Liquid biopsy-based microRNA models as potential biomarkers of bowel conditions.

MicroRNAs detected in liquid biopsies have demonstrated to have potential as biomarkers of benign and malignant bowel conditions. Numerous studies have reported on the combination of different microRNAs to improve the diagnostic capabilities in the detection of bowel conditions. This editorial will be reviewing the best scientific evidence available to this date on studies showing microRNA models with sensitivity and specificity values. Also, the current challenges on how to implement microRNAs in the routine clinical practice are exposed.

Integrity and quantity of salivary cell-free DNA as a potential molecular biomarker in oral cancer: A preliminary study.

BACKGROUND: Differences in cell-free DNA (cfDNA) fragments have been described as a valuable tool to distinguish cancer patients from healthy individuals. We aim to investigate the concentration and integrity of cfDNA fragments in saliva from oral squamous cell carcinoma (OSCC) patients and healthy individuals in order to explore their value as diagnostic biomarkers. METHODS: Saliva samples were collected from a total of 34 subjects (19 OSCC patients and 15 healthy controls). The total concentration of salivary cfDNA (scfDNA) was determined using a fluorometry method and quantitative real-time polymerase chain reaction (qPCR). To evaluate the scfDNA quantity and integrity, qPCR targeting Arthobacter luteus (ALU) sequences at three amplicons of different lengths (60, 115, and 247 bp, respectively) was carried out. ScfDNA integrity indexes (ALU115/ALU60 and ALU247/ALU60) were calculated as the ratio between the absolute concentration of the longer amplicons 115 bp and 247 bp and the total scfDNA amount (amplicon 60 bp). RESULTS: The total scfDNA concentration (ALU60) was higher in OSCC than in healthy donors, but this trend was not statistically significant. The medians of scfDNA integrity indexes, ALU115/ALU60 and ALU247/ALU60, were significantly higher in OSCC, showing area under the curve values of 0.8211 and 0.7018, respectively. CONCLUSION: Our preliminary results suggest that scfDNA integrity indexes (ALU115/ALU60 and ALU247/ALU60) have potential as noninvasive diagnostic biomarkers for OSCC.

Genome-wide DNA methylation profiling in tongue squamous cell carcinoma.

OBJECTIVES: To provide a comprehensive characterization of DNA methylome of oral tongue squamous cell carcinoma (OTSCC) and identify novel tumor-specific DNA methylation markers for early detection using saliva. MATERIAL AND METHODS: Genome-wide DNA methylation analysis including six OTSCC matched adjacent non-tumoral tissue and saliva was performed using Infinium MethylationEPIC array. Differentially methylated levels of selected genes in our OTSCC cohort were further validated using OTSCC methylation data from The Cancer Genome Atlas database (TCGA). The methylation levels of a set of tumor-specific hypermethylated genes associated with a downregulated expression were evaluated in saliva. Receiver operating characteristic (ROC) curves were performed to assess the diagnostic value of DNA methylation markers. RESULTS: A total of 25,890 CpGs (20,505 hypomethylated and 5385 hypermethylated) were differentially methylated (DMCpGs) between OTSCC and adjacent non-tumoral tissue. Hypermethylation of 11 tumor-specific genes was validated in OTSCC TCGA cohort. Of these 11 genes, A2BP1, ANK1, ALDH1A2, GFRA1, TTYH1, and PDE4B were also hypermethylated in saliva. These six salivary methylated genes showed high diagnostic accuracy (≥0.800) for discriminating patients from controls. CONCLUSIONS: This is the first largest genome-wide DNA methylation study on OTSCC that identifies a group of novel tumor-specific DNA methylation markers with diagnostic potential in saliva.

Methods for the Detection of Circulating Biomarkers in Cancer Patients.

