Chemoradiation therapy changes oral microbiome and metabolomic profiles in patients with oral cavity cancer and oropharyngeal cancer.

BACKGROUND: Patients with oral cavity cancer (OCC) and oropharyngeal cancer (OPC) are often seen with locoregionally advanced disease requiring complex multimodality treatments. These treatments may have detrimental effects on the oral microbiome, which is critical to maintaining physiological balance and health. METHODS: The effects of different OCC and OPC treatment types on the oral microbiome and metabolomic profiles for 24-month post-treatment in patients with OCC and OPC were investigated using 16S rRNA gene amplicon next-generation sequencing and gas chromatography-mass spectrometry (GC-MS), respectively. RESULTS: Chemoradiation resulted in oral dysbiosis with specific depletion of genera which regulate the enterosalivary nitrate-nitrite-nitric oxide pathway. These data also correlate with the oral metabolomic profiles with nitric oxide-related precursor, modulator, or catalyst significantly downregulated in saliva samples from patients' postchemoradiation. CONCLUSIONS: Together, we have shown that oral dysbiosis due to the effects of chemoradiation could potentially have an impact on OCC and OPC patient's quality of life post-treatment.

The Use of Three-Dimensional DNA Fluorescent In Situ Hybridization (3D DNA FISH) for the Detection of Anaplastic Lymphoma Kinase (ALK) in Non-Small Cell Lung Cancer (NSCLC) Circulating Tumor Cells.

Tumor tissue biopsy is often limited for non-small cell lung cancer (NSCLC) patients and alternative sources of tumoral information are desirable to determine molecular alterations such as anaplastic lymphoma kinase (ALK) rearrangements. Circulating tumor cells (CTCs) are an appealing component of liquid biopsies, which can be sampled serially over the course of treatment. In this study, we enrolled a cohort of ALK-positive (n = 8) and ALK-negative (n = 12) NSCLC patients, enriched for CTCs using spiral microfluidic technology and performed DNA fluorescent in situ hybridization (FISH) for ALK. CTCs were identified in 12/20 NSCLC patients ranging from 1 to 26 CTCs/7.5 mL blood. Our study revealed that 3D imaging of CTCs for ALK translocations captured a well-defined separation of 3' and 5' signals indicative of ALK translocations and overlapping 3'/5' signal was easily resolved by imaging through the nuclear volume. This study provides proof-of-principle for the use of 3D DNA FISH in the determination of CTC ALK translocations in NSCLC.

Chronic High-Fat Diet Induces Early Barrett's Esophagus in Mice through Lipidome Remodeling.

Esophageal adenocarcinoma (EAC) incidence has been rapidly increasing, potentially associated with the prevalence of the risk factors gastroesophageal reflux disease (GERD), obesity, high-fat diet (HFD), and the precursor condition Barrett's esophagus (BE). EAC development occurs over several years, with stepwise changes of the squamous esophageal epithelium, through cardiac metaplasia, to BE, and then EAC. To establish the roles of GERD and HFD in initiating BE, we developed a dietary intervention model in C57/BL6 mice using experimental HFD and GERD (0.2% deoxycholic acid, DCA, in drinking water), and then analyzed the gastroesophageal junction tissue lipidome and microbiome to reveal potential mechanisms. Chronic (9 months) HFD alone induced esophageal inflammation and metaplasia, the first steps in BE/EAC pathogenesis. While 0.2% deoxycholic acid (DCA) alone had no effect on esophageal morphology, it synergized with HFD to increase inflammation severity and metaplasia length, potentially via increased microbiome diversity. Furthermore, we identify a tissue lipid signature for inflammation and metaplasia, which is characterized by elevated very-long-chain ceramides and reduced lysophospholipids. In summary, we report a non-transgenic mouse model, and a tissue lipid signature for early BE. Validation of the lipid signature in human patient cohorts could pave the way for specific dietary strategies to reduce the risk of BE in high-risk individuals.

The Performance of an Oral Microbiome Biomarker Panel in Predicting Oral Cavity and Oropharyngeal Cancers.

