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A set of four salivary miRNAs that can be used in discriminating healthy controls from the early stage (Stages I&II) of HPV-negative HNSCC patients. Significant differences are *P<0.05 and ***P<0.001.
Source publication
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-posi...
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Citations
... For example, salivary miR-122, miR-124, miR-205 and miR-146a are potential differentiators between HPV-positive and HPV-negative cancers [69]. Other studies aimed at differentiating HPV-positive and HPV-negative patients reported the discriminatory value of salivary miR-9, miR-134, miR-196b, miR-210 and miR-455 [70], as well as salivary miR-486-5p and miR-20-5p isolated in exosomes of both head and neck cancers and HPV-positive/p16 cervical cancers [71,72]. These data provide clear evidence for the existence of novel circulating molecules that allow to discriminate between HPVassociated tumors. ...
... For example, salivary miR-122, miR-124, miR-205 and miR-146a are potential differentiators between HPV-positive and HPV-negative cancers [69]. Other studies aimed at differentiating HPV-positive and HPV-negative patients reported the discriminatory value of salivary miR-9, miR-134, miR-196b, miR-210 and miR-455 [70], as well as salivary miR-486-5p and miR-20-5p isolated in exosomes of both head and neck cancers and HPVpositive/p16 cervical cancers [71,72]. These data provide clear evidence for the existence of novel circulating molecules that allow to discriminate between HPV-associated tumors. ...
Human papillomavirus (HPV)-positive Head and Neck Squamous Cell Carcinomas (HNSCC) comprise a particular cancer entity traditionally associated with better clinical outcomes. Around 25% of HNSCC are HPV positive, HPV16 being the most prevalent type. Nevertheless, close to 30% of the HPV-positive patients have an unfavorable prognosis, revealing that this type of tumor exhibits great heterogeneity leading to different clinical behaviors. Efforts have been made to identify RNA molecules with prognostic value associated with the clinical outcome of patients with HPV-positive HNSCC, with the aim of identifying patients at high risk of metastasis, disease recurrence, and poor survival, who would require closer clinical follow-up and timely intervention. Moreover, the molecular identification of those HPV-positive HNSCC patients with good prognosis will allow the implementation of de-escalating therapeutic strategies, aiming to reduce side effects, resulting in a better quality of life. This review compiles a series of recent studies addressing different methodological and conceptual approaches aimed at searching for potential gene expression-based biomarkers associated with the prognosis of patients with HPV-positive HNSCC.
... Extraction of miRNAs was performed as previously reported, 300 µL of saliva sample was used for isolation. 18,42 Tissue samples were homogenised using a sterilised mortar and pestle. Samples were crushed into a fine powder and mixed with 1.5 mL of Qiazol (Qiagen) and extraction of miRNAs was followed as for saliva samples. ...
Oral cancer (OC) is the most common form of head and neck cancer. Despite the high incidence and unfavourable patient outcomes, currently, there are no biomarkers for the early detection of OC. This study aims to discover, develop, and validate a novel saliva-based microRNA signature for early diagnosis and prediction of OC risk in oral potentially malignant disorders (OPMD). The Cancer Genome Atlas (TCGA) miRNA sequencing data and small RNA sequencing data of saliva samples were used to discover differentially expressed miRNAs. Identified miRNAs were validated in saliva samples of OC ( n = 50), OPMD ( n = 52), and controls ( n = 60) using quantitative real-time PCR. Eight differentially expressed miRNAs (miR-7-5p, miR-10b-5p, miR-182-5p, miR-215-5p, miR-431-5p, miR-486-3p, miR-3614-5p, and miR-4707-3p) were identified in the discovery phase and were validated. The efficiency of our eight-miRNA signature to discriminate OC and controls was: area under curve (AUC): 0.954, sensitivity: 86%, specificity: 90%, positive predictive value (PPV): 87.8% and negative predictive value (NPV): 88.5% whereas between OC and OPMD was: AUC: 0.911, sensitivity: 90%, specificity: 82.7%, PPV: 74.2% and NPV: 89.6%. We have developed a risk probability score to predict the presence or risk of OC in OPMD patients. We established a salivary miRNA signature that can aid in diagnosing and predicting OC, revolutionising the management of patients with OPMD. Together, our results shed new light on the management of OC by salivary miRNAs to the clinical utility of using miRNAs derived from saliva samples.
