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Oral cavity cancer (OCC) is the predominant subtype of head and neck cancer (HNC) and has up to 50% mortality. Genome-wide microRNA (miR) sequencing data indicates overexpression of miR-9-5p in HNC tumours, however, the biological role of miR-9-5p in OCC is complex; it can either act as a tumour suppressor or an oncomir, regulating many target genes at the post-transcriptional level. We have investigated the overexpression of miR-9-5p in three OCC cell lines. We have evaluated its expression levels and Galectin-3 as potential biomarkers in saliva samples collected from controls and OCC patients. We found that over expression of miR-9-5p in OCC cell lines resulted in a significant reduction in cell proliferation and migration, and an increase in apoptosis, which was paralleled by an increase in Galectin-3 secretion and export of Galectin-3 protein. Our data are consistent with miR-9-5p being a modulator of Galectin-3 via the AKT/γ-catenin pathway. In addition, the positive correlation between the levels of miR-9-5p expression and secreted Galectin-3 in saliva reflects a similar relationship in vivo, and supports the utility of their integrative evaluation in OCC. Our findings indicate that both miR-9-5p and Galectin-3 are critical biomolecules in the progression of OCC.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every corner of the globe, causing societal instability. The resultant coronavirus disease 2019 (COVID-19) leads to fever, sore throat, cough, chest and muscle pain, dyspnoea, confusion, anosmia, ageusia and headache. These can progress to life-threatening respiratory insufficiency, also affecting the heart, kidney, liver and nervous systems. The diagnosis of SARS-CoV-2 infection is often confused with that of influenza and seasonal upper respiratory tract viral infections. Due to available treatment strategies and required containments, rapid diagnosis is mandated. This Review brings clarity to the rapidly growing body of available and in-development diagnostic tests, including nanomaterial-based tools. It serves as a resource guide for scientists, physicians, students and the public at large.

The COVID-19 pandemic has affected more than 38 million people world-wide by person to person transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therapeutic and preventative strategies for SARS-CoV-2 remains a significant challenge. Within the past several months, effective treatment options have emerged and now include repurposed antivirals, corticosteroids and virus-specific antibodies. The latter has included convalescence plasma and monoclonal antibodies. Complete viral eradication will be achieved through an effective, safe and preventative vaccine. To now provide a comprehensive summary for each of the pharmacotherapeutics and preventative strategies being offered or soon to be developed for SARS-CoV-2.

Biological markers that evaluate physical healing as well as psychological impact of a burn are essential for effective treatment of paediatric burns. The objective of this review is to summarize the evidence supporting the use of biomarkers in children with burns. An extensive review of the literature was performed using PubMed. A total of 59 biomarkers were identified relating to burn presence, specifically relating to processes involved in inflammation, wound healing, growth and metabolism. In addition, biomarkers involved in the stress response cascade following a burn trauma were also identified. Although many biomarkers have been identified that are potentially associated with burn-related physical and psychological trauma, an understanding of burn biology is still lacking in children. We propose that future research in the field of children's burns should be conducted using broad screening methods for identifying potential biomarkers, examine the biological interactions of different biomarkers, utilize child-appropriate biological fluids such as urine or saliva, and include a range of different severity burns. Through further research, the biological response to burn injury may be fully realized and clinically relevant diagnostic tests and treatment therapies utilizing these biomarkers could be developed, for the improvement of healing outcomes in paediatric burn patients., (© The Author(s) 2021. Published by Oxford University Press.)

Background: Immune checkpoint inhibitors (ICI) have shown durable and long-term benefits in a subset of head and neck squamous cell carcinoma (HNSCC) patients. To identify patient-responders from non-responders, biomarkers are needed which are predictive of outcome to ICI therapy. Cues in the tumor microenvironment (TME) have been informative in understanding the tumor-immune contexture., Methods: In this preliminary study, the NanoString GeoMx™ Digital Spatial Profiling (DSP) technology was used to determine the immune marker and compartment specific measurements in a cohort of HNSCC tumors from patients receiving ICI therapy., Results: Our data revealed that markers involved with immune cell infiltration (CD8 T-cells) were not predictive of outcome to ICI therapy. Rather, a number of immune cell types and protein markers (CD4, CD68, CD45, CD44, CD66b) were found to correlate with progressive disease. Cross platform comparison with the Opal Vectra (Perkin Elmer) for a number of markers across similar regions of interest demonstrated concordance for pan-cytokeratin, CD8, and PD-L1., Conclusion: This study, to our knowledge, represents the first digital spatial analysis of HNSCC tumors. A larger cohort of HNSCC will be required to orthogonally validate the findings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kulasinghe, Taheri, O’Byrne, Hughes, Kenny and Punyadeera.)

Exosomes are small extracellular vesicles released by cells under physiological and pathological conditions. There is emerging evidence associating exosomes with tumorigenesis. They carry cargo (DNA, RNA, miRNA and protein) pertaining to the cell of origin and play a key role in intercellular communication, influencing several cellular processes. Moreover, exosomes can be shed and found in almost all body fluids, providing a source of biomarkers for tumor diagnosis and prognosis. In addition, the use of exosomes for cancer therapeutics is another research area that is gaining attention. This book chapter aims to explore the role of exosomes in tumor biogenesis, progression and clinical applications, comprehensively compiling the research for three tumor types, namely head and neck cancer, lung cancer and glioblastoma., (© 2021 Elsevier Inc. All rights reserved.)