The oral microbiome can play a role in the instigation and progression of oral diseases that can manifest into other systemic conditions. These associations encourage the exploration of oral dysbiosis leading to the pathogenesis of cancers. In this study, oral rinse was used to characterize the oral microbiome fluctuation associated with oral cavity cancer (OCC) and oropharyngeal cancers (OPC). The study cohort consists of normal healthy controls (n = 10, between 20 and 30 years of age; n = 10, above 50 years of age), high-risk individuals (n = 11, above 50 years of age with bad oral hygiene and/or oral diseases) and OCC and OPC patients (n = 31, HPV-positive; n = 21, HPV-negative). Oral rinse samples were analyzed using 16S rRNA gene amplicon sequencing on the MiSeq platform. Kruskal-Wallis rank test was used to identify genera associated with OCC and OPC. A logistic regression analysis was carried out to determine the performance of these genera as a biomarker panel to predict OCC and OPC. In addition, a two-fold cross-validation with a bootstrap procedure was carried out in R to investigate how well the panel would perform in an emulated clinical scenario. Our data indicate that the oral microbiome is able to predict the presence of OCC and OPC with sensitivity and specificity of 100 and 90%, respectively. With further validation, the panel could potentially be implemented into clinical diagnostic and prognostic workflows for OCC and OPC.

Oral Microbiome: A New Biomarker Reservoir for Oral and Oropharyngeal Cancers.

Current biomarkers (DNA, RNA and protein) for oral cavity and oropharyngeal cancers demonstrate biological variations between individuals, rendering them impractical for clinical translation. Whilst these biomarkers originate from the host, there is not much information in the literature about the influence of oral microbiota on cancer pathogenesis, especially in oral cancers. Oral microbiotas are known to participate in disease initiation and progression not only limited to the oral cavity, but also at other distant sites. Due to the close proximity of oral microbiota and oral cavity and oropharyngeal tumours, abundance changes in oral microbiota may provide useful information on tumourigenesis. This review aims to highlight information on the role of oral microbiota in oral cavity and oropharyngeal cancers. An in-depth analysis into the oral microbiota may provide a new avenue to diagnose and treat these patients.

The saliva microbiome profiles are minimally affected by collection method or DNA extraction protocols.

Saliva has attracted attention as a diagnostic fluid due to the association of oral microbiota with systemic diseases. However, the lack of standardised methods for saliva collection has led to the slow uptake of saliva in microbiome research. The aim of this study was to systematically evaluate the potential effects on salivary microbiome profiles using different methods of saliva collection, storage and gDNA extraction. Three types of saliva fractions were collected from healthy individuals with or without the gDNA stabilising buffer. Subsequently, three types of gDNA extraction methods were evaluated to determine the gDNA extraction efficiencies from saliva samples. The purity of total bacterial gDNA was evaluated using the ratio of human ß-globin to bacterial 16S rRNA PCR while 16S rRNA gene amplicon sequencing was carried out to identify the bacterial profiles present in these samples. The quantity and quality of extracted gDNA were similar among all three gDNA extraction methods and there were no statistically significant differences in the bacterial profiles among different saliva fractions at the genus-level of taxonomic classification. In conclusion, saliva sampling, processing and gDNA preparation do not have major influence on microbiome profiles.

A Pilot Study into the Association between Oral Health Status and Human Papillomavirus-16 Infection.

BACKGROUND: Over the next 20 years, oropharyngeal cancers (OPC) will represent the majority of head and neck cancers (HNCs) in the United States. It is estimated that human papillomavirus (HPV) may account for as much as 70% to 80% of OPCs in North America and in certain parts of Europe. It is hence crucial to understand the disease risk factors and natural history of oral HPV infections. We hypothesized that poor oral health (by measures such as poor oral hygiene and periodontal disease) leads to a higher degree of oral HPV-16 infections within a patient cohort from a dental school clinic. This study aims to test this hypothesis and gauge possible disease associations before larger scale studies. SUBJECTS AND METHODS: 223 participants were recruited in this study from the University of Queensland Dental School clinic. Clinical oral health parameters (such as oral hygiene measures and periodontal disease measurements) have been examined and determined by dental professionals. We have collected oral rinse samples from these volunteers. RESULTS: 10 (4.5%) out of 223 participants were found to have HPV-16 DNA in their oral rinse samples using NB2 endpoint PCR and Sanger sequencing. Within the HPV-16 DNA positive subjects, 7 (70%) and 3 (30%) were associated with poor oral hygiene and periodontal disease, respectively. CONCLUSION: Our results show a trend towards a positive correlation between oral HPV-16 infection and poor clinical oral health status.

