Developing and Validating a Multivariable Prognostic-Predictive Classifier for Treatment Escalation of Oropharyngeal Squamous Cell Carcinoma: The PREDICTR-OPC Study.

PURPOSE: While there are several prognostic classifiers, to date, there are no validated predictive models that inform treatment selection for oropharyngeal squamous cell carcinoma (OPSCC).Our aim was to develop clinical and/or biomarker predictive models for patient outcome and treatment escalation for OPSCC. EXPERIMENTAL DESIGN: We retrospectively collated clinical data and samples from a consecutive cohort of OPSCC cases treated with curative intent at ten secondary care centers in United Kingdom and Poland between 1999 and 2012. We constructed tissue microarrays, which were stained and scored for 10 biomarkers. We then undertook multivariable regression of eight clinical parameters and 10 biomarkers on a development cohort of 600 patients. Models were validated on an independent, retrospectively collected, 385-patient cohort. RESULTS: A total of 985 subjects (median follow-up 5.03 years, range: 4.73-5.21 years) were included. The final biomarker classifier, comprising p16 and survivin immunohistochemistry, high-risk human papillomavirus (HPV) DNA in situ hybridization, and tumor-infiltrating lymphocytes, predicted benefit from combined surgery + adjuvant chemo/radiotherapy over primary chemoradiotherapy in the high-risk group [3-year overall survival (OS) 63.1% vs. 41.1%, respectively, HR = 0.32; 95% confidence interval (CI), 0.16-0.65; P = 0.002], but not in the low-risk group (HR = 0.4; 95% CI, 0.14-1.24; P = 0.114). On further adjustment by propensity scores, the adjusted HR in the high-risk group was 0.34, 95% CI = 0.17-0.67, P = 0.002, and in the low-risk group HR was 0.5, 95% CI = 0.1-2.38, P = 0.384. The concordance index was 0.73. CONCLUSIONS: We have developed a prognostic classifier, which also appears to demonstrate moderate predictive ability. External validation in a prospective setting is now underway to confirm this and prepare for clinical adoption.

Feasibility of mass cytometry proteomic characterisation of circulating tumour cells in head and neck squamous cell carcinoma for deep phenotyping.

BACKGROUND: Circulating tumour cells (CTCs) are a potential cancer biomarker, but current methods of CTC analysis at single-cell resolution are limited. Here, we describe high-dimensional single-cell mass cytometry proteomic analysis of CTCs in HNSCC. METHODS: Parsortix microfluidic-enriched CTCs from 14 treatment-naïve HNSCC patients were analysed by mass cytometry analysis using 41 antibodies. Immune cell lineage, epithelial-mesenchymal transition (EMT), stemness, proliferation and immune checkpoint expression was assessed alongside phosphorylation status of multiple signalling proteins. Patient-matched tumour gene expression and CTC EMT profiles were compared. Standard bulk CTC RNAseq was performed as a baseline comparator to assess mass cytometry data. RESULTS: CTCs were detected in 13/14 patients with CTC counts of 2-24 CTCs/ml blood. Unsupervised clustering separated CTCs into epithelial, early EMT and advanced EMT groups that differed in signalling pathway activation state. Patient-specific CTC cluster patterns separated into immune checkpoint low and high groups. Patient tumour and CTC EMT profiles differed. Mass cytometry outperformed bulk RNAseq to detect CTCs and characterise cell phenotype. DISCUSSION: We demonstrate mass cytometry allows high-plex proteomic characterisation of CTCs at single-cell resolution and identify common CTC sub-groups with potential for novel biomarker development and immune checkpoint inhibitor treatment stratification.

Lead Time to Recurrence After Posttreatment Plasma and Saliva HPV DNA Testing in Patients With Low-Risk HPV Oropharynx Cancer.