Glycosylation is the most common post-translational modification (PTM) of proteins. Malignant tumour cells frequently undergo an alteration in surface protein glycosylation. This phenomenon is also common in cancers of the head and neck, most of which are squamous cell carcinomas (HNSCC). It affects cell functions, including proliferation, motility and invasiveness, thus increasing the propensity to metastasise. HNSCC represents the sixth most frequent malignancy worldwide. These neoplasms, which arise from the mucous membranes of the various anatomical subsites of the upper aero-digestive tract, are heterogeneous in terms of aetiology and clinico-pathologic features. With current treatments, only about 50% of HNSCC patients survive beyond 5-years. Therefore, there is the pressing need to dissect NHSCC heterogeneity to inform treatment choices. In particular, reliable biomarkers of predictive and prognostic value are eagerly needed. This review describes the current state of the art and bio-pathological meaning of glycosylation signatures associated with HNSCC and explores the possible role of tumour specific glycoproteins as potential biomarkers and attractive therapeutic targets. We have also compiled data relating to altered glycosylation and the nature of glycoproteins as tools for the identification of circulating tumour cells (CTCs) in the new era of liquid biopsy., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hepatocellular carcinoma (HCC) is a common cause of cancer-related deaths worldwide. Despite advances in systemic therapies, patient survival remains low due to late diagnosis and frequent underlying liver diseases. HCC diagnosis generally relies on imaging and liver tissue biopsy. Liver biopsy presents limitations because it is invasive, potentially risky for patients and it frequently misrepresents tumour heterogeneity. Recently, liquid biopsy has emerged as a way to monitor cancer progression in a non-invasive manner. Tumours shed content into the bloodstream, such as circulating tumour cells (CTCs), circulating nucleic acids, extracellular vesicles and proteins, that can be isolated from biological fluids of patients with HCC. These biomarkers provide knowledge regarding the genetic landscape of tumours and might be used for diagnostic or prognostic purposes. In this review, we summarize recent literature on circulating biomarkers for HCC, namely CTCs, circulating tumour DNA (ctDNA), RNA, extracellular vesicles and proteins, and their clinical relevance in HCC., (Copyright © 2020. Published by Elsevier B.V.)

Given that oropharyngeal squamous cell carcinoma (OPSCC) have now surpassed cervical cancer as the most common human papillomavirus (HPV)-driven cancer, there is an interest in developing non-invasive predictive biomarkers to early detect HPV-driven OPSCC. In total, 665 cancer-free individuals were recruited from Queensland, Australia. Oral HPV16 DNA positivity in those individuals was determined by our in-house developed sensitive PCR method. Individuals with (n = 9) or without (n = 12) oral HPV16 infections at baseline were followed for a median duration of 24 mo. Individuals with persistent oral HPV16 infection (≥ 30 mo) were invited for clinical examination of their oral cavity and oropharynx by an otolaryngologist. Oral HPV16 DNA was detected in 12 out of 650 cancer-free individuals (1.8%; 95% confidence interval [CI]: 1.0-3.2). Of the 3 individuals with persistent oral HPV16 infection, the first individual showed no clinical evidence of pathology. The second individual was diagnosed with a 2 mm invasive squamous cell carcinoma (T1N0M0) positive for both p16INK4a expression and HPV16 DNA. The third individual was found to have a mildly dysplastic lesion in the tonsillar region that was negative for p16INK4a expression and HPV16 DNA and she continues to have HPV16 DNA in her saliva. Taken together, our data support the value of using an oral HPV16 DNA assay as a potential screening tool for the detection of microscopic HPV-driven OPSCC. Larger multicenter studies across various geographic regions recruiting populations at a higher risk of developing HPV-driven OPSCC are warranted to extend and confirm the results of the current investigation., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Tumour tissue-based information is limited. Liquid biopsy can provide valuable real-time information through circulating tumour cells (CTCs). Profiling and expanding CTCs may provide avenues to study transient metastatic disease., Methods: Seventy non-small cell lung cancer (NSCLC) patients were recruited. CTCs were enriched using the spiral microfluidic chip and a RosetteSep™ using bloods from NSCLC patients. CTC cultures were carried out using the Clevers media under hypoxic conditions. CTCs were characterized using immunofluorescence and mutation-specific antibodies for samples with known mutation profiles. Exome sequencing was used to characterized CTC cultures., Results: CTCs (>2 cells) were detected in 38/70 (54.3%) of patients ranging from 0 to 385 CTCs per 7.5 mL blood. In 4/5 patients where primary tumours harboured an EGFR exon 19 deletion, this EGFR mutation was also captured in CTCs. ALK translocation was confirmed on CTCs from a patient harbouring an ALK-rearrangement in the primary tumour. Short term CTC cultures were successfully generated in 9/70 NSCLC patients. Whole exome sequencing (WES) confirmed the presence of somatic mutations in the CTC cultures with mutational signatures consistent with NSCLC., Conclusions: We were able to detect CTCs in >50% of NSCLC patients. NSCLC patients with >2 CTCs had a poor prognosis. The short-term CTC culture success rate was 12.9%. Further optimization of this culture methodology may provide a means by which to expand CTCs derived from NSCLC patient's bloods. CTC cultures allow for expansion of cells to a critical mass, allowing for functional characterization of CTCs with the goal of drug sensitivity testing and the creation of CTC cell lines., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tlcr-20-521). KOB serves as an unpaid editorial board member of Translational Lung Cancer Research. The other authors have no conflicts of interest to declare., (2020 Translational Lung Cancer Research. All rights reserved.)

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.

Background: Patients with HF are at a higher risk of rehospitalisation and, as such, significant costs to our healthcare system. A non-invasive method to collect body fluids and measure Gal-3 could improve the current management of HF. In this study, we investigated the potential prognostic utility of salivary Galectin-3 (Gal-3) in patients with heart failure (HF)., Methods: We collected saliva samples from patients with HF (n = 105) either at hospital discharge or during routine clinical visits. Gal-3 concentrations in saliva samples were measured by ELISA. The Kaplan-Meier survival curve analysis and Cox proportional regression model were used to determine the potential prognostic utility of salivary Gal-3 concentrations., Results: The primary end point was either cardiovascular death or hospitalisation. Salivary Gal-3 concentrations were significantly higher (p < 0.05) in patients with HF who subsequently experienced the primary endpoint compared to those who did not. HF patients with salivary Gal-3 concentrations > 172.58 ng/mL had a significantly (p < 0.05) higher cumulative risk of the primary endpoint compared to those with lower salivary Gal-3 concentrations. In patients with HF, salivary Gal-3 concentration was a predictor of the primary endpoint even after adjusting for other covariates., Conclusions: In our pilot study, HF patients with salivary Gal-3 concentrations of > 172.58 ng/mL demonstrated a higher cumulative risk of the primary outcome compared to those with lower Gal-3 levels, even after adjusting for other variables. Confirming our findings in a larger multi-centre clinical trial in the future would enable salivary Gal-3 measurements to form part of routine management for patients with HF.