Salivary DNA methylation panel to diagnose HPV-positive and HPV-negative head and neck cancers.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumours with a typical 5 year survival rate of <40 %. DNA methylation in tumour-suppressor genes often occurs at an early stage of tumorigenesis, hence DNA methylation can be used as an early tumour biomarker. Saliva is an ideal diagnostic medium to detect early HNSCC tumour activities due to its proximity to tumour site, non-invasiveness and ease of sampling. We test the hypothesis that the surveillance of DNA methylation in five tumour-suppressor genes (RASSF1α, p16 INK4a , TIMP3, PCQAP/MED15) will allow us to diagnose HNSCC patients from a normal healthy control group as well as to discriminate between Human Papillomavirus (HPV)-positive and HPV-negative patients. METHODS: Methylation-specific PCR (MSP) was used to determine the methylation levels of RASSF1α, p16 INK4a , TIMP3 and PCQAP/MED15 in DNA isolated from saliva. Statistical analysis was carried out using non-parametric Mann-Whitney's U-test for individually methylated genes. A logistic regression analysis was carried out to determine the assay sensitivity when combing the five genes. Further, a five-fold cross-validation with a bootstrap procedure was carried out to determine how well the panel will perform in a real clinical scenario. RESULTS: Salivary DNA methylation levels were not affected by age. Salivary DNA methylation levels for RASSF1α, p16 INK4a , TIMP3 and PCQAP/MED15 were higher in HPV-negative HNSCC patients (n = 88) compared with a normal healthy control group (n = 122) (sensitivity of 71 % and specificity of 80 %). Conversely, DNA methylation levels for these genes were lower in HPV-positive HNSCC patients (n = 45) compared with a normal healthy control group (sensitivity of 80 % and specificity of 74 %), consistent with the proposed aetiology of HPV-positive HNSCCs. CONCLUSIONS: Salivary DNA tumour-suppressor methylation gene panel has the potential to detect early-stage tumours in HPV-negative HNSCC patients. HPV infection was found to deregulate the methylation levels in HPV-positive HNSCC patients. Large-scale double-blinded clinical trials are crucial before this panel can potentially be integrated into a clinical setting.

A pilot study to compare the detection of HPV-16 biomarkers in salivary oral rinses with tumour p16(INK4a) expression in head and neck squamous cell carcinoma patients.

BACKGROUND: Human papilloma virus-16 (HPV-16) infection is a major risk factor for a subset of head and neck squamous cell carcinoma (HNSCC), in particular oropharyngeal squamous cell carcinoma (OPSCC). Current techniques for assessing the HPV-16 status in HNSCC include the detection of HPV-16 DNA and p16(INK4a) expression in tumor tissues. When tumors originate from hidden anatomical sites, this method can be challenging. A non-invasive and cost-effective alternative to biopsy is therefore desirable for HPV-16 detection especially within a community setting to screen at-risk individuals. METHODS: The present study compared detection of HPV-16 DNA and RNA in salivary oral rinses with tumor p16(INK4a) status, in 82 HNSCC patients using end-point and quantitative polymerase chain reaction (PCR). RESULTS: Of 42 patients with p16(INK4a)-positive tumours, 39 (sensitivity = 92.9 %, PPV = 100 % and NPV = 93 %) had oral rinse samples with detectable HPV-16 DNA, using end-point and quantitative PCR. No HPV-16 DNA was detected in oral rinse samples from 40 patients with p16(INK4a) negative tumours, yielding a test specificity of 100 %. For patients with p16(INK4a) positive tumours, HPV-16 mRNA was detected using end-point reverse transcription PCR (RT-PCR) in 24/40 (sensitivity = 60 %, PPV = 100 % and NPV = 71 %), and using quantitative RT-PCR in 22/40 (sensitivity = 55 %, PPV = 100 % and NPV = 69 %). No HPV-16 mRNA was detected in oral rinse samples from the p16(INK4a)-negative patients, yielding a specificity of 100 %. CONCLUSIONS: We demonstrate that the detection of HPV-16 DNA in salivary oral rinse is indicative of HPV status in HNSCC patients and can potentially be used as a diagnostic tool in addition to the current methods.

Salivary epigenetic biomarkers in head and neck squamous cell carcinomas.

The early detection of head and neck squamous cell carcinoma (HNSCC) continues to be a challenge to the clinician. Saliva as a diagnostic medium carries significant advantages including its close proximity to the region of interest, ease of collection and noninvasive nature. While the identification of biomarkers continues to carry significant diagnostic and prognostic utility in HNSCC, epigenetic alterations present a novel opportunity to serve this purpose. With the developments of novel and innovative technologies, epigenetic alterations are now emerging as attractive candidates in HNSCC. As such, this review will focus on two commonly aberrant epigenetic alterations: DNA methylation and microRNA expression in HNSCC and their potential clinical utility. Identification and validation of these salivary epigenetic biomarkers would not only enable early diagnosis but will also facilitate in the clinical management.