Liquid biopsy has emerged as one of the main pillars for personalized oncology. The term englobes body-fluid samples which contain tumor-derived material such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and circulating extracellular vesicles (cEVs). Potential clinical application of liquid biopsy analyses includes cancer screening, detection of minimal residual disease and recurrence, therapy selection, and evaluation of acquired resistance. Despite the great developments of technology focused on circulating biomarkers characterization only cfDNA testing is nowadays implemented for the therapy selection in some advanced tumors. This can be partially explained by the fact that there is still a lack of global standardization of procedures both in the pre-analytical and analytical steps. In the present chapter, we summarize the different strategies for addressing the study of liquid biopsy taking into account their pros and cons to be applied in a clinical context and we also discuss the main technical and clinical challenges in the field of circulating biomarkers and personalized oncology.

Liquid Biopsy in Oral Cancer.

Oral cancer is one of the most prevalent forms of cancer worldwide. Carcinogenesis is a complex process, in which heterogeneity plays an important role in the development and progression of the disease. This review provides an overview of the current biological and clinical significance of circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), and exosomes for diagnosis and prognosis of oral cancer. We highlight the importance of liquid biopsy­using blood and saliva­which represents a potential alternative to solid biopsy for diagnosis and prognosis. Moreover, liquid biomarkers allow for the real-time monitoring of tumour evolution and therapeutic responses, initiating the era of personalized medicine. However, in oral cancer, the impact of liquid biopsies in clinical settings is still limited, requiring further studies to discover the best scenario for its clinical use.

Anatomical variations of the greater palatine canal in cone-beam computed tomography.

PURPOSE: To analyze anatomical variations of the greater palatine canal (GPC) using cone beam computed tomography (CBCT) images. METHODS: This study included 110 CBCTs. Axial slices were used to determine the shape of GPC and the number of lesser palatine foramina (LPF). Sagittal slices were used to assess the shape of the GPC and the number of lesser palatine canals (LPCs). RESULTS: The most prevalent axial and sagittal GPC shapes were oval (46.36%) and hourglass (23.64%). Most GPCs presented one LPF (47.27%) and one LPC (90.91%). CONCLUSIONS: GPC anatomy is highly variable. CBCT is a useful tool for evaluating the anatomical variations of GPC.

Cancer Salivary Biomarkers for Tumours Distant to the Oral Cavity.

The analysis of saliva as a diagnostic approach for systemic diseases was proposed just two decades ago, but recently great interest in the field has emerged because of its revolutionary potential as a liquid biopsy and its usefulness as a non-invasive sampling method. Multiple molecules isolated in saliva have been proposed as cancer biomarkers for diagnosis, prognosis, drug monitoring and pharmacogenetic studies. In this review, we focus on the current status of the salivary diagnostic biomarkers for different cancers distant to the oral cavity, noting their potential use in the clinic and their applicability in personalising cancer therapies.

Morphometric differences of nasopalatine canal based on 3D classifications: descriptive analysis on CBCT.

PURPOSE: This descriptive retrospective study analyzed differences among sagittal, coronal and axial NC groups based on the dimensions of nasopalatine canal (NC), buccal bone plate (BBP) and palatal bone plate (PBP) to canal. METHODS: Measurements were made on 224 CBCTs for NC, BBP and PBP on the three anatomic planes at three levels: level 1, when the incisive foramen is completely closed on the axial plane; level 2, at the midpoint of NC length (NCL) on the sagittal plane; and level 3, at the foramina of Stenson on the sagittal plane. ANOVA tests with post hoc tests were used. The intraclass correlation coefficient and Kappa test were used for evaluating the intraobserver agreement. RESULTS: Regarding coronal classification, these significant differences were found: BBP length (BL)level 1 was lower for the two parallel canals group; PBP length (PL)level 1 was lower for single canal group; and NCL was lower for Y-type canal group. Regarding axial classification, these significant differences were found: LPlevel 1 was lower for 3.1-3 group; PBP width (PW)level 3 was the greatest for 3.1-3; and LPlevel 3 was lower for 1.1. CONCLUSIONS: Presurgical evaluation with CBCT in premaxillae region should include analysis on coronal and axial planes and not only on sagittal plane seeing as morphometric differences were found on coronal and axial planes. Following the morphological coronal classification, two parallel canals presented a higher NCL, a higher LP and a lower LV at inferior edge of alveolar ridge.