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Disruption of DNA methylation patterns is one of the hallmarks of cancer. Similar to other cancer types, human papillomavirus (HPV)-driven head and neck cancer (HNC) also reveals alterations in its methylation profile. The intrinsic ability of HPV oncoproteins E6 and E7 to interfere with DNA methyltransferase activity contributes to these methylation changes. There are many genes that have been reported to be differentially methylated in HPV-driven HNC. Some of these genes are involved in major cellular pathways, indicating that DNA methylation, at least in certain instances, may contribute to the development and progression of HPV-driven HNC. Furthermore, the HPV genome itself becomes a target of the cellular DNA methylation machinery. Some of these methylation changes appearing in the viral long control region (LCR) may contribute to uncontrolled oncoprotein expression, leading to carcinogenesis. Consistent with these observations, demethylation therapy appears to have significant effects on HPV-driven HNC. This review article comprehensively summarizes DNA methylation changes and their diagnostic and therapeutic indications in HPV-driven HNC., Competing Interests: The authors declare no conflict of interest.

Introduction : Saliva has gained attention as an important diagnostic fluid because it contains biomolecules that have the potential to detect early-stage cancer or to monitor the response to treatment in patients. Several saliva-based proteins have been proposed as potential biomarkers for head and neck cancers (HNC). Areas covered : This review aims to provide an update on saliva-based protein biomarkers for HNC, often studied in observational research and clinical trials. Expert opinion : Despite the increasing number of studies relating to salivary proteins as biomarkers for HNC, there is no consensus regarding which proteins have the best clinical utility. Most studies have analyzed individual proteins and not a protein panel approach. It must be considered that combining different proteins as a panel can increase the accuracy and will have the potential to change the current clinical practice for HNC patients.

The role of human papillomavirus type 16 (HPV16) in oral potentially malignant disorders (OPMD) and oral cavity carcinoma (OC) is still under debate. We investigated HPV16 prevalence in unstimulated saliva, oral rinse samples, oral swabs and tumour biopsies collected from OPMD ( n = 83) and OC ( n = 106) patients. HPV16 genotype, viral load, physical status (episomal vs. integrated) and tumour p16INK4a expression were determined. Oral HPV16 prevalence was higher in OC than in OPMD, but this difference was not statistically significant (7.5% (8/106) versus 3.6% (3/83), odds ratio (OR): 2.18, 95% confidence interval (CI): 0.56, 8.48, p = 0.26). There was a significant association ( p < 0.05) between oral HPV16 infection and heavy tobacco consumption. Real-time PCR results indicated that no integration events occurred in either OPMD or OC cases based on the HPV16 E2/E6 ratio. HPV16 positive OPMD and OC patients had similar HPV16 E2 and E6 viral loads. The inter-rater agreement between tumour p16INK4a expression and oral HPV16 infection was considered as fair (k = 0.361) for OC. Our data suggest that the involvement of HPV16 in oral carcinogenesis is limited., Competing Interests: The authors declare no conflict of interest.

Gliomas are the most common tumours of the central nervous system and the most aggressive form is glioblastoma (GBM). Despite advances in treatment, patient survival remains low. GBM diagnosis typically relies on imaging techniques and postoperative pathological diagnosis; however, both procedures have their inherent limitations. Imaging modalities cannot differentiate tumour progression from treatment-related changes that mimic progression, known as pseudoprogression, which might lead to misinterpretation of therapy response and delay clinical interventions. In addition to imaging limitations, tissue biopsies are invasive and most of the time cannot be performed over the course of treatment to evaluate 'real-time' tumour dynamics. In an attempt to address these limitations, liquid biopsies have been proposed in the field. Blood sampling is a minimally invasive procedure for a patient to endure and could provide tumoural information to guide therapy. Tumours shed tumoural content, such as circulating tumour cells, cell-free nucleic acids, proteins and extracellular vesicles, into the circulation, and these biomarkers are reported to cross the blood-brain barrier. The use of liquid biopsies is emerging in the field of GBM. In this review, we aim to summarise the current literature on circulating biomarkers, namely circulating tumour cells, circulating tumour DNA and extracellular vesicles as potential non-invasively sampled biomarkers to manage the treatment of patients with GBM.

Saliva has become one of the more attractive body fluids for protein biomarker discovery studies because of its ease of collection, non-invasiveness and multiple samples can be collected from an individual at a single time point. With the development of modern data acquisition strategies, such as Sequential Window Acquisition of all Theoretical Mass Spectrometry (SWATH-MS), the quality of saliva sample preparation has become a crucial factor for a successful protein identification and quantification. Several sample preparation methods have been proposed, but there has been no systematic evaluation conducted to date that compared each of these methods. We have therefore, performed an extensive assessment using technical and biological repeats to evaluate the number of protein IDs and repeatability of three most commonly used techniques, in-solution digestion, filter-aided sample preparation (FASP) and in stage-tip (iST) digestion. We discovered that in the case of human saliva sample, FASP provided the highest number of proteins (human and microbial) identifiable from a pool saliva sample, and there were no significant differences in terms of repeatability among the three methods investigated., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction : Local and distant metastatic disease occurs in approximately half of head and neck squamous cell carcinoma (HNSCC) patients, representing an ongoing cause for treatment failure. Circulating tumor cells (CTCs) are transient cancer cells which have the capacity to metastasize to distant sites such as the lungs and liver in HNSCC. When metastatic disease is radiographically evident, the patient prognosis is often poor. Therefore, methodologies to assess micrometastatic disease are needed to (1) identify patients likely to develop metastatic disease and (2) treat and monitor these patients more aggressively. Whilst CTCs are well documented in other tumor streams such as breast, colorectal cancer and prostate cancers, the data and clinical utility in HNSCC remains limited. Areas covered : Here we summarize the recent advances of CTCs and applications in HNSCC. Expert opinion : CTC enumeration can be prognostic in HNSCC; further studies are warranted to investigate the role of CTC clusters in HNSCC; CTC culture ( in vivo / ex vivo ) may present a possibility to expand these rare cells to a critical mass for functional testing; PD-L1 expression of HNSCC CTCs may present a means by which to determine patients likely to respond to therapy; a HNSCC CTC-specific marker is warranted.