Importance: Head and neck squamous cell carcinoma is a highly lethal cancer that is often associated with human papillomavirus (HPV). Recent studies have shown promise in the use of HPV DNA detection in salivary rinses and plasma as a factor associated with a future diagnosis of HPV-positive oropharynx cancer (HPVOPC). However, the use of plasma and salivary HPV DNA detection in defining risk for recurrence in the context of a prospective, phase 3, clinical trial coupled with standardized clinical surveillance has not been reported. Objective: To identify patients with low-risk HPVOPC at risk for recurrence by detection of HPV16 DNA in plasma and salivary rinses. Design, Setting, and Participants: In this cohort study, 233 low-risk patients were recruited from 32 head and neck treatment centers in Ireland (1 [3.1%]), the Netherlands (1 [3.1%]), and the UK (30 [93.8%]) as part of the DE-ESCALATE HPV trial, an open-label, phase 3 randomized clinical trial examining treatment with cetuximab vs cisplatin for HPVOPC. Patients were assayed for the presence of HPV16 DNA in plasma and salivary rinse via a quantitative polymerase chain reaction-based assay. Main Outcomes and Measures: Assay results were associated with risk of recurrence and lead time from HPV16 DNA detection to recurrence. Results: Of 233 patients, 45 (19.3%) were women, and the mean (SD) age was 57.01 (8.45) years. A total 1040 salivary or blood samples were collected during the course of the study. With a median follow-up of 760 days, the sensitivity and specificity of combined plasma and salivary rinse HPV DNA assays for detecting recurrence were 65% and 87%, respectively. There was a median lead time of positive test to event/recurrence date of 19 days (range, 0-536 days) and mean (SD) of 122 (169.8) days. Conclusion and Relevance: The results of this cohort study suggest that in the setting of a randomized, prospective, phase 3 trial for low-risk patients with HPVOPC, posttreatment presence of HPV DNA in plasma and salivary rinses is associated with recurrence; a lead time between test positivity and clinical recurrence offers a potential opportunity for earlier detection of recurrence.

Prognostic implications of p16 and HPV discordance in oropharyngeal cancer (HNCIG-EPIC-OPC): a multicentre, multinational, individual patient data analysis.

BACKGROUND: p16INK4a (p16) immunohistochemistry is the most widely used biomarker assay for inferring HPV causation in oropharyngeal cancer in clinical and trial settings. However, discordance exists between p16 and HPV DNA or RNA status in some patients with oropharyngeal cancer. We aimed to clearly quantify the extent of discordance, and its prognostic implications. METHODS: In this multicentre, multinational individual patient data analysis, we did a literature search in PubMed and Cochrane database for systematic reviews and original studies published in English between Jan 1, 1970, and Sept 30, 2022. We included retrospective series and prospective cohorts of consecutively recruited patients previously analysed in individual studies with minimum cohort size of 100 patients with primary squamous cell carcinoma of the oropharynx. Patient inclusion criteria were diagnosis with a primary squamous cell carcinoma of oropharyngeal cancer; data on p16 immunohistochemistry and on HPV testing; information on age, sex, tobacco, and alcohol use; staging by TNM 7th edition; information on treatments received; and data on clinical outcomes and follow-up (date of last follow-up if alive, date of recurrence or metastasis, and date and cause of death). There were no limits on age or performance status. The primary outcomes were the proportion of patients of the overall cohort who showed the different p16 and HPV result combinations, as well as 5-year overall survival and 5-year disease-free survival. Patients with recurrent or metastatic disease or who were treated palliatively were excluded from overall survival and disease-free survival analyses. Multivariable analysis models were used to calculate adjusted hazard ratios (aHR) for different p16 and HPV testing methods for overall survival, adjusted for prespecified confounding factors. FINDINGS: Our search returned 13 eligible studies that provided individual data for 13 cohorts of patients with oropharyngeal cancer from the UK, Canada, Denmark, Sweden, France, Germany, the Netherlands, Switzerland, and Spain. 7895 patients with oropharyngeal cancer were assessed for eligibility. 241 were excluded before analysis, and 7654 were eligible for p16 and HPV analysis. 5714 (74·7%) of 7654 patients were male and 1940 (25·3%) were female. Ethnicity data were not reported. 3805 patients were p16-positive, 415 (10·9%) of whom were HPV-negative. This proportion differed significantly by geographical region and was highest in the areas with lowest HPV-attributable fractions (r=-0·744, p=0·0035). The proportion of patients with p16+/HPV- oropharyngeal cancer was highest in subsites outside the tonsil and base of tongue (29·7% vs 9·0%, p<0·0001). 5-year overall survival was 81·1% (95% CI 79·5-82·7) for p16+/HPV+, 40·4% (38·6-42·4) for p16-/HPV-, 53·2% (46·6-60·8) for p16-/HPV+, and 54·7% (49·2-60·9) for p16+/HPV-. 5-year disease-free survival was 84·3% (95% CI 82·9-85·7) for p16+/HPV+, 60·8% (58·8-62·9) for p16-/HPV-; 71·1% (64·7-78·2) for p16-/HPV+, and 67·9% (62·5-73·7) for p16+/HPV-. Results were similar across all European sub-regions, but there were insufficient numbers of discordant patients from North America to draw conclusions in this cohort. INTERPRETATION: Patients with discordant oropharyngeal cancer (p16-/HPV+ or p16+/HPV-) had a significantly worse prognosis than patients with p16+/HPV+ oropharyngeal cancer, and a significantly better prognosis than patients with p16-/HPV- oropharyngeal cancer. Along with routine p16 immunohistochemistry, HPV testing should be mandated for clinical trials for all patients (or at least following a positive p16 test), and is recommended where HPV status might influence patient care, especially in areas with low HPV-attributable fractions. FUNDING: European Regional Development Fund, Generalitat de Catalunya, National Institute for Health Research (NIHR) UK, Cancer Research UK, Medical Research Council UK, and The Swedish Cancer Foundation and the Stockholm Cancer Society.