Anatomical characteristics and visibility of mental foramen and accessory mental foramen: Panoramic radiography vs. cone beam CT.

BACKGROUND: The mental foramen (MF) is a small foramen located in the anterolateral region of the mandible through which the mental nerve and vessels emerge. The knowledge on the anatomic characteristics and variations of MF is very important in surgical procedures involving that area. The aim of this study was two-fold: firstly, to analyze the anatomic characteristics of MF and the presence of accessory mental foramen (AMF) using CBCT and, secondly, to compare the capability of CBCT and PAN in terms of MF and AMF visualization, as well as influencing factors. MATERIAL AND METHODS: A sample of 344 CBCT scans was analyzed for presence and characteristics (i.e. diameter, area, shape, exit angle) of MF and AMF. Subsequently, corresponding PANs were analyzed to ascertain whether MF and AMF were visible. RESULTS: Out of the 344 patients, 344 (100%) MFs and 45 (13%) AMFs were observed on CBCT. Regarding gender, MF diameter and area, MF-MIB and MF-MSB distances, and exit angle were all significantly higher in males. Also, statistically significant differences were found in terms of age and dental status. Statistically significant differences in MF long and short diameters and MF area were found with respect to AMF presence (p=.021, p=.008, p=.021). Only 83.87% of the MFs and 45.83% of the AMFs identified on CBCT were also visible on PANs. MF diameter, shape, exit angle, and age had a significant influence on MF visualization on PAN (B=.43, p=.005; B=-.55, p=.020; B=.20, p=.008; B=.61, p=.005). CONCLUSIONS: PAN is not an adequate technique to properly identify MF and AMF. Diameter, shape, exit angle, and age are all factors influencing MF visualization on PAN images. For surgery involving the MF anatomical region, a preoperative radiological study with CBCT is of crucial importance to avoid complications.

Haemodynamic response and psychometric test measuring dental anxiety in a spanish population in galicia.

PURPOSE: To evaluate haemodynamic changes during dental checkups and preventive treatments and establish the relationship between haemodynamic parameters and psychometric tests. MATERIALS AND METHODS: Seventy-one paediatric patients (39 boys and 32 girls ages 6 to 14 years, mean age 8.04) scheduled to receive dental procedures fulfilled the inclusion criteria for this prospective study. Anxiety was measured at three time points by using the following haemodynamic parameters: heart rate (HR), systolic and diastolic blood pressure (SBP and DBP) and oxygen saturation (SaO2). The Children's Fear Survey Schedule Dental Subscale (CFSS-DS) and the Facial Image Scale (FIS) were used as psychometric tests before and after child attendance, respectively. RESULTS: Variations in HR and BP during dental procedures were statistically significant while SaO2 values were not. The highest mean HR, SBP and DBP values were obtained during dental procedures, while the lowest SBP and DBP were recorded at baseline. HR and BP changes (r = 0.32, P < 0.01) were statistically correlated. The most anxious children based on both CFSS-DS and FIS scales also had the highest mean HR. Changes in BP and oxygen saturation parameters were found to have no relationship with anxiety groups in either scale. CONCLUSIONS: Dental checkups and preventive treatments cause significant changes in HR and BP. However, the CFSS-DS questionnaire does not adequately predict these changes.

Liquid biopsies based on cell-free DNA as a potential biomarker in head and neck cancer.

In the era of 'precision medicine', liquid biopsies based on cell-free DNA (cfDNA) have emerged as a promising tool in the oncology field. cfDNA from cancer patients is a mixture of tumoral (ctDNA) and non-tumoral DNA originated from healthy, cancer and tumor microenvironmental cells. Apoptosis, necrosis, and active secretion from extracellular vesicles represent the main mechanisms of cfDNA release into the physiological body fluids. Focused on HNC, two main types of cfDNA can be identified: the circulating cfDNA (ccfDNA) and the salivary cfDNA (scfDNA). Numerous studies have reported on the potential of cfDNA analysis as potential diagnostic, prognostic, and monitoring biomarker for HNC. Thus, ctDNA has emerged as an attractive strategy to detect cancer specific genetic and epigenetic alterations including DNA somatic mutations and DNA methylation patterns. This review aims to provide an overview of the up-to-date studies evaluating the value of the analysis of total cfDNA, cfDNA fragment length, and ctDNA analysis at DNA mutation and methylation level in HNC patients.