Screening for systolic heart failure (SHF) has been problematic. Heart failure management guidelines suggest screening for structural heart disease and SHF prevention strategies should be a top priority. We developed a multi-protein biomarker panel using saliva as a diagnostic medium to discriminate SHF patients and healthy controls. We collected saliva samples from healthy controls (n = 88) and from SHF patients (n = 100). We developed enzyme linked immunosorbent assays to quantify three specific proteins/peptide (Kallikrein-1, Protein S100-A7, and Cathelicidin antimicrobial peptide) in saliva samples. The analytical and clinical performances and predictive value of the proteins were evaluated. The analytical performances of the immunoassays were all within acceptable analytical ranges. The multi-protein panel was able to significantly ( p < 0.001) discriminate saliva samples collected from patients with SHF from controls. The multi-protein panel demonstrated good performance with an overall diagnostic accuracy of 81.6% (sensitivity of 79.2% and specificity of 85.7%) when distinguishing SHF patients from healthy individuals. In conclusion, we have developed immunoassays to measure the salivary concentrations of three proteins combined as a panel to accurately distinguish SHF patients from healthy controls. While this requires confirmation in larger cohorts, our findings suggest that this three-protein panel has the potential to be used as a biomarker for early detection of SHF.

Fanconi anemia (FA) is a rare inherited genetic condition that may lead to bone marrow failure, leukemia, and/or solid tumors. It is caused by the loss of function of at least 1 gene of the FA/BRCA pathway, which is necessary for DNA repair. Patients with FA have a 200-fold to 1000-fold risk of developing head and neck cancer, mainly oral squamous cell carcinoma (OSCC), and of doing so at a much younger age than individuals within the general population. Also, patients who have FA with OSCC have poor overall survival rates, reinforcing the necessity to detect OSCC early. The scope of the current review is to provide an update on OSCC in patients with FA., (© 2019 American Cancer Society.)

Background: Accumulating evidence has suggested the utility of salivary oral rinse as a diagnostic fluid to detect oral human papillomavirus (HPV) DNA, but there are many methods for collecting saliva., Methods: Salivary oral rinse and unstimulated whole mouth saliva samples were collected from 45 oropharyngeal cancer (OPC) patients., Results: We show a positive correlation of HPV-16 E2 (r = 0.95, P < 0.0001) and E6/7 (r = 0.93, P < 0.0001) relative copy number as well as HPV genotypes in both sample methods. There was a significant correlation between the two sample methods in the ratio of HPV16 E2 to E6/7 DNA (r = 0.46, P < 0.01). Consistent with previous studies, a mixed HPV-16 form (episomal and integrated) was commonly found in both saliva and tumor samples., Conclusion: Detection of HPV in saliva samples collected by either method yielded comparable results, and showed good sensitivity for detection of HPV derived from OPC., (© 2018 Wiley Periodicals, Inc.)

Silencing of tumor-suppressor genes (TSGs) by DNA promoter hypermethylation is an early event in carcinogenesis; hence, TSGs may serve as early tumor biomarkers. We determined the promoter methylation levels of p16 INK4a , RASSF1A , TIMP3 , and PCQAP/MED15 TSGs in salivary DNA from oral cancer (OC) and oropharyngeal cancer (OPC) patients, using methylation-specific PCR coupled with densitometry analysis. We assessed the association between DNA methylation of individual TSGs with OC and OPC risk factors. The performance and the clinical validity of this quadruple-methylation marker panel were evaluated in discriminating OC and OPC patients from healthy controls using the CombiROC web tool. Our study reports that RASSF1A , TIMP3 , and PCQAP/MED15 TSGs were significantly hypermethylated in OC and OPC cases compared to healthy controls. DNA methylation levels of TSGs were significantly augmented by smoking, alcohol use, and betel quid chewing, indicating the fact that frequent exposure to risk factors may drive oral and oropharyngeal carcinogenesis through TSG promoter hypermethylation. Also, this quadruple-methylation marker panel of p16 INK4a , RASSF1A , TIMP3 , and PCQAP/MED15 TSGs demonstrated excellent diagnostic accuracy in the early detection of OC at 91.7% sensitivity and 92.3% specificity and of OPC at 99.8% sensitivity and 92.1% specificity from healthy controls., Competing Interests: The authors declare no conflicts of interest.