Characterizing the epithelial-mesenchymal transition status of circulating tumor cells in head and neck squamous cell carcinoma.

BACKGROUND: Circulating tumor cells (CTCs), in particular those undergoing an epithelial-mesenchymal transition (EMT), are a promising source of biomarkers in head and neck squamous cell carcinoma (HNSCC). Our aim was to validate a protocol using microfluidic enrichment (Parsortix platform) with flow-cytometry CTC characterization. METHOD: Blood samples from 20 treatment naïve HNSCC patients underwent Parsortix enrichment and flow cytometry analysis to quantify CTCs and identify epithelial or EMT subgroups-correlated to clinical outcomes and EMT gene-expression in tumor tissue. RESULTS: CTCs were detected in 65% of patients (mean count 4 CTCs/ml). CTCs correlated with advanced disease (p = 0.0121), but not T or N classification. Epithelial or EMT CTCs did not correlate with progression-free or overall survival. Tumor mesenchymal gene-expression did not correlate with CTC EMT expression (p = 0.347). DISCUSSION: Microfluidic enrichment and flow cytometry successfully characterizes EMT CTCs in HNSCC. The lack of association between tumor and CTC EMT profile suggests CTCs may undergo an adaptive EMT in response to stimuli within the circulation.

A digital score of tumour-associated stroma infiltrating lymphocytes predicts survival in head and neck squamous cell carcinoma.

The infiltration of T-lymphocytes in the stroma and tumour is an indication of an effective immune response against the tumour, resulting in better survival. In this study, our aim was to explore the prognostic significance of tumour-associated stroma infiltrating lymphocytes (TASILs) in head and neck squamous cell carcinoma (HNSCC) through an AI-based automated method. A deep learning-based automated method was employed to segment tumour, tumour-associated stroma, and lymphocytes in digitally scanned whole slide images of HNSCC tissue slides. The spatial patterns of lymphocytes and tumour-associated stroma were digitally quantified to compute the tumour-associated stroma infiltrating lymphocytes score (TASIL-score). Finally, the prognostic significance of the TASIL-score for disease-specific and disease-free survival was investigated using the Cox proportional hazard analysis. Three different cohorts of haematoxylin and eosin (H&E)-stained tissue slides of HNSCC cases (n = 537 in total) were studied, including publicly available TCGA head and neck cancer cases. The TASIL-score carries prognostic significance (p = 0.002) for disease-specific survival of HNSCC patients. The TASIL-score also shows a better separation between low- and high-risk patients compared with the manual tumour-infiltrating lymphocytes (TILs) scoring by pathologists for both disease-specific and disease-free survival. A positive correlation of TASIL-score with molecular estimates of CD8+ T cells was also found, which is in line with existing findings. To the best of our knowledge, this is the first study to automate the quantification of TASILs from routine H&E slides of head and neck cancer. Our TASIL-score-based findings are aligned with the clinical knowledge, with the added advantages of objectivity, reproducibility, and strong prognostic value. Although we validated our method on three different cohorts (n = 537 cases in total), a comprehensive evaluation on large multicentric cohorts is required before the proposed digital score can be adopted in clinical practice. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Immediate Sample Fixation Increases Circulating Tumour Cell (CTC) Capture and Preserves Phenotype in Head and Neck Squamous Cell Carcinoma: Towards a Standardised Approach to Microfluidic CTC Biomarker Discovery.