Somatic mutations in tumor and plasma of locoregional recurrent and/or metastatic head and neck cancer using a next-generation sequencing panel: A preliminary study.

BACKGROUND: We explore the utility of TruSight Tumor 170 panel (TST170) for detecting somatic mutations in tumor and cfDNA from locoregional recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC). METHODS: Targeted NGS of tumor DNA and plasma cfDNA was performed using TST170 panel. In addition, a set of somatic mutations previously described in HNSCC were selected for validating in tumor, plasma, and saliva by digital droplet PCR. RESULTS: The TST170 panel identified 13 non-synonymous somatic mutations, of which five were detected in tumoral tissue, other five in plasma cfDNA, and three in both tissue and plasma cfDNA. Of the eight somatic mutations identified in tissue, three were also identified in plasma cfDNA, showing an overall concordance rate of 37.5%. CONCLUSIONS: This preliminary study shows the possibility to detect somatic mutations in tumor and plasma of HNSCC patients using a single assay that would facilitate the clinical implementation of personalized medicine in the clinic.

Dentin as a suitable bone substitute comparable to ß-TCP--an experimental study in mice.

Microvascular supply is of fundamental importance to the survival and integration of grafting. Since the autogenous bone is still the gold standard for osseous augmentation, the aim of this study was to analyze the initial osseous, angiogenic and inflammatory response and subsequent osseointegration after implantation of dentin and beta-tricalcium phosphate (ß-TCP) scaffolds into the calvaria chamber of balb/c mice comparing with bone. The vascularisation of perforated implants of dentin (n=8), ß-TCP (n=8) and isogenic calvarial bone (n=8) displaying pores similar in size and structure was analyzed in vivo using intravital fluorescence microscopy. In additional animals (n=24) the osseointegration of dentin, ß-TCP and bone implants was assessed by fluorochrome sequential labelling of growing bone for up to 12 weeks. Animals without implants served as controls. Intravital fluorescence microscopy revealed that implantation of bone substitutes caused an only mild inflammatory response. Comparable to isogenic bone both dentin and ß-TCP scaffolds were found nearly completely vascularized by day 22 and osseointegrated within 12 weeks. In conclusion, dentin and ß-TCP scaffolds are similar to isogenic bone in terms of inflammatory and neovascularization response, highlighting their potential utility in regeneration of bone defects.

Age Estimation Using Maxillary Central Incisor Analysis on Cone Beam Computed Tomography Human Images.

Forensic dentistry plays an important role in human identification, and dental age estimation is an important part of the process. Secondary dentin deposition throughout an individual's lifetime and consequent modification in teeth anatomy is an important parameter for age estimation procedures. The aim of the present study was to develop regression equations to determine age in adults by means of linear measurements and ratios on sagittal, coronal and axial slices of maxillary central incisors using cone bean computed tomography (CBCT). Multiplanar measurements of upper central incisors were taken for a sample of 373 CBCTs. Subsequently, one-way analysis of variance (ANOVA) and multivariate linear regressions were performed for age estimation. The equations obtained from axial linear measurements and ratios presented a standard error of the estimate (SEE) of ±10.9 years (R2 = 0.49), and a SEE of ±10.8 years (R2 = 0.50), respectively. The equation obtained for multiplanar linear measurements presented a SEE of ±10.9 years (R2 = 0.52), while the equation for multiplanar ratios presented a SEE of ±10.7 years (R2 = 0.51). Thus, CBCT measurements on upper central incisors were found to be an acceptable method for age estimation. Horizontal measurements, especially pulp measurements, improve the accuracy of age estimate equations.

Salivary DNA Methylation as an Epigenetic Biomarker for Head and Neck Cancer. Part II: A Cancer Risk Meta-Analysis.