The incidence of human papillomavirus (HPV)-positive oropharyngeal cancer (OPC) is rising in high-income countries, including Australia. Increasing evidence suggests that accurate HPV testing is pivotal for clinical decision making and treatment planning in these patients. Recently, the eighth edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) tumor-node-metastasis (TNM) staging system for OPC (based on the p16INK4a (p16) status) was proposed and has been implemented. However, the applicability of this new staging system is still far from clear. In our study, n = 127 OPC patients from Queensland, Australia were recruited, and the tumor p16 expression in these patients was examined using immunohistochemical (IHC) analysis. HPV-16 genotyping, viral load, and physical status (episomal versus integrated) in the saliva samples of OPC patients were determined using the qPCR method. A good inter-rater agreement ( k = 0.612) was found between tumor p16 expression and oral HPV-16 infection in OPC. Importantly, according to the eighth edition staging system, HPV-16 DNA viral load (>10 copies/50 ng) was significantly associated with the advanced stages of OPC. In concordance with previous studies, a mixed HPV-16 form (partially or fully integrated) was predominately found in OPC patients. Taken together, our data support HPV-16 detection in saliva as a screening biomarker to identify people within the community who are at risk of developing OPC., Competing Interests: Kurt Baeten and Gert Scheper are employees of Janssen Pharmaceuticals and may be stockholders of Johnson & Johnson.

Objectives: In non-small cell lung cancers (NSCLC), tumour biopsy can often be an invasive procedure. The development of a non-invasive methodology to study genetic changes via circulating tumour cells (CTCs) is an appealing concept. Whilst CTCs typically remain as rare cells, improvements in epitope-independent CTC isolation techniques has given rise to a greater capture of CTCs. In this cross sectional study, we demonstrate the capture and characterization of NSCLC CTCs for the clinically actionable markers epidermal growth factor receptor (EGFR) alterations, anaplastic lymphoma kinase (ALK) rearrangements and programmed death ligand-1 (PD-L1) expression. The study identified CTCs/CTC clusters in 26/35 Stage IV NSCLC patients, and subsequently characterized the CTCs for EGFR mutation, ALK status and PD-L1 status. This pilot study demonstrates the potential of a non-invasive fluid biopsy to determine clinically relevant biomarkers in NSCLC., Competing Interests: The authors declare no conflict of interest.

Circulating tumor cells (CTCs) carry a wealth of information on primary and metastatic tumors critical for precise cancer detection, monitoring, and treatment. Numerous microfluidic platforms have been developed in the past few years to capture these rare cells in patient bloodstream for deciphering the critical information needed. However, the practical need for a high-quality method of CTC isolation remains to be met. Herein, we demonstrate a novel multi-flow microfluidic device that is able to sensitively provide high purity (>87%) of separation outcome without labeling. Our device is constructed and configured based on the phenomenal effect of size-dependent inertial migration. The recovery rate of >93% has been achieved using spiked cancer cells at clinically relevant concentrations (10 cells per 5 mL and above). We have also successfully detected CTCs from 6 out of 8 non-small-cell-lung-cancer (NSCLC) patients, while none for 5 healthy control subjects. With these results, we envision our approach is a promising alternative for reliable CTC capture, and thus for facilitating the progress of extracting information from CTCs to personalize treatment strategies for solid tumor patients., Competing Interests: The authors declare that they have no conflict of interest.

Circulating tumour cells (CTCs) are the metastatic precursors to distant disease in head and neck cancers (HNCs). Whilst the prognostic and predictive value of single CTCs have been well documented, the role of CTC clusters, which potentially have a higher metastatic capacity are limited. In this study, the authors used a novel straight microfluidic chip to focus and capture CTCs. The chip offers high cell recoveries with clinically relevant numbers (10⁻500 cells/mL) without the need for further purification. Single CTCs were identified in 10/21 patient samples (range 2⁻24 CTCs/mL), CTC clusters in 9/21 patient samples (range 1⁻6 CTC clusters/mL) and circulating tumour microemboli (CTM) in 2/21 samples. This study demonstrated that CTC clusters contain EGFR amplified single CTCs within the cluster volume. This novel microfluidic chip demonstrates the efficient sorting and preservation of single CTCs, CTC clusters and CTMs. The authors intend to expand this study to a larger cohort to determine the clinical implication of the CTC subsets in HNC.

Background: p16 has often been found to be overexpressed in patients oral squamous cell carcinoma (OSCC), but its prognostic value between anatomic subsites is still unclear., Objective: The aim of this study was to investigate the diagnostic and prognostic values of p16 in OSCC originating from tongue, gingiva or buccal mucosa., Methods: A total of 147 OSCC patients with tumors arising from the tongue, gingiva or buccal mucosa were enrolled in this study. p16 expression was detected using immunohistochemistry (IHC), and the presence of HPV16 was determined by real-time PCR in p16 positive patients. The correlation of p16 expression with the clinical parameters was evaluated., Results: Only one p16 positive patient with a cut off value of 25% and 75% was HPV16 positive. Although overall survival (OS), recurrence free survival (RFS) and metastasis free survival (MFS) had no significant differences between the p16 positive and negative patients, p16 negative patients (cut off value 25%) had more RFS in the buccal mucosa cancer (p= 0.03) than the p16-positive patients., Conclusions: The prevalence of HPV16 in Chinese OSCC patients was low. p16 overexpression decoupled from HPV infection was not a prognostic marker for OSCC patients except for patients with the buccal mucosa cancer.

Metastasis is responsible for 90% of cancer-related deaths. The study of circulating tumor cells (CTCs) enables the study of the units of disease responsible for the process of metastasis. While the biology of the primary tissue is relatively known, little is understood about the cells en route to distant sites. Here we describe the isolation of CTCs using the spiral microfluidic technology for the efficient sorting of CTCs from head and neck cancer (HNC) patient blood samples. Furthermore, the molecular characterization of CTCs can aid in stratifying patients for targeted therapy such as immunotherapy, which is having a profound impact in the treatment of metastatic HNC.