INTRODUCTION: Research demonstrates strong evidence that circulating tumour cells (CTCs) can provide diagnostic and/or prognostic biomarkers in head and neck squamous cell carcinoma (HNSCC) and a potential tool for therapeutic stratification. However, the question still remains as to the optimum method of CTC enrichment and how this can be translated into clinical practice. We aimed to evaluate the Parsortix microfluidic device for CTC enrichment and characterisation in HNSCC, seeking to optimise a sample collection and processing protocol that preserves CTC integrity and phenotype. METHOD: Spiking experiments of the FaDu and SCC040 HNSCC cell lines were used to determine the Parsortix capture rate of rare "CTC-like" cells. Capture rates of cancer cells spiked into EDTA blood collections tubes (BCTs) were compared to the Transfix fixative BCT and Cytodelics whole blood freezing protocol. The Lexogen Quantseq library preparation was used to profile gene expression of unfixed cells before and after microfluidic enrichment and enriched cell line spiked Transfix blood samples. An antibody panel was optimised to enable immunofluorescence microscopy CTC detection in HNSCC patient Transfix blood samples, using epithelial (EpCAM) and mesenchymal (N-cadherin) CTC markers. RESULTS: Across a spiked cell concentration range of 9-129 cells/mL, Parsortix demonstrated a mean cell capture rate of 53.5% for unfixed cells, with no significant relationship between spiked cell concentration and capture rate. Samples preserved in Transfix BCTs demonstrated significantly increased capture rates at 0 h (time to processing) compared to EDTA BCTs (65.3% vs. 51.0%). Capture rates in Transfix BCTs were maintained at 24 h and 72 h timepoints, but dropped significantly in EDTA BCTs. Gene expression profiling revealed that microfluidic enrichment of unfixed cell lines caused downregulation of RNA processing/binding gene pathways and upregulation of genes involved in cell injury, apoptosis and oxidative stress. RNA was successfully extracted and sequenced from Transfix preserved cells enriched using Parsortix, demonstrating epithelial specific transcripts from spiked cells. In a proof-of-concept cohort of four patients with advanced HNSCC, CTCs were successfully identified and visualised with epithelial and epithelial-mesenchymal phenotypes. CONCLUSION: We have optimised a protocol for detection of CTCs in HNSCC with the Parsortix microfluidic device, using Transfix BCTs. We report a significant benefit, both in terms of cell capture rates and preserving cell phenotype, for using a fixative BCT- particularly if samples are stored before processing. In the design of large cohort multi-site clinical trials, such data are of paramount importance.

Upper Aerodigestive Tract Squamous Cell Carcinomas Show Distinct Overall DNA Methylation Profiles and Different Molecular Mechanisms behind WNT Signaling Disruption.

Upper aerodigestive tract (UADT) tumors present different biological behavior and prognosis, suggesting specific molecular mechanisms underlying their development. However, they are rarely considered as single entities (particularly head and neck subsites) and share the most common genetic alterations. Therefore, there is a need for a better understanding of the global DNA methylation differences among UADT tumors. We performed a genome-wide DNA methylation analysis of esophageal (ESCC), laryngeal (LSCC), oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinomas, and their non-tumor counterparts. The unsupervised analysis showed that non-tumor tissues present markedly distinct DNA methylation profiles, while tumors are highly heterogeneous. Hypomethylation was more frequent in LSCC and OPSCC, while ESCC and OSCC presented mostly hypermethylation, with the latter showing a CpG island overrepresentation. Differentially methylated regions affected genes in 127 signaling pathways, with only 3.1% of these being common among different tumor subsites, but with different genes affected. The WNT signaling pathway, known to be dysregulated in different epithelial tumors, is a frequent hit for DNA methylation and gene expression alterations in ESCC and OPSCC, but mostly for genetic alterations in LSCC and OSCC. UADT tumor subsites present differences in genome-wide methylation regarding their profile, intensity, genomic regions and signaling pathways affected.

Mitochondrial Succinate Metabolism and Reactive Oxygen Species Are Important but Not Essential for Eliciting Carotid Body and Ventilatory Responses to Hypoxia in the Rat.