... The NucleoSpin miRNA isolation kit (Macherey-Nagel) was used to isolate miRNA from saliva and tissue samples. Extraction of miRNAs was performed as previously reported, 300µL of saliva sample was used for isolation 16,39 . Tissue samples were homogenized using a sterilized mortar and pestle. ...
Oral cancer (OC) is the most common form of head and neck cancer. Despite the high incidence and unfavourable patient outcomes, currently, there are no biomarkers for the early detection of OC. This study aims to discover, develop, and validate a novel saliva-based microRNA signature for early diagnosis and prediction of OC risk in oral potentially malignant disorders (OPMD).
The Cancer Genome Atlas (TCGA) miRNA sequencing data and small RNA sequencing data of saliva samples were used to discover differentially expressed miRNAs. Identified miRNAs were validated in saliva samples of OC (n=50), OPMD (n=52), and controls (n=60) using quantitative real-time PCR.
Eight differentially expressed miRNAs (miR-7-5p, miR-10b-5p, miR-182-5p, miR-215-5p, miR-431-5p, miR-486-3p, miR-3614-5p, and miR-4707-3p) were identified in the discovery phase and were validated. The efficiency of our eight-miRNA signature to discriminate OC and controls was: Area Under Curve (AUC): 0.954, sensitivity: 86%, specificity: 90%, positive predictive value (PPV): 87.8% and negative predictive value (NPV): 88.5% whereas between OC and OPMD was: AUC: 0.911, sensitivity: 90%, specificity: 82.7%, PPV: 74.2% and NPV: 89.6%. We have developed a risk probability score to predict the development of OC in OPMD patients.
We established a salivary miRNA signature that can aid in diagnosing and predicting OC, revolutionizing the management of patients with OPMD. Together, our results shed new light on the management of OC by salivary miRNAs to the clinical utility of using miRNAs derived from saliva samples.
... During the COVID-19 pandemic, the urgency for rapid, non-invasive, remote testing has come to the forefront, in which salivary diagnostics is showing promise as an alternative diagnostic medium to blood and tumour tissue testing [68]. Salivary diagnostics is still in a research phase but is expected to transform healthcare practice because of its ease of collection and the ability to be done at the conform of one's home [160]. ...
Head and Neck cancers (HNC) are a heterogeneous group of upper aero-digestive tract cancer and account for 931,922 new cases and 467,125 deaths worldwide. About 90% of these cancers are of squamous cell origin (HNSCC). HNSCC is associated with excessive tobacco and alcohol consumption and infection with oncogenic viruses. Genotyping tumour tissue to guide clinical decision-making is becoming common practice in modern oncology, but in the management of patients with HNSCC, cytopathology or histopathology of tumour tissue remains the mainstream for diagnosis and treatment planning. Due to tumour heterogeneity and the lack of access to tumour due to its anatomical location, alternative methods to evaluate tumour activities are urgently needed. Liquid biopsy approaches can overcome issues such as tumour heterogeneity, which is associated with the analysis of small tissue biopsy. In addition, liquid biopsy offers repeat biopsy sampling, even for patients with tumours with access limitations. Liquid biopsy refers to biomarkers found in body fluids, traditionally blood, that can be sampled to provide clinically valuable information on both the patient and their underlying malignancy. To date, the majority of liquid biopsy research has focused on blood-based biomarkers, such as circulating tumour DNA (ctDNA), circulating tumour cells (CTCs), and circulating microRNA. In this review, we will focus on ctDNA as a biomarker in HNSCC because of its robustness, its presence in many body fluids, adaptability to existing clinical laboratory-based technology platforms, and ease of collection and transportation. We will discuss mechanisms of ctDNA release into circulation, technological advances in the analysis of ctDNA, ctDNA as a biomarker in HNSCC management, and some of the challenges associated with translating ctDNA into clinical and future perspectives. ctDNA provides a minimally invasive method for HNSCC prognosis and disease surveillance and will pave the way in the future for personalized medicine, thereby significantly improving outcomes and reducing healthcare costs.