Oral premalignant disorders (OPMD) have relatively high malignant transformation rates to Oral Cancers (OC). Oral carcinogenesis is a multistep process that originates as epithelial hyperplasia followed by epithelial dysplasia, leading to fully malignant phenotypes. Early detection can be lifesaving but is currently not possible due to the lack of early diagnostic tools. The current diagnostic methods such as biopsy sampling, tumor tissue staining, and imaging techniques require skilled personnel and are invasive, painful, time-consuming, and expensive. Saliva has gained momentum as the diagnostic fluid of the future due to its noninvasive nature, ease of sampling, multiple samples can be collected with ease and more importantly does not require skilled personnel. The use of saliva in cancer diagnostics is an emerging and an expanding field. MicroRNA (miRNA) play a role in cancer initiation and progression and the expression changes of miRNA have been investigated as a potential biomarker in cancer studies. In this chapter, we describe a robust and cost-effective protocol to isolate and enrich miRNA from saliva samples. Profiling miRNAs in saliva samples can form part of the clinical management of OPMD and OC patients in the future.

Tumor biopsy is the gold standard for the assessment of clinical biomarkers for treatment. However, tumors change dynamically in response to therapy, and there remains a need for a more representative biomarker that can be assayed over the course of treatment. Circulating tumor cells (CTCs) may provide clinically important and comprehensive tumoral information that is predictive of treatment response and outcome. Blood samples were processed for CTCs from 56 patients using the ClearCell FX system. Captured cells were phenotyped for CTC clusters and markers for immunotherapy (PD-L1) CTC chromosomal architecture (ALK, EGFR). CTCs were isolated in 11/23 (47.8%) of head and neck cancer (HNC) patients and 17/33 (51.5%) of non-small-cell lung cancer (NSCLC) patients. CTCs were determined to be PD-L1-positive in 6/11 (54.4%) HNC and 11/17 (64.7%) NSCLC cases, respectively. 3D chromosomal DNA FISH for ALK and EGFR molecular targets showed better resolution than in 2D when imaging CTCs. HNC CTC-positive patients had shorter progression-free survival (PFS) (hazard ratio[HR]: 4.946; 95% confidence internal[CI]:1.571-15.57; P = 0.0063), and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P = 0.0485). In the advanced stage NSCLC patient cohort, PFS was not found to be associated with CTCs prior to therapy ([HR]:2.246; 95% [CI]:0.9565-5.273; P = 0.0632), nor the presence of PD-L1 expression ([HR]:1.646; 95% [CI]:0.5128-5.283; P = 0.4023). This study demonstrated that CTCs are predictive of poorer outcomes in HNC and provides distinct and separate utility for CTCs in HNC and NSCLC, which may be more representative of the disease burden and overall survival than the parameters used to measure them., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: PIK3CA pathways are the most frequently mutated oncogenic pathway in head and neck squamous cell carcinoma (HNSCC), including virally driven HNCs. PIK3CA is involved in the PI3K-PTEN-mTOR signalling pathway. PIK3CA has been implicated in HNSCC progression and PIK3CA mutations may serve as predictive biomarkers for therapy selection. Circulating tumour DNA (ctDNA) derived from necrotic and apoptotic tumour cells are thought to harbour tumour-specific genetic alterations. As such, the detection of PIK3CA alterations detected by ctDNA holds promise as a potential biomarker in HNSCC., Methods: Blood samples from treatment naïve HNSCC patients ( n = 29) were interrogated for a commonly mutated PIK3CA hotspot mutation using low cost allele-specific Plex-PCR TM technology., Results: In this pilot, cross sectional study, PIK3CA E545K mutation was detected in the plasma samples of 9/29 HNSCC patients using the Plex-PCR TM technology., Conclusion: The results of this pilot study support the notion of using allele-specific technologies for cost-effective testing of ctDNA, and further assert the potential utility of ctDNA in HNSCC.

Over the past decade, there has been emerging research in the field of extracellular vesicles, especially those originating from endosomes, referred to as 'exosomes. Exosomes are membrane-bound nanovesicles secreted by most cell types upon fusion of multivesicular bodies (MVBs) to the cell plasma membrane. These vesicles are present in almost all body fluids such as blood, urine, saliva, breast milk, cerebrospinal and peritoneal fluids. Exosomes participate in intercellular communication by transferring the biologically active molecules like proteins, nucleic acids, and lipids to neighboring cells. Exosomes are enriched in the tumour microenvironment and growing evidence demonstrates that exosomes mediate cancer progression and metastasis. Given the important biological role played by these nanovesicles in cancer pathogenesis, these can be used as ideal non-invasive biomarkers in detecting and monitoring tumours as well as therapeutic targets. The scope of the current review is to provide an overview of exosomes with a special focus on salivary exosomes as potential biomarkers in head and neck cancers., (Copyright © 2018. Published by Elsevier Ltd.)

Lung cancer affects over 1. 8 million people worldwide and is the leading cause of cancer related mortality globally. Currently, diagnosis of lung cancer involves a combination of imaging and invasive biopsies to confirm histopathology. Non-invasive diagnostic techniques under investigation include "liquid biopsies" through a simple blood draw to develop predictive and prognostic biomarkers. A better understanding of circulating tumor cell (CTC) dissemination mechanisms offers promising potential for the development of techniques to assist in the diagnosis of lung cancer. Enumeration and characterization of CTCs has the potential to act as a prognostic biomarker and to identify novel drug targets for a precision medicine approach to lung cancer care. This review will focus on the current status of CTCs and their potential diagnostic and prognostic utility in this setting.

There is growing awareness for the need of early diagnostic tools to aid in point-of-care testing in cancer. Tumor biopsy remains the conventional means in which to sample a tumor and often presents with challenges and associated risks. Therefore, alternative sources of tumor biomarkers is needed. Liquid biopsy has gained attention due to its non-invasive sampling of tumor tissue and ability to serially assess disease via a simple blood draw over the course of treatment. Among the leading technologies developing liquid biopsy solutions, microfluidics has recently come to the fore. Microfluidic platforms offer cellular separation and analysis platforms that allow for high throughout, high sensitivity and specificity, low sample volumes and reagent costs and precise liquid controlling capabilities. These characteristics make microfluidic technology a promising tool in separating and analyzing circulating tumor biomarkers for diagnosis, prognosis and monitoring. In this review, the characteristics of three kinds of circulating tumor markers will be described in the context of cancer, circulating tumor cells (CTCs), exosomes, and circulating tumor DNA (ctDNA). The review will focus on how the introduction of microfluidic technologies has improved the separation and analysis of these circulating tumor markers.