Reflex increases in breathing in response to acute hypoxia are dependent on activation of the carotid body (CB)-A specialised peripheral chemoreceptor. Central to CB O2-sensing is their unique mitochondria but the link between mitochondrial inhibition and cellular stimulation is unresolved. The objective of this study was to evaluate if ex vivo intact CB nerve activity and in vivo whole body ventilatory responses to hypoxia were modified by alterations in succinate metabolism and mitochondrial ROS (mitoROS) generation in the rat. Application of diethyl succinate (DESucc) caused concentration-dependent increases in chemoafferent frequency measuring approximately 10-30% of that induced by severe hypoxia. Inhibition of mitochondrial succinate metabolism by dimethyl malonate (DMM) evoked basal excitation and attenuated the rise in chemoafferent activity in hypoxia. However, approximately 50% of the response to hypoxia was preserved. MitoTEMPO (MitoT) and 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SKQ1) (mitochondrial antioxidants) decreased chemoafferent activity in hypoxia by approximately 20-50%. In awake animals, MitoT and SKQ1 attenuated the rise in respiratory frequency during hypoxia, and SKQ1 also significantly blunted the overall hypoxic ventilatory response (HVR) by approximately 20%. Thus, whilst the data support a role for succinate and mitoROS in CB and whole body O2-sensing in the rat, they are not the sole mediators. Treatment of the CB with mitochondrial selective antioxidants may offer a new approach for treating CB-related cardiovascular-respiratory disorders.

Lack of predictive tools for conventional and targeted cancer therapy: Barriers to biomarker development and clinical translation.

Predictive tools, utilising biomarkers, aim to objectively assessthe potentialresponse toa particular clinical intervention in order to direct treatment.Conventional cancer therapy remains poorly served by predictive biomarkers, despite being the mainstay of treatment for most patients. In contrast, targeted therapy benefits from a clearly defined protein target for potential biomarker assessment. We discuss potential data sources of predictive biomarkers for conventional and targeted therapy, including patient clinical data andmulti-omicbiomarkers (genomic, transcriptomic and protein expression).Key examples, either clinically adopted or demonstrating promise for clinical translation, are highlighted. Following this, we provide an outline of potential barriers to predictive biomarker development; broadly discussing themes of approaches to translational research and study/trial design, and the impact of cellular and molecular tumor heterogeneity. Future avenues of research are also highlighted.

Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA.

Abnormal CpG methylation in cancer is ubiquitous and generally detected in tumour specimens using a variety of techniques at a resolution encompassing single CpG loci to genome wide coverage. Analysis of samples with very low DNA inputs, such as formalin fixed (FFPE) biopsy specimens from clinical trials or circulating tumour DNA is challenging at the genome-wide level because of lack of available input. We present the results of low input experiments into the Illumina Infinium HD methylation assay on FFPE specimens and ctDNA samples. METHODS: For all experiments, the Infinium HD assay for methylation was used. In total, forty-eight FFPE specimens were used at varying concentrations (lowest input 50 ng); eighteen blood derived specimens (lowest input 10 ng) and six matched ctDNA input (lowest input 10 ng)/fresh tumour specimens (lowest input 250 ng) were processed. Downstream analysis was performed in R/Bioconductor for quality control metrics and differential methylation analysis as well as copy number calls. RESULTS: Correlation coefficients for CpG methylation were high at the probe level averaged R2 = 0.99 for blood derived samples and R2 > 0.96 for the FFPE samples. When matched ctDNA/fresh tumour samples were compared, R2 > 0.91 between the two. Results of differential methylation analysis did not vary significantly by DNA input in either the blood or FFPE groups. There were differences seen in the ctDNA group as compared to their paired tumour sample, possibly because of enrichment for tumour material without contaminating normal. Copy number variants observed in the tumour were generally also seen in the paired ctDNA sample with good concordance via DQ plot. CONCLUSIONS: The Illumina Infinium HD methylation assay can robustly detect methylation across a range of sample types, including ctDNA, down to an input of 10 ng. It can also reliably detect oncogenic methylation changes and copy number variants in ctDNA. These findings demonstrate that these samples can now be accessed by methylation array technology, allowing analysis of these important sample types.

The anti-tumour activity of DNA methylation inhibitor 5-aza-2'-deoxycytidine is enhanced by the common analgesic paracetamol through induction of oxidative stress.