... A single miRNA can bind to many mRNA sequences and each mRNA sequence is a target of many miRNAs. Compelling evidence has demonstrated that the expression of miRNA is commonly dysregulated in multiple disease states, including FA [10][11][12]. However, there are few studies analyzing miRNAs expression levels in tumor tissues, plasma or serum of FA patients [13,14]. ...
The overarching goal of this study is to predict the risk of developing oral squamous cell carcinoma (OSCC) in Fanconi anemia (FA) patients. We have compared the microRNA (miRNA, miR) expression levels in saliva samples from FA patients (n = 50) who are at a low-moderate and/or high risk of developing OSCC to saliva samples from healthy controls (n = 16). The miRNA expression levels in saliva samples were quantified using qPCR. We observed that miR-744, miR-150-5P, and miR-146B-5P had the best discriminatory capacity between FA patients and controls, with an area under the curve (AUC) of 94.0%, 92.9% and 85.3%, respectively. Our data suggest that miR-1, miR-146B-5P, miR-150-5P, miR-155-5P, and miR-744 could be used as panel to predict the risk of developing OSCC in FA patients, with a 89.3% sensitivity and a 68.2% specificity (AUC = 81.5%). Our preliminary data support the notion that the expression levels of salivary miRNAs have the potential to predict the risk of developing OSCC in FA patients and in the future may reduce deaths associated with OSCC.
... OSCC and OPSCC diagnosis was performed with biopsy and examination of formalin-fixed paraffin-embedded (FFPE) tissue sections by routine (Hematoxylin and eosin) stain using standard methodology. Most patients with OPSCC (>97 %) were HPV positive as tested by salivary HPV-16 and published by our team [7,[27][28][29]. Patients with oral premalignant disorders (OPMD) or cancer-free participants could be clinically adjudicated by a primary physician. ...
Objective:
Oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) can go undetected resulting in late detection and poor outcomes. We describe the development and validation of CancerDetect for Oral & Throat cancer™ (CDOT), to detect markers of OSCC and/or OPSCC within a high-risk population.
Material and methods:
We collected saliva samples from 1,175 individuals who were 50 years or older, or adults with a tobacco use history. 945 of those were used to train a classifier using machine learning methods, resulting in a salivary microbial and human metatranscriptomic signature. The classifier was then independently validated on the 230 remaining samples prospectively collected and unseen by the classifier, consisting of 20 OSCC (all stages), 76 OPSCC (all stages), and 134 negatives (including 14 pre-malignant).
Results:
On the validation cohort, the specificity of the CDOT test was 94 %, sensitivity was 90 % for participants with OSCC, and 84.2 % for participants with OPSCC. Similar classification results were observed among people in early stage (stages I & II) vs late stage (stages III & IV).
Conclusions:
CDOT is a non-invasive test that can be easily administered in dentist offices, primary care centres and specialised cancer clinics for early detection of OPSCC and OSCC. This test, having received FDA's breakthrough designation for accelerated review, has the potential to enable early diagnosis, saving lives and significantly reducing healthcare expenditure.
... 19,24 2.6 | miRNA isolation miRNA isolation was carried out using the NucleoSpin® miRNA isolation kit (Machnery-Nagel) according to a previously published protocol with a few minor modifications. 25,26 The fraction containing 200 μL of saliva with 800 μL of QIAzol (Qiagen) was used. The mixture was vortexed and incubated at room temperature for 5 min. ...