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.

Rationale: Saliva as a sample matrix is rapidly gaining interest for disease diagnosis and point-of-care assays because it is easy to collect (non-invasive) and contains many health-related biomarkers. However, saliva poses particular problems relative to more common urine and blood matrices, which includes low analyte concentrations, lack of understanding of biomolecule transportation and inherent viscosity variability in human samples. While several studies have sought to improve assay sensitivity, few have addressed sample viscosity specifically. The goal of this study is to minimize the effect of sample viscosity on paper-based analytical devices (PADs) for the measurement of pH and nitrite in human saliva. Methods: PADs were used to measure salivary pH from 5.0 to 10.0 with a universal indicator consisting of chlorophenol red, phenol red and phenolphthalein. Nitrite determination was performed using the Griess reaction. Artificial saliva with viscosity values between 1.54 and 5.10 mPa∙s was tested on the proposed PAD. To ensure the proposed PADs can be tailored for use in-field analysis, the devices were shipped to Australia and tested with human specimens. Results: Initial experiments showed that viscosity had a significant impact on the calibration curve for nitrite; however, a more consistent curve could be generated when buffer was added after the sample, irrespective of sample viscosity. The linear range for nitrite detection was 0.1 to 2.4 mg/dL using the improved method. The nitrite measurement in artificial saliva also showed a good correlation with the standard spectrophotometry method ( p =0.8484, paired sample t -test, n=20). Measured pH values from samples with varying viscosities correlated well with the results from our pH meter. Conclusions: The inherent variation of salivary viscosity that impacts nitrite and pH results can be addressed using a simple washing step on the PAD without the need for complex procedures., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Distant metastasis (DM) from head and neck cancers (HNC) portends a poor patient prognosis. Despite its important biological role, little is known about the cells which seed these DM. Circulating tumour cells (CTCs) represent a transient cancer cell population, which circulate in HNC patients' peripheral blood and seed at distant sites. Capture and analysis of CTCs offers insights into tumour metastasis and can facilitate treatment strategies. Whilst the data on singular CTCs have shown clinical significance, the role of CTC clusters in metastasis remains limited. In this pilot study, we assessed 60 treatment naïve HNC patients for CTCs with disease ranging from early to advanced stages, for CTC clusters utilizing spiral CTC enrichment technology. Single CTCs were isolated in 18/60-30% (Ranging from Stage I-IV), CTC clusters in 15/60-25% (exclusively Stage IV) with 3/15-20% of CTC clusters also containing leukocytes. The presence of CTC clusters associated with the development of distant metastatic disease(P = 0.0313). This study demonstrates that CTC clusters are found in locally advanced patients, and this may be an important prognostic marker. In vivo and in vitro studies are warranted to determine the role of these CTC clusters, in particular, whether leukocyte involvement in CTC clusters has clinical relevance.

Immune checkpoint inhibitors have gained traction over the last few years in the treatment of metastatic/recurrent head and neck squamous cell carcinoma (HNSCC) patients. Monoclonal antibodies that block the programmed death 1 (PD-1) receptor and its major ligand, PD-L1, have shown durable responses and low toxicity profiles. There are currently no validated predictive biomarkers to select patients likely to respond to anti-PD-1/PD-L1 therapy to avoid unwanted side effects and to reduce healthcare expenditure. A circulating tumour cell (CTC) PD-L1 assay could be developed as a companion diagnostic tool to potentially predict the efficacy of immune checkpoint blockade treatments., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Head and neck squamous cell carcinomas (HNSCC) are a heterogeneous group of tumours that originate predominantly from the oral cavity, pharynx and larynx. Our aim was to determine whether salivary miRNA expression levels can diagnose these cancer subtypes. Saliva samples were collected from healthy controls (n=113, smoker and non-smokers), HPV-positive (n=54) and HPV-negative (n=47) HNSCC patients. The miRNA expression levels in saliva was quantified using qPCR. The potential of salivary miRNAs to discriminate these groups of patients was evaluated using multiple logistic regression with ROC analysis and a 10-fold cross-validation analysis. Salivary miRNA-9, -127, -134, -191, -222 and -455 were shown to discriminate a control group from a HPV-negative HNSCC patient group with a sensitivity of 60% and a specificity of 94%; whilst salivary miRNA-9,-134, -196b, -210, and -455 were the most parsimonious subset discriminating a control group from a HPV-positive HNSCC group, with a sensitivity of 65% and a specificity of 95%. Furthermore, miRNA-9, -134, -196b, -210 and -455 as a panel, was the most parsimonious subset to discriminate HPV-positive HNSCC patients from HPV-negative HNSCC patients. In addition, the expression levels of miRNA-9, -127, -196a, -196b, -210, -222 and -455 were significantly increased in the saliva collected from early stage HNSCC patients compared to controls. A future multi-centre confirmatory study is warranted to test the diagnostic performance of these salivary miRNA prior to clinical implementation., Competing Interests: CONFLICTS OF INTEREST CP has a PCT patent filed. The other authors disclose no potential conflicts of interest.

