, 1.8), chromium-VI at >75th percentile cumulative exposure (OR = 1.9, 95% CI = 1.2, 3.0), and chromium-VI and nickel combined at 20-29 years duration (OR = 1.5, 95% CI = 1.1, 2.2). CONCLUSIONS: These findings support hypotheses of causal links between four lung carcinogens (asbestos, respirable crystalline silica, chromium-VI, and nickel) and laryngeal cancer.

Prediction of survival of HPV16-negative, p16-negative oral cavity cancer patients using a 13-gene signature: A multicenter study using FFPE samples.

OBJECTIVES: To test the performance of an oral cancer prognostic 13-gene signature for the prediction of survival of patients diagnosed with HPV-negative and p16-negative oral cavity cancer. MATERIALS AND METHODS: Diagnostic formalin-fixed paraffin-embedded oral cavity cancer tumor samples were obtained from the Fred Hutchinson Cancer Research Center/University of Washington, University of Calgary, University of Michigan, University of Utah, and seven ARCAGE study centers coordinated by the International Agency of Research on Cancer. RNA from 638 Human Papillomavirus (HPV)-negative and p16-negative samples was analyzed for the 13 genes using a NanoString assay. Ridge-penalized Cox regressions were applied to samples randomly split into discovery and validation sets to build models and evaluate the performance of the 13-gene signature in predicting 2-year oral cavity cancer-specific survival overall and separately for patients with early and late stage disease. RESULTS: Among AJCC stage I/II patients, including the 13-gene signature in the model resulted in substantial improvement in the prediction of 2-year oral cavity cancer-specific survival. For models containing age and sex with and without the 13-gene signature score, the areas under the Receiver Operating Characteristic Curve (AUC) and partial AUC were 0.700 vs. 0.537 (p < 0.001), and 0.046 vs. 0.018 (p < 0.001), respectively. Improvement in predicting prognosis for AJCC stage III/IV disease also was observed, but to a lesser extent. CONCLUSIONS: If confirmed using tumor samples from a larger number of early stage oral cavity cancer patients, the 13-gene signature may inform personalized treatment of early stage HPV-negative and p16-negative oral cavity cancer patients.

The microbiome and oral cancer: More questions than answers.

Recent advances in DNA sequencing technology have facilitated rapid advances in the analysis of the human microbiome and its role in human disease. Several studies have now shown that OSCC and some oral premalignant conditions are associated with alterations in the oral microbiome. These studies raise questions regarding the role of the oral microbiome in the progression of oral malignancies and whether microbiome change is a significant risk factor in the development of oral cancer. This short review summarises current knowledge in the field and highlights questions that require further investigation.

Genetic Contributions to The Association Between Adult Height and Head and Neck Cancer: A Mendelian Randomization Analysis.

With the aim to dissect the effect of adult height on head and neck cancer (HNC), we use the Mendelian randomization (MR) approach to test the association between genetic instruments for height and the risk of HNC. 599 single nucleotide polymorphisms (SNPs) were identified as genetic instruments for height, accounting for 16% of the phenotypic variation. Genetic data concerning HNC cases and controls were obtained from a genome-wide association study. Summary statistics for genetic association were used in complementary MR approaches: the weighted genetic risk score (GRS) and the inverse-variance weighted (IVW). MR-Egger regression was used for sensitivity analysis and pleiotropy evaluation. From the GRS analysis, one standard deviation (SD) higher height (6.9 cm; due to genetic predisposition across 599 SNPs) raised the risk for HNC (Odds ratio (OR), 1.14; 95% Confidence Interval (95%CI), 0.99-1.32). The association analyses with potential confounders revealed that the GRS was associated with tobacco smoking (OR = 0.80, 95% CI (0.69-0.93)). MR-Egger regression did not provide evidence of overall directional pleiotropy. Our study indicates that height is potentially associated with HNC risk. However, the reported risk could be underestimated since, at the genetic level, height emerged to be inversely associated with smoking.

Oral lymphangiectasias and Crohn's disease: two case reports.

Lymphangiectasias, or acquired lymphangiomas, are rare in the oral cavity, more typically occurring on the skin or the genital area and, to our knowledge, have not been reported previously in association with Crohn's disease. Lymphangiectasias can occur at any age and develop secondary to chronic obstruction of the lymphatics. This differentiates them from congenital lymphangiomas, which are congenital malformations of the lymphatic system. We present 2 cases of oral lymphangiectasias associated with Crohn's disease, one of which was treated successfully with cryotherapy.