The DNA demethylating agent 5-aza-2'-deoxycytidine (DAC, decitabine) has anti-cancer therapeutic potential, but its clinical efficacy is hindered by DNA damage-related side effects and its use in solid tumours is debated. Here we describe how paracetamol augments the effects of DAC on cancer cell proliferation and differentiation, without enhancing DNA damage. Firstly, DAC specifically upregulates cyclooxygenase-2-prostaglandin E2 pathway, inadvertently providing cancer cells with survival potential, while the addition of paracetamol offsets this effect. Secondly, in the presence of paracetamol, DAC treatment leads to glutathione depletion and finally to accumulation of ROS and/or mitochondrial superoxide, both of which have the potential to restrict tumour growth. The benefits of combined treatment are demonstrated here in head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukaemia cell lines, further corroborated in a HNSCC xenograft mouse model and through mining of publicly available DAC and paracetamol responses. The sensitizing effect of paracetamol supplementation is specific to DAC but not its analogue 5-azacitidine. In summary, the addition of paracetamol could allow for DAC dose reduction, widening its clinical usability and providing a strong rationale for consideration in cancer therapy.

Circulating Tumour Cell Expression of Immune Markers as Prognostic and Therapeutic Biomarkers in Head and Neck Squamous Cell Carcinoma: A Systematic Review and Meta-Analysis.

Rates of loco-regional recurrence and distant metastasis remain high among head and neck squamous cell carcinoma (HNSCC) patients, despite advancing cancer treatment modalities and therapeutic agents. One area that has generated considerable interest is the immune landscape of the tumour, heralding a wave of immune checkpoint inhibitors with notable efficacy in recurrent/metastatic HNSCC patients. However, HNSCC remains poorly served by biomarkers that can direct treatment in a personalised fashion to target the tumour heterogeneity seen between patients. Detection and analysis of circulating tumour cells (CTCs) in HNSCC has provided a previously unseen view of the metastasis forming cells that are potentially contributing to poor clinical outcomes. In particular, identifying CTC expression of phenotypic and druggable protein markers has allowed CTC sub-populations to be defined that hold prognostic value or are potential therapeutic targets themselves. The aim of this systematic review was to examine the role of CTC immune-marker expression as prognostic/therapeutic biomarkers in HNSCC by evaluating progress to date and discussing areas for future research. Our results highlight how few studies have been able to demonstrate prognostic significance of immune-marker expression in CTCs. As expected, the immune checkpoint PD-L1 was the most widely investigated marker. However, no studies evaluated CTC target immune marker expression in immunotherapy cohorts. Despite these findings, the data presented demonstrate promise that CTCs may be a source of future biomarkers for immunotherapy and will provide valuable information regarding the potential immune evasion of these metastasis forming cells.

G-Protein-Coupled Receptor (GPCR) Signaling in the Carotid Body: Roles in Hypoxia and Cardiovascular and Respiratory Disease.

The carotid body (CB) is an important organ located at the carotid bifurcation that constantly monitors the blood supplying the brain. During hypoxia, the CB immediately triggers an alarm in the form of nerve impulses sent to the brain. This activates protective reflexes including hyperventilation, tachycardia and vasoconstriction, to ensure blood and oxygen delivery to the brain and vital organs. However, in certain conditions, including obstructive sleep apnea, heart failure and essential/spontaneous hypertension, the CB becomes hyperactive, promoting neurogenic hypertension and arrhythmia. G-protein-coupled receptors (GPCRs) are very highly expressed in the CB and have key roles in mediating baseline CB activity and hypoxic sensitivity. Here, we provide a brief overview of the numerous GPCRs that are expressed in the CB, their mechanism of action and downstream effects. Furthermore, we will address how these GPCRs and signaling pathways may contribute to CB hyperactivity and cardiovascular and respiratory disease. GPCRs are a major target for drug discovery development. This information highlights specific GPCRs that could be targeted by novel or existing drugs to enable more personalized treatment of CB-mediated cardiovascular and respiratory disease.

Acoustofluidic Salivary Exosome Isolation: A Liquid Biopsy Compatible Approach for Human Papillomavirus-Associated Oropharyngeal Cancer Detection.