Background:
Despite the rising incidence, particularly of the human papillomavirus (HPV)-associated fraction of oropharyngeal cancer (OPC), there are no early detection methods for OPC. Considering the close association between saliva and head and neck cancers, this study was designed to investigate salivary micro RNA (miRNAs) associated with OPC, especially focusing on HPV-positive OPC.
Methods:
Saliva was collected from OPC patients at diagnosis and patients were clinically followed up ≤5 years. Salivary small RNA isolated from HPV-positive OPC patients (N = 6), and HPV-positive (N = 4) and negative controls (N = 6) were analysed by next-generation sequencing to identify dysregulated miRNAs. Discovered miRNAs were validated by quantitative PCR using two different assays in a separate cohort of patients (OPC = 91, controls = 92). The relative expression was calculated considering SNORD-96A as the normalizer. Candidate miRNAs with diagnostic and prognostic potential were evaluated by generalized logistic regression.
Results:
A panel consisting of nine miRNAs was identified to have the best diagnostic performance to discriminate HPV-positive OPC from HPV-positive controls (AUC- validation-1 = 94.8%, validation-2 = 98%). Further, a panel consisting of six miRNAs were identified to discriminate OPC from controls regardless of the HPV status (AUC- validation-1 = 77.2%, validation-2 = 86.7%). In addition, the downregulation of hsa-miR-7-5p was significantly associated with poor overall survival of OPC patients (HR = 0.638). A panel consisting of nine miRNAs were identified for the prediction of the overall survival of the OPC patients (log-rank test-p = 0.0008).
Conclusion:
This study highlights that salivary miRNAs can play an essential role in the detection and prognostication of OPC.
... Saliva was collected using two different methods, unstimulated whole mouth saliva ('drool', UWMS) and oral rinse, as previously described. [33][34][35][36] Briefly, before saliva collection, patients were requested to fast and to rinse their mouths. For unstimulated saliva, volunteers were asked to seat comfortably with the head slightly tilted forward for about 2-5 min, and then collect their saliva in 50 mL falcon tubes. ...
Background:
Despite aggressive treatment, more than 90% of glioblastoma (GBM) patients experience recurrences. GBM response to therapy is currently assessed by imaging techniques and tissue biopsy. However, difficulties with these methods may cause misinterpretation of treatment outcomes. Currently, no validated therapy response biomarkers are available for monitoring GBM progression. Metabolomics holds potential as a complementary tool to improve the interpretation of therapy responses to help in clinical interventions for GBM patients.
Methods:
Saliva and blood from GBM patients were collected pre and postoperatively. Patients were stratified conforming their progression-free survival (PFS) into favourable or unfavourable clinical outcomes (>9 months or PFS ≤ 9 months, respectively). Analysis of saliva (whole-mouth and oral rinse) and plasma samples was conducted utilising LC-QqQ-MS and LC-QTOF-MS to determine the metabolomic and lipidomic profiles. The data were investigated using univariate and multivariate statistical analyses and graphical LASSO-based graphic network analyses.
Results:
Altogether, 151 metabolites and 197 lipids were detected within all saliva and plasma samples. Among the patients with unfavourable outcomes, metabolites such as cyclic-AMP, 3-hydroxy-kynurenine, dihydroorotate, UDP and cis-aconitate were elevated, compared to patients with favourable outcomes during pre-and post-surgery. These metabolites showed to impact the pentose phosphate and Warburg effect pathways. The lipid profile of patients who experienced unfavourable outcomes revealed a higher heterogeneity in the abundance of lipids and fewer associations between markers in contrast to the favourable outcome group.
Conclusion:
Our findings indicate that changes in salivary and plasma metabolites in GBM patients can potentially be employed as less invasive prognostic biomarkers/biomarker panel but validation with larger cohorts is required.