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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: Over 26 million people suffer from heart failure (HF) globally. Current diagnosis of HF relies on clinical evaluation, blood assays and imaging techniques. Our aim is to develop a diagnostic assay to detect HF in at risk individuals within the community using human saliva as a medium, potentially leading to a simple, safe early warning system., Methods: Saliva samples were collected from healthy controls (n=36) and HF patients (n=75). Salivary proteome profiles were analysed by Sequential Window Acquisition of All Theoretical fragment ion spectra - Mass Spectrometry (SWATH-MS). A total of 738 proteins were quantified and 177 proteins demonstrated significant differences between HF patients and healthy controls. Candidate biomarkers were chosen based on their abundance and difference between the two cohorts. A multi-protein panel was developed using logistic regression analysis. The diagnostic performance of the multi-protein panel was assessed using receiver operative characteristic curves. The candidate proteins were further confirmed, using western blot analysis, and validated technically, using an independent biological cohort., Results: A group of six proteins were chosen in the discovery phase as potential candidates based on their differences in the abundance between the two cohorts. During the validation phase, two of the proteins were not detected with western blotting and as such were removed. The final panel consists of four proteins with sensitivity of 83.3%, specificity of 62.5% with an area under ROC curve of 0.78 in discriminating healthy controls from NYHA class I/II HF patients, and was validated in a second independent cohort study., Conclusion: Analysis of salivary proteome using SWATH-MS revealed novel HF-specific protein candidates yielding high diagnostic performance. A multi-centre longitudinal clinical trial will be the next step before clinical implementation of this panel., Competing Interests: Competing Interests: XZ, BS and CP are inventors of a PCT patent application Australian provisional patent application number is 2016903387.

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 β-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.

Background: Cytokeratin (CK) intermediate filaments are demonstrated to have enormous potential in regulating cellular motility and cancer progression. There are more than 20 divergent CKs that have been identified, of which CK 8, 17, 18 and 19 are reported to be elevated in the tumour biopsies of head and neck cancer squamous cell carcinoma (HNSCC) patients. However, CK expression profiles in the saliva of HNSCC patients have not been investigated. We aim to investigate the mRNA expression profiles of CKs in saliva collected from healthy controls, HPV-negative and -positive HNSCC patients., Methods: Oral rinse samples were collected from 42 cancer-free healthy controls (age-matched) and patients who have been diagnosed with HPV-negative (n = 20) and -positive (n = 48) HNSCC., Results: Here, we report that the mRNA expression profiles of CKs differed in saliva collected from healthy controls and HNSCC patients. The mRNA expression levels of CK 8 and 18 were significantly elevated in saliva collected from HPV-negative HNSCC patients; whilst, CK 17 and 19 were expressed at a higher mRNA level in saliva collected from HPV-positive HNSCC patients compared to healthy controls. Importantly, receiver operating characteristic (ROC) analysis showed salivary CK 8 and 18 to have superior sensitivity and specificity in discriminating the HPV-negative HNSCC patients from healthy controls (80% and 86%) as well as between HPV-negative and -positive HNSCC patients (75% and 81%)., Conclusion: In summary, we have demonstrated that an aberrant expression of salivary CKs may serve as a potential non-invasive diagnostic biomarker in HNSCC., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Background: Blockade of the PD-1/PD-L1 immune checkpoint pathway is emerging as a promising immunotherapeutic approach for the management and treatment of head and neck cancer patients who do not respond to 1st/2nd line therapy. However, as checkpoint inhibitors are cost intensive, identifying patients who would most likely benefit from anti PD-L1 therapy is required. Developing a non-invasive technique would be of major benefit to the patient and to the health care system., Case Presentation: We report the case of a 56 year old man affected by a supraglottic squamous cell carcinoma (SCC). A CT scan showed a 20 mm right jugulodigastric node and suspicious lung lesions. The lung lesion was biopsied and confirmed to be consistent with SCC. The patient was offered palliative chemotherapy. At the time of presentation, a blood sample was taken for circulating tumour cell (CTC) analysis. The dissemination of cancer was confirmed by the detection of CTCs in the peripheral blood of the patient, measured by the CellSearch System (Janssen Diagnostics). Using marker-independent, low-shear spiral microfluidic technology combined with immunocytochemistry, CTC clusters were found in this patient at the same time point, expressing PD-L1., Conclusion: This report highlights the potential use of CTCs to identify patients which might respond to anti PD-L1 therapy.

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.

Whilst locoregional control of head and neck cancers (HNCs) has improved over the last four decades, long-term survival has remained largely unchanged. A possible reason for this is that the rate of distant metastasis has not changed. Such disseminated disease is reflected in measurable levels of cancer cells in the blood of HNC patients, referred to as circulating tumour cells (CTCs). Numerous marker-independent techniques have been developed for CTC isolation and detection. Recently, microfluidics-based platforms have come to the fore to avoid molecular bias. In this pilot, proof of concept study, we evaluated the use of the spiral microfluidic chip for CTC enrichment and subsequent detection in HNC patients. CTCs were detected in 13/24 (54%) HNC patients, representing both early to late stages of disease. Importantly, in 7/13 CTC-positive patients, CTC clusters were observed. This is the first study to use spiral microfluidics technology for CTC enrichment in HNC.

Saliva is an easily accessible sample and offers practical and noninvasive biomarker solutions as an alternative to blood and urine-based diagnostics. Saliva contains a plethora of biomolecules such as nucleic acids, hormones, proteins, and electrolytes. On the other hand, little is known on the extent to which the biomolecules in saliva vary over time within a given person. This baseline information is crucial for future development of robust saliva-based diagnostics. We have collected unstimulated whole mouth saliva from 20 healthy young men at four times during the day, including before and after a meal. We measured the salivary cortisol, testosterone, C-reactive protein (CRP), stability of genomic DNA (gDNA) and DNA methylation levels of APC, P16 INK4a , and PCQAP in these samples. We found that the salivary CRP, DNA methylation, and CD44 gDNA levels did not vary significantly across four time points (p > 0.05) while the salivary cortisol and testosterone levels significantly varied from the morning collection to the afternoon collection (p < 0.05). Furthermore, salivary cortisol levels were significantly affected by eating (p < 0.05). Our study offers a within-person baseline temporal assessment of several clinically relevant biomolecules and diagnostics, and suggests that salivary cortisol and testosterone levels vary over time in a given day whereas CRP and DNA methylation of tumor suppressor genes and CD44 amplification are stable throughout the day. Future research and clinical applications of salivary biomarkers and diagnostics should take into consideration their temporal variations.