Previous efforts to evaluate the detection of human papilloma viral (HPV) DNA in whole saliva as a diagnostic measure for HPV-associated oropharyngeal cancer (HPV-OPC) have not shown sufficient clinical performance. We hypothesize that salivary exosomes are packaged with HPV-associated biomarkers, and efficient enrichment of salivary exosomes through isolation can enhance diagnostic and prognostic performance for HPV-OPC. In this study, an acoustofluidic (the fusion of acoustics and microfluidics) platform was developed to perform size-based isolation of salivary exosomes. These data showed that this platform is capable of consistently isolating exosomes from saliva samples, regardless of viscosity variation and collection method. Compared with the current gold standard, differential centrifugation, droplet digital RT-PCR analysis showed that the average yield of salivary exosomal small RNA from the acoustofluidic platform is 15 times higher. With this high-yield exosome isolation platform, we show that HPV16 DNA could be detected in isolated exosomes from the saliva of HPV-associated OPC patients at 80% concordance with tissues/biopsies positive for HPV16. Overall, these data demonstrated that the acoustofluidic platform can achieve high-purity and high-yield salivary exosome isolation for downstream salivary exosome-based liquid biopsy applications. Additionally, HPV16 DNA sequences in HPV-OPC patients are packaged in salivary exosomes and their isolation will enhance the detection of HPV16 DNA.

Repurposed quinacrine synergizes with cisplatin, reducing the effective dose required for treatment of head and neck squamous cell carcinoma.

Despite highly toxic treatments, head and neck squamous cell carcinoma (HNSCC) have poor outcomes. There is an unmet need for more effective, less toxic therapies. Repurposing of clinically-approved drugs, with known safety profiles, may provide a time- and cost-effective approach to address this need. We have developed the AcceleraTED platform to repurpose drugs for HNSCC treatment; using in vitro assays (cell viability, clonogenic survival, apoptosis) and in vivo models (xenograft tumors in NOD/SCID/gamma mice). Screening a library of clinically-approved drugs identified the anti-malarial agent quinacrine as a candidate, which significantly reduced viability in a concentration dependent manner in five HNSCC cell lines (IC50 0.63-1.85 µM) and in six primary HNSCC samples (IC50 ~2 µM). Decreased clonogenic survival, increased apoptosis and accumulation of LC3-II (indicating altered autophagy) were also observed. Effects were additional to those resulting from standard treatments (cisplatin +/- irradiation) alone. In vivo, daily treatment with 100 mg/kg oral quinacrine plus cisplatin significantly inhibited tumor outgrowth, extending median time to reach maximum tumor volume from 20 to 32 days (p < 0.0001) versus control, and from 28 to 32 days versus 2 mg/kg cisplatin alone. Importantly, combination therapy enabled the dose of cisplatin to be halved to 1 mg/kg, whilst maintaining the same impairment of tumor growth. Treatment was well tolerated; murine plasma levels reached a steady concentration of 0.5 µg/mL, comparable to levels achievable and tolerated in humans. Consequently, due to its favorable toxicity profile and proven safety, quinacrine may be particularly useful in reducing cisplatin dose, especially in frail and older patients; warranting a clinical trial.

Circulating Tumour Cell Biomarkers in Head and Neck Cancer: Current Progress and Future Prospects.

Head and neck cancer (HNC) continues to carry a significant burden of disease both for patients and health services. Facilitating biomarker-led treatment decisions is critical to improve outcomes in this group and deliver therapy tailored to the individual tumour biological profile. One solution to develop such biomarkers is a liquid biopsy analysing circulating tumour cells (CTCs)-providing a non-invasive and dynamic assessment of tumour specific alterations in 'real-time'. A major obstacle to implementing such a test is the standardisation of CTC isolation methods and subsequent down-stream analysis. Several options are available, with a recent shift in vogue from positive-selection marker-dependent isolation systems to marker-independent negative-selection techniques. HNC single-CTC characterisation, including single-cell sequencing, to identify actionable mutations and gene-expression signatures has the potential to both guide the understanding of patient tumour heterogeneity and support the adoption of personalised medicine strategies. Microfluidic approaches for isolating CTCs and cell clusters are emerging as novel technologies which can be incorporated with computational platforms to complement current diagnostic and prognostic strategies. We review the current literature to assess progress regarding CTC biomarkers in HNC and potential avenues for future translational research and clinical implementation.

Development and Validation of a Combined Hypoxia and Immune Prognostic Classifier for Head and Neck Cancer.