... Saliva-based POC testing is one of the best options to increase accessibility, reduce expenses through early diagnosis of diseases and enable early treatment. The transition to salivary diagnostics is attractive because while upholding current testing standards, sample collection is non-invasive and risk free when compared to blood-based methods, leading to an increase in patient compliance for testing [14][15][16]. As seen during the current pandemic, it is clear that testing is required to be done in a remote or field setting to minimize the widespread of infection. ...
... Salivary diagnostics is attracting increasing attention in the field of POC due to its non-invasive nature, reduces the discomfort and risk posed to the patient, can be collected without specialised training (or self-collected by the patient) and stored at room temperature for transportation, allowing greater accessibility for screening in rural, Indigenous, and regional communities [14]. Saliva is also a more stable and a less complex matrix compared to blood and as such, is ideal for field testing [52]. ...
There has been a long-standing interest in point-of-care (POC) diagnostics as a tool to improve patient care because it can provide rapid, actionable results near the patient. Some of the successful examples of POC testing include lateral flow assays, urine dipsticks, and glucometers. Unfortunately, POC analysis is somewhat limited by the ability to manufacture simple devices to selectively measure disease specific biomarkers and the need for invasive biological sampling. Next generation POCs are being developed that make use of microfluidic devices to detect biomarkers in biological fluids in a non-invasive manner, addressing the above-mentioned limitations. Microfluidic devices are desirable because they can provide the ability to perform additional sample processing steps not available in existing commercial diagnostics. As a result, they can provide more sensitive and selective analysis. While most POC methods make use of blood or urine as a sample matrix, there has been a growing push to use saliva as a diagnostic medium. Saliva represents an ideal non-invasive biofluid for detecting biomarkers because it is readily available in large quantities and analyte levels reflect those in blood. However, using saliva in microfluidic devices for POC diagnostics is a relatively new and an emerging field. The overarching aim of this review is to provide an update on recent literature focused on the use of saliva as a biological sample matrix in microfluidic devices. We will first cover the characteristics of saliva as a sample medium and then review microfluidic devices that are developed for the analysis of salivary biomarkers.
... Infection with HPV has been associated with an increased risk of developing HNSCC. It is well known that the HPVpositive HNSCC and HPV-negative HNSCC are biologically and clinically different [26,27]. We then next investigated whether the presence of HPV genome or fragments thereof could impact the overall HNSCC cell N-glycome. ...
Glycosylation is the most common post-translational modification of proteins, and glycosylation changes at cell surfaces are frequently associated with malignant epithelia including head and neck squamous cell carcinoma (HNSCC). In HNSCC, 5-year survival remains poor, averaging around 50% globally: this is partly related to late diagnosis. Specific protein glycosylation signatures on malignant keratinocytes have promise as diagnostic and prognostic biomarkers and as therapeutic targets. Nevertheless, HNSCC-specific glycome is to date largely unknown. Herein, we tested six established HNSCC cell lines to capture the qualitative and semi-quantitative N-glycome using porous graphitized carbon liquid chromatography coupled to electrospray ionisation tandem mass spectrometry. Oligomannose-type N-glycans were the predominant features in all HNSCC cell lines analysed (57.5–70%). The levels of sialylated N-glycans showed considerable cell line-dependent differences ranging from 24 to 35%. Importantly, α2-6 linked sialylated N-glycans were dominant across most HNSCC cell lines except in SCC-9 cells where similar levels of α2-6 and α2-3 sialylated N-glycans were observed. Furthermore, we found that HPV-positive cell lines contained higher levels of phosphorylated oligomannose N-glycans, which hint towards an upregulation of lysosomal pathways. Almost all fucose-type N-glycans carried core-fucose residues with just minor levels (< 4%) of Lewis-type fucosylation identified. We also observed paucimannose-type N-glycans (2–5.5%), though in low levels. Finally, we identified oligomannose N-glycans carrying core-fucose residues and confirmed their structure by tandem mass spectrometry. This first systematic mapping of the N-glycome revealed diverse and specific glycosylation features in HNSCC, paving the way for further studies aimed at assessing their possible diagnostic relevance.