Aberrant methylation of tumor suppressor genes has been reported as an important epigenetic silencer in head and neck cancer (HNC) pathogenesis. Here, we performed a comprehensive meta-analysis to evaluate the overall and specific impact of salivary gene promoter methylation on HNC risk. The methodological quality was assessed using the Newcastle-Ottawa scale (NOS). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the association and Egger's and Begg's tests were applied to detect publication bias. The frequency of salivary DNA promoter methylation was significantly higher in HNC patients than in healthy controls (OR: 8.34 (95% CI = 6.10-11.39; p < 0.01). The pooled ORs showed a significant association between specific tumor-related genes and HNC risk: p16 (3.75; 95% CI = 2.51-5.60), MGMT (5.72; 95% CI = 3.00-10.91), DAPK (5.34; 95% CI = 2.18-13.10), TIMP3 (3.42; 95% CI = 1.99-5.88), and RASSF1A (7.69; 95% CI = 3.88-15.23). Overall, our meta-analysis provides precise evidence on the association between salivary DNA promoter hypermethylation and HNC risk. Thus, detection of promoter DNA methylation in saliva is a potential biomarker for predicting HNC risk.

The Dental Aesthetic Index and Its Association with Dental Caries, Dental Plaque and Socio-Demographic Variables in Schoolchildren Aged 12 and 15 Years.

The Dental Aesthetic Index (DAI) was determined in 12- and 15-year-old schoolchildren to ascertain the prevalence of malocclusion and to assess its association with dental caries experience, dental plaque accumulation, and socio-demographic variables. We performed a cross-sectional study with a stratified two-stage sampling design. An oral health survey and oral examination were conducted, and socio-demographic data were recorded. The sample comprised 1453 schoolchildren aged 12 (868) and 15 (585). These two samples were analyzed separately because statistically significant differences were found: the 12-year-old age group displayed a higher frequency of schoolchildren who attended state-run public schools (p = 0.004) and belonged to a lower social class (p = 0.001); the 15-year-old age group registered higher levels of caries (p = 0.001) and lower levels of dental plaque (p < 0.001). The malocclusion was 9.5% higher (p = 0.001), and the global mean DAI score was likewise higher among the 12-year-olds (p < 0.001). The multivariate regression analysis not only showed that caries and dental plaque were the variables that were the most strongly associated with malocclusion, but that caries (OR = 1.5) and dental plaque (OR > 2) were also risk factors for malocclusion in both groups. In conclusion, this study revealed a higher prevalence of malocclusion and dental plaque at age 12. A higher risk of caries and dental plaque was found to be related to the presence of malocclusion in both age groups.

Detection of the Accessory Mental Foramina on Human Mandibles Using Cone-beam Computed Tomography: A Systematic Review and Meta-analysis.

INTRODUCTION: This study aimed to perform a systematic review and meta-analysis on accessory mental foramen (AMF) research using cone-beam computed tomographic (CBCT) imaging. METHODS: A systematic review was performed in PubMed, Embase, Thomas Reuter's Web of Science, Scopus, and ScienceDirect databases according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis statement. Articles focusing on AMF prevalence and location using CBCT imaging were selected without language restrictions. Studies reporting pooled results only or presenting any pathology in the area surrounding the mental foramen (MF) were excluded. A meta-analysis using random effects was performed. RESULTS: The present meta-analysis included a total of 46 articles involving 21,761 subjects. The overall pooled AMF prevalence was 7.87% (95% confidence interval [CI], 6.69-9.24) in subjects and 4.75% (95% CI, 3.79-5.95) in hemimandibles (n = 31,158). AMF presence was most commonly unilateral, reaching 90.15% (95% CI, 82.98-94.49). AMFs were significantly more frequent in right hemimandibles (χ2 = 5.20, P < .05) and were most commonly located posterior and inferior to the MF. However, AMFs superior to the MF were also observed in 47.43% (95% CI, 38.45-56.58) of cases. The studies conducted over the last 3 years showed significantly higher AMF prevalence levels (χ2 = 5.12, P < .05). CONCLUSIONS: Our meta-analysis demonstrates that AMF prevalence is considerable and should not be underestimated. AMFs are most frequently located in right hemimandibles. The presence of AMFs superior to the MF is frequent. Around 3% of people present superior AMFs. This fact puts those patients at greater risk for injury when performing periapical surgery in this area.