PURPOSE: Intratumoral hypoxia and immunity have been correlated with patient outcome in various tumor settings. However, these factors are not currently considered for treatment selection in head and neck cancer (HNC) due to lack of validated biomarkers. Here we sought to develop a hypoxia-immune classifier with potential application in patient prognostication and prediction of response to targeted therapy. EXPERIMENTAL DESIGN: A 54-gene hypoxia-immune signature was constructed on the basis of literature review. Gene expression was analyzed in silico using the The Cancer Genome Atlas (TCGA) HNC dataset (n = 275) and validated using two independent cohorts (n = 130 and 123). IHC was used to investigate the utility of a simplified protein signature. The spatial distribution of hypoxia and immune markers was examined using multiplex immunofluorescence staining. RESULTS: Unsupervised hierarchical clustering of TCGA dataset (development cohort) identified three patient subgroups with distinct hypoxia-immune phenotypes and survival profiles: hypoxialow/immunehigh, hypoxiahigh/immunelow, and mixed, with 5-year overall survival (OS) rates of 71%, 51%, and 49%, respectively (P = 0.0015). The prognostic relevance of the hypoxia-immune gene signature was replicated in two independent validation cohorts. Only PD-L1 and intratumoral CD3 protein expression were associated with improved OS on multivariate analysis. Hypoxialow/immunehigh and hypoxiahigh/immunelow tumors were overrepresented in "inflamed" and "immune-desert" microenvironmental profiles, respectively. Multiplex staining demonstrated an inverse correlation between CA-IX expression and prevalence of intratumoral CD3+ T cells (r = -0.5464; P = 0.0377), further corroborating the transcription-based classification. CONCLUSIONS: We developed and validated a hypoxia-immune prognostic transcriptional classifier, which may have clinical application to guide the use of hypoxia modification and targeted immunotherapies for the treatment of HNC.

Human Papillomavirus (HPV) Vaccine Effectiveness and Potential Herd Immunity for Reducing Oncogenic Oropharyngeal HPV-16 Prevalence in the United Kingdom: A Cross-sectional Study.

BACKGROUND: Oropharyngeal cancer incidence is rapidly rising due to human papillomavirus (HPV) type 16 infection. The dearth of data on effectiveness of national female-only vaccination programs in preventing oral HPV infection and potential herd immunity in unvaccinated males has resulted in considerable controversy regarding the need to vaccinate males, especially in countries with high female vaccination coverage. METHODS: Subjects aged 0-65 years undergoing tonsillectomy for nonmalignant indications were recruited in 6 hospitals in the United Kingdom. Oral samples were collected as follows: oral rinse, tongue base, and pharyngeal wall brushes, then tonsil tissue (tonsillectomy). Vaccination data were obtained from regional health authorities. All samples were centrally tested for HPV DNA by polymerase chain reaction. RESULTS: Of 940 subjects, 243 females and 69 males were aged 12-24 years (median age, 18.6 years), with 189 (78%) females and no males vaccinated against HPV. Overall, oropharyngeal HPV-16 prevalence was significantly lower in vaccinated versus unvaccinated females (0.5% vs 5.6%, P = .04). In contrast, prevalence of any oropharyngeal HPV type was similar in vaccinated and unvaccinated females (19% vs 20%, P = .76). Oropharyngeal HPV-16 prevalence in unvaccinated males was similar to vaccinated females (0% vs 0.5%, P > .99), and lower than unvaccinated females (0% vs 5.6%, P = .08). CONCLUSIONS: Our findings indicate that the UK female-only vaccination program is associated with significant reductions in oropharyngeal HPV-16 infections. These are also the first data to suggest potential herd immunity from female-only vaccination against oropharyngeal HPV infection in contemporaneously aged males.

The Clinical Utility of Cell-Free DNA Measurement in Differentiated Thyroid Cancer: A Systematic Review.

BACKGROUND: Cell-free DNA (cfDNA) can be detected in the circulation of healthy individuals, but is found in higher concentrations in cancer patients. Furthermore, mutations in tumor cells can be identified in circulating DNA fragments. This has been the subject of significant interest in the field of cancer research, but little has been published in thyroid cancer. OBJECTIVES: To assess all available evidence on the use of circulating cfDNA in the diagnosis, management and surveillance of patients with differentiated thyroid cancer, and collate it into a systematic review to guide future research. METHODS: A comprehensive literature search on the measurement of cfDNA in thyroid cancer was undertaken, and results from relevant studies collated into a systematic review. RESULTS: Nine studies were identified, with varying methodologies and findings. Key techniques and findings are summarized. CONCLUSION: There is limited but promising evidence that somatic mutations in thyroid cancer can be detected in circulating cfDNA and are associated with more advanced disease. Further research is required to develop a clinically useful tool based on cfDNA to improve the management of thyroid cancers.