Saliva Gene Promoter Hypermethylation as a Biomarker in Oral Cancer.

Oral carcinogenesis is a multistep process characterized by a summation of multiple genetic and epigenetic alterations in key regulatory genes. The silencing of genes by aberrant promoter hypermethylation is thought to be an important epigenetic event in cancer development and progression which has great potential as a biomarker for early diagnosis, tumor molecular subtyping, prognosis, monitoring, and therapy. Aberrant DNA methylation has been detected in different liquid biopsies, which may represent a potential alternative to solid biopsies. The detection of methylated genes in saliva may have clinical application for noninvasive oral cancer screening and early diagnosis. Here, we review the current evidence on gene promoter hypermethylation in saliva.

Salivary DNA Methylation as an Epigenetic Biomarker for Head and Neck Cancer. Part I: A Diagnostic Accuracy Meta-Analysis.

DNA hypermethylation is an important epigenetic mechanism for gene expression inactivation in head and neck cancer (HNC). Saliva has emerged as a novel liquid biopsy representing a potential source of biomarkers. We performed a comprehensive meta-analysis to evaluate the overall diagnostic accuracy of salivary DNA methylation for detecting HNC. PubMed EMBASE, Web of Science, LILACS, and the Cochrane Library were searched. Study quality was assessed by the Quality Assessment for Studies of Diagnostic Accuracy-2, and sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (dOR), and their corresponding 95% confidence intervals (CIs) were calculated using a bivariate random-effect meta-analysis model. Meta-regression and subgroup analyses were performed to assess heterogeneity. Eighty-four study units from 18 articles with 8368 subjects were included. The pooled sensitivity and specificity of salivary DNA methylation were 0.39 and 0.87, respectively, while PLR and NLR were 3.68 and 0.63, respectively. The overall area under the curve (AUC) was 0.81 and the dOR was 8.34. The combination of methylated genes showed higher diagnostic accuracy (AUC, 0.92 and dOR, 36.97) than individual gene analysis (AUC, 0.77 and dOR, 6.02). These findings provide evidence regarding the potential clinical application of salivary DNA methylation for HNC diagnosis.

Salivary biomarkers for cancer diagnosis: a meta-analysis.

Background: Saliva represents a promising non-invasive source of novel biomarkers for diagnosis and prognosis cancer. This meta-analysis evaluates the diagnostic value of salivary biomarkers for detection of malignant non-oral tumours to better define the value of saliva as an alternative liquid biopsy.Materials and methods: We performed a systematic review and meta-analysis. PubMed, Embase, LILACS and the Cochrane Library were searched to identify articles that examined the potential of salivary biomarkers for the diagnosis of malignant non-oral tumours. To assess the overall accuracy, we calculated the diagnostic odds ratio (DOR), area under hierarchical summary receiver operating characteristic (AUC) curve, sensitivity, specificity, positive likelihood ratio (PLR) and negative likelihood ratio (NLR) using a random- or fixed-effects model. Heterogeneity and publication bias were assessed. Statistical tests were two-sided.Results: One hundred fifty-five study units from 29 articles with 11,153 subjects were included. The pooled sensitivity, specificity, PLR, NLR, DOR and AUC were 0.76 (95% confidence intervals (CI), 0.74-0.77), 0.76 (95% CI, 0.75-0.77), 3.22 (95% CI, 2.92-3.55), 0.31 (95% CI, 0.28-0.34), 13.42 (95% CI, 12.28-15.96) and 0.85 (95% CI, 0.84-0.87), respectively.Conclusion: Salivary biomarkers may be potentially used for non-invasive diagnosis of malignant non-oral tumours.Key messagesThis meta-analysis evaluates the diagnostic value of salivary biomarkers for detection of malignant non-oral tumours to better define the role of saliva as an alternative liquid biopsy.Salivary biomarkers showed 85% accuracy for cancer distant to the oral cavity.Saliva represents a promising non-invasive source of novel biomarkers in